U.S. patent application number 13/406154 was filed with the patent office on 2013-07-04 for gene therapy for sulfatase deficiency.
This patent application is currently assigned to SHIRE HUMAN GENETIC THERAPIES, INC.. The applicant listed for this patent is Andrea Ballabio, Maria Pia Cosma, Thomas Dierks, Michael W. Heartlein, Bernhard Schmidt, Kurt von Figura. Invention is credited to Andrea Ballabio, Maria Pia Cosma, Thomas Dierks, Michael W. Heartlein, Bernhard Schmidt, Kurt von Figura.
Application Number | 20130172403 13/406154 |
Document ID | / |
Family ID | 32869644 |
Filed Date | 2013-07-04 |
United States Patent
Application |
20130172403 |
Kind Code |
A1 |
von Figura; Kurt ; et
al. |
July 4, 2013 |
GENE THERAPY FOR SULFATASE DEFICIENCY
Abstract
This invention relates to methods and compositions for the
diagnosis and treatment of Multiple Sulfatase Deficiency (MSD) as
well as other sulfatase deficiencies. More specifically, the
invention relates to isolated molecules that modulate
post-translational modifications on sulfatases. Such modifications
are essential for proper sulfatase function.
Inventors: |
von Figura; Kurt;
(Gottingen, DE) ; Schmidt; Bernhard; (Gottingen,
DE) ; Dierks; Thomas; (Gottingen, DE) ;
Heartlein; Michael W.; (Boxborough, MA) ; Cosma;
Maria Pia; (Naples, IT) ; Ballabio; Andrea;
(Naples, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
von Figura; Kurt
Schmidt; Bernhard
Dierks; Thomas
Heartlein; Michael W.
Cosma; Maria Pia
Ballabio; Andrea |
Gottingen
Gottingen
Gottingen
Boxborough
Naples
Naples |
MA |
DE
DE
DE
US
IT
IT |
|
|
Assignee: |
SHIRE HUMAN GENETIC THERAPIES,
INC.
Lexington
MA
|
Family ID: |
32869644 |
Appl. No.: |
13/406154 |
Filed: |
February 27, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10775678 |
Feb 10, 2004 |
8227212 |
|
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13406154 |
|
|
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60447747 |
Feb 11, 2003 |
|
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Current U.S.
Class: |
514/44R ;
435/320.1; 435/455 |
Current CPC
Class: |
A61P 19/08 20180101;
C12N 9/0071 20130101; C12Y 108/99 20130101; A61K 38/44 20130101;
A61P 3/00 20180101; A61P 15/08 20180101; A61K 2039/53 20130101;
A61P 15/00 20180101; A61P 19/00 20180101; Y02A 50/30 20180101; A61P
25/00 20180101; C12Y 301/06013 20130101; A61P 25/02 20180101; C12N
9/0051 20130101; A61P 43/00 20180101; A61K 38/465 20130101; A61K
38/00 20130101; A61P 17/00 20180101; C12N 9/16 20130101; A61K
48/0066 20130101 |
Class at
Publication: |
514/44.R ;
435/455; 435/320.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00 |
Claims
1.-85. (canceled)
86. A gene therapy method of treating a sulfatase deficiency in a
subject, the method comprising delivering to a cell of the subject
a vector comprising a polynucleotide encoding a Formylglycine
Generating Enzyme (FGE) comprising an amino acid sequence having at
least 80% identity to the amino acid sequence of amino acids 34-374
of SEQ ID NO:2, wherein the FGE has C.sub..alpha.-formylglycine
generating activity.
87. The gene therapy method of claim 86, wherein the FGE comprises
an amino acid sequence having at least 95% identity to the amino
acid sequence of amino acids 34-374 of SEQ ID NO:2.
88. The gene therapy method of claim 86, wherein the FGE comprises
the amino acid sequence of amino acids 34-374 of SEQ ID NO:2.
89. The gene therapy method of claim 86, wherein the sulfatase
deficiency is selected from Mucopolysaccharidosis II (MPS II;
Hunter Syndrome), Mucopolysaccharidosis IIIA (MPS IIIA; Sanfilippo
Syndrome A), Mucopolysaccharidosis VIII (MPS VIII),
Mucopolysaccharidosis IVA (MPS IVA; Morquio Syndrome A),
Mucopolysaccharidosis VI (MPS VI; Maroteaux-Lamy Syndrome),
Metachromatic Leukodystrophy (MLD), X-linked Recessive
Chondrodysplasia Punctata 1, or X-linked Ichthyosis (Steroid
Sulfatase Deficiency).
90. The gene therapy method of claim 89, wherein the sulfatase
deficiency is Mucopolysaccharidosis IIIA (MPS IIIA; Sanfilippo
Syndrome A).
91. The gene therapy method of claim 89, wherein the sulfatase
deficiency is Mucopolysaccharidosis II (MPS II; Hunter
Syndrome).
92. The gene therapy method of claim 89, wherein the sulfatase
deficiency is Metachromatic Leukodystrophy (MLD).
93. The gene therapy method of claim 86, wherein the method further
comprises delivering to the cell a polynucleotide encoding a
cysteine-type sulfatase.
94. The gene therapy method of claim 93, wherein the polynucleotide
encoding the cysteine-type sulfatase is delivered on a separate
vector.
95. The gene therapy method of claim 93, wherein the cysteine-type
sulfatase is selected from the group consisting of Iduronate
2-Sulfatase, Sulfamidase, N-Acetylgalactosamine 6-Sulfatase,
N-Acetylglucosamine 6-Sulfatase, Arylsulfatase A, Arylsulfatase B,
Arylsulfatase C, Arylsulfatase D, Arylsulfatase E, Arylsulfatase F,
Arylsulfatase G, HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, and
HSulf-6.
96. The gene therapy method of claim 86, wherein the vector is a
viral vector.
97. The gene therapy method of claim 96, wherein the viral vector
comprises nucleic acid sequences from adenovirus, adeno-associated
virus, or retrovirus.
98. The gene therapy method of claim 97, wherein the viral vector
comprises nucleic acid sequences from adeno-associated virus.
99. The gene therapy method of claim 86, wherein the delivering
step comprises in vivo transduction of the cell of the subject.
100. The gene therapy method of claim 99, wherein the cell is
selected from the group consisting of fibroblasts, keratinocytes,
epithelial cells, endothelial cells, glial cells, neural cells,
blood cells, lymphocytes, bone marrow cells, muscle cells, and
precursors thereof.
101. The gene therapy method of claim 99, wherein the cell is a
neuron.
102. The gene therapy method of claim 86, wherein the delivering
step comprises in vitro transduction of the cell of the
subject.
103. The gene therapy method of claim 102, wherein the cell is a
primary cell isolated from the subject.
104. The gene therapy method of claim 86, wherein the
polynucleotide encoding the FGE is operably linked to an gene
expression regulatory sequence.
105. The gene therapy method of claim 86, wherein the gene
expression regulatory sequence is a mammalian or viral
promoter.
106. A composition for gene therapy for a sulfatase deficiency
comprising a vector comprising a polynucleotide encoding a
Formylglycine Generating Enzyme (FGE) comprising an amino acid
sequence having at least 80% identity to the amino acid sequence of
amino acids 34-374 of SEQ ID NO:2, wherein the FGE has
C.sub..alpha.-formylglycine generating activity.
107. The composition of claim 106, wherein the FGE comprises an
amino acid sequence having at least 95% identity to the amino acid
sequence of amino acids 34-374 of SEQ ID NO:2.
108. The composition of claim 106, wherein the FGE comprises the
amino acid sequence of amino acids 34-374 of SEQ ID NO:2.
109. The composition of claim 106, wherein the composition further
comprises a polynucleotide encoding a cysteine-type sulfatase.
110. The composition of claim 109, wherein the polynucleotide
encoding the cysteine-type sulfatase is encoded by a separate
vector.
111. The composition of claim 109, wherein the cysteine-type
sulfatase is selected from the group consisting of Iduronate
2-Sulfatase, Sulfamidase, N-Acetylgalactosamine 6-Sulfatase,
N-Acetylglucosamine 6-Sulfatase, Arylsulfatase A, Arylsulfatase B,
Arylsulfatase C, Arylsulfatase D, Arylsulfatase E, Arylsulfatase F,
Arylsulfatase G, HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, and
HSulf-6
112. The composition of claim 106, wherein the vector is a viral
vector.
113. The composition of claim 112, wherein the viral vector
comprises nucleic acid sequences from adenovirus, adeno-associated
virus, or retrovirus.
114. The composition of claim 113, wherein the viral vector
comprises nucleic acid sequences from adeno-associated virus.
115. The composition of claim 106, wherein the polynucleotide
encoding the FGE is operably linked to an gene expression
regulatory sequence.
116. The composition of claim 115, wherein the gene expression
regulatory sequence is a mammalian or viral promoter.
117. The composition of claim 106, wherein the sulfatase deficiency
is selected from Mucopolysaccharidosis II (MPS II; Hunter
Syndrome), Mucopolysaccharidosis IIIA (MPS IIIA; Sanfilippo
Syndrome A), Mucopolysaccharidosis VIII (MPS VIII),
Mucopolysaccharidosis IVA (MPS IVA; Morquio Syndrome A),
Mucopolysaccharidosis VI (MPS VI; Maroteaux-Lamy Syndrome),
Metachromatic Leukodystrophy (MLD), X-linked Recessive
Chondrodysplasia Punctata 1, or X-linked Ichthyosis (Steroid
Sulfatase Deficiency).
118. The composition of claim 117, wherein the sulfatase deficiency
is Mucopolysaccharidosis IIIA (MPS IIIA; Sanfilippo Syndrome
A).
119. The composition of claim 117, wherein the sulfatase deficiency
is Mucopolysaccharidosis II (MPS II; Hunter Syndrome).
120. The composition of claim 117, wherein the sulfatase deficiency
is Metachromatic Leukodystrophy (MLD).
121. A gene therapy vector comprising a polynucleotide encoding a
Formylglycine Generating Enzyme (FGE) comprising an amino acid
sequence having at least 80% identity to the amino acid sequence of
amino acids 34-374 of SEQ ID NO:2, wherein the FGE has
C.sub..alpha.-formylglycine generating activity.
122. The gene therapy vector of claim 121, wherein the FGE
comprises an amino acid sequence having at least 95% identity to
the amino acid sequence of amino acids 34-374 of SEQ ID NO:2.
123. The gene therapy vector of claim 121, wherein the
polynucleotide encoding the FGE is operably linked to an gene
expression regulatory sequence.
124. The gene therapy vector of claim 123, wherein the gene
expression regulatory sequence is a mammalian or viral
promoter.
125. A cell transduced with a gene therapy vector comprising a
polynucleotide encoding a Formylglycine Generating Enzyme (FGE)
comprising an amino acid sequence having at least 80% identity to
the amino acid sequence of amino acids 34-374 of SEQ ID NO:2,
wherein the FGE has C.sub..alpha.-formylglycine generating
activity.
126. The cell of claim 125, wherein the cell is a primary cell.
127. The cell of claim 125, wherein the cell is a secondary
cell.
128. The cell of claim 125, wherein the cell is selected from the
group consisting of fibroblasts, keratinocytes, epithelial cells,
endothelial cells, glial cells, neural cells, blood cells,
lymphocytes, bone marrow cells, muscle cells, and precursors
thereof.
129. The cell of claim 125, wherein the vector is integrated into
the genome of the cell.
130. The cell of claim 125, wherein the vector functions in an
extrachromosomal fashion.
131. The cell of claim 125, wherein the polynucleotide encoding the
FGE is operably linked to an gene expression regulatory
sequence.
132. The cell of claim 131, wherein the gene expression regulatory
sequence is a mammalian or viral promoter.
133. The cell of claim 132, wherein the mammalian promoter is an
endogenous promoter of the cell.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of prior filed, co-pending
application Ser. No. 10/775,678, filed Feb. 10, 2004, which claims
priority under 35 U.S.C. .sctn.119(e) from Provisional U.S. Patent
Application Ser. No. 60/447,747, filed Feb. 11, 2003, and entitled
DIAGNOSIS AND TREATMENT OF MULTIPLE SULFATASE DEFICIENCY AND OTHER
SULFATASE DEFICIENCIES. The contents of each application are hereby
expressly incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates to methods and compositions for the
diagnosis and treatment of Multiple Sulfatase Deficiency (MSD) as
well as other sulfatase deficiencies. More specifically, the
invention relates to isolated molecules that modulate
post-translational modifications on sulfatases. Such modifications
are essential for proper sulfatase function.
BACKGROUND OF THE INVENTION
[0003] Sulfatases are members of a highly conserved gene family,
sharing extensive sequence homology (Franco, B., et al., Cell,
1995, 81:15-25; Parenti, G., et al., Curr. Opin. Gen. Dev., 1997,
7:386-391), a high degree of structural similarity (Bond, C. S., et
al., Structure, 1997, 5:277-289; Lukatela, G., et al.,
Biochemistry, 1998, 37:3654-64), and a unique post-translational
modification that is essential for sulfate ester cleavage (Schmidt,
B., et al., Cell, 1995, 82:271-278; Selmer, T., et al., Eur. J.
Biochem., 1996, 238:341-345). The post-translational modification
involves the oxidation of a conserved cysteine (in eukaryotes) or
serine (in certain prokaryotes) residue, at C.sub..beta., yielding
L-C.sub..alpha.-formylglycine (a.k.a. FGly; 2-amino-3-oxopropanoic
acid) in which an aldehyde group replaces the thiomethyl group of
the side chain. The aldehyde is an essential part of the catalytic
site of the sulfatase and likely acts as an aldehyde hydrate. One
of the geminal hydroxyl groups accepts the sulfate during sulfate
ester cleavage leading to the formation of a covalently sulfated
enzyme intermediate. The other hydroxyl is required for the
subsequent elimination of the sulfate and regeneration of the
aldehyde group. This modification occurs in the endoplasmic
reticulum during, or shortly after, import of the nascent sulfatase
polypeptide and is directed by a short linear sequence surrounding
the cysteine (or serine) residue to be modified. This highly
conserved sequence is hexapeptide L/V-C(S)-X-P-S-R (SEQ ID NO:32),
present in the N-terminal region of all eukaryotic sulfatases and
most frequently carries a hydroxyl or thiol group on residue X
(Dierks, T., et al., Proc. Natl. Acad. Sci. U.S.A., 1997,
94:11963-11968).
[0004] To date thirteen sulfatase genes have been identified in
humans. They encode enzymes with different substrate specificity
and subcellular localization such as lysosomes, Golgi and ER. Four
of these genes, ARSC, ARSD, ARSE, and ARSF, encoding arylsulfatase
C, D, E and F, respectively, are located within the same
chromosomal region (Xp22.3). They share significant sequence
similarity and a nearly identical genomic organization, indicating
that they arose from duplication events that occurred recently
during evolution (Franco B, et al., Cell, 1995, 81:15-25; Meroni G,
et al., Hum Mol Genet, 1996, 5:423-31).
[0005] The importance of sulfatases in human metabolism is
underscored by the identification of at least eight human monogenic
diseases caused by the deficiency of individual sulfatase
activities. Most of these conditions are lysosomal storage
disorders in which phenotypic consequences derive from the type and
tissue distribution of the stored material. Among them are five
different types of mucopolysaccharidoses (MPS types II, IIIA, II1D,
IVA, and VI) due to deficiencies of sulfatases acting on the
catabolism of glycosaminoglycans (Neufeld and Muenzer, 2001, The
mucopolysaccharidoses, In The Metabolic and Molecular Bases of
Inherited Disease, C. R. Scriver, A. L. Beaudet, W. S. Sly, D.
Valle, B. Childs, K. W. Kinzler and B. Vogelstein, eds. New York:
Mc Graw-Hill, pp. 3421-3452), and metachromatic leukodystrophy
(MLD), which is characterized by the storage of sulfolipids in the
central and peripheral nervous systems leading to severe and
progressive neurologic deterioration. Two additional human diseases
are caused by deficiencies of non-lysosomal sulfatases. These
include X-linked ichthyosis, a skin disorder due to steroid
sulfatase (STS/ARSC) deficiency, and chondrodysplasia punctata, a
disorder affecting bone and cartilage due to arylsulfatase E (ARSE)
deficiency. Sulfatases are also implicated in drug-induced human
malformation syndromes, such as Warfarin embryopathy, caused by
inhibition of ARSE activity due to in utero exposure to warfarin
during pregnancy.
[0006] In an intriguing human monogenic disorder, multiple
sulfatase deficiency (MSD), all sulfatase activities are
simultaneously defective. Consequently, the phenotype of this
severe multisystemic disease combines the features observed in
individual sulfatase deficiencies. Cells from patients with MSD are
deficient in sulfatase activities even after transfection with
cDNAs encoding human sulfatases, suggesting the presence of a
common mechanism required for the activity of all sulfatases
(Rommerskirch and von Figura, Proc. Natl. Acad. Sci., USA, 1992,
89:2561-2565). The post-translational modification of sulfatases
was found to be defective in one patient with MSD, suggesting that
this disorder is caused by a mutation in a gene, or genes,
implicated in the cysteine-to-formylglycine conversion machinery
(Schmidt, B., et al., Cell, 1995, 82:271-278). In spite of intense
biological and medical interest, efforts aimed at the
identification of this gene(s) have been hampered by the rarity of
MSD patients and consequent lack of suitable familial cases to
perform genetic mapping.
SUMMARY OF THE INVENTION
[0007] This invention provides methods and compositions for the
diagnosis and treatment of Multiple Sulfatase Deficiency (MIM
272200), and the treatment of other sulfatase deficiencies. More
specifically, we have identified a gene that encodes Formylglycine
Generating Enzyme (FGE), an enzyme responsible for the unique
post-translational modification occurring on sulfatases that is
essential for sulfatase function (formation of
L-C.sub..alpha.-formylglycine; a.k.a. FGly and/or
2-amino-3-oxopropanoic acid). It has been discovered, unexpectedly,
that mutations in the FGE gene lead to the development of Multiple
Sulfatase Deficiency (MSD) in subjects. It has also been
discovered, unexpectedly, that FGE enhances the activity of
sulfatases, including, but not limited to, Iduronate 2-Sulfatase,
Sulfamidase, N-Acetylgalactosamine 6-Sulfatase, N-Acetylglucosamine
6-Sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C,
Arylsulfatase D, Arylsulfatase E, Arylsulfatase F, Arylsulfatase G,
HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, and HSulf-6. In view
of these discoveries, the molecules of the present invention can be
used in the diagnosis and treatment of Multiple Sulfatase
Deficiency as well as other sulfatase deficiencies.
[0008] Methods for using the molecules of the invention in the
diagnosis of Multiple Sulfatase Deficiency, are provided.
[0009] Additionally, methods for using these molecules in vivo or
in vitro for the purpose of modulating FGly formation on
sulfatases, methods for treating conditions associated with such
modification, and compositions useful in the preparation of
therapeutic preparations for the treatment of Multiple Sulfatase
Deficiency, as well as other sulfatase deficiencies, are also
provided.
[0010] The present invention thus involves, in several aspects,
polypeptides modulating FGly formation on sulfatases, isolated
nucleic acids encoding those polypeptides, functional modifications
and variants of the foregoing, useful fragments of the foregoing,
as well as therapeutics and diagnostics, research methods,
compositions and tools relating thereto.
[0011] According to one aspect of the invention, an isolated
nucleic acid molecule selected conditions to a molecule consisting
of a nucleotide sequence set forth as SEQ ID NO:1 and which code
for a Formylglycine Generating Enzyme (FGE) polypeptide having
C.sub..alpha.-formylglycine generating activity, (b) nucleic acid
molecules that differ from the nucleic acid molecules of (a) in
codon sequence due to the degeneracy of the genetic code, and (c)
complements of (a) or (b), is provided. In certain embodiments, the
isolated nucleic acid molecule comprises the nucleotide sequence
set forth as SEQ ID NO:1. In some embodiments, the isolated nucleic
acid molecule consists of the nucleotide sequence set forth as SEQ
ID NO:3 or a fragment thereof.
[0012] The invention in another aspect provides an isolated nucleic
acid molecule selected from the group consisting of (a) unique
fragments of a nucleotide sequence set forth as SEQ ID NO:1, and
(b) complements of (a), provided that a unique fragment of (a)
includes a sequence of contiguous nucleotides which is not
identical to any sequence selected from the sequence group
consisting of: (1) sequences identical to SEQ ID NO. 4 and/or
nucleotides 20-1141 of SEQ ID NO. 4, and (2) complements of (1). In
any of the foregoing embodiments, complements refer to full-length
complements.
[0013] In one embodiment, the sequence of contiguous nucleotides is
selected from the group consisting of (1) at least two contiguous
nucleotides nonidentical to the sequence group, (2) at least three
contiguous nucleotides nonidentical to the sequence group, (3) at
least four contiguous nucleotides nonidentical to the sequence
group, (4) at least five contiguous nucleotides nonidentical to the
sequence group, (5) at least six contiguous nucleotides
nonidentical to the sequence group, and (6) at least seven
contiguous nucleotides nonidentical to the sequence group.
[0014] In another embodiment, the fragment has a size selected from
the group consisting of at least: 8 nucleotides, 10 nucleotides, 12
nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20,
nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28
nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 75
nucleotides, 100 nucleotides, 200 nucleotides, 1000 nucleotides and
every integer length therebetween.
[0015] According to another aspect, the invention provides
expression vectors, and host cells transformed or transfected with
such expression vectors, comprising the nucleic acid molecules
described above.
[0016] According to still another aspect, the invention provides
cells expressing activated forms of the endogenous FGE gene. In one
embodiment, activation of the endogenous FGE gene occurs via
homologous recombination.
[0017] According to another aspect of the invention, an isolated
polypeptide is provided. The isolated polypeptide is encoded by the
foregoing nucleic acid molecules of the invention. In some
embodiments, the isolated polypeptide is encoded by the nucleic
acid of SEQ ID NO:1, giving rise to a polypeptide having the
sequence of SEQ ID NO:2 that has C.sub..alpha.-formylglycine
generating activity. In other embodiments, the isolated polypeptide
may be a fragment or variant of the foregoing of sufficient length
to represent a sequence unique within the human genome, and
identifying with a polypeptide that has C.sub..alpha.-formylglycine
generating activity, provided that the fragment includes a sequence
of contiguous amino acids which is not identical to any sequence
encoded for by a nucleic acid sequence having SEQ ID NO. 4. In
another embodiment, immunogenic fragments of the polypeptide
molecules described above are provided. The immunogenic fragments
may or may not have C.sub..alpha.-formylglycine generating
activity.
[0018] According to another aspect of the invention, isolated
binding polypeptides are provided which selectively bind a
polypeptide encoded by the foregoing nucleic acid molecules of the
invention. Preferably the isolated binding polypeptides selectively
bind a polypeptide which comprises the sequence of SEQ ID NO:2,
fragments thereof, or a polypeptide belonging to the family of
isolated polypeptides having C.alpha.-formylglycine generating
activity described elsewhere herein. In preferred embodiments, the
isolated binding polypeptides include antibodies and fragments of
antibodies (e.g., Fab, F(ab).sub.2, Fd and antibody fragments which
include a CDR3 region which binds selectively to the FGE
polypeptide). In certain embodiments, the antibodies are human. In
some embodiments, the antibodies are monoclonal antibodies. In one
embodiment, the antibodies are polyclonal antisera. In further
embodiments, the antibodies are humanized. In yet further
embodiments, the antibodies are chimeric.
[0019] According to another aspect of the invention, a family of
isolated polypeptides having C.sub..alpha.-formylglycine generating
activity, are provided. Each of said polypeptides comprises from
amino terminus to carboxyl terminus: (a) an amino-terminal
subdomain 1; a subdomain 2; a carboxy-terminal subdomain 3
containing from 35 to 45 amino acids; and wherein subdomain 3 has
at least about 75% homology and a length approximately equal to
subdomain 3 of a polypeptide selected from the group consisting of
SEQ ID NO. 2, 5, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,
70, 72, 74, 76, and 78. In important embodiments, subdomain 2
contains from 120 to 140 amino acids. In further important
embodiments, at least 5% of the amino acids of subdomain 2 are
Tryptophans. In some embodiments, subdomain 2 has at least about
50% homology to subdomain 2 of a polypeptide selected from the
group consisting of SEQ ID NO. 2, 5, 46, 48, 50, 52, 54, 56, 58,
60, 62, 64, 66, 68, 70, 72, 74, 76, and 78. In certain embodiments,
subdomain 3 of each of the polypeptides has at least between about
80% and about 100% homology to subdomain 3 of a polypeptide
selected from the group consisting of SEQ ID NO. 2, 5, 46, 48, 50,
52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78.
[0020] According to a further aspect of the invention, a method for
determining the level of FGE expression in a subject, is provided.
The method involves measuring expression of FGE in a test sample
from a subject to determine the level of FGE expression in the
subject. In certain embodiments, the measured FGE expression in the
test sample is compared to FGE expression in a control containing a
known level of FGE expression. Expression is defined as FGE mRNA
expression, FGE polypeptide expression, or FGE
C.sub..alpha.-formylglycine generating activity as defined
elsewhere herein. Various methods can be used to measure
expression. Preferred embodiments of the invention include PCR and
Northern blotting for measuring mRNA expression, FGE monoclonal
antibodies or FGE polyclonal antisera as reagents to measure FGE
polypeptide expression, as well as methods for measuring FGE
C.sub..alpha.-formylglycine generating activity.
[0021] In certain embodiments, test samples such as biopsy samples,
and biological fluids such as blood, are used as test samples. FGE
expression in a test sample of a subject is compared to FGE
expression in control.
[0022] According to another aspect of the invention, a method for
identifying an agent useful in modulating generating activity of a
molecule, is provided. The method involves (a) contacting a
molecule having C.sub..alpha.-formylglycine generating activity
with a candidate agent, (b) measuring C.sub..alpha.-formylglycine
generating activity of the molecule, and (c) comparing the measured
C.sub..alpha.-formylglycine generating activity of the molecule to
a control to determine whether the candidate agent modulates
C.sub..alpha.-formylglycine generating activity of the molecule,
wherein the molecule is a nucleic acid molecule having the
nucleotide sequence selected from the group consisting of SEQ ID
NO: 1, 3, 4, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69,
71, 73, 75, 77, and 80-87, or an expression product thereof (e.g.,
a peptide having a sequence selected from the group consisting of
SEQ ID NO. 2, 5, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,
70, 72, 74, 76, and 78). In certain embodiments, the control is
C.sub..alpha.-formylglycine generating activity of the molecule
measured in the absence of the candidate agent.
[0023] According to still another aspect of the invention, a method
of diagnosing Multiple Sulfatase Deficiency in a subject, is
provided. The method involves contacting a biological sample from a
subject suspected of having Multiple Sulfatase Deficiency with an
agent, said agent specifically binding to a molecule selected from
the group consisting of: (i) a FGE nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1, 3, or 4, (ii) an expression
product of the nucleic acid molecule of (i), or (iii) a fragment of
the expression product of (ii); and measuring the amount of bound
agent and determining therefrom if the expression of said nucleic
acid molecule or of an expression product thereof is aberrant,
aberrant expression being diagnostic of the Multiple Sulfatase
Deficiency in the subject.
[0024] According to still another aspect of the invention, a method
for diagnosing a condition characterized by aberrant expression of
a nucleic acid molecule or an expression product thereof, is
provided. The method involves contacting a biological sample from a
subject with an agent, wherein said agent specifically binds to
said nucleic acid molecule, an expression product thereof, or a
fragment of an expression product thereof; and measuring the amount
of bound agent and determining therefrom if the expression of said
nucleic acid molecule or of an expression product thereof is
aberrant, aberrant expression being diagnostic of the condition,
wherein the nucleic acid molecule has the nucleotide sequence of
SEQ ID NO:1 and the condition is Multiple Sulfatase Deficiency.
[0025] According to another aspect of the invention, a method for
determining Multiple Sulfatase Deficiency in a subject
characterized by aberrant expression of a nucleic acid molecule or
an expression product thereof, is provided. The method involves
monitoring a sample from a patient for a parameter selected from
the group consisting of (i) a nucleic acid molecule having the
nucleotide sequence of SEQ ID NO:1, 3, 4, or a nucleic acid
molecule having a sequence derived from the FEG genomic locus, (ii)
a polypeptide encoded by the nucleic acid molecule, (iii) a peptide
derived from the polypeptide, and (iv) an antibody which
selectively binds the polypeptide or peptide, as a determination of
Multiple Sulfatase Deficiency in the subject. In some embodiments,
the sample is a biological fluid or a tissue as described in any of
the foregoing embodiments. In certain embodiments the step of
monitoring comprises contacting the sample with a detectable agent
selected from the group consisting of (a) an isolated nucleic acid
molecule which selectively hybridizes under stringent conditions to
the nucleic acid molecule of (i), (b) an antibody which selectively
binds the polypeptide of (ii), or the peptide of (iii), and (c) a
polypeptide or peptide which binds the antibody of (iv). The
antibody, polypeptide, peptide, or nucleic acid can be labeled with
a radioactive label or an enzyme. In further embodiments, the
method further comprises assaying the sample for the peptide.
[0026] According to another aspect of the invention, a kit is
provided. The kit comprises a package containing an agent that
selectively binds to any of the foregoing FGE isolated nucleic
acids, or expression products thereof, and a control for comparing
to a measured value of binding of said agent any of the foregoing
FGE isolated nucleic acids or expression products thereof. In some
embodiments, the control is a predetermined value for comparing to
the measured value. In certain embodiments, the control comprises
an epitope of the expression product of any of the foregoing FGE
isolated nucleic acids. In one embodiment, the kit further
comprises a second agent that selectively binds to a polypeptide
selected from the group consisting of Iduronate 2-Sulfatase,
Sulfamidase, N-Acetylgalactosamine 6-Sulfatase, N-Acetylglucosamine
6-Sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C,
Arylsulfatase D, Arylsulfatase E, Arylsulfatase F, Arylsulfatase G,
HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, and HSulf-6, or a
peptide thereof, and a control for comparing to a measured value of
binding of said second agent to said polypeptide or peptide
thereof.
[0027] According to a further aspect of the invention, a method of
treating Multiple Sulfatase Deficiency, is provided. The method
involves administering to a subject in need of such treatment an
agent that modulates C.sub..alpha.-formylglycine generating
activity, in an amount effective to treat Multiple Sulfatase
Deficiency in the subject. In some embodiments, the method further
comprises co-administering an agent selected from the group
consisting of a nucleic acid molecule encoding Iduronate
2-Sulfatase, Sulfamidase, N-Acetylgalactosamine 6-Sulfatase,
N-Acetylglucosamine 6-Sulfatase, Arylsulfatase A, Arylsulfatase B,
Arylsulfatase C, Arylsulfatase D, Arylsulfatase E, Arylsulfatase F,
Arylsulfatase G, HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, or
HSulf-6, an expression product of the nucleic acid molecule, and a
fragment of the expression product of the nucleic acid molecule. In
certain embodiments, the agent that modulates
C.sub..alpha.-formylglycine generating activity is an isolated
nucleic acid molecule of the invention (e.g., a nucleic acid
molecule as claimed in claims 1-8, or a nucleic acid having a
sequence selected from the group consisting of SEQ ID NO: 1, 3, 4,
45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
and 80-87). In important embodiments, the agent that modulates
C.sub..alpha.-formylglycine generating activity is a peptide of the
invention (e.g., a peptide as claimed in claims 11-15, 19, 20, or a
peptide having a sequence selected from the group consisting of SEQ
ID NO. 2, 5, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
72, 74, 76, and 78). The agent that modulates
C.sub..alpha.-formylglycine generating activity may be produced by
a cell expressing an endogenous and/or exogenous FGE nucleic acid
molecule. In important embodiments, the endogenous FGE nucleic acid
molecule may be activated.
[0028] According to one aspect of the invention, a method for
increasing C.sub..alpha.-formylglycine generating activity in a
subject, is provided. The method involves administering an isolated
FGE nucleic acid molecule of the invention (e.g., a nucleic acid
molecule as claimed in claims 1-8, or a nucleic acid having a
sequence selected from the group consisting of SEQ ID NO: 1, 3, 4,
45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
and 80-87), and/or an expression product thereof, to a subject, in
an amount effective to increase C.sub..alpha.-formylglycine
generating activity in the subject.
[0029] According to one aspect of the invention, a method for
treating a subject with Multiple Sulfatase Deficiency, is provided.
The method involves administering to a subject in need of such
treatment an agent that modulates C.sub..alpha.-formylglycine
generating activity, in an amount effective to increase
C.sub..alpha.-formylglycine generating activity in the subject. In
some embodiments, the agent that modulates
C.sub..alpha.-formylglycine generating activity is a sense nucleic
acid of the invention (e.g., a nucleic acid molecule as claimed in
claims 1-8, or a nucleic acid having a sequence selected from the
group consisting of SEQ ID NO: 1, 3, 4, 45, 47, 49, 51, 53, 55, 57,
59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 80-87). In certain
embodiments, the agent that modulates C.sub..alpha.-formylglycine
generating activity is an isolated polypeptide of the invention
(e.g., a polypeptide as claimed in claims 11-15, 19, 20, or a
peptide having a sequence selected from the group consisting of SEQ
ID NO. 2, 5, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70,
72, 74, 76, and 78).
[0030] According to still another aspect of the invention, a method
for increasing C.sub..alpha.-formylglycine generating activity in a
cell, is provided. The method involves contacting the cell with an
isolated nucleic acid molecule of the invention (e.g., a nucleic
acid molecule as claimed in claims 1-8, or a nucleic acid having a
sequence selected from the group consisting of SEQ ID NO: 1, 3, 4,
45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
and 80-87), or an expression product thereof, in an amount
effective to increase C.sub..alpha.-formylglycine generating
activity in the cell. In important embodiments, the method involves
activating the endogenous FGE gene to increase
C.sub..alpha.-formylglycine generating activity in the cell.
[0031] According to a further aspect of the invention, a
pharmaceutical composition is provided. The composition comprises
an agent comprising an isolated nucleic acid molecule of the
invention (e.g., an isolated nucleic acid molecule as claimed in
any one of claims 1-8, an FGE nucleic acid molecule having a
sequence selected from the group consisting of SEQ ID NO: 1, 3, 4,
45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
and 80-87), or an expression product thereof, in a pharmaceutically
effective amount to treat Multiple Sulfatase Deficiency, or an
expression product thereof, in a pharmaceutically effective amount
to treat Multiple Sulfatase Deficiency, and a pharmaceutically
acceptable carrier.
[0032] According to one aspect of the invention, a method for
identifying a candidate agent useful in the treatment of Multiple
Sulfatase Deficiency, is provided. The method involves determining
expression of a set of nucleic acid molecules in a cell or tissue
under conditions which, in the absence of a candidate agent, permit
a first amount of expression of the set of nucleic acid molecules,
wherein the set of nucleic acid molecules comprises at least one
nucleic acid molecule selected from the group consisting of: (a)
nucleic acid molecules which hybridize under stringent conditions
to a molecule consisting of a nucleotide sequence set forth as SEQ
ID NO:1 and which code for a polypeptide having
C.sub..alpha.-formylglycine generating activity (FGE), (b) nucleic
acid molecules that differ from the nucleic acid molecules of (a)
or (b) in codon sequence due to the degeneracy of the genetic code,
(c) a nucleic acid molecule having a sequence selected from the
group consisting of SEQ ID NO: 1, 3, 4, 45, 47, 49, 51, 53, 55, 57,
59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 80-87, and (d)
complements of (a) or (b) or (c), contacting the cell or tissue
with the candidate agent, and detecting a test amount of expression
of the set of nucleic acid molecules, wherein an increase in the
test amount of expression in the presence of the candidate agent
relative to the first amount of expression indicates that the
candidate agent is useful in the treatment of the Multiple
Sulfatase Deficiency.
[0033] According to a further aspect of the invention, methods for
preparing medicaments useful in the treatment of Multiple Sulfatase
Deficiency and/or other sulfatase deficiencies, are provided.
[0034] According to still another aspect of the invention, a
solid-phase nucleic acid molecule array, is provided. The array
consists essentially of a set of nucleic acid molecules, expression
products thereof, or fragments (of either the nucleic acid or the
polypeptide molecule) thereof, each nucleic acid molecule encoding
for a polypeptide selected from the group consisting of SEQ ID NO.
2, 5, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74,
76, and 78, Iduronate 2-Sulfatase, Sulfamidase,
N-Acetylgalactosamine 6-Sulfatase, N-Acetylglucosamine 6-Sulfatase,
Arylsulfatase A, Arylsulfatase B, Arylsulfatase C, Arylsulfatase D,
Arylsulfatase E, Arylsulfatase F, Arylsulfatase G, HSulf-1,
HSulf-2, HSulf-3, HSulf-4, HSulf-5, and HSulf-6, fixed to a solid
substrate. In some embodiments, the solid-phase array further
comprises at least one control nucleic acid molecule. In certain
embodiments, the set of nucleic acid molecules comprises at least
one, at least two, at least three, at least four, or even at least
five nucleic acid molecules, each selected from the group
consisting of SEQ ID NO. 2, 5, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, 72, 74, 76, and 78, Iduronate 2-Sulfatase,
Sulfamidase, N-Acetylgalactosamine 6-Sulfatase, N-Acetylglucosamine
6-Sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C,
Arylsulfatase D, Arylsulfatase E, Arylsulfatase F, Arylsulfatase G,
HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, and HSulf-6.
[0035] According to a further aspect of the invention, a method for
treating a sulfatase deficiency in a subject, is provided. The
method involves administering to a subject in need of such
treatment a sulfatase that has been produced according to the
invention, in an amount effective to treat the sulfatase deficiency
in the subject and the sulfatase deficiency is not Multiple
Sulfatase Deficiency. In important embodiments, the sulfatase is
produced by a cell that has been contacted with an an agent that
modulates C.sub..alpha.-formylglycine generating activity. In
certain embodiments, the sulfatase deficiency includes, but is not
limited to, Mucopolysaccharidosis II (MPS II; Hunter Syndrome),
Mucopolysaccharidosis IIIA (MPS IIIA; Sanfilippo Syndrome A),
Mucopolysaccharidosis VIII (MPS VIII), Mucopolysaccharidosis IVA
(MPS IVA; Morquio Syndrome A), Mucopolysaccharidosis VI (MPS VI;
Maroteaux-Lamy Syndrome), Metachromatic Leukodystrophy (MLD),
X-linked Recessive Chondrodysplasia Punctata 1, or X-linked
Ichthyosis (Steroid Sulfatase Deficiency). In certain embodiments,
the agent that modulates C.sub..alpha.-formylglycine generating
activity can be a nucleic acid molecule or peptide of the
invention. In one embodiment, the sulfatase and the agent that
modulates C.sub..alpha.-formylglycine generating activity are
co-expressed in the same cell. The sulfatase and/or the agent that
modulates C.sub..alpha.-formylglycine generating activity can be
endogenous or exogenous in origin. If endogenous in origin it can
be activated (e.g., by insertion of strong promoter and/or other
elements at the appropriates places known in the art). If
exogenous, its expression can be driven by elements on the
expression vector, or it can be targeted to appropriated places
within the cell genome that will allow for its enhanced expression
(e.g., downstream of a strong promoter).
[0036] According to another aspect of the invention, a
pharmaceutical composition, is provided. The composition comprises
an agent comprising an isolated nucleic acid molecule of the
invention, or an expression product thereof, in a pharmaceutically
effective amount to treat a sulfatase deficiency, and a
pharmaceutically acceptable carrier.
[0037] According to a still further aspect of the invention, a
method for increasing sulfatase activity in a cell, is provided.
The method involves contacting a cell expressing a sulfatase with
an isolated nucleic acid molecule of the invention (e.g., an
isolated nucleic acid molecule as claimed in any one of claims 1-8,
an FGE nucleic acid molecule having a sequence selected from the
group consisting of SEQ ID NO: 1, 3, 4, 45; 47, 49, 51, 53, 55, 57,
59, 61, 63, 65, 67, 69, 71, 73, 75, 77, and 80-87), or an
expression product thereof (e.g., a polypeptide as claimed in
claims 11-15, 19, 20, or a peptide having a sequence selected from
the group consisting of SEQ ID NO. 2, 5, 46, 48, 50, 52, 54, 56,
58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78), in an amount
effective to increase sulfatase activity in the cell. The cell may
express an endogenous and/or an exogenous sulfatase. In important
embodiments, the endogenous sulfatase is activated. In certain
embodiments, the sulfatase is Iduronate 2-Sulfatase, Sulfamidase,
N-Acetylgalactosamine 6-Sulfatase, N-Acetylglucosamine 6-Sulfatase,
Arylsulfatase A, Arylsulfatase B, Arylsulfatase C, Arylsulfatase D,
Arylsulfatase E, Arylsulfatase F, Arylsulfatase G, HSulf-1,
HSulf-2, HSulf-3, HSulf-4, HSulf-5, and/or HSulf-6. In certain
embodiments the cell is a mammalian cell.
[0038] According to another aspect of the invention, a
pharmaceutical composition, is provided. The composition comprises
a sulfatase that is produced by cell, in a pharmaceutically
effective amount to treat a sulfatase deficiency, and a
pharmaceutically acceptable carrier, wherein said cell has been
contacted with an agent comprising an isolated nucleic acid
molecule of the invention (e.g., as claimed in claims 1-8, or a
nucleic acid molecule having a sequence selected from the group
consisting of SEQ ID NO: 1, 3, 4, 45, 47, 49, 51, 53, 55, 57, 59,
61, 63, 65, 67, 69, 71, 73, 75, 77, and 80-87), or an expression
product thereof (e.g., a peptide selected from the group consisting
of SEQ ID NO. 2, 5, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,
70, 72, 74, 76, and 78).
[0039] According to still another aspect of the invention, an
isolated variant allele of a human FGE gene which encodes a variant
FGE polypeptide, is provided. The isolated variant allele comprises
an amino acid sequence comprising at least one variation in SEQ ID
NO:2, wherein the at least one variation comprises: MetlArg;
MetlVal; Leu20Phe; Ser155Pro; Ala177Pro; Cys218Tyr; Arg224Trp;
Asn259Ile; Pro266Leu; Ala279Val; Arg327Stop; Cys336Arg; Arg345Cys;
Ala348Pro; Arg349G1n; Arg349Trp; Arg349Trp; Ser359Stop; or a
combination thereof.
[0040] According to yet another aspect of the invention, an
isolated variant human FGE polypeptide, is provided. The isolated
variant human FGE polypeptide comprises an amino acid sequence
comprising at least one variation in SEQ ID NO:2, wherein the at
least one variation comprises: MetlArg; MetlVal; Leu20Phe;
Ser155Pro; Ala177Pro; Cys218Tyr; Arg224Trp; Asn259Ile; Pro266Leu;
Ala279Val; Arg327Stop; Cys336Arg; Arg345Cys; Ala348Pro; Arg349G1n;
Arg349Trp; Arg349Trp; Ser359Stop; or a combination thereof.
[0041] Antibodies having any of the foregoing variant human FGE
polypeptides as an immunogen are also provided. Such antibodies
include polyclonal antisera, monoclonal, chimeric, and can also be
detectably labeled. A detectable label may comprise a radioactive
element, a chemical which fluoresces, or an enzyme.
[0042] According to another aspect of the invention, a
sulfatase-producing cell wherein the ratio of active sulfatase to
total sulfatase produced by the cell is increased, is provided. The
cell comprises: (i) a sulfatase with an increased expression, and
(ii) a Formylglycine Generating Enzyme with an increased
expression, wherein the ratio of active sulfatase to total
sulfatase (i.e., the specific activity of the sulfatase) produced
by the cell is increased by at least 5% over the ratio of active
sulfatase to total sulfatase produced by the cell in the absence of
the Formylglycine Generating Enzyme. In certain embodiments, the
ratio of active sulfatase to total sulfatase produced by the cell
is increased by at least 10%, 15%, 20%, 50%, 100%, 200%, 500%,
1000%, over the ratio of active sulfatase to total sulfatase
produced by the cell in the absence of the Formylglycine Generating
Enzyme.
[0043] According to a further aspect of the invention, an improved
method for treating a sulfatase deficiency in a subject is
provided. The method involves administering to a subject in need of
such treatment a sulfatase in an effective amount to treat the
sulfatase deficiency in the subject, wherein the sulfatase is
contacted with a Formylglycine Generating Enzyme in an amount
effective to increase the specific activity of the sulfatase. In an
important embodiment, the sulfatase is selected from the group
consisting of Iduronate 2-Sulfatase, Sulfamidase,
N-Acetylgalactosamine 6-Sulfatase, N-Acetylglucosamine 6-Sulfatase,
Arylsulfatase A, Arylsulfatase B, Arylsulfatase C, Arylsulfatase D,
Arylsulfatase E, Arylsulfatase F, Arylsulfatase G, HSulf-1,
HSulf-2, HSulf-3, HSulf-4, HSulf-5, and HSulf-6. In certain
embodiments, the Formylglycine Generating Enzyme is encoded by a
nucleic acid molecule as claimed in claims 1-8, or a nucleic acid
having a sequence selected from the group consisting of SEQ ID NO:
1, 3, 4, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,
73, 75, 77, and 80-87. In some embodiments, the Formylglycine
Generating Enzyme is a peptide as claimed in claims 11-15, 19, 20,
or a peptide having a sequence selected from the group consisting
of SEQ ID NO. 2, 5, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,
70, 72, 74, 76, and 78.
[0044] These and other objects of the invention will be described
in further detail in connection with the detailed description of
the invention.
Brief Description of the Sequences
[0045] SEQ ID NO:1 is the nucleotide sequence of the human FGE
cDNA.
[0046] SEQ ID NO:2 is the predicted amino acid sequence of the
translation product of human FGE cDNA (SEQ ID NO:1).
[0047] SEQ ID NO:3 is the nucleotide sequence of the human FGE cDNA
encoding the polypeptide of SEQ ID NO:2 (i.e., nucleotides 20-1141
of SEQ ID NO:1).
[0048] SEQ ID NO:4 is the nucleotide sequence of GenBank Acc. No.
AK075459.
[0049] SEQ ID NO:5 is the predicted amino acid sequence of the
translation product of SEQ ID NO:4, an unnamed protein product
having GenBank Acc. No. BAC 11634.
[0050] SEQ ID NO:6 is the nucleotide sequence of the human
Iduronate 2-Sulfatase cDNA (GenBank Acc. No. M58342).
[0051] SEQ ID NO:7 is the predicted amino acid sequence of the
translation product of human Iduronate 2-Sulfatase cDNA (SEQ ID
NO:6).
[0052] SEQ ID NO:8 is the nucleotide sequence of the human
Sulfamidase cDNA (GenBank Acc. No. U30894).
[0053] SEQ ID NO:9 is the predicted amino acid sequence of the
translation product of human Sulfamidase cDNA (SEQ ID NO:8).
[0054] SEQ ID NO:10 is the nucleotide sequence of the human
N-Acetylgalactosamine 6-Sulfatase cDNA (GenBank Acc. No.
U06088).
[0055] SEQ ID NO:11 is the predicted amino acid sequence of the
translation product of human N-Acetylgalactosamine 6-Sulfatase cDNA
(SEQ ID NO:10).
[0056] SEQ ID NO:12 is the nucleotide sequence of the human
N-Acetylglucosamine 6-Sulfatase cDNA (GenBank Acc. No. Z12173).
[0057] SEQ ID NO:13 is the predicted amino acid sequence of the
translation product of human N-Acetylglucosamine 6-Sulfatase cDNA
(SEQ ID NO:12).
[0058] SEQ ID NO:14 is the nucleotide sequence of the human
Arylsulfatase A cDNA (GenBank Acc. No. X52151).
[0059] SEQ ID NO:15 is the predicted amino acid sequence of the
translation product of human Arylsulfatase A cDNA (SEQ ID
NO:14).
[0060] SEQ ID NO:16 is the nucleotide sequence of the human
Arylsulfatase B cDNA (GenBank Acc. No. J05225).
[0061] SEQ ID NO:17 is the predicted amino acid sequence of the
translation product of human Arylsulfatase B cDNA (SEQ ID
NO:16).
[0062] SEQ ID NO:18 is the nucleotide sequence of the human
Arylsulfatase C cDNA (GenBank Acc. No. J04964).
[0063] SEQ ID NO:19 is the predicted amino acid sequence of the
translation product of human Arylsulfatase C cDNA (SEQ ID
NO:18).
[0064] SEQ ID NO:20 is the nucleotide sequence of the human
Arylsulfatase D cDNA (GenBank Acc. No. X83572).
[0065] SEQ ID NO:21 is the predicted amino acid sequence of the
translation product of human Arylsulfatase D cDNA (SEQ ID
NO:20).
[0066] SEQ ID NO:22 is the nucleotide sequence of the human
Arylsulfatase E cDNA (GenBank Acc. No. X83573).
[0067] SEQ ID NO:23 is the predicted amino acid sequence of the
translation product of human Arylsulfatase E cDNA (SEQ ID
NO:22).
[0068] SEQ ID NO:24 is the nucleotide sequence of the human
Arylsulfatase F cDNA (GenBank Acc. No. X97868).
[0069] SEQ ID NO:25 is the predicted amino acid sequence of the
translation product of human Arylsulfatase F cDNA (SEQ ID
NO:24).
[0070] SEQ ID NO:26 is the nucleotide sequence of the human
Arylsulfatase G cDNA (GenBank Acc. No. BC012375).
[0071] SEQ ID NO:27 is the predicted amino acid sequence of the
translation product of the human Arylsulfatase G (SEQ ID
NO:26).
[0072] SEQ ID NO:28 is the nucleotide sequence of the HSulf-1 cDNA
(GenBank Acc. No. AY101175).
[0073] SEQ ID NO:29 is the predicted amino acid sequence of the
translation product of HSulf-1 cDNA (SEQ ID NO:28).
[0074] SEQ ID NO:30 is the nucleotide sequence of the HSulf-2 cDNA
(GenBank Acc. No. AY 101176).
[0075] SEQ ID NO:31 is the predicted amino acid sequence of the
translation product of HSulf-2 cDNA (SEQ ID NO:30).
[0076] SEQ ID NO:32 is the highly conserved hexapeptide
LN-FGly-X-P-S-R present on sulfatases.
[0077] SEQ ID NO:33 is a synthetic FGly formation substrate; its
primary sequence is derived from human Arylsulfatase A.
[0078] SEQ ID NO:34 is scrambled oligopeptide PVSLPTRSCAALLTGR.
[0079] SEQ ID NO:35 is Ser69 oligopeptide PVSLSTPSRAALLTGR.
[0080] SEQ ID NO:36 is human FGE-specific primer 1199nc.
[0081] SEQ ID NO:37 is human FGE-specific forward primer 1c.
[0082] SEQ ID NO:38 is human FGE-specific reverse primer 1182c.
[0083] SEQ ID NO:39 is human 5'-FGE-specific primer containing
EcoRI site. SEQ ID NO:40 is a HA-specific primer.
[0084] SEQ ID NO:41 is a c-myc-specific primer.
[0085] SEQ ID NO:42 is a RGS-His6-specific primer.
[0086] SEQ ID NO:43 is tryptic oligopeptide SQNTPDSSASNLGFR from a
human FGE preparation.
[0087] SEQ ID NO:44 is tryptic oligopeptide MVPIPAGVFTMGTDDPQIK
from a human FGE preparation.
[0088] SEQ ID NO:45 is the nucleotide sequence of the human FGE2
paralog (GenBank GI: 24308053).
[0089] SEQ ID NO:46 is the predicted amino acid sequence of the
translation product of the human FGE2 paralog (SEQ ID NO:45).
[0090] SEQ ID NO:47 is the nucleotide sequence of the mouse FGE
paralog (GenBank GI: 26344956).
[0091] SEQ ID NO:48 is the predicted amino acid sequence of the
translation product of the mouse FGE paralog (SEQ ID NO:47).
[0092] SEQ ID NO:49 is the nucleotide sequence of the mouse FGE
ortholog (GenBank GI: 22122361).
[0093] SEQ ID NO:50 is the predicted amino acid sequence of the
translation product of the mouse FGE ortholog (SEQ ID NO:49).
[0094] SEQ ID NO:51 is the nucleotide sequence of the fruitfly FGE
ortholog (GenBank GI: 20130397).
[0095] SEQ ID NO:52 is the predicted amino acid sequence of the
translation product of the fruitfly FGE ortholog (SEQ ID
NO:51).
[0096] SEQ ID NO:53 is the nucleotide sequence of the mosquito FGE
ortholog (GenBank GI: 21289310).
[0097] SEQ ID NO:54 is the predicted amino acid sequence of the
translation product of the mosquito FGE ortholog (SEQ ID
NO:53).
[0098] SEQ ID NO:55 is the nucleotide sequence of the closely
related S. coelicolor FGE ortholog (GenBank GI: 21225812).
[0099] SEQ ID NO:56 is the predicted amino acid sequence of the
translation S. coelicolor FGE ortholog (SEQ ID NO:55).
[0100] SEQ ID NO:57 is the nucleotide sequence of the closely
related C. ortholog (GenBank GI: 25028125).
[0101] SEQ ID NO:58 is the predicted amino acid sequence of the
translation C. efficiens FGE ortholog (SEQ ID NO:57).
[0102] SEQ ID NO:59 is the nucleotide sequence of the N.
aromaticivorans (GenBank GI: 23108562).
[0103] SEQ ID NO:60 is the predicted amino acid sequence of the
translation N. aromaticivorans FGE ortholog (SEQ ID NO:59).
[0104] SEQ ID NO:61 is the nucleotide sequence of the M. loti FGE
ortholog 13474559).
[0105] SEQ ID NO:62 is the predicted amino acid sequence of the
translation M. loti FGE ortholog (SEQ ID NO:61).
[0106] SEQ ID NO:63 is the nucleotide sequence of the B. fungorum
(GenBank GI: 22988809).
[0107] SEQ ID NO:64 is the predicted amino acid sequence of the
translation B. fungorum FGE ortholog (SEQ ID NO:63).
[0108] SEQ ID NO:65 is the nucleotide sequence of the S. meliloti
FGE orth GI: 16264068).
[0109] SEQ ID NO:66 is the predicted amino acid sequence of the
translation S. meliloti FGE ortholog (SEQ ID NO:65).
[0110] SEQ ID NO:67 is the nucleotide sequence of the Microscilla
sp. (GenBank GI: 14518334).
[0111] SEQ ID NO:68 is the predicted amino acid sequence of the
translation Microscilla sp. FGE ortholog (SEQ ID NO:67).
[0112] SEQ ID NO:69 is the nucleotide sequence of the P. putida
KT2440 (GenBank GI: 26990068).
[0113] SEQ ID NO:70 is the predicted amino acid sequence of the
translation P. putida KT2440 FGE ortholog (SEQ ID NO:69).
[0114] SEQ ID NO:71 is the nucleotide sequence of the R.
metallidurans (GenBank GI: 22975289).
[0115] SEQ ID NO:72 is the predicted amino acid sequence of the
translation R. metallidurans FGE ortholog (SEQ ID NO:71).
[0116] SEQ ID NO:73 is the nucleotide sequence of the P. marinus
FGE ortholog (GenBank GI: 23132010).
[0117] SEQ ID NO:74 is the predicted amino acid sequence of the
translation product of the P. marinus FGE ortholog (SEQ ID
NO:73).
[0118] SEQ ID NO:75 is the nucleotide sequence of the C. crescentus
CB15 FGE ortholog (GenBank GI: 16125425).
[0119] SEQ ID NO:76 is the predicted amino acid sequence of the
translation product of the C. crescentus CB15 FGE ortholog (SEQ ID
NO:75).
[0120] SEQ ID NO:77 is the nucleotide sequence of the M.
tuberculosis Ht37Rv FGE ortholog (GenBank GI: 15607852).
[0121] SEQ ID NO:78 is the predicted amino acid sequence of the
translation product of the M. tuberculosis Ht37Rv FGE ortholog (SEQ
ID NO:77).
[0122] SEQ ID NO:79 is the highly conserved heptapeptide present on
subdomain 3 of FGE orthologs and paralogs.
[0123] SEQ ID NO:80 is the nucleotide sequence of FGE ortholog EST
fragment having GenBank Acc. No.: CA379852.
[0124] SEQ ID NO:81 is the nucleotide sequence of FGE ortholog EST
fragment having GenBank Acc. No.: A1721440.
[0125] SEQ ID NO:82 is the nucleotide sequence of FGE ortholog EST
fragment having GenBank Acc. No.: BJ505402.
[0126] SEQ ID NO:83 is the nucleotide sequence of FGE ortholog EST
fragment having GenBank Acc. No.: BJ054666.
[0127] SEQ ID NO:84 is the nucleotide sequence of FGE ortholog EST
fragment having GenBank Acc. No.: AL892419.
[0128] SEQ ID NO:85 is the nucleotide sequence of FGE ortholog EST
fragment having GenBank Acc. No.: CA064079.
[0129] SEQ ID NO:86 is the nucleotide sequence of FGE ortholog EST
fragment having GenBank Acc. No.: BF189614.
[0130] SEQ ID NO:87 is the nucleotide sequence of FGE ortholog EST
fragment having GenBank Acc. No.: AV609121.
[0131] SEQ ID NO:88 is the nucleotide sequence of the HSulf-3
cDNA.
[0132] SEQ ID NO:89 is the predicted amino acid sequence of the
translation product of HSulf-3 cDNA (SEQ ID NO:88).
[0133] SEQ ID NO:90 is the nucleotide sequence of the HSulf-4
cDNA.
[0134] SEQ ID NO:91 is the predicted amino acid sequence of the
translation product of HSulf-4 cDNA (SEQ ID NO:90).
[0135] SEQ ID NO:92 is the nucleotide sequence of the HSulf-5
cDNA.
[0136] SEQ ID NO:93 is the predicted amino acid sequence of the
translation product of HSulf-5 cDNA (SEQ ID NO:92).
[0137] SEQ ID NO:94 is the nucleotide sequence of the HSulf-6
cDNA.
[0138] SEQ ID NO:95 is the predicted amino acid sequence of the
translation product of HSulf-6 cDNA (SEQ ID NO:94).
BRIEF DESCRIPTION OF THE DRAWINGS
[0139] FIG. 1: A MALDI-TOF mass spectra schematic of P23 after
incubation in the absence (A) or presence (B) of a soluble extract
from bovine testis microsomes.
[0140] FIG. 2: A phylogenetic tree derived from an alignment of
human FGE and 21 proteins of the PFAM-DUF323 seed.
[0141] FIG. 3: Organisation of the human and murine FGE gene locus.
Exons are shown to scale as boxes and bright boxes (murine locus).
The numbers above the intron lines indicate the size of the introns
in kilobases.
[0142] FIG. 4: Diagram showing a map of FGE Expression Plasmid
pXMG.1.3
[0143] FIG. 5: Bar graph depicting N-Acetylgalactosamine
6-Sulfatase Activity in 36F Cells Transiently Transfected with FGE
Expression Plasmid.
[0144] FIG. 6: Bar graph depicting N-Acetylgalactosamine
6-Sulfatase Specific Activity in 36F Cells Transiently Transfected
with FGE Expression Plasmid.
[0145] FIG. 7: Bar graph depicting N-Acetylgalactosamine
6-Sulfatase Production in 36F Cells Transiently Transfected with
FGE Expression Plasmid.
[0146] FIG. 8: Graph depicting Iduronate 2-Sulfatase Activity in
3006 Cells Transiently Transfected with FGE Expression Plasmid.
[0147] FIG. 9: Depicts a kit embodying features of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0148] The invention involves the discovery of the gene that
encodes Formylglycine Generating Enzyme (FGE), an enzyme
responsible for the unique post-translational modification
occurring on sulfatases that is essential for sulfatase function:
the formation of L-C.sub..alpha.formylglycine (a.k.a. FGly and/or
2-amino-3-oxopropanoic acid). It has been discovered, unexpectedly,
that mutations in the FGE gene lead to the development of Multiple
Sulfatase Deficiency (MSD) in subjects. It has also been
discovered, unexpectedly, that FGE enhances the activity of
sulfatases, including, but not limited to, Iduronate 2-Sulfatase,
Sulfamidase, N-Acetylgalactosamine 6-Sulfatase, N-Acetylglucosamine
6-Sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C,
Arylsulfatase D, Arylsulfatase E, Arylsulfatase F, Arylsulfatase G,
HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, and HSulf-6, and
sulfatases described in U.S. Provisional applications with
publication numbers 20030073118, 20030147875, 20030148920,
20030162279, and 20030166283 (the contents of which are expressly
incorporated herein). In view of these discoveries, the molecules
of the present invention can be used in the diagnosis and/or
treatment of Multiple Sulfatase Deficiency, as well as the
treatment of other sulfatase deficiencies.
[0149] Methods for using the molecules of the invention in the
diagnosis of Multiple Sulfatase Deficiency are provided.
[0150] Additionally, methods for using these molecules in vivo or
in vitro for the purpose of modulating FGly formation on
sulfatases, methods for treating conditions associated with such
modification, and compositions useful in the preparation of
therapeutic preparations for the treatment of Multiple Sulfatase
Deficiency as well as other sulfatase deficiencies, are also
provided.
[0151] The present invention thus involves, in several aspects,
polypeptides modulating FGly formation on sulfatases, isolated
nucleic acids encoding those polypeptides, functional modifications
and variants of the foregoing, useful fragments of the foregoing,
as well as therapeutics and diagnostics, research methods,
compositions and tools relating thereto.
[0152] "C.sub..alpha.-formylglycine generating activity" refers to
the ability of a molecule to form, or enhance the formation of,
FGly on a substrate. The substrate may be a sulfatase as described
elsewhere herein, or a synthetic oligopeptide (see, e.g., SEQ ID
NO:33, and the Examples). The substrate preferably contains the
conserved hexapeptide of SEQ ID NO:32 [I./V-C(S)-X-P-S-R]. Methods
for assaying FGly formation are as described in the art (see, e.g.,
Dierks, T., et al., Proc. Natl. Acad. Sci. U.S.A., 1997,
94:11963-11968), and elsewhere herein (see, e.g., the Examples). A
"molecule," as used herein, embraces both "nucleic acids" and
"polypeptides." FGE molecules are capable of forming, or
enhancing/increasing formation of, FGly both in vivo and in
vitro.
[0153] "Enhancing (or "increasing")" C.sub..alpha.-formylglycine
generating activity, as used herein, typically refers to increased
expression of FGE and/or its encoded polypeptide. Increased
expression refers to increasing (i.e., to a detectable extent)
replication, transcription, and/or translation of any of the
nucleic acids of the invention (FGE nucleic acids as described
elsewhere herein), since upregulation of any of these processes
results in concentration/amount increase of the polypeptide encoded
by the gene (nucleic acid). Enhancing (or increasing)
C.sub..alpha.-formylglycine generating activity also refers to
preventing or inhibiting FGE degradation (e.g., via increased
ubiquitinization), downregulation, etc., resulting, for example, in
increased or stable FGE molecule t.sub.1/2 (half-life) when
compared to a control. Downregulation or decreased expression
refers to decreased expression of a gene and/or its encoded
polypeptide. The upregulation or downregulation of gene expression
can be directly determined by detecting an increase or decrease,
respectively, in the level of mRNA for the gene (e.g, FGE), or the
level of protein expression of the gene-encoded polypeptide, using
any suitable means known to the art, such as nucleic acid
hybridization or antibody detection methods, respectively, and in
comparison to controls. Upregulation or downregulation of FGE gene
expression can also be determined indirectly by detecting a change
in C.sub..alpha.-formylglycine generating activity.
[0154] "Expression," as used herein, refers to nucleic acid and/or
polypeptide expression, as well as to activity of the polypeptide
molecule (e.g., C.sub..alpha.-formylglycine generating activity of
the molecule).
[0155] One aspect of the invention involves the cloning of a cDNA
encoding FGE. FGE according to the invention is an isolated nucleic
acid molecule that comprises a nucleic acid molecule of SEQ ID
NO:1, and codes for a polypeptide with C.sub..alpha.-formylglycine
generating activity. The sequence of the human FGE cDNA is
presented as SEQ ID NO:1, and the predicted amino acid sequence of
this cDNA's encoded protein product is presented as SEQ ID
NO:2.
[0156] As used herein, a subject is a mammal or a non-human mammal.
In all embodiments human FGE and human subjects are preferred.
[0157] The invention thus involves in one aspect an isolated FGE
polypeptide, the cDNA encoding this polypeptide, functional
modifications and variants of the foregoing, useful fragments of
the foregoing, as well as diagnostics and therapeutics relating
thereto.
[0158] As used herein with respect to nucleic acids, the term
"isolated" means: (i) amplified in vitro by, for example,
polymerase chain reaction (PCR); (ii) recombinantly produced by
cloning; (iii) purified, as by cleavage and gel separation; or (iv)
synthesized by, for example, chemical synthesis. An isolated
nucleic acid is one which is readily manipulated by recombinant DNA
techniques well known in the art. Thus, a nucleotide sequence
contained in a vector in which 5' and 3' restriction sites are
known or for which polymerase chain reaction (PCR) primer sequences
have been disclosed is considered isolated but a nucleic acid
sequence existing in its native state in its natural host is not.
An isolated nucleic acid may be substantially purified, but need
not be. For example, a nucleic acid that is isolated within a
cloning or expression vector is not pure in that it may comprise
only a tiny percentage of the material in the cell in which it
resides. Such a nucleic acid is isolated, however, as the term is
used herein because it is readily manipulated by standard
techniques known to those of ordinary skill in the art.
[0159] As used herein with respect to polypeptides, the term
"isolated" means separated from its native environment in
sufficiently pure form so that it can be manipulated or used for
any one of the purposes of the invention. Thus, isolated means
sufficiently pure to be used (i) to raise and/or isolate
antibodies, (ii) as a reagent in an assay, (iii) for sequencing,
(iv) as a therapeutic, etc.
[0160] According to the invention, isolated nucleic acid molecules
that code for a FGE polypeptide having C.sub..alpha.-formylglycine
generating activity include: (a) nucleic acid molecules which
hybridize under stringent conditions to a molecule consisting of a
nucleic acid of SEQ ID NO:1 and which code for a FGE polypeptide
having C.sub..alpha.-formylglycine generating activity, (b)
deletions, additions and substitutions of (a) which code for a
respective FGE polypeptide having C.sub..alpha.-formylglycine
generating activity, (c) nucleic acid molecules that differ from
the nucleic acid molecules of (a) or (b) in codon sequence due to
the degeneracy of the genetic code, and (d) complements of (a), (b)
or (c). "Complements," as used herein, includes "full-length
complementary strands or 100% complementary strands of (a), (b) or
(c).
[0161] Homologs and alleles of the FGE nucleic acids of the
invention also having C.sub..alpha.-formylglycine generating
activity are encompassed by the present invention. Homologs, as
described herein, include the molecules identified elsewhere herein
(see e.g., SEQ ID NOs:4, 5, 45-78, and 80-87) i.e. orthologs and
paralogs. Further homologs can be identified following the
teachings of the present invention as well as by conventional
techniques. Since the FGE homologs described herein all share
C.sub..alpha.-formylglycine generating activity, they can be used
interchangeably with the human FGE molecule in all aspects of the
invention.
[0162] Thus, an aspect of the invention is those nucleic acid
sequences which code for FGE polypeptides and which hybridize to a
nucleic acid molecule consisting of the coding region of SEQ ID
NO:1, under stringent conditions. In an important embodiment, the
term "stringent conditions," as used herein, refers to parameters
with which the art is familiar. With nucleic acids, hybridization
conditions are said to be stringent typically under conditions of
low ionic strength and a temperature just below the melting
temperature (T.sub.m of the DNA hybrid complex (typically, about
3.degree. C. below the T.sub.m of the hybrid). Higher stringency
makes for a more specific correlation between the probe sequence
and the target. Stringent conditions used in the hybridization of
nucleic acids are well known in the art and may be found in
references which compile such methods, e.g. Molecular Cloning: A
Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or
Current Protocols in Molecular Biology, F. M. Ausubel, et al.,
eds., John Wiley & Sons, Inc., New York. An example of
"stringent conditions" is hybridization at 65.degree. C. in
6.times.SSC. Another example of stringent conditions is
hybridization at 65.degree. C. in hybridization buffer that
consists of 3.5.times.SSC, 0.02% Ficoll, 0.02% polyvinyl
pyrolidone, 0.02% Bovine Serum Albumin, 2.5 mM
NaH.sub.2PO.sub.4[pH7], 0.5% SDS, 2 mM EDTA. (SSC is 0.15M sodium
chloride/0.15M sodium citrate, pH7; SDS is sodium dodecyl sulphate;
and EDTA is ethylenediaminetetracetic acid). After hybridization,
the membrane upon which the DNA is transferred is washed at
2.times.SSC at room temperature and then at
0.1.times.SSC/0.1.times.SDS at temperatures up to 68.degree. C. In
a further example, an alternative to the use of an aqueous
hybridization solution is the use of a formamide hybridization
solution. Stringent hybridization conditions can thus be achieved
using, for example, a 50% formamide solution and 42.degree. C.
There are other conditions, reagents, and so forth which can be
used, and would result in a similar degree of stringency. The
skilled artisan will be familiar with such conditions, and thus
they are not given here. It will be understood, however, that the
skilled artisan will be able to manipulate the conditions in a
manner to permit the clear identification of homologs and alleles
of FGE nucleic acids of the invention. The skilled artisan also is
familiar with the methodology for screening cells and libraries for
expression of such molecules which then are routinely isolated,
followed by isolation of the pertinent nucleic acid molecule and
sequencing.
[0163] In general homologs and alleles typically will share at
least 40% nucleotide identity and/or at least 50% amino acid
identity to SEQ ID NO:1 and SEQ ID NO:2, respectively, in some
instances will share at least 50% nucleotide identity and/or at
least 65% amino acid identity and in still other instances will
share at least 60% nucleotide identity and/or at least 75% amino
acid identity. In further instances, homologs and alleles typically
will share at least 90%, 95%, or even 99% nucleotide identity
and/or at least 95%, 98%, or even 99% amino acid identity to SEQ ID
NO:1 and SEQ ID NO:2, respectively. The homology can be calculated
using various, publicly available software tools developed by NCBI
(Bethesda, Md.). Exemplary tools include the heuristic algorithm of
Altschul S F, et al., (J Mol Biol, 1990, 215:403-410), also known
as BLAST. Pairwise and ClustalW alignments (BLOSUM30 matrix
setting) as well as Kyte-Doolittle hydropathic analysis can be
obtained using public (EMBL, Heidelberg, Germany) and commercial
(e.g., the MacVector sequence analysis software from Oxford
Molecular Group/enetics Computer Group, Madison, Wis.).
Watson-Crick complements of the foregoing nucleic acids also are
embraced by the invention.
[0164] In screening for FGE related genes, such as homologs and
alleles of FGE, a Southern blot may be performed using the
foregoing conditions, together with a radioactive probe. After
washing the membrane to which the DNA is finally transferred, the
membrane can be placed against X-ray film or a phosphoimager plate
to detect the radioactive signal.
[0165] Given the teachings herein of a full-length human FGE cDNA
clone, other mammalian sequences such as the mouse cDNA clone
corresponding to the human FGE gene can be isolated from a cDNA
library, using standard colony hybridization techniques.
[0166] The invention also includes degenerate nucleic acids which
include alternative codons to those present in the native
materials. For example, serine residues are encoded by the codons
TCA, AGT, TCC, TCG, TCT and AGC. Thus, it will be apparent to one
of ordinary skill in the art that any of the serine-encoding
nucleotide triplets may be employed to direct the protein synthesis
apparatus, in vitro or in vivo, to incorporate a serine residue
into an elongating FGE polypeptide. Similarly, nucleotide sequence
triplets which encode other amino acid residues include, but are
not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC,
CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT
(threonine codons); AAC and AAT (asparagine codons); and ATA, ATC
and ATT (isoleucine codons). Other amino acid residues may be
encoded similarly by multiple nucleotide sequences. Thus, the
invention embraces degenerate nucleic acids that differ from the
biologically isolated nucleic acids in codon sequence due to the
degeneracy of the genetic code.
[0167] The invention also provides isolated unique fragments of SEQ
ID NO:1 or SEQ ID NO:3 or complements of thereof. A unique fragment
is one that is a `signature` for the larger nucleic acid. For
example, the unique fragment is long enough to assure that its
precise sequence is not found in molecules within the human genome
outside of the FGE nucleic acids defined above (and human alleles).
Those of ordinary skill in the art may apply no more than routine
procedures to determine if a fragment is unique within the human
genome. Unique fragments, however, exclude fragments completely
composed of the nucleotide sequences selected from the group
consisting of SEQ ID NO:4, and/or other previously published
sequences as of the filing date of this application.
[0168] A fragment which is completely composed of the sequence
described in the foregoing GenBank deposits is one which does not
include any of the nucleotides unique to the sequences of the
invention. Thus, a unique fragment according to the invention must
contain a nucleotide sequence other than the exact sequence of
those in the GenBank deposits or fragments thereof. The difference
may be an addition, deletion or substitution with respect to the
GenBank sequence or it may be a sequence wholly separate from the
GenBank sequence.
[0169] Unique fragments can be used as probes in Southern and
Northern blot assays to identify such nucleic acids, or can be used
in amplification assays such as those employing PCR. As known to
those skilled in the art, large probes such as 200, 250, 300 or
more nucleotides are preferred for certain uses such as Southern
and Northern blots, while smaller fragments will be preferred for
uses such as PCR. Unique fragments also can be used to produce
fusion proteins for generating antibodies or determining binding of
the polypeptide fragments, as demonstrated in the Examples, or for
generating immunoassay components. Likewise, unique fragments can
be employed to produce nonfused fragments of the FGE polypeptides,
useful, for example, in the preparation of antibodies, immunoassays
or therapeutic applications. Unique fragments further can be used
as antisense molecules to inhibit the expression of FGE nucleic
acids and polypeptides respectively.
[0170] As will be recognized by those skilled in the art, the size
of the unique fragment will depend upon its conservancy in the
genetic code. Thus, some regions of SEQ ID NO:1 or SEQ ID NO:3 and
complements will require longer segments to be unique while others
will require only short segments, typically between 12 and 32
nucleotides long (e.g. 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 bases) or more, up to the
entire length of the disclosed sequence. As mentioned above, this
disclosure intends to embrace each and every fragment of each
sequence, beginning at the first nucleotide, the second nucleotide
and so on, up to 8 nucleotides short of the end, and ending
anywhere from nucleotide number 8, 9, 10 and so on for each
sequence, up to the very last nucleotide, (provided the sequence is
unique as described above). Virtually any segment of the region of
SEQ ID NO:1 beginning at nucleotide 1 and ending at nucleotide
1180, or SEQ ID NO:3 beginning at nucleotide 1 and ending at
nucleotide 1122, or complements thereof, that is 20 or more
nucleotides in length will be unique. Those skilled in the art are
well versed in methods for selecting such sequences, typically on
the basis of the ability of the unique fragment to selectively
distinguish the sequence of interest from other sequences in the
human genome of the fragment to those on known databases typically
is all that is necessary, although in vitro confirmatory
hybridization and sequencing analysis may be performed.
[0171] As mentioned above, the invention embraces antisense
oligonucleotides that selectively bind to a nucleic acid molecule
encoding a FGE polypeptide, to decrease FGE activity.
[0172] As used herein, the term "antisense oligonucleotide" or
"antisense" describes an oligonucleotide that is an
oligoribonucleotide, oligodeoxyribonucleotide, modified
oligoribonucleotide, or modified oligodeoxyribonucleotide which
hybridizes under physiological conditions to DNA comprising a
particular gene or to an mRNA transcript of that gene and, thereby,
inhibits the transcription of that gene and/or the translation of
that mRNA. The antisense molecules are designed so as to interfere
with transcription or translation of a target gene upon
hybridization with the target gene or transcript. Those skilled in
the art will recognize that the exact length of the antisense
oligonucleotide and its degree of complementarity with its target
will depend upon the specific target selected, including the
sequence of the target and the particular bases which comprise that
sequence. It is preferred that the antisense oligonucleotide be
constructed and arranged so as to bind selectively with the target
under physiological conditions, i.e., to hybridize substantially
more to the target sequence than to any other sequence in the
target cell under physiological conditions. Based upon SEQ ID NO:1
or upon allelic or homologous genomic and/or cDNA sequences, one of
skill in the art can easily choose and synthesize any of a number
of appropriate antisense molecules for use in accordance with the
present invention. In order to be sufficiently selective and potent
for inhibition, such antisense oligonucleotides should comprise at
least 10 and, more preferably, at least 15 consecutive bases which
are complementary to the target, although in certain cases modified
oligonucleotides as short as 7 bases in length have been used
successfully as antisense oligonucleotides (Wagner et al., Nat.
Med, 1995, 1(11):1116-1118; Nat. Biotech., 1996, 14:840-844). Most
preferably, the antisense oligonucleotides comprise a complementary
sequence of 20-30 bases. Although oligonucleotides may be chosen
which are antisense to any region of the gene or mRNA transcripts,
in preferred embodiments the antisense oligonucleotides correspond
to N-terminal or 5' upstream sites such as translation initiation,
transcription initiation or promoter sites. In addition,
3'-untranslated regions may be targeted by antisense
oligonucleotides. Targeting to mRNA splicing sites has also been
used in the art but may be less preferred if alternative mRNA
splicing occurs. In addition, the antisense is targeted,
preferably, to sites in which mRNA secondary structure is not
expected (see, e.g., Sainio et al., Cell Mol. Neurobiol.
14(5):439-457, 1994) and at which proteins are not expected to
bind. Finally, although, SEQ ID No:1 discloses a cDNA sequence, one
of ordinary skill in the art may easily derive the genomic DNA
corresponding to this sequence. Thus, the present invention also
provides for antisense oligonucleotides which are complementary to
the genomic DNA corresponding to SEQ ID NO:1. Similarly, antisense
to allelic or homologous FGE cDNAs and genomic DNAs are enabled
without undue experimentation.
[0173] In one set of embodiments, the antisense oligonucleotides of
the invention may be composed of "natural" deoxyribonucleotides,
ribonucleotides, or any combination thereof. That is, the 5' end of
one native nucleotide and the 3' end of another native nucleotide
may be covalently linked, as in natural systems, via a
phosphodiester internucleoside linkage. These oligonucleotides may
be prepared by art recognized methods which may be carried out
manually or by an automated synthesizer. They also may be produced
recombinantly by vectors.
[0174] In preferred embodiments, however, the antisense
oligonucleotides of the invention also may include "modified"
oligonucleotides. That is, the oligonucleotides may be modified in
a number of ways which do not prevent them from hybridizing to
their target but which enhance their stability or targeting or
which otherwise enhance their therapeutic effectiveness.
[0175] The term "modified oligonucleotide" as used herein describes
an oligonucleotide in which (1) at least two of its nucleotides are
covalently linked via a synthetic internucleoside linkage (i.e., a
linkage other than a phosphodiester linkage between the 5' end of
one nucleotide and the 3' end of another nucleotide) and/or (2) a
chemical group not normally associated with nucleic acids has been
covalently attached to the oligonucleotide. Preferred synthetic
internucleoside linkages are phosphorothioates, alkylphosphonates,
phosphorodithioates, phosphate esters, alkylphosphonothioates,
phosphoramidates, carbamates, carbonates, phosphate triesters,
acetamidates, carboxymethyl esters and peptides.
[0176] The term "modified oligonucleotide" also encompasses
oligonucleotides with a covalently modified base and/or sugar. For
example, modified oligonucleotides include oligonucleotides having
backbone sugars which are covalently attached to low molecular
weight organic groups other than a hydroxyl group at the 3'
position and other than a phosphate group at the 5' position. Thus
modified oligonucleotides may include a 2'43-alkylated ribose
group. In addition, modified oligonucleotides may include sugars
such as arabinose instead of ribose. The present invention, thus,
contemplates pharmaceutical preparations containing modified
antisense molecules that are complementary to and hybridizable
with, under physiological conditions, nucleic acids encoding FGE
polypeptides, together with pharmaceutically acceptable carriers.
Antisense oligonucleotides may be administered as part of a
pharmaceutical composition. Such a pharmaceutical composition may
include the antisense oligonucleotides in combination with any
standard physiologically and/or pharmaceutically acceptable
carriers which are known in the art. The compositions should be
sterile and contain a therapeutically effective amount of the
antisense oligonucleotides in a unit of weight or volume suitable
for administration to a patient. The term "pharmaceutically
acceptable" means a non-toxic material that does not interfere with
the effectiveness of the biological activity of the active
ingredients. The term "physiologically acceptable" refers to a
non-toxic material that is compatible with a biological system such
as a cell, cell culture, tissue, or organism. The characteristics
of the carrier will depend on the route of administration.
Physiologically and pharmaceutically acceptable carriers include
diluents, fillers, salts, buffers, stabilizers, solubilizers, and
other materials which are well known in the art.
[0177] The invention also involves methods for increasing
C.sub..alpha.-formylglycine generating activity in a cell. In
important embodiments, this is accomplished by the use of vectors
("expression vectors" and/or "targeting vectors").
[0178] "Vectors," as used herein, may be any of a number of nucleic
acids into which a desired sequence may be inserted by restriction
and ligation for transport between different genetic environments
or for expression in a host cell. Vectors are typically composed of
DNA although RNA vectors are also available. Vectors include, but
are not limited to, plasmids, phagemids and virus genomes. A
cloning vector is one which is able to replicate in a host cell,
and which is further characterized by one or more endonuclease
restriction sites at which the vector may be cut in a determinable
fashion and into which a desired DNA sequence may be ligated such
that the new recombinant vector retains its ability to replicate in
the host cell. In the case of plasmids, replication of the desired
sequence may occur many times as the plasmid increases in copy
number within the host bacterium or just a single time per host
before the host reproduces by mitosis. In the case of phage,
replication may occur actively during a lytic phase or passively
during a lysogenic phase. An "expression vector" is one into which
a desired DNA sequence (e.g., the FGE cDNA of SEQ ID NO:3) may be
inserted by restriction and ligation such that it is operably
joined to regulatory sequences and may be expressed as an RNA
transcript. Vectors may further contain one or more marker
sequences suitable for use in the identification of cells which
have or have not been transformed or transfected with the vector.
Markers include, for example, genes encoding proteins which
increase or decrease either resistance or sensitivity to
antibiotics or other compounds, genes which encode enzymes whose
activities are detectable by standard assays known in the art
(e.g., .beta.-galactosidase or alkaline phosphatase), and genes
which visibly affect the phenotype of transformed or transfected
cells, hosts, colonies or plaques (e.g., green fluorescent
protein).
[0179] A "targeting vector" is one which typically contains
targeting constructs/sequences that are used, for example, to
insert a regulatory sequence within an endogenous gene (e.g.,
within the sequences of an exon and/or intron), within the
endogenous gene promoter sequences, or upstream of the endogenous
gene promoter sequences. In another example, a targeting vector may
contain the gene of interest (e.g., encoded by the cDNA of SEQ ID
NO:1) and other sequences necessary for the targeting of the gene
to a preferred location in. the genome (e.g., a trascriptionally
active location, for example downstream of an enogenous promoter of
an unrelated gene). Construction of targeting constructs and
vectors are described in detail in U.S. Pat. Nos. 5,641,670 and
6,270,989, and which are expressly incorporated herein by
reference.
[0180] Virtually any cells, prokaryotic or eukaryotic, which can be
transformed with heterologous DNA or RNA and which can be grown or
maintained in culture, may be used in the practice of the
invention. Examples include bacterial cells such as Escherichia
coli, insect cells, and mammalian cells such as human, mouse,
hamster, pig, goat, primate, etc. They may be primary or secondary
cell strains (which exhibit a finite number of mean population
doublings in culture and are not immortalized) and immortalized
cell lines (which exhibit an apparently unlimited lifespan in
culture). Primary and secondary cells include, for example,
fibroblasts, keratinocytes, epithelial cells (e.g., mammary
epithelial cells, intestinal epithelial cells), endothelial cells,
glial cells, neural cells, formed elements of the blood (e.g.,
lymphocytes, bone marrow cells), muscle cells and precursors of
these somatic cell types including embryonic stem cells. Where the
cells are to be used in gene therapy, primary cells are preferably
obtained from the individual to whom the manipulated cells are
administered. However, primary cells can be obtained from a donor
(other than the recipient) of the same species. Examples of
immortalized human cell lines which may be used with the DNA
constructs and methods of the present invention include, but are
not limited to, HT-1080 cells (ATCC CCL 121), HeLa cells and
derivatives of HeLa cells (ATCC CCL 2, 2.1 and 2.2), MCF-7 breast
cancer cells (ATCC BTH 22), K-562 leukemia cells (ATCC CCL 243), KB
carcinoma cells (ATCC CCL 17), 2780AD ovarian carcinoma cells (Van
der Blick, A. M. et al., Cancer Res, 48:5927-5932 (1988), Raji
cells (ATCC CCL 86), WiDr colon adenocarcinoma cells (ATCC CCL
218), SW620 colon adenocarcinoma cells (ATCC CCL 227), Jurkat cells
(ATCC TIB 152), Namalwa cells (ATCC CRL1432), HL-60 cells (ATCC CCL
240), Daudi cells (ATCC CCL 213), RPMI 8226 cells (ATCC CCL 155),
U-937 cells (ATCC CRL 1593), Bowes Melanoma cells (ATCC CRL 9607),
WI-38VA13 subline 2R4 cells (ATCC CLL 75.1), and MOLT-4 cells (ATCC
CRL 1582), CHO cells, and COS cells, as well as heterohybridoma
cells produced by fusion of human cells and cells of another
species. Secondary human fibroblast strains, such as WI-38 (ATCC
CCL 75) and MRC-5 (ATCC CCL 171) may also be used. Further
discussion of the types of cells that may be used in practicing the
methods of the present invention are described in U.S. Pat. Nos.
5,641,670 and 6,270,989. Cell-free transcription systems also may
be used in lieu of cells.
[0181] The cells of the invention are maintained under conditions,
as are known in the art, which result in expression of the FGE
protein or functional fragments thereof. Proteins expressed using
the methods described may be purified from cell lysates or cell
supernatants. Proteins made according to this method can be
prepared as a pharmaceutically-useful formulation and delivered to
a human or non-human animal by conventional pharmaceutical routes
as is known in the art (e.g., oral, intravenous, intramuscular,
intranasal, intratracheal or subcutaneous). As described elsewhere
herein, the recombinant cells can be immortalized, primary, or
secondary cells, preferably human. The use of cells from other
species may be desirable in cases where the non-human cells are
advantageous for protein production purposes where the non-human
FGE produced is useful therapeutically.
[0182] As used herein, a coding sequence and regulatory sequences
are said to be "operably" joined when they are covalently linked in
such a way as to place the expression or transcription of the
coding sequence under the influence or control of the regulatory
sequences. If it is desired that the coding sequences be translated
into a functional protein, two DNA sequences are said to be
operably joined if induction of a promoter in the 5' regulatory
sequences results in the transcription of the coding sequence and
if the nature of the linkage between the two DNA sequences does not
(1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the promoter region to direct the
transcription of the coding sequences, or (3) interfere with the
ability of the corresponding RNA transcript to be translated into a
protein. Thus, a promoter region would be operably joined to a
coding sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting
transcript might be translated into the desired protein or
polypeptide.
[0183] The precise nature of the regulatory sequences needed for
gene expression may vary between species or cell types, but shall
in general include, as necessary, 5' non-transcribed and 5'
non-translated sequences involved with the initiation of
transcription and translation respectively, such as a TATA box,
capping sequence, CAAT sequence, and the like. Especially, such 5'
non-transcribed regulatory sequences will include a promoter region
which includes a promoter sequence for transcriptional control of
the operably joined gene. Regulatory sequences may also include
enhancer sequences or upstream activator sequences as desired. The
vectors of the invention may optionally include 5' leader or signal
sequences. The choice and design of an appropriate vector is within
the ability and discretion of one of ordinary skill in the art.
Expression vectors containing all the necessary elements for
expression are commercially available and known to those skilled in
the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, 1989. Cells are genetically engineered by the introduction
into the cells of heterologous DNA (RNA) encoding FGE polypeptide
or fragment or variant thereof. That heterologous DNA (RNA) is
placed under operable control of transcriptional elements to permit
the expression of the heterologous DNA in the host cell.
[0184] Preferred systems for mRNA expression in mammalian cells are
those such as pRc/CMV (available from Invitrogen, Carlsbad, Calif.)
that contain a selectable marker such as a gene that confers G418
resistance (which facilitates the selection of stably transfected
cell lines) and the human cytomegalovirus (CMV) enhancer-promoter
sequences. Additionally, suitable for expression in primate or
canine cell lines is the pCEP4 vector (Invitrogen, Carlsbad,
Calif.), which contains an Epstein Barr virus (EBV) origin of
replication, facilitating the maintenance of plasmid as a multicopy
extrachromosomal element. Another expression vector is the pEF-BOS
plasmid containing the promoter of polypeptide Elongation Factor
1a, which stimulates efficiently transcription in vitro. The
plasmid is described by Mishizuma and Nagata (Nuc. Acids Res.
18:5322, 1990), and its use in transfection experiments is
disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716,
1996). Still another preferred expression vector is an adenovirus,
described by Stratford-Perricaudet, which is defective for E1 and
E3 proteins (J. Clin. Invest. 90:626-630, 1992). The use of the
adenovirus as an Adeno.P1A recombinant is disclosed by Warnier et
al., in intradermal injection in mice for immunization against P1A
(Int. J. Cancer, 67:303-310, 1996).
[0185] The invention also embraces so-called expression kits, which
allow the artisan to prepare a desired expression vector or
vectors. Such expression kits include at least separate portions of
each of the previously discussed coding sequences. Other components
may be added, as desired, as long as the previously mentioned
sequences, which are required, are included.
[0186] It will also be recognized that the invention embraces the
use of the above described, FGE cDNA sequence containing expression
vectors, to transfect host cells and cell lines, be these
prokaryotic (e.g., Escherichia coli), or eukaryotic (e.g., CHO
cells, COS cells, yeast expression systems and recombinant
baculovirus expression in insect cells). Especially useful are
mammalian cells such as human, mouse, hamster, pig, goat, primate,
etc. They may be of a wide variety of tissue types, and include
primary cells and immortalized cell lines as described elsewhere
herein. Specific examples include HT-1080 cells, CHO cells,
dendritic cells, U293 cells, peripheral blood leukocytes, bone
marrow stem cells, embryonic stem cells, and insect cells. The
invention also permits the construction of FGE gene "knock-outs" in
cells and in animals, providing materials for studying certain
aspects of FGE activity.
[0187] The invention also provides isolated polypeptides (including
whole proteins and partial proteins), encoded by the foregoing FGE
nucleic acids, and include the polypeptide of SEQ ID NO:2 and
unique fragments thereof. Such polypeptides are useful, for
example, alone or as part of fusion proteins to generate
antibodies, as components of an immunoassay, etc. Polypeptides can
be isolated from biological samples including tissue or cell
homogenates, and can also be expressed recombinantly in a variety
of prokaryotic and eukaryotic expression systems by constructing an
expression vector appropriate to the expression system, introducing
the expression vector into the expression system, and isolating the
recombinantly expressed protein. Short polypeptides, including
antigenic peptides (such as are presented by MHC molecules on the
surface of a cell for immune recognition) also can be synthesized
chemically using well-established methods of peptide synthesis.
[0188] A unique fragment of a FGE polypeptide, in general, has the
features and characteristics of unique fragments as discussed above
in connection with nucleic acids. As will be recognized by those
skilled in the art, the size of the unique fragment will depend
upon factors such as whether the fragment constitutes a portion of
a conserved protein domain. Thus, some regions of SEQ ID NO:2 will
require longer segments to be unique while others will require only
short segments, typically between 5 and 12 amino acids (e.g. 5, 6,
7, 8, 9, 10, 11 and 12 amino acids long or more, including each
integer up to the full length, 287 amino acids long).
[0189] Unique fragments of a polypeptide preferably are those
fragments which retain a distinct functional capability of the
polypeptide. Functional capabilities which can be retained in a
unique fragment of a polypeptide include interaction with
antibodies, interaction with other polypeptides or fragments
thereof, interaction with other molecules, etc. One important
activity is the ability to act as a signature for identifying the
polypeptide. Those skilled in the art are well versed in methods
for selecting unique amino acid sequences, typically on the basis
of the ability of the unique fragment to selectively distinguish
the sequence of interest from non-family members. A comparison of
the sequence of the fragment to those on known databases typically
is all that is necessary.
[0190] The invention embraces variants of the FGE polypeptides
described above. As used herein, a "variant" of a FGE polypeptide
is a polypeptide which contains one or more modifications to the
primary amino acid sequence of a FGE polypeptide. Modifications
which create a FGE polypeptide variant are typically made to the
nucleic acid which encodes the FGE polypeptide, and can include
deletions, point mutations, truncations, amino acid substitutions
and addition of amino acids or non-amino acid moieties to: 1)
reduce or eliminate an activity of a FGE polypeptide; 2) enhance a
property of a FGE polypeptide, such as protein stability in an
expression system or the stability of protein-ligand binding; 3)
provide a novel activity or property to a FGE polypeptide, such as
addition of an antigenic epitope or addition of a detectable
moiety; or 4) to provide equivalent or better binding to a FGE
polypeptide receptor or other molecule. Alternatively,
modifications can be made directly to the polypeptide, such as by
cleavage, addition of a linker molecule, addition of a detectable
moiety, such as biotin, addition of a fatty acid, and the like.
Modifications also embrace fusion proteins comprising all or part
of the FGE amino acid sequence. One of skill in the art will be
familiar with methods for predicting the effect on protein
conformation of a change in protein sequence, and can thus "design"
a variant FGE polypeptide according to known methods. One example
of such a method is described by Dahiyat and Mayo in Science
278:82-87, 1997, whereby proteins can be designed de novo. The
method can be applied to a known protein to vary only a portion of
the polypeptide sequence. By applying the computational methods of
Dahiyat and Mayo, specific variants of the FGE polypeptide can be
proposed and tested to determine whether the variant retains a
desired conformation.
[0191] Variants can include FGE polypeptides which are modified
specifically to alter a feature of the polypeptide unrelated to its
physiological activity. For example, cysteine residues can be
substituted or deleted to prevent unwanted disulfide linkages.
Similarly, certain amino acids can be changed to enhance expression
of a FGE polypeptide by eliminating proteolysis by proteases in an
expression system (e.g., dibasic amino acid residues in yeast
expression systems in which KEX2 protease activity is present).
[0192] Mutations of a nucleic acid which encodes a FGE polypeptide
preferably preserve the amino acid reading frame of the coding
sequence, and preferably do not create regions in the nucleic acid
which are likely to hybridize to form secondary structures, such a
hairpins or loops, which can be deleterious to expression of the
variant polypeptide.
[0193] Mutations can be made by selecting an amino acid
substitution, or by random mutagenesis of a selected site in a
nucleic acid which encodes the polypeptide. Variant polypeptides
are then expressed and tested for one or more activities to
determine which mutation provides a variant polypeptide with the
desired properties. Further mutations can be made to variants (or
to non-variant FGE polypeptides) which are silent as to the amino
acid sequence of the polypeptide, but which provide preferred
codons for translation in a particular host, or alter the structure
of the mRNA to, for example, enhance stability and/or expression.
The preferred codons for translation of a nucleic acid in, e.g.,
Escherichia coli, mammalian cells, etc. are well known to those of
ordinary skill in the art. Still other mutations can be made to the
noncoding sequences of a FGE gene or cDNA clone to enhance
expression of the polypeptide.
[0194] The skilled artisan will realize that conservative amino
acid substitutions may be made in FGE polypeptides to provide
functionally equivalent variants of the foregoing polypeptides,
i.e, the variants retain the functional capabilities of the FGE
polypeptides. As used herein, a "conservative amino acid
substitution" refers to an amino acid substitution which does not
significantly alter the tertiary structure and/or activity of the
polypeptide. Variants can be prepared according to methods for
altering polypeptide sequence known to one of ordinary skill in the
art, and include those that are found in references which compile
such methods, e.g. Molecular Cloning: A Laboratory Manual, J.
Sambrook, et al., eds., Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current
Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John
Wiley & Sons, Inc., New York. Exemplary functionally equivalent
variants of the FGE polypeptides include conservative amino acid
substitutions of SEQ ID NO:2. Conservative substitutions of amino
acids include substitutions made amongst amino acids within the
following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A,
G; (e) S, T; (f) Q, N; and (g) E, D.
[0195] Thus functionally equivalent variants of FGE polypeptides,
i.e., variants of FGE polypeptides which retain the function of the
natural FGE polypeptides, are contemplated by the invention.
Conservative amino-acid substitutions in the amino acid sequence of
FGE polypeptides to produce functionally equivalent variants of FGE
polypeptides typically are made by alteration of a nucleic acid
encoding FGE polypeptides (SEQ ID NOs:1, 3). Such substitutions can
be made by a variety of methods known to one of ordinary skill in
the art. For example, amino acid substitutions may be made by
PCR-directed mutation, site-directed mutagenesis according to the
method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,
1985), or by chemical synthesis of a gene encoding a FGE
polypeptide. The activity of functionally equivalent fragments of
FGE polypeptides can be tested by cloning the gene encoding the
altered FGE polypeptide into a bacterial or mammalian expression
vector, introducing the vector into an appropriate host cell,
expressing the altered FGE polypeptide, and testing for a
functional capability of the FGE polypeptides as disclosed herein
(e.g., C.sub..alpha.-formylglycine generating activity, etc.).
[0196] The invention as described herein has a number of uses, some
of which are described elsewhere herein. First, the invention
permits isolation of FGE polypeptides. A variety of methodologies
well-known to the skilled practitioner can be utilized to obtain
isolated FGE molecules. The polypeptide may be purified from cells
which naturally produce the polypeptide by chromatographic means or
immunological recognition. Alternatively, an expression vector may
be introduced into cells to cause production of the polypeptide. In
another method, mRNA transcripts may be microinjected or otherwise
introduced into cells to cause production of the encoded
polypeptide. Translation of FGE mRNA in cell-free extracts such as
the reticulocyte lysate system also may be used to produce FGE
polypeptides. Those skilled in the art also can readily follow
known methods for isolating FGE polypeptides. These include, but
are not limited to, immunochromatography, HPLC, size-exclusion
chromatography, ion-exchange chromatography and immune-affinity
chromatography.
[0197] The invention also provides, in certain embodiments,
"dominant negative" polypeptides derived from FGE polypeptides. A
dominant negative polypeptide is an inactive variant of a protein,
which, by interacting with the cellular machinery, displaces an
active protein from its interaction with the cellular machinery or
competes with the active protein, thereby reducing the effect of
the active protein. For example, a dominant negative receptor which
binds a ligand but does not transmit a signal in response to
binding of the ligand can reduce the biological effect of
expression of the ligand. Likewise, a dominant negative
catalytically-inactive kinase which interacts normally with target
proteins but does not phosphorylate the target proteins can reduce
phosphorylation of the target proteins in response to a cellular
signal. Similarly, a dominant negative transcription factor which
binds to a promoter site in the control region of a gene but does
not increase gene transcription can reduce the effect of a normal
transcription factor by occupying promoter binding sites without
increasing transcription.
[0198] The end result of the expression of a dominant negative
polypeptide in a cell is a reduction in function of active
proteins. One of ordinary skill in the art can assess the potential
for a dominant negative variant of a protein, and use standard
mutagenesis techniques to create one or more dominant negative
variant polypeptides. See, e.g., U.S. Pat. No. 5,580,723 and
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press, 1989. The skilled
artisan then can test the population of mutagenized polypeptides
for diminution in a selected activity and/or for retention of such
an activity. Other similar methods for creating and testing
dominant negative variants of a protein will be apparent to one of
ordinary skill in the art.
[0199] The isolation of the FGE cDNA also makes it possible for the
artisan to diagnose a disorder characterized by an aberrant
expression of FGE. These methods involve determining expression of
the FGE gene, and/or FGE polypeptides derived therefrom. In the
former situation, such determinations can be carried out via any
standard nucleic acid determination assay, including the polymerase
chain reaction, or assaying with labeled hybridization probes as
exemplified below. In the latter situation, such determination can
be carried out via any standard immunological assay using, for
example, antibodies which bind to the secreted FGE protein. A
preferred disorder that can be diagnosed according to the invention
is Multiple Sulfatase Deficiency.
[0200] The invention also embraces isolated peptide binding agents
which, for example, can be antibodies or fragments of antibodies
("binding polypeptides"), having the ability to selectively bind to
FGE polypeptides. Antibodies include polyclonal and monoclonal
antibodies, prepared according to conventional methodology. In
certain embodiments, the invention excludes binding agents (e.g.,
antibodies) that bind to the polypeptides encoded by the nucleic
acids of SEQ ID NO:4.
[0201] Significantly, as is well-known in the art, only a small
portion of an antibody molecule, the paratope, is involved in the
binding of the antibody to its epitope (see, in general, Clark, W.
R. (1986) The Experimental Foundations of Modern Immunology Wiley
& Sons, Inc., New York; Roitt, I. (1991) Essential Immunology,
7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and
Fc regions, for example, are effectors of the complement cascade
but are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab').sub.2
fragment, retains both of the antigen binding sites of an intact
antibody. Similarly, an antibody from which the Fc region has been
enzymatically cleaved, or which has been produced without the Fc
region, designated an Fab fragment, retains one of the antigen
binding sites of an intact antibody molecule. Proceeding further,
Fab fragments consist of a covalently bound antibody light chain
and a portion of the antibody heavy chain denoted Fd. The Fd
fragments are the major determinant of antibody specificity (a
single Fd fragment may be associated with up to ten different light
chains without altering antibody specificity) and Fd fragments
retain epitope-binding ability in isolation.
[0202] Within the antigen-binding portion of an antibody, as is
well-known in the art, there are complementarity determining
regions (CDRs), which directly interact with the epitope of the
antigen, and framework regions (FRs), which maintain the tertiary
structure of the paratope (see, in general, Clark, 1986; Roitt,
1991). In both the heavy chain Fd fragment and the light chain of
IgG immunoglobulins, there are four framework regions (FR1 through
FR4) separated respectively by three complementarity determining
regions (CDR1 through CDR3). The CDRs, and in particular the CDR3
regions, and more particularly the heavy chain CDR3, are largely
responsible for antibody specificity.
[0203] It is now well-established in the art that the non-CDR
regions of a mammalian antibody may be replaced with similar
regions of conspecific or heterospecific antibodies while retaining
the epitopic specificity of the original antibody. This is most
clearly manifested in the development and use of "humanized"
antibodies in which non-human CDRs are covalently joined to human
FR and/or Fc/pFc' regions to produce a functional antibody. See,
e.g., U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,762 and
5,859,205. Thus, for example, PCT International Publication Number
WO 92/04381 teaches the production and use of humanized murine RSV
antibodies in which at least a portion of the murine FR regions
have been replaced by FR regions of human origin. Such antibodies,
including fragments of intact antibodies with antigen-binding
ability, are often referred to as "chimeric" antibodies.
[0204] Thus, as will be apparent to one of ordinary skill in the
art, the present invention also provides for F(ab').sub.2, Fab, Fv
and Fd fragments; chimeric antibodies in which the Fc and/or FR
and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been
replaced by homologous human or non-human sequences; chimeric
F(ab').sub.2 fragment antibodies in which the FR and/or CDR1 and/or
CDR2 and/or light chain CDR3 regions have been replaced by
homologous human or non-human sequences; chimeric Fab fragment
antibodies in which the FR and/or CDR1 and/or CDR2 and/or light
chain CDR3 regions have been replaced by homologous human or
non-human sequences; and chimeric Fd fragment antibodies in which
the FR and/or CDR 1 and/or CDR2 regions have been replaced by
homologous human or non-human sequences. The present invention also
includes so-called single chain antibodies.
[0205] Thus, the invention involves polypeptides of numerous size
and type that bind specifically to FGE polypeptides, and complexes
of both FGE polypeptides and their binding partners. These
polypeptides may be derived also from sources other than antibody
technology. For example, such polypeptide binding agents can be
provided by degenerate peptide libraries which can be readily
prepared in solution, in immobilized form, as bacterial flagella
peptide display libraries or as phage display libraries.
Combinatorial libraries also can be synthesized of peptides
containing one or more amino acids. Libraries further can be
synthesized of peptides and non-peptide synthetic moieties.
[0206] Phage display can be particularly effective in identifying
binding peptides useful according to the invention. Briefly, one
prepares a phage library (using e.g. m13, fd, or lambda phage),
displaying inserts from 4 to about 80 amino acid residues using
conventional procedures. The inserts may represent, for example, a
completely degenerate or biased array. One then can select
phage-bearing inserts which bind to the FGE polypeptide or a
complex of FGE and a binding partner. This process can be repeated
through several cycles of reselection of phage that bind to the FGE
polypeptide or complex. Repeated rounds lead to enrichment of phage
bearing particular sequences. DNA sequence analysis can be
conducted to identify the sequences of the expressed polypeptides.
The minimal linear portion of the sequence that binds to the FGE
polypeptide or complex can be determined. One can repeat the
procedure using a biased library containing inserts containing part
or all of the minimal linear portion plus one or more additional
degenerate residues upstream or downstream thereof. Yeast
two-hybrid screening methods also may be used to identify
polypeptides that bind to the FGE polypeptides. Thus, the FGE
polypeptides of the invention, or a fragment thereof, or complexes
of FGE and a binding partner can be used to screen peptide
libraries, including phage display libraries, to identify and
select peptide binding partners of the FGE polypeptides of the
invention. Such molecules can be used, as described, for screening
assays, for purification protocols, for interfering directly with
the functioning of FGE and for other purposes that will be apparent
to those of ordinary skill in the art.
[0207] An FGE polypeptide, or a fragment thereof, also can be used
to isolate their native binding partners. Isolation of binding
partners may be performed according to well-known methods. For
example, isolated FGE polypeptides can be attached to a substrate,
and then a solution suspected of containing a FGE binding partner
may be applied to the substrate. If the binding partner for FGE
polypeptides is present in the solution, then it will bind to the
substrate-bound FGE polypeptide. The binding partner then may be
isolated. Other proteins which are binding partners for FGE, may be
isolated by similar methods without" undue experimentation. A
preferred binding partner is a sulfatase.
[0208] The invention also provides methods to measure the level of
FGE expression in a subject. This can be performed by first
obtaining a test sample from the subject. The test sample can be
tissue or biological fluid. Tissues include brain, heart, serum,
breast, colon, bladder, uterus, prostate, stomach, testis, ovary,
pancreas, pituitary gland, adrenal gland, thyroid gland, salivary
gland, mammary gland, kidney, liver, intestine, spleen, thymus,
blood vessels, bone marrow, trachea, and lung. In certain
embodiments, test samples originate from heart and blood vessel
tissues, and biological fluids include blood, saliva and urine.
Both invasive and non-invasive techniques can be used to obtain
such samples and are well documented in the art. At the molecular
level both PCR and Northern blotting can be used to determine the
level of FGE mRNA using products of this invention described
herein, and protocols well known in the art that are found in
references which compile such methods. At the protein level, FGE
expression can be determined using either polyclonal or monoclonal
anti-FGE sera in combination with standard immunological assays.
The preferred methods will compare the measured level of FGE
expression of the test sample to a control. A control can include a
known amount of a nucleic acid probe, a FGE epitope (such as a FGE
expression product), or a similar test sample of a subject with a
control or `normal` level of FGE expression.
[0209] FGE polypeptides preferably are produced recombinantly,
although such polypeptides may be isolated from biological
extracts. Recombinantly produced FGE polypeptides include chimeric
proteins comprising a fusion of a FGE protein with another
polypeptide, e.g., a polypeptide capable of providing or enhancing
protein-protein binding, sequence specific nucleic acid binding
(such as GAL4), enhancing stability of the FGE polypeptide under
assay conditions, or providing a detectable moiety, such as green
fluorescent protein. A polypeptide fused to a FGE polypeptide or
fragment may also provide means of readily detecting the fusion
protein, e.g., by immunological recognition or by fluorescent
labeling.
[0210] The invention also is useful in the generation of transgenic
non-human animals. As used herein, "transgenic non-human animals"
includes non-human animals having one or more exogenous nucleic
acid molecules incorporated in germ line cells and/or somatic
cells. Thus the transgenic animals include "knockout" animals
having a homozygous or heterozygous gene disruption by homologous
recombination, animals having episomal or chromosomally
incorporated expression vectors, etc. Knockout animals can be
prepared by homologous recombination using embryonic stem cells as
is well known in the art. The recombination may be facilitated
using, for example, the cre/lox system or other recombinase systems
known to one of ordinary skill in the art. In certain embodiments,
the recombinase system itself is expressed conditionally, for
example, in certain tissues or cell types, at certain embryonic or
post-embryonic developmental stages, is induced by the addition of
a compound which increases or decreases expression, and the like.
In general, the conditional expression vectors used in such systems
use a variety of promoters which confer the desired gene expression
pattern (e.g., temporal or spatial). Conditional promoters also can
be operably linked to FGE nucleic acid molecules to increase
expression of FGE in a regulated or conditional manner.
Trans-acting negative regulators of FGE activity or expression also
can be operably linked to a conditional promoter as described
above. Such trans-acting regulators include antisense FGE nucleic
acids molecules, nucleic acid molecules which encode dominant
negative FGE molecules, ribozyme molecules specific for FGE nucleic
acids, and the like. The transgenic non-human animals are useful in
experiments directed toward testing biochemical or physiological
effects of diagnostics or therapeutics for conditions characterized
by increased or decreased FGE expression. Other uses will be
apparent to one of ordinary skill in the art.
[0211] The invention also contemplates gene therapy. The procedure
for performing ex vivo gene therapy is outlined in U.S. Pat. No.
5,399,346 and in exhibits submitted in the file history of that
patent, all of which are publicly available documents. In general,
it involves introduction in vitro of a functional copy of a gene
into a cell(s) of a subject which contains a defective copy of the
gene, and returning the genetically engineered cell(s) to the
subject. The functional copy of the gene is under operable control
of regulatory elements which permit expression of the gene in the
genetically engineered cell(s). Numerous transfection and
transduction techniques as well as appropriate expression vectors
are well known to those of ordinary skill in the art, some of which
are described in PCT application WO95/00654. In vivo gene therapy
using vectors such as adenovirus, retroviruses, herpes virus, and
targeted liposomes also is contemplated according to the
invention.
[0212] The invention further provides efficient methods of
identifying agents or lead compounds for agents active at the level
of a FGE or FGE fragment dependent cellular function. In
particular, such functions include interaction with other
polypeptides or fragments. Generally, the screening methods involve
assaying for compounds which interfere with FGE activity (such as
C.alpha.-formylglycine generating activity), although compounds
which enhance FGE C.sub..alpha.-formylglycine generating activity
also can be assayed using the screening methods. Such methods are
adaptable to automated, high throughput screening of compounds.
Target indications include cellular processes modulated by FGE such
as C.sub..alpha.-formylglycine generating activity.
[0213] A wide variety of assays for candidate (pharmacological)
agents are provided, including, labeled in vitro protein-ligand
binding assays, electrophoretic mobility shift assays,
immunoassays, cell-based assays such as two- or three-hybrid
screens, expression assays, etc. The transfected nucleic acids can
encode, for example, combinatorial peptide libraries or cDNA
libraries. Convenient reagents for such assays, e.g., GAL4 fusion
proteins, are known in the art. An exemplary cell-based assay
involves transfecting a cell with a nucleic acid encoding a FGE
polypeptide fused to a GAL4 DNA binding domain and a nucleic acid
encoding a reporter gene operably linked to a gene expression
regulatory region, such as one or more GAL4 binding sites.
Activation of reporter gene transcription occurs when the FGE and
reporter fusion polypeptide binds such as to enable transcription
of the reporter gene. Agents which modulate a FGE polypeptide
mediated cell function are then detected through a change in the
expression of reporter gene. Methods for determining changes in the
expression of a reporter gene are known in the art.
[0214] FGE fragments used in the methods, when not produced by a
transfected nucleic acid are added to an assay mixture as an
isolated polypeptide. FGE polypeptides preferably are produced
recombinantly, although such polypeptides may be isolated from
biological extracts. Recombinantly produced FGE polypeptides
include chimeric proteins comprising a fusion of a FGE protein with
another polypeptide, e.g., a polypeptide capable of providing or
enhancing protein-protein binding, sequence specific nucleic acid
binding (such as GAL4), enhancing stability of the FGE polypeptide
under assay conditions, or providing a detectable moiety, such as
green fluorescent protein or Flag epitope.
[0215] The assay mixture is comprised of a natural intracellular
FGE binding target capable of interacting with FGE. While natural
FGE binding targets may be used, it is frequently preferred to use
portions (e.g., peptides--see e.g., the peptide of SEQ ID NO:33--or
nucleic acid fragments) or analogs (i.e., agents which mimic the
FGE binding properties of the natural binding target for purposes
of the assay) of the FGE binding target so long as the portion or
analog provides binding affinity and avidity to the FGE fragment
measurable in the assay.
[0216] The assay mixture also comprises a candidate agent.
Typically, a plurality of assay mixtures are run in parallel with
different agent concentrations to obtain a different response to
the various concentrations. Typically, one of these concentrations
serves as a negative control, i.e., at zero concentration of agent
or at a concentration of agent below the limits of assay detection.
Candidate agents encompass numerous chemical classes, although
typically they are organic compounds. Preferably, the candidate
agents are small organic compounds, i.e., those having a molecular
weight of more than 50 yet less than about 2500, preferably less
than about 1000 and, more preferably, less than about 500.
Candidate agents comprise functional chemical groups necessary for
structural interactions with polypeptides and/or nucleic acids, and
typically include at least an amine, carbonyl, hydroxyl or carboxyl
group, preferably at least two of the functional chemical groups
and more preferably at least three of the functional chemical
groups. The candidate agents can comprise cyclic carbon or
heterocyclic structure and/or aromatic or polyaromatic structures
substituted with one or more of the above-identified functional
groups. Candidate agents also can be biomolecules such as peptides,
saccharides, fatty acids, sterols, isoprenoids, purines,
pyrimidines, derivatives or structural analogs of the above, or
combinations thereof and the like. Where the agent is a nucleic
acid, the agent typically is a DNA or RNA molecule, although
modified nucleic acids as defined herein are also contemplated.
[0217] Candidate agents are obtained from a wide variety of sources
including libraries of synthetic or natural compounds. For example,
numerous means are available for random and directed synthesis of a
wide variety of organic compounds and biomolecules, including
expression of randomized oligonucleotides, synthetic organic
combinatorial libraries, phage display libraries of random
peptides, and the like. Alternatively, libraries of natural
compounds in the form of bacterial, fungal, plant and animal
extracts are available or readily produced. Additionally, natural
and synthetically produced libraries and compounds can be modified
through conventional chemical, physical, and biochemical means.
Further, known (pharmacological) agents may be subjected to
directed or random chemical modifications such as acylation,
alkylation, esterification, amidification, etc. to produce
structural analogs of the agents.
[0218] A variety of other reagents also can be included in the
mixture. These include reagents such as salts, buffers, neutral
proteins (e.g., albumin), detergents, etc. which may be used to
facilitate optimal protein-protein and/or protein-nucleic acid
binding. Such a reagent may also reduce non-specific or background
interactions of the reaction components. Other reagents that
improve the efficiency of the assay such as protease, inhibitors,
nuclease inhibitors, antimicrobial agents, and the like may also be
used.
[0219] The mixture of the foregoing assay materials is incubated
under conditions whereby, but for the presence of the candidate
agent, the FGE polypeptide specifically binds a cellular binding
target, a portion thereof or analog thereof. The order of addition
of components, incubation temperature, time of incubation, and
other parameters of the assay may be readily determined. Such
experimentation merely involves optimization of the assay
parameters, not the fundamental composition of the assay.
Incubation temperatures typically are between 4.degree. C. and
40.degree. C. Incubation times preferably are minimized to
facilitate rapid, high throughput screening, and typically are
between 0.1 and 10 hours.
[0220] After incubation, the presence or absence of specific
binding between the FGE polypeptide and one or more binding targets
is detected by any convenient method available to the user. For
cell free binding type assays, a separation step is often used to
separate bound from unbound components. The separation step may be
accomplished in a variety of ways. Conveniently, at least one of
the components is immobilized on a solid substrate, from which the
unbound components may be easily separated. The solid substrate can
be made of a wide variety of materials and in a wide variety of
shapes, e.g., microtiter plate, microbead, dipstick, resin
particle, etc. The substrate preferably is chosen to maximum signal
to noise ratios, primarily to minimize background binding, as well
as for ease of separation and cost.
[0221] Separation may be effected for example, by removing a bead
or dipstick from a reservoir, emptying or diluting a reservoir such
as a microtiter plate well, rinsing a bead, particle,
chromatograpic column or filter with a wash solution or solvent.
The separation step preferably includes multiple rinses or washes.
For example, when the solid substrate is a microtiter plate, the
wells may be washed several times with a washing solution, which
typically includes those components of the incubation mixture that
do not participate in specific bindings such as salts, buffer,
detergent, non-specific protein, etc. Where the solid substrate is
a magnetic bead, the beads may be washed one or more times with a
washing solution and isolated using a magnet.
[0222] Detection may be effected in any convenient way for
cell-based assays such as two- or three-hybrid screens. The
transcript resulting from a reporter gene transcription assay of
FGE polypeptide interacting with a target molecule typically
encodes a directly or indirectly detectable product, e.g.,
.beta.-galactosidase activity, luciferase activity, and the like.
For cell free binding assays, one of the components usually
comprises, or is coupled to, a detectable label. A wide variety of
labels can be used, such as those that provide direct detection
(e.g., radioactivity, luminescence, optical or electron density,
etc), or indirect detection (e.g., epitope tag such as the FLAG
epitope, enzyme tag such as horseseradish peroxidase, etc.). The
label may be bound to a FGE binding partner, or incorporated into
the structure of the binding partner.
[0223] A variety of methods may be used to detect the label,
depending on the nature of the label and other assay components.
For example, the label may be detected while bound to the solid
substrate or subsequent to separation from the solid substrate.
Labels may be directly detected through optical or electron
density, radioactive emissions, nonradiative energy transfers, etc.
or indirectly detected with antibody conjugates,
streptavidin-biotin conjugates, etc. Methods for detecting the
labels are well known in the art.
[0224] The invention provides FGE-specific binding agents, methods
of identifying and making such agents, and their use in diagnosis,
therapy and pharmaceutical development. For example, FGE-specific
pharmacological agents are useful in a variety of diagnostic and
therapeutic applications, especially where disease or disease
prognosis is associated with altered FGE binding characteristics
such as in Multiple Sulfatase Deficiency. Novel FGE-specific
binding agents include FGE-specific antibodies, cell surface
receptors, and other natural intracellular and extracellular
binding agents identified with assays such as two hybrid screens,
and non-natural intracellular and extracellular binding agents
identified in screens of chemical libraries and the like.
[0225] In general, the specificity of FGE binding to a specific
molecule is determined by binding equilibrium constants. Targets
which are capable of selectively binding a FGE polypeptide
preferably have binding equilibrium constants of at least about
10.sup.7 M.sup.-1 more preferably at least about 10.sup.8 M.sup.-1,
and most preferably at least about 10.sup.9 M.sup.-1. A wide
variety of cell based and cell free assays may be used to
demonstrate FGE-specific binding. Cell based assays include one,
two and three hybrid screens, assays in which FGE-mediated
transcription is inhibited or increased, etc. Cell free assays
include FGE-protein binding assays, immunoassays, etc. Other assays
useful for screening agents which bind FGE polypeptides include
fluorescence resonance energy transfer (FRET), and electrophoretic
mobility shift analysis (EMSA).
[0226] According to another aspect of the invention, a method for
identifying an agent useful in modulating
C.sub..alpha.-formylglycine generating activity of a molecule of
the invention, is provided. The method involves (a) contacting a
molecule having C.sub..alpha.-formylglycine generating activity
with a candidate agent, (b) measuring C.sub..alpha.-formylglycine
generating activity of the molecule, and (c) comparing the measured
C.sub..alpha.-formylglycine generating activity of the molecule to
a control to determine whether the candidate agent modulates
C.sub..alpha.-formylglycine generating activity of the molecule,
wherein the molecule is an FGE nucleic acid molecule of the
invention, or an expression product thereof "Contacting" refers to
both direct and indirect contacting of a molecule having
C.sub..alpha.-formylglycine generating activity with the candidate
agent. "Indirect" contacting means that the candidate agent exerts
its effects on the C.sub..alpha.-formylglycine generating activity
of the molecule via a third agent (e.g., a messenger molecule, a
receptor, etc.). In certain embodiments, the control is
C.sub..alpha.-formylglycine generating activity of the molecule
measured in the absence of the candidate agent. Assaying methods
and candidate agents are as described above in the foregoing
embodiments with respect to FGE.
[0227] According to still another aspect of the invention, a method
of diagnosing a disorder characterized by aberrant expression of a
nucleic acid molecule, an expression product thereof, or a fragment
of an expression product thereof, is provided. The method involves
contacting a biological sample isolated from a subject with an
agent that specifically binds to the nucleic acid molecule, an
expression product thereof, or a fragment of an expression product
thereof, and determining the interaction between the agent and the
nucleic acid molecule or the expression product as a determination
of the disorder, wherein the nucleic acid molecule is an FGE
molecule according to the invention. The disorder is Multiple
Sulfatase Deficiency. Mutations in the FGE gene that cause the
aberrant expression of FGE molecules result in the following amino
acid changes on SEQ ID NO:2: MetlArg; Met 1Val; Leu20Phe;
Ser155Pro; Ala177Pro; Cys218Tyr; Arg224Trp; Asn259Ile; Pro266Leu;
Ala279Val; Arg327Stop; Cys336Arg; Arg345Cys; Ala348Pro; Arg349G1n;
Arg349Trp; Arg349Trp; Ser359Stop; or a combination thereof.
[0228] In the case where the molecule is a nucleic acid molecule,
such determinations can be carried out via any standard nucleic
acid determination assay, including the polymerase chain reaction,
or assaying with labeled hybridization probes as exemplified
herein. In the case where the molecule is an expression product of
the nucleic acid molecule, or a fragment of an expression product
of the nucleic acid molecule, such determination can be carried out
via any standard immunological assay using, for example, antibodies
which bind to any of the polypeptide expression products.
[0229] "Aberrant expression" refers to decreased expression
(underexpression) or increased expression (overexpression) of FGE
molecules (nucleic acids and/or polypeptides) in comparison with a
control (i.e., expression of the same molecule in a healthy or
"normal" subject). A "healthy subject", as used herein, refers to a
subject who, according to standard medical standards, does not have
or is at risk for developing Multiple Sulfatase Deficiency. Healthy
subjects also do not otherwise exhibit symptoms of disease. In
other words, such subjects, if examined by a medical professional,
would be characterized as healthy and free of symptoms of a
Multiple Sulfatase Deficiency. These include features of
metachromatic leukodystrophy and of a mucopolysaccharidosis, such
as increased amounts of acid mucopolysaccharides in several
tissues, mild `gargoylism`, rapid neurologic deterioration,
excessive presence of mucopolysaccharide and sulfatide in the
urine, increased cerebrospinal fluid protein, and metachromatic
degeneration of myelin in peripheral nerves.
[0230] The invention also provides novel kits which could be used
to measure the levels of the nucleic acids of the invention, or
expression products of the invention.
[0231] In one embodiment, a kit comprises a package containing an
agent that selectively binds to any of the foregoing FGE isolated
nucleic acids, or expression products thereof, and a control for
comparing to a measured value of binding of said agent any of the
foregoing FGE isolated nucleic acids or expression products
thereof. In some embodiments, the control is a predetermined value
for comparing to the measured value. In certain embodiments, the
control comprises an epitope of the expression product of any of
the foregoing FGE isolated nucleic acids. In one embodiment, the
kit further comprises a second agent that selectively binds to a
polypeptide selected from the group consisting of Iduronate
2-Sulfatase, Sulfamidase, N-Acetylgalactosamine 6-Sulfatase,
N-Acetylglucosamine 6-Sulfatase, Arylsulfatase A, Arylsulfatase B,
Arylsulfatase C, Arylsulfatase D, Arylsulfatase E, Arylsulfatase F,
Arylsulfatase G, HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, and
HSulf-6, or a peptide thereof, and a control for comparing to a
measured value of binding of said second agent to said polypeptide
or peptide thereof.
[0232] In the case of nucleic acid detection, pairs of primers for
amplifying a nucleic acid molecule of the invention can be
included. The preferred kits would include controls such as known
amounts of nucleic acid probes, epitopes (such as Iduronate
2-Sulfatase, Sulfamidase, N-Acetylgalactosamine 6-Sulfatase,
N-Acetylglucosamine 6-Sulfatase, Arylsulfatase A, Arylsulfatase B,
Arylsulfatase C, Arylsulfatase D, Arylsulfatase E, Arylsulfatase F,
Arylsulfatase G, HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, and
HSulf-6, expression products) or anti-epitope antibodies, as well
as instructions or other printed material. In certain embodiments
the printed material can characterize risk of developing a
sulfatase deficiency condition based upon the outcome of the assay.
The reagents may be packaged in containers and/or coated on wells
in predetermined amounts, and the kits may include standard
materials such as labeled immunological reagents (such as labeled
anti-IgG antibodies) and the like. One kit is a packaged
polystyrene microtiter plate coated with FGE protein and a
container containing labeled anti-human IgG antibodies. A well of
the plate is contacted with, for example, a biological fluid,
washed and then contacted with the anti-IgG antibody. The label is
then detected. A kit embodying features of the present invention,
generally designated by the numeral 11, is illustrated in FIG. 25.
Kit 11 is comprised of the following major elements: packaging 15,
an agent of the invention 17, a control agent 19 and instructions
21. Packaging 15 is a box-like structure for holding a vial (or
number of vials) containing an agent of the invention 17, a vial
(or number of vials) containing a control agent 19, and
instructions 21. Individuals skilled in the art can readily modify
packaging 15 to suit individual needs.
[0233] The invention also embraces methods for treating Multiple
Sulfatase Deficiency in a subject. The method involves
administering to a subject in need of such treatment an agent that
modulates C.sub..alpha.-formylglycine generating activity, in an
amount effective to increase C.sub..alpha.-formylglycine generating
activity in the subject. In some embodiments, the method further
comprises co-administering an agent selected from the group
consisting of a nucleic acid molecule encoding Iduronate
2-Sulfatase, Sulfamidase, N-Acetylgalactosamine 6-Sulfatase,
N-Acetylglucosamine 6-Sulfatase, Arylsulfatase A, Arylsulfatase B,
Arylsulfatase C, Arylsulfatase D, Arylsulfatase E, Arylsulfatase F,
Arylsulfatase G, HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, and
HSulf-6, an expression product of the nucleic acid molecule, and/or
a fragment of the expression product of the nucleic acid
molecule.
[0234] "Agents that modulate expression" of a nucleic acid or a
polypeptide, as used herein, are known in the art, and refer to
sense and antisense nucleic acids, dominant negative nucleic acids,
antibodies to the polypeptides, and the like. Any agents that
modulate expression of a molecule (and as described herein,
modulate its activity), are useful according to the invention. In
certain embodiments, the agent that modulates
C.sub..alpha.-formylglycine generating activity is an isolated
nucleic acid molecule of the invention (e.g., a nucleic acid of SEQ
ID NO.3). In important embodiments, the agent that modulates
C.sub..alpha.-formylglycine generating activity is a peptide of the
invention (e.g., a peptide of SEQ ID NO.2). In some embodiments,
the agent that modulates C.sub..alpha.-formylglycine generating
activity is a sense nucleic acid of the invention.
[0235] According to one aspect of the invention, a method for
increasing C.sub..alpha.-formylglycine generating activity in a
subject, is provided. The method involves administering an isolated
FGE nucleic acid molecule of the invention, and/or an expression
product thereof, to a subject, in an amount effective to increase
C.sub..alpha.-formylglycine generating activity in the subject.
[0236] According to still another aspect of the invention, a method
for increasing C.sub..alpha.-formylglycine generating activity in a
cell, is provided. The method involves contacting the cell with an
isolated nucleic acid molecule of the invention (e.g., a nucleic
acid of SEQ ID NO.1), or an expression product thereof (e.g., a
peptide of SEQ ID NO.2), in an amount effective to increase
C.sub..alpha.-formylglycine generating activity in the cell. In
important embodiments, the method involves activating the
endogenous FGE gene to increase C.sub..alpha.-formylglycine
generating activity in the cell.
[0237] In any of the foregoing embodiments the nucleic acid may be
operatively coupled to a gene expression sequence which directs the
expression of the nucleic acid molecule within a eukaryotic cell
such as an HT-1080 cell. The "gene expression sequence" is any
regulatory nucleotide sequence, such as a promoter sequence or
promoter-enhancer combination, which facilitates the efficient
transcription and translation of the nucleic acid to which it is
operably linked. The gene expression sequence may, for example, be
a mammalian or viral promoter, such as a constitutive or inducible
promoter. Constitutive mammalian promoters include, but are not
limited to, the promoters for the following genes: hypoxanthine
phosphoribosyl transferase (HPTR), adenosine deaminase, pyruvate
kinase, cc-actin promoter and other constitutive promoters.
Exemplary viral promoters which function constitutively in
eukaryotic cells include, for example, promoters from the simian
virus, papilloma virus, adenovirus, human immunodeficiency virus
(HIV), Rous sarcoma virus, cytomegalovirus, the long terminal
repeats (LTR) of moloney leukemia virus and other retroviruses, and
the thymidine kinase promoter of herpes simplex virus. Other
constitutive promoters are known to those of ordinary skill in the
art. The promoters useful as gene expression sequences of the
invention also include inducible promoters. Inducible promoters are
activated in the presence of an inducing agent. For example, the
metallothionein promoter is activated to increase transcription and
translation in the presence of certain metal ions. Other inducible
promoters are known to those of ordinary skill in the art.
[0238] In general, the gene expression sequence shall include, as
necessary, 5' non-transcribing and 5' non-translating sequences
involved with the initiation of transcription and translation,
respectively, such as a TATA box, capping sequence, CAAT sequence,
and the like. Especially, such 5' non-transcribing sequences will
include a promoter region which includes a promoter sequence for
transcriptional control of the operably joined nucleic acid. The
gene expression sequences optionally includes enhancer sequences or
upstream activator sequences as desired.
[0239] Preferably, any of the FGE nucleic acid molecules of the
invention is linked to a gene expression sequence which permits
expression of the nucleic acid molecule in a cell of a specific
cell lineage, e.g., a neuron. A sequence which permits expression
of the nucleic acid molecule in a cell such as a neuron, is one
which is selectively active in such a cell type, thereby causing
expression of the nucleic acid molecule in these cells. The
synapsin-1 promoter, for example, can be used to express any of the
foregoing nucleic acid molecules of the invention in a neuron; and
the von Willebrand factor gene promoter, for example, can be used
to express a nucleic acid molecule in a vascular endothelial cell.
Those of ordinary skill in the art will be able to easily identify
alternative promoters that are capable of expressing a nucleic acid
molecule in any of the preferred cells of the invention.
[0240] The nucleic acid sequence and the gene expression sequence
are said to be "operably linked" when they are covalently linked in
such a way as to place the transcription and/or translation of the
nucleic acid coding sequence (e.g, in the case of FGE, SEQ ID NO.
3) under the influence or control of the gene expression sequence.
If it is desired that the nucleic acid sequence be translated into
a functional protein, two DNA sequences are said to be operably
linked if induction of a promoter in the 5' gene expression
sequence results in the transcription of the nucleic acid sequence
and if the nature of the linkage between the two DNA sequences does
not (1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the promoter region to direct the
transcription of the nucleic acid sequence, and/or (3) interfere
with the ability of the corresponding RNA transcript to be
translated into a protein. Thus, a gene expression sequence would
be operably linked to a nucleic acid sequence if the gene
expression sequence were capable of effecting transcription of that
nucleic acid sequence such that the resulting transcript might be
translated into the desired protein or polypeptide.
[0241] The molecules of the invention can be delivered to the
preferred cell types of the invention alone or in association with
a vector (see also earlier discussion on vectors). In its broadest
sense (and consistent with the description of expression and
targeting vectors elsewhere herein), a "vector" is any vehicle
capable of facilitating: (1) delivery of a molecule to a target
cell and/or (2) uptake of the molecule by a target cell.
Preferably, the delivery vectors transport the molecule into the
target cell with reduced degradation relative to the extent of
degradation that would result in the absence of the vector.
Optionally, a "targeting ligand" can be attached to the vector to
selectively deliver the vector to a cell which expresses on its
surface the cognate receptor for the targeting ligand. In this
manner, the vector (containing a nucleic acid or a protein) can be
selectively delivered to a neuron. Methodologies for targeting
include conjugates, such as those described in U.S. Pat. No.
5,391,723 to Priest. Another example of a well-known targeting
vehicle is a liposome. Liposomes are commercially available from
Gibco BRL. Numerous methods are published for making targeted
liposomes.
[0242] In general, the vectors useful in the invention include, but
are not limited to, plasmids, phagemids, viruses, other vehicles
derived from viral or bacterial sources that have been manipulated
by the insertion or incorporation of the nucleic acid sequences of
the invention, and additional nucleic acid fragments (e.g.,
enhancers, promoters) which can be attached to the nucleic acid
sequences of the invention. Viral vectors are a preferred type of
vector and include, but are not limited to, nucleic acid sequences
from the following viruses: adenovirus; adeno-associated virus;
retrovirus, such as moloney murine leukemia virus; harvey murine
sarcoma virus; murine mammary tumor virus; rouse sarcoma virus;
SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma
viruses; herpes virus; vaccinia virus; polio virus; and RNA virus
such as a retrovirus. One can readily employ other vectors not
named but known in the art.
[0243] A particularly preferred virus for certain applications is
the adeno-associated virus, a double-stranded. DNA virus. The
adeno-associated virus is capable of infecting a wide range of cell
types and species and can be engineered to be
replication-deficient. It further has advantages, such as heat and
lipid solvent stability, high transduction frequencies in cells of
diverse lineages, including hematopoietic cells, and lack of
superinfection inhibition thus allowing multiple series of
transductions. Reportedly, the adeno-associated virus can integrate
into human cellular DNA in a site-specific manner, thereby
minimizing the possibility of insertional mutagenesis and
variability of inserted gene expression. In addition, wild-type
adeno-associated virus infections have been followed in tissue
culture for greater than 100 passages in the absence of selective
pressure, implying that the adeno-associated virus genomic
integration is a relatively stable event. The adeno-associated
virus can also function in an extrachromosomal fashion.
[0244] In general, other preferred viral vectors are based on
non-cytopathic eukaryotic viruses in which non-essential genes have
been replaced with the gene of interest. Non-cytopathic viruses
include retroviruses, the life cycle of which involves reverse
transcription of genomic viral RNA into DNA with subsequent
proviral integration into host cellular DNA. Adenoviruses and
retroviruses have been approved for human gene therapy trials. In
general, the retroviruses are replication-deficient (i.e., capable
of directing synthesis of the desired proteins, but incapable of
manufacturing an infectious particle). Such genetically altered
retroviral expression vectors have general utility for the
high-efficiency transduction of genes in vivo. Standard protocols
for producing replication-deficient retroviruses (including the
steps of incorporation of exogenous genetic material into a
plasmid, transfection of a packaging cell lined with plasmid,
production of recombinant retroviruses by the packaging cell line,
collection of viral particles from tissue culture media, and
infection of the target cells with viral particles) are provided in
Kriegler, M., "Gene Transfer and Expression, A Laboratory Manual,"
W.H. Freeman C.O., New York (1990) and Murry, E. J. Ed. "Methods in
Molecular Biology," vol. 7, Humana Press, Inc., Cliffton, N.J.
(1991).
[0245] Another preferred retroviral vector is the vector derived
from the moloney murine leukemia virus, as described in Nabel, E.
G., et al., Science, 1990, 249:1285-1288. These vectors reportedly
were effective for the delivery of genes to all three layers of the
arterial wall, including the media. Other preferred vectors are
disclosed in Flugelman, et al., Circulation, 1992, 85:1110-1117.
Additional vectors that are useful for delivering molecules of the
invention are described in U.S. Pat. No. 5,674,722 by Mulligan, et.
al.
[0246] In addition to the foregoing vectors, other delivery methods
may be used to deliver a molecule of the invention to a cell such
as a neuron, liver, fibroblast, and/or a vascular endothelial cell,
and facilitate uptake thereby.
[0247] A preferred such delivery method of the invention is a
colloidal dispersion system. Colloidal dispersion systems include
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles, and liposomes. A preferred colloidal system of the
invention is a liposome. Liposomes are artificial membrane vessels
which are useful as a delivery vector in vivo or in vitro. It has
been shown that large unilamellar vessels (LUV), which range in
size from 0.2-4.0 .mu.m can encapsulate large macromolecules. RNA,
DNA, and intact virions can be encapsulated within the aqueous
interior and be delivered to cells in a biologically active form
(Fraley, et al., Trends Biochem. Sci., 1981, 6:77). In order for a
liposome to be an efficient gene transfer vector, one or more of
the following characteristics should be present: (1) encapsulation
of the gene of interest at high efficiency with retention of
biological activity; (2) preferential and substantial binding to a
target cell in comparison to non-target cells; (3) delivery of the
aqueous contents of the vesicle to the target cell cytoplasm at
high efficiency; and (4) accurate and effective expression of
genetic information.
[0248] Liposomes may be targeted to a particular tissue, such as
the myocardium or the vascular cell wall, by coupling the liposome
to a specific ligand such as a monoclonal antibody, sugar,
glycolipid, or protein. Ligands which may be useful for targeting a
liposome to the vascular wall include, but are not limited to the
viral coat protein of the Hemagglutinating virus of Japan.
Additionally, the vector may be coupled to a nuclear targeting
peptide, which will direct the nucleic acid to the nucleus of the
host cell.
[0249] Liposomes are commercially available from Gibco BRL, for
example, as LIPOFECTIN.TM. and LIPOFECTACE.TM., which are formed of
cationic lipids such as N-[1-(2, dioleyloxy)-propyl]-N,N,
N-trimethylammonium chloride (DOTMA) and dimethyl
dioctadecylammonium bromide (DDAB). Methods for making liposomes
are well known in the art and have been described in many
publications. Liposomes also have been reviewed by Gregoriadis, G.
in Trends in Biotechnology, V. 3, p. 235-241 (1985). Novel
liposomes for the intracellular delivery of macromolecules,
including nucleic acids, are also described in PCT International
application no. PCT/US96/07572 (Publication NO. WO 96/40060,
entitled "Intracellular Delivery of Macromolecules").
[0250] In one particular embodiment, the preferred vehicle is a
biocompatible micro particle or implant that is suitable for
implantation into the mammalian recipient. Exemplary bioerodible
implants that are useful in accordance with this method are
described in PCT International application no. PCT/US/03307
(Publication No. WO 95/24929, entitled "Polymeric Gene Delivery
System", claiming priority to U.S. patent application serial no.
213,668, filed Mar. 15, 1994). PCT/US/0307 describes a
biocompatible, preferably biodegradable polymeric matrix for
containing an exogenous gene under the control of an appropriate
promoter. The polymeric matrix is used to achieve sustained release
of the exogenous gene in the patient. In accordance with the
instant invention, the nucleic acids described herein are
encapsulated or dispersed within the biocompatible, preferably
biodegradable polymeric matrix disclosed in PCT/US/03307. The
polymeric matrix preferably is in the form of a micro particle such
as a micro sphere (wherein a nucleic acid is dispersed throughout a
solid polymeric matrix) or a microcapsule (wherein a nucleic acid
is stored in the core of a polymeric shell). Other forms of the
polymeric matrix for containing the nucleic acids of the invention
include films, coatings, gels, implants, and stents. The size and
composition of the polymeric matrix device is selected to result in
favorable release kinetics in the tissue into which the matrix
device is implanted. The size of the polymeric matrix devise
further is selected according to the method of delivery which is to
be used, typically injection into a tissue or administration of a
suspension by aerosol into the nasal and/or pulmonary areas. The
polymeric matrix composition can be selected to have both favorable
degradation rates and also to be formed of a material which is
bioadhesive, to further increase the effectiveness of transfer when
the devise is administered to a vascular surface. The matrix
composition also can be selected not to degrade, but rather, to
release by diffusion over an extended period of time.
[0251] Both non-biodegradable and biodegradable polymeric matrices
can be used to deliver the nucleic acids of the invention to the
subject. Biodegradable matrices are preferred. Such polymers may be
natural or synthetic polymers. Synthetic polymers are preferred.
The polymer is selected based on the period of time over which
release is desired, generally in the order of a few hours to a year
or longer. Typically, release over a period ranging from between a
few hours and three to twelve months is most desirable. The polymer
optionally is in the form of a hydrogel that can absorb up to about
90% of its weight in water and further, optionally is cross-linked
with multi-valent ions or other polymers.
[0252] In general, the nucleic acids of the invention are delivered
using the bioerodible implant by way of diffusion, or more
preferably, by degradation of the polymeric matrix. Exemplary
synthetic polymers which can be used to form the biodegradable
delivery system include: polyamides, polycarbonates, polyalkylenes,
polyalkylene glycols, polyalkylene oxides, polyalkylene
terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl
esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides,
polysiloxanes, polyurethanes and co-polymers thereof, alkyl
cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, polymers of acrylic and methacrylic
esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,
hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,
cellulose acetate, cellulose propionate, cellulose acetate
butyrate, cellulose acetate phthalate, carboxylethyl cellulose,
cellulose triacetate, cellulose sulphate sodium salt, poly(methyl
methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),
poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl
acetate, poly vinyl chloride, polystyrene and
polyvinylpyrrolidone.
[0253] Examples of non-biodegradable polymers include ethylene
vinyl acetate, poly(meth) acrylic acid, polyamides, copolymers and
mixtures thereof.
[0254] Examples of biodegradable polymers include synthetic
polymers such as polymers of lactic acid and glycolic acid,
polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid),
poly(valeric acid), and poly(lactide-cocaprolactone), and natural
polymers such as alginate and other polysaccharides including
dextran and cellulose, collagen, chemical derivatives thereof
(substitutions, additions of chemical groups, for example, alkyl,
alkylene, hydroxylations, oxidations, and other modifications
routinely made by those skilled in the art), albumin and other
hydrophilic proteins, zein and other prolamines and hydrophobic
proteins, copolymers and mixtures thereof. In general, these
materials degrade either by enzymatic hydrolysis or exposure to
water in vivo, by surface or bulk erosion.
[0255] Bioadhesive polymers of particular interest include
bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and
J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of
which are incorporated herein, polyhyaluronic acids, casein,
gelatin, glutin, polyanhydrides, polyacrylic acid, alginate,
chitosan, poly(methyl methacrylates), poly(ethyl methacrylates),
poly(butylmethacrylate), poly(isobutyl methacrylate),
poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl
methacrylate), poly(phenyl methacrylate), poly(methyl acrylate),
poly(isopropyl acrylate), poly(isobutyl acrylate), and
poly(octadecyl acrylate). Thus, the invention provides a
composition of the above-described molecules of the invention for
use as a medicament, methods for preparing the medicament and
methods for the sustained release of the medicament in vivo.
[0256] Compaction agents also can be used in combination with a
vector of the invention. A "compaction agent", as used herein,
refers to an agent, such as a histone, that neutralizes the
negative charges on the nucleic acid and thereby permits compaction
of the nucleic acid into a fine granule. Compaction of the nucleic
acid facilitates the uptake of the nucleic acid by the target cell.
The compaction agents can be used alone, i.e., to deliver an
isolated nucleic acid of the invention in a form that is more
efficiently taken up by the cell or, more preferably, in
combination with one or more of the above-described vectors.
[0257] Other exemplary compositions that can be used to facilitate
uptake by a target cell of the nucleic acids of the invention
include calcium phosphate and other chemical mediators of
intracellular transport, microinjection compositions, and
electroporation.
[0258] The invention embraces methods for increasing sulfatase
activity in a cell. Such methods involve contacting a cell
expressing a sulfatase with an isolated nucleic acid molecule of
the invention (e.g., an isolated nucleic acid molecule as claimed
in any one of claims 1-8, an FGE nucleic acid molecule having a
sequence selected from the group consisting of SEQ ID NO: 1, 3, 4,
45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,
and 80-87), or an expression product thereof (e.g., a polypeptide
as claimed in claims 11-15, 19, 20, or a peptide having a sequence
selected from the group consisting of SEQ ID NO. 2, 5, 46, 48, 50,
52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78), in an
amount effective to increase sulfatase activity in the cell.
"Increasing" sulfatase activity, as used herein, refers to
increased affinity for, and/or conversion of, the specific
substrate for the sulfatase, typically the result of an increase in
FGly formation on the sulfatase molecule. In one embodiment, the
cell expresses a sulfatase at levels higher than those of wild type
cells.
[0259] By "increasing sulfatase activity in a cell" also refers to
increasing activity of a sulfatase that is secreted by the cell.
The cell may express an endogenous and/or an exogenous sulfatase.
Said contacting of the FGE molecule also refers to activating the
cells's endogenous FGE gene. In important embodiments, the
endogenous sulfatase is activated. In certain embodiments, the
sulfatase is Iduronate 2-Sulfatase, Sulfamidase,
N-Acetylgalactosamine 6-Sulfatase, N-Acetylglucosamine 6-Sulfatase,
Arylsulfatase A, Arylsulfatase B, Arylsulfatase C, Arylsulfatase D,
Arylsulfatase E, Arylsulfatase F, Arylsulfatase G, HSulf-1,
HSulf-2, HSulf-3, HSulf-4, HSulf-5, and/or HSulf-6. In certain
embodiments the cell is a mammalian cell.
[0260] According to another aspect of the invention, a
pharmaceutical composition, is provided. The composition comprises
a sulfatase that is produced by cell, in a pharmaceutically
effective amount to treat a sulfatase deficiency, and a
pharmaceutically acceptable carrier, wherein said cell has been
contacted with an agent comprising an isolated nucleic acid
molecule of the invention (e.g., as claimed in claims 1-8, or a
nucleic acid molecule having a sequence selected from the group
consisting of SEQ ID NO: 1, 3, 4, 45, 47, 49, 51, 53, 55, 57, 59,
61, 63, 65, 67, 69, 71, 73, 75, 77, and 80-87), or an expression
product thereof (e.g., a peptide selected from the group consisting
of SEQ ID NO. 2, 5, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,
70, 72, 74, 76, and 78). In important embodiments, the sulfatase is
expressed at higher levels than normal/control cells.
[0261] The invention also embraces a sulfatase producing cell
wherein the ratio of active sulfatase to total sulfatase produced
by the cell is increased. The cell comprises: (i) a sulfatase with
an increased activity compared to a control, and (ii) a
Formylglycine Generating Enzyme with an increased activity compared
to a control, wherein the ratio of active sulfatase to total
sulfatase produced by the cell is increased by at least 5% over the
ratio of active sulfatase to total sulfatase produced by the cell
in the absence of the Formylglycine Generating Enzyme. It is known
in the art that overexpression of sulfatases can decrease the
activity of endogenous sulfatases (Anson et al., Biochem. J., 1993,
294:657-662). Furthermore, only a fraction of the recombinant
sulfatases is active. We have discovered, unexpectedly, that
increased expression/activity of FGE in a cell with increased
expression/activity of a sulfatase results in the production of a
sulfatase that is more active. Since the presence of FGly on a
sulfatase molecule is associated with sulfatase activity, "active
sulfatase" can be quantitated by determining the presence of FGly
on the sulfatase cell product using MALDI-TOF mass spectrometry, as
described elsewhere herein. The ratio with total sulfatase can then
be easily determined.
[0262] The invention also provides methods for the diagnosis and
therapy of sulfatase deficiencies. Such disorders include, but are
not limited to, Multiple Sulfatase Deficiency,
Mucopolysaccharidosis II (MPS II; Hunter Syndrome),
Mucopolysaccharidosis IIIA (MPS IIIA; Sanfilippo Syndrome A),
Mucopolysaccharidosis VIII (MPS VIII), Mucopolysaccharidosis IVA
(MPS IVA; Morquio Syndrome A), Mucopolysaccharidosis VI (MPS VI;
Maroteaux-Lamy Syndrome), Metachromatic Leukodystrophy (MLD),
X-linked Recessive Chondrodysplasia Punctata 1, and X-linked
Ichthyosis (Steroid Sulfatase Deficiency).
[0263] The methods of the invention are useful in both the acute
and the prophylactic treatment of any of the foregoing conditions.
As used herein, an acute treatment refers to the treatment of
subjects having a particular condition. Prophylactic treatment
refers to the treatment of subjects at risk of having the
condition, but not presently having or experiencing the symptoms of
the condition.
[0264] In its broadest sense, the terms "treatment" or "to treat"
refer to both acute and prophylactic treatments. If the subject in
need of treatment is experiencing a condition (or has or is having
a particular condition), then treating the condition refers to
ameliorating, reducing or eliminating the condition or one or more
symptoms arising from the condition. In some preferred embodiments,
treating the condition refers to ameliorating, reducing or
eliminating a specific symptom or a specific subset of symptoms
associated with the condition. If the subject in need of treatment
is one who is at risk of having a condition, then treating the
subject refers to reducing the risk of the subject having the
condition.
[0265] The mode of administration and dosage of a therapeutic agent
of the invention will vary with the particular stage of the
condition being treated, the age and physical condition of the
subject being treated, the duration of the treatment, the nature of
the concurrent therapy (if any), the specific route of
administration, and the like factors within the knowledge and
expertise of the health practitioner.
[0266] As described herein, the agents of the invention are
administered in effective amounts to treat any of the foregoing
sulfatase deficiencies. In general, an effective amount is any
amount that can cause a beneficial change in a desired tissue of a
subject. Preferably, an effective amount is that amount sufficient
to cause a favorable phenotypic change in a particular condition
such as a lessening, alleviation or elimination of a symptom or of
a condition as a whole.
[0267] In general, an effective amount is that amount of a
pharmaceutical preparation that alone, or together with further
doses, produces the desired response. This may involve only slowing
the progression of the condition temporarily, although more
preferably, it involves halting the progression of the condition
permanently or delaying the onset of or preventing the condition
from occurring. This can be monitored by routine methods.
Generally, doses of active compounds would be from about 0.01 mg/kg
per day to 1000 mg/kg per day. It is expected that doses ranging
from 50 .mu.g-500 mg/kg will be suitable, preferably orally and in
one or several administrations per day.
[0268] Such amounts will depend, of course, on the particular
condition being treated, the severity of the condition, the
individual patient parameters including age, physical condition,
size and weight, the duration of the treatment, the nature of
concurrent therapy (if any), the specific route of administration
and like factors within the knowledge and expertise of the health
practitioner. Lower doses will result from certain forms of
administration, such as intravenous administration. In the event
that a response in a subject is insufficient at the initial doses
applied, higher doses (or effectively higher doses by a different,
more localized delivery route) may be employed to the extent that
patient tolerance permits. Multiple doses per day are contemplated
to achieve appropriate systemic levels of compounds. It is
preferred generally that a maximum dose be used, that is, the
highest safe dose according to sound medical judgment. It will be
understood by those of ordinary skill in the art, however, that a
patient may insist upon a lower dose or tolerable dose for medical
reasons, psychological reasons or for virtually any other
reasons.
[0269] The agents of the invention may be combined, optionally,
with a pharmaceutically-acceptable carrier to form a pharmaceutical
preparation. The term "pharmaceutically-acceptable carrier" as used
herein means one or more compatible solid or liquid fillers,
diluents or encapsulating substances which are suitable for
administration into a human. The term "carrier" denotes an organic
or inorganic ingredient, natural or synthetic, with which the
active ingredient is combined to facilitate the application. The
components of the pharmaceutical compositions also are capable of
being co-mingled with the molecules of the present invention, and
with each other, in a manner such that there is no interaction
which would substantially impair the desired pharmaceutical
efficacy. In some aspects, the pharmaceutical preparations comprise
an agent of the invention in an amount effective to treat a
disorder.
[0270] The pharmaceutical preparations may contain suitable
buffering agents, including: acetic acid in a salt; citric acid in
a salt; boric acid in a salt; or phosphoric acid in a salt. The
pharmaceutical compositions also may contain, optionally, suitable
preservatives, such as: benzalkonium chloride; chlorobutanol;
parabens or thimerosal.
[0271] A variety of administration routes are available. The
particular mode selected will depend, of course, upon the
particular drug selected, the severity of the condition being
treated and the dosage required for therapeutic efficacy. The
methods of the invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of the active
compounds without causing clinically unacceptable adverse effects.
Such modes of administration include oral, rectal, topical, nasal,
intradermal, transdermal, or parenteral routes. The term
"parenteral" includes subcutaneous, intravenous, intraomental,
intramuscular, or infusion. Intravenous or intramuscular routes are
not particularly suitable for long-term therapy and prophylaxis. As
an example, pharmaceutical compositions for the acute treatment of
subjects having a migraine headache may be formulated in a variety
of different ways and for a variety of administration modes
including tablets, capsules, powders, suppositories, injections and
nasal sprays.
[0272] The pharmaceutical preparations may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well-known in the art of pharmacy. All methods include the
step of bringing the active agent into association with a carrier
which constitutes one or more accessory ingredients. In general,
the compositions are prepared by uniformly and intimately bringing
the active compound into association with a liquid carrier, a
finely divided solid carrier, or both, and then, if necessary,
shaping the product.
[0273] Compositions suitable for oral administration may be
presented as discrete units, such as capsules, tablets, lozenges,
each containing a predetermined amount of the active compound.
Other compositions include suspensions in aqueous liquids or
non-aqueous liquids such as a syrup, elixir or an emulsion.
[0274] Compositions suitable for parenteral administration
conveniently comprise a sterile aqueous preparation of an agent of
the invention, which is preferably isotonic with the blood of the
recipient. This aqueous preparation may be formulated according to
known methods using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation also may be a
sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1,3-butane diol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose any bland fixed oil may be employed including
synthetic mono- or di-glycerides. In addition, fatty acids such as
oleic acid may be used in the preparation of injectables.
Formulations suitable for oral, subcutaneous, intravenous,
intramuscular, etc. administrations can be found in Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.
[0275] According to one aspect of the invention, a method for
increasing C.sub..alpha.-formylglycine generating activity in a
cell, is provided. The method involves contacting the cell with an
isolated nucleic acid molecule of the invention (e.g., a nucleic
acid of SEQ ID NO.1), or an expression product thereof (e.g., a
peptide of SEQ ID NO.2), in an amount effective to increase
C.sub..alpha.-formylglycine generating activity in the cell. In
important embodiments, the method involves activating the
endogenous FGE gene to increase C.sub..alpha.-formylglycine
generating activity in the cell. In some embodiments, the
contacting is performed under conditions that permit entry of a
molecule of the invention into the cell.
[0276] The term "permit entry" of a molecule into a cell according
to the invention has the following meanings depending upon the
nature of the molecule. For an isolated nucleic acid it is meant to
describe entry of the nucleic acid through the cell membrane and
into the cell nucleus, where upon the "nucleic acid transgene" can
utilize the cell machinery to produce functional polypeptides
encoded by the nucleic acid. By "nucleic acid transgene" it is
meant to describe all of the nucleic acids of the invention with or
without the associated vectors. For a polypeptide, it is meant to
describe entry of the polypeptide through the cell membrane and
into the cell cytoplasm, and if necessary, utilization of the cell
cytoplasmic machinery to functionally modify the polypeptide (e.g.,
to an active form).
[0277] Various techniques may be employed for introducing nucleic
acids of the invention into cells, depending on whether the nucleic
acids are introduced in vitro or in vivo in a host. Such techniques
include transfection of nucleic acid-CaPO.sub.4 precipitates,
transfection of nucleic acids associated with DEAE, transfection
with a retrovirus including the nucleic acid of interest, liposome
mediated transfection, and the like. For certain uses, it is
preferred to target the nucleic acid to particular cells. In such
instances, a vehicle used for delivering a nucleic acid of the
invention into a cell (e.g., a retrovirus, or other virus; a
liposome) can have a targeting molecule attached thereto. For
example, a molecule such as an antibody specific for a surface
membrane protein on the target cell or a ligand for a receptor on
the target cell can be bound to or incorporated within the nucleic
acid delivery vehicle. For example, where liposomes are employed to
deliver the nucleic acids of the invention, proteins which bind to
a surface membrane protein associated with endocytosis may be
incorporated into the liposome formulation for targeting and/or to
facilitate uptake. Such proteins include capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular half
life, and the like. Polymeric delivery systems also have been used
successfully to deliver nucleic acids into cells, as is known by
those skilled in the art. Such systems even permit oral delivery of
nucleic acids.
[0278] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of an agent of the present
invention, increasing convenience to the subject and the physician.
Many types of release delivery systems are available and known to
those of ordinary skill in the art. They include polymer base
systems such as poly(lactide-glycolide), copolyoxalates,
polycaprolactones, polyesteramides, polyorthoesters,
polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the
foregoing polymers containing drugs are described in, for example,
U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer
systems that are: lipids including sterols such as cholesterol,
cholesterol esters and fatty acids or neutral fats such as mono-
di- and tri-glycerides; hydrogel release systems; sylastic systems;
peptide based systems; wax coatings; compressed tablets using
conventional binders and excipients; partially fused implants; and
the like. Specific examples include, but are not limited to: (a)
erosional systems in which an agent of the invention is contained
in a form within a matrix such as those described in U.S. Pat. Nos.
4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in
which an active component permeates at a controlled rate from a
polymer such as described in U.S. Pat. Nos. 3,854,480, 5,133,974
and 5,407,686. In addition, pump-based hardware delivery systems
can be used, some of which are adapted for implantation.
[0279] Use of a long-term sustained release implant may be
desirable. Long-term release, as used herein, means that the
implant is constructed and arranged to deliver therapeutic levels
of the active ingredient for at least 30 days, and preferably 60
days. Long-term sustained release implants are well-known to those
of ordinary skill in the art and include some of the release
systems described above. Specific examples include, but are not
limited to, long-term sustained release implants described in U.S.
Pat. No. 4,748,024, and Canadian Patent No. 1330939.
[0280] The invention also involves the administration, and in some
embodiments co-administration, of agents other than the FGE
molecules of the invention that when administered in effective
amounts can act cooperatively, additively or synergistically with a
molecule of the invention to: (i) modulate
C.sub..alpha.-formylglycine generating activity, and (ii) treat any
of the conditions in which C.sub..alpha.-formylglycine generating
activity of a molecule of the invention is involved (e.g., a
sulfatase deficiency including MSD). Agents other than the
molecules of the invention include Iduronate 2-Sulfatase,
Sulfamidase, N-Acetylgalactosamine 6-Sulfatase, N-Acetylglucosamine
6-Sulfatase, Arylsulfatase A, Arylsulfatase B, Arylsulfatase C,
Arylsulfatase D, Arylsulfatase E, Arylsulfatase F, Arylsulfatase G,
HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5, or HSulf-6, (nucleic
acids and polypeptides, and/or fragments thereof), and/or
combinations thereof.
[0281] "Co-administering," as used herein, refers to administering
simultaneously two or more compounds of the invention (e.g., an FGE
nucleic acid and/or polypeptide, and an agent known to be
beneficial in the treatment of, for example, a sulfatase
deficiency--e.g., Iduronate 2-Sulfatase in the treatment of
MPSII--), as an admixture in a single composition, or sequentially,
close enough in time so that the compounds may exert an additive or
even synergistic effect.
[0282] The invention also embraces solid-phase nucleic acid
molecule arrays. The array consists essentially of a set of nucleic
acid molecules, expression products thereof, or fragments (of
either the nucleic acid or the polypeptide molecule) thereof, each
nucleic acid molecule selected from the group consisting of FGE,
Iduronate 2-Sulfatase, Sulfamidase, N-Acetylgalactosamine
6-Sulfatase, N-Acetylglucosamine 6-Sulfatase, Arylsulfatase A,
Arylsulfatase B, Arylsulfatase C, Arylsulfatase D, Arylsulfatase E,
Arylsulfatase F, Arylsulfatase G, HSulf-1, HSulf-2, HSulf-3,
HSulf-4, HSulf-5, and HSulf-6, fixed to a solid substrate. In some
embodiments, the solid-phase array further comprises at least one
control nucleic acid molecule. In certain embodiments, the set of
nucleic acid molecules comprises at least one, at least two, at
least three, at least four, or even at least five nucleic acid
molecules, each selected from the group consisting of FGE,
Iduronate 2-Sulfatase, Sulfamidase, N-Acetylgalactosamine
6-Sulfatase, N-Acetylglucosamine 6-Sulfatase, Arylsulfatase A,
Arylsulfatase B, Arylsulfatase C, Arylsulfatase D, Arylsulfatase E,
Arylsulfatase F, Arylsulfatase G, HSulf-1, HSulf-2, HSulf-3,
HSulf-4, HSulf-5, and HSulf-6. In preferred embodiments, the set of
nucleic acid molecules comprises a maximum number of 100 different
nucleic acid molecules. In important embodiments, the set of
nucleic acid molecules comprises a maximum number of 10 different
nucleic acid molecules.
[0283] According to the invention, standard hybridization
techniques of microarray technology are utilized to assess patterns
of nucleic acid expression and identify nucleic acid expression.
Microarray technology, which is also known by other names
including: DNA chip technology, gene chip technology, and
solid-phase nucleic acid array technology, is well known to those
of ordinary skill in the art and is based on, but not limited to,
obtaining an array of identified nucleic acid probes (e.g.,
molecules described elsewhere herein such as of FGE, Iduronate
2-Sulfatase, Sulfamidase, N-Acetylgalactosamine 6-Sulfatase,
N-Acetylglucosamine 6-Sulfatase, Arylsulfatase A, Arylsulfatase B,
Arylsulfatase C, Arylsulfatase D, Arylsulfatase E, Arylsulfatase F,
Arylsulfatase G, HSulf-1, HSulf-2, HSulf-3, HSulf-4, HSulf-5,
and/or HSulf-6) on a fixed substrate, labeling target molecules
with reporter molecules (e.g., radioactive, chemiluminescent, or
fluorescent tags such as fluorescein, Cye3-dUTP, or CyeS-dUTP),
hybridizing target nucleic acids to the probes, and evaluating
target-probe hybridization. A probe with a nucleic acid sequence
that perfectly matches the target sequence will, in general, result
in detection of a stronger reporter-molecule signal than will
probes with less perfect matches. Many components and techniques
utilized in nucleic acid microarray technology are presented in The
Chipping Forecast, Nature Genetics, Vol. 21, January 1999, the
entire contents of which is incorporated by reference herein.
[0284] According to the present invention, microarray substrates
may include but are not limited to glass, silica, aluminosilicates,
borosilicates, metal oxides such as alumina and nickel oxide,
various clays, nitrocellulose, or nylon. In all embodiments a glass
substrate is preferred. According to the invention, probes are
selected from the group of nucleic acids including, but not limited
to: DNA, genomic DNA, cDNA, and oligonucleotides; and may be
natural or synthetic. Oligonucleotide probes preferably are 20 to
25-mer oligonucleotides and DNA/cDNA probes preferably are 500 to
5000 bases in length, although other lengths may be used.
Appropriate probe length may be determined by one of ordinary skill
in the art by following art-known procedures. In one embodiment,
preferred probes are sets of two or more of the nucleic acid
molecules set forth as SEQ ID NOs: 1, 3, 4, 6, 8, 10, and/or 12.
Probes may be purified to remove contaminants using standard
methods known to those of ordinary skill in the art such as gel
filtration or precipitation.
[0285] In one embodiment, the microarray substrate may be coated
with a compound to enhance synthesis of the probe on the substrate.
Such compounds include, but are not limited to, oligoethylene
glycols. In another embodiment, coupling agents or groups on the
substrate can be used to covalently link the first nucleotide or
olignucleotide to the substrate. These agents or groups may
include, but are not limited to: amino, hydroxy, bromo, and carboxy
groups. These reactive groups are preferably attached to the
substrate through a hydrocarbyl radical such as an alkylene or
phenylene divalent radical, one valence position occupied by the
chain bonding and the remaining attached to the reactive groups.
These hydrocarbyl groups may contain up to about ten carbon atoms,
preferably up to about six carbon atoms. Alkylene radicals are
usually preferred containing two to four carbon atoms in the
principal chain. These and additional details of the process are
disclosed, for example, in U.S. Pat. No. 4,458,066, which is
incorporated by reference in its entirety.
[0286] In one embodiment, probes are synthesized directly on the
substrate in a predetermined grid pattern using methods such as
light-directed chemical synthesis, photochemical deprotection, or
delivery of nucleotide precursors to the substrate and subsequent
probe production.
[0287] In another embodiment, the substrate may be coated with a
compound to enhance binding of the probe to the substrate. Such
compounds include, but are not limited to: polylysine, amino
silanes, amino-reactive silanes (Chipping Forecast, 1999) or
chromium (Gwynne and Page, 2000). In this embodiment,
presynthesized probes are applied to the substrate in a precise,
predetermined volume and grid pattern, utilizing a
computer-controlled robot to apply probe to the substrate in a
contact-printing manner or in a non-contact manner such as ink jet
or piezo-electric delivery. Probes may be covalently linked to the
substrate with methods that include, but are not limited to,
UV-irradiation. In another embodiment probes are linked to the
substrate with heat.
[0288] Targets are nucleic acids selected from the group, including
but not limited to: DNA, genomic DNA, cDNA, RNA, mRNA and may be
natural or synthetic. In all embodiments, nucleic acid molecules
from subjects suspected of developing or having a sulfatase
deficiency, are preferred. In certain embodiments of the invention,
one or more control nucleic acid molecules are attached to the
substrate. Preferably, control nucleic acid molecules allow
determination of factors including but not limited to: nucleic acid
quality and binding characteristics; reagent quality and
effectiveness; hybridization success; and analysis thresholds and
success. Control nucleic acids may include, but are not limited to,
expression products of genes such as housekeeping genes or
fragments thereof.
[0289] To select a set of sulfatase deficiency disease markers, the
expression data generated by, for example, microarray analysis of
gene expression, is preferably analyzed to determine which genes in
different categories of patients (each category of patients being a
different sulfatase deficiency disorder), are significantly
differentially expressed. The significance of gene expression can
be determined using Permax computer software, although any standard
statistical package that can discriminate significant differences
is expression may be used. Permax performs permutation 2-sample
t-tests on large arrays of data. For high dimensional vectors of
observations, the Permax software computes t-statistics for each
attribute, and assesses significance using the permutation
distribution of the maximum and minimum overall attributes. The
main use is to determine the attributes (genes) that are the most
different between two groups (e.g., control healthy subject and a
subject with a particular sulfatase deficiency), measuring "most
different" using the value of the t-statistics, and their
significance levels.
[0290] Expression of sulfatase deficiency disease related nucleic
acid molecules can also be determined using protein measurement
methods to determine expression of SEQ ID NOs: 2, e.g., by
determining the expression of polypeptides encoded by SEQ ID NOs:
1, and/or 3. Preferred methods of specifically and quantitatively
measuring proteins include, but are not limited to: mass
spectroscopy-based methods such as surface enhanced laser
desorption ionization (SELDI; e.g., Ciphergen ProteinChip System),
non-mass spectroscopy-based methods, and immunohistochemistry-based
methods such as 2-dimensional gel electrophoresis.
[0291] SELDI methodology may, through procedures known to those of
ordinary skill in the art, be used to vaporize microscopic amounts
of protein and to create a "fingerprint" of individual proteins,
thereby allowing simultaneous measurement of the abundance of many
proteins in a single sample. Preferably SELDI-based assays may be
utilized to characterize multiple sulfatase deficiency as well as
stages of such conditions. Such assays preferably include, but are
not limited to the following examples. Gene products discovered by
RNA microarrays may be selectively measured by specific (antibody
mediated) capture to the SELDI protein disc (e.g., selective
SELDI). Gene products discovered by protein screening (e.g., with
2-D gels), may be resolved by "total protein SELDI" optimized to
visualize those particular markers of interest from among SEQ ID
NOs: 1, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, and/or 28.
Predictive models of a specific sulfatase deficiency from SELDI
measurement of multiple markers from among SEQ ID NOs: 1, 6, 8, 10,
12, 14, 16, 18, 20, 22, 24, 26, and/or 28, may be utilized for the
SELDI strategies.
[0292] The use of any of the foregoing microarray methods to
determine expression of a sulfatase deficiency disease related
nucleic acids can be done with routine methods known to those of
ordinary skill in the art and the expression determined by protein
measurement methods may be correlated to predetermined levels of a
marker used as a prognostic method for selecting treatment
strategies for sulfatase deficiency disease patients.
[0293] The invention also embraces a sulfatase-producing cell
wherein the ratio of active sulfatase to total sulfatase produced
(i.e., the specific activity) by the cell is increased. The cell
comprises: (i) a sulfatase with an increased expression, and (ii) a
Formylglycine Generating Enzyme with an increased expression,
wherein the ratio of active sulfatase to total sulfatase produced
by the cell is increased by at least 5% over the ratio of active
sulfatase to total sulfatase produced by the cell in the absence of
the Formylglycine Generating Enzyme.
[0294] A "sulfatase with an increased expression," as used herein,
typically refers to increased expression of a sulfatase and/or its
encoded polypeptide compared to a control. Increased expression
refers to increasing (i.e., to a detectable extent) replication,
transcription, and/or translation of any of the sulfatase nucleic
acids (sulfatase nucleic acids of the invention as described
elsewhere herein), since upregulation of any of these processes
results in concentration/amount increase of the polypeptide encoded
by the gene (nucleic acid). This can be accomplished using a number
of methods known in the art, also described elsewhere herein, such
as transfection of a cell with the sulfatase cDNA, and/or genomic
DNA encompassing the sulfatase locus, activating the endogenous
sulfatase gene by placing, for example, a strong promoter element
upstream of the endogenous sulfatase gene genomic locus using
homologous recombination (see, e.g., the gene activation technology
described in detail in U.S. Pat. Nos. 5,733,761, 6,270,989, and
6,565,844, all of which are expressly incorporated herein by
reference), etc. A typical control would be an identical cell
transfected with a vector plasmid(s). Enhancing (or increasing)
sulfatase activity also refers to preventing or inhibiting
sulfatase degradation (e.g., via increased ubiquitinization),
downregulation, etc., resulting, for example, in increased or
stable sulfatase molecule t1/2 (half-life) when compared to a
control. Downregulation or decreased expression refers to decreased
expression of a gene and/or its encoded polypeptide. The
upregulation or downregulation of gene expression can be directly
determined by detecting an increase or decrease, respectively, in
the level of mRNA for the gene (e.g, a sulfatase), or the level of
protein expression of the gene-encoded polypeptide, using any
suitable means known to the art, such as nucleic acid hybridization
or antibody detection methods, respectively, and in comparison to
controls. Upregulation or downregulation of sulfatase gene
expression can also be determined indirectly by detecting a change
in sulfatase activity.
[0295] Similarily, a "Formylglycine Generating Enzyme with an
increased expression," as used herein, typically refers to
increased expression of an FGE nucleic acid of the invention and/or
its encoded polypeptide compared to a control. Increased expression
refers to increasing (i.e., to a detectable extent) replication,
transcription, and/or translation of any of the FGE nucleic acids
of the invention (as described elsewhere herein), since
upregulation of any of these processes results in
concentration/amount increase of the polypeptide encoded by the
gene (nucleic acid). This can be accomplished using the methods
described above (for the sulfatases), and elsewhere herein.
[0296] In certain embodiments, the ratio of active sulfatase to
total sulfatase produced by the cell is increased by at least 10%,
15%, 20%, 50%, 100%, 200%, 500%, 1000%, over the ratio of active
sulfatase to total sulfatase produced by the cell in the absence of
the Formylglycine Generating Enzyme.
[0297] The invention further embraces an improved method for
treating a sulfatase deficiency in a subject. The method involves
administering to a subject in need of such treatment a sulfatase in
an effective amount to treat the sulfatase deficiency in the
subject, wherein the sulfatase is contacted with a Formylglycine
Generating Enzyme in an amount effective to increase the specific
activity of the sulfatase. As described elsewhere herein, "specific
activity" refers to the ratio of active sulfatase to total
sulfatase produced. "Contacted," as used herein, refers to FGE
post-translationally modifying the sulfatase as described elsewhere
herein. It would be apparent to one of ordinary skill in the art
that an FGE can contact a sulfatase and modify it if nucleic acids
encoding FGE and a sulfatase are co-expressed in a cell, or even if
an isolated FGE polypeptide contacts an isolated sulfatase
polypeptide in vivo or in vitro. Even though an isolated FGE
polypeptide can be co-administered with an isolated sulfatase
polypeptide to a subject to treat a sulfatase deficiency in the
subject, it is preferred that the contact between FGE and the
sulfatase takes place in vitro prior to administration of the
sulfatase to the subject. This improved method of treatment is
beneficial to a subject since lower amounts of the sulfatase need
to be administered, and/or with less frequency, since the sulfatase
is of higher specific activity.
[0298] The invention will be more fully understood by reference to
the following examples. These examples, however, are merely
intended to illustrate the embodiments of the invention and are not
to be construed to limit the scope of the invention.
EXAMPLES
Example 1
Multiple Sulfatase Deficiency is Caused by Mutations in the Gene
Encoding the Human C.alpha.-Formylglycine Generating Enzyme
(FGE)
Experimental Procedures
Materials and Methods
In Vitro Assay for FGE
[0299] For monitoring the activity of FGE, the N-acetylated and
C-amidated 23mer peptide P23 (MTDFYVPVSLCTPSRAALLTGRS) (SEQ ID
NO:33) was used as substrate. The conversion of the Cysteine
residue in position 11 to FGly was monitored by MALDI-TOF mass
spectrometry. A 6 .mu.M stock solution of P23 in 30% acetonitrile
and 0.1% trifluoroacetic acid (TFA) was prepared. Under standard
conditions 6 pmol of P23 were incubated at 37.degree. C. with up to
10 .mu.l enzyme in a final volume of 30 .mu.l 50 mM Tris/HCl, pH
9.0, containing 67 mM NaCl, 15 .mu.M CaCl.sub.2, 2 mM DTT, and 0.33
mg/ml bovine serum albumin. To stop the enzyme reaction 1.5 .mu.l
10% TFA were added. P23 then was bound to ZipTip C18 (Millipore),
washed with 0.1% TFA and eluted in 3 .mu.l 50% acetonitrile, 0.1%
TFA. 0.5 .mu.l of the eluate was mixed with 0.5 .mu.l of matrix
solution (5 mg/ml a-cyano-4-hydroxy-cinnamic acid (Bruker
Daltonics, Billerica, Mass.) in 50% acetonitrile, 0.1% TFA) on a
stainless steel target. MALDI-TOF mass spectrometry was performed
with a Reflex III (Bruker Daltonics) using reflectron mode and
laser energy just above the desorption/ionization threshold. All
spectra were averages of 200-300 shots from several spots on the
target. The mass axis was calibrated using peptides of molecular
masses ranging from 1000 to 3000 Da as external standards.
Monoisotopic MR.sup.+ of P23 is 2526.28 and of the FGly containing
product 2508.29. Activity (pmol product/h) was calculated on the
basis of the peak height of the product divided by the sum of the
peak heights of P23 and the product.
Purification of FGE from Bovine Testis
[0300] Bovine testes were obtained from the local slaughter house
and stored for up to 20 h on ice. The parenchyme was freed from
connective tissue and homogenized in a waring blendor and by three
rounds of motor pottering. Preparation of rough microsomes (RM) by
cell fractionation of the obtained homogenate was performed as
described (Meyer et al., J. Biol. Chem., 2000, 275:14550-14557)
with the following modifications. Three differential centrifugation
steps, 20 minutes each at 4.degree. C., were performed at 500 g
(JA10 rotor), 3000 g (JA10) and 10000 g (JA20). From the last
supernatant the RM membranes were sedimented (125000 g, Ti45 rotor,
45 min, 4.degree. C.), homogenized by motor pottering and layered
on a sucrose cushion (50 mM Hepes, pH 7.6, 50 mM KAc, 6 mM
MgAc.sub.2, 1 mM EDTA, 1.3 M sucrose, 5 mM .beta.-mercaptoethanol).
RMs were recovered from the pellet after spinning for 210 minutes
at 45000 rpm in a Ti45 rotor at 4.degree. C. Usually 100000-150000
equivalents RM, as defined by Walter and Blobel (Methods Enzymol.,
1983, 96:84-93), were obtained from 1 kg of testis tissue. The
reticuloplasm, i.e. the luminal content of the RM, was obtained by
differential extraction at low concentrations of deoxy Big Chap, as
described (Fey et al., J. Biol. Chem., 2001, 276:47021-47028). For
FGE purification, 95 ml of reticuloplasm were dialyzed for 20 h at
4.degree. C. against 20 mM Tris/HCl, pH 8.0, 2.5 mM DTT, and
cleared by centrifugation at 125000 g for 1 h. 32 ml-aliquots of
the cleared reticuloplasm were loaded on a MonoQ HR10/10 column
(Amersham Biosciences, Piscataway, N.J.) at room temperature,
washed and eluted at 2 ml/min with a linear gradient of 0 to 0.75 M
NaCl in 80 ml of the Tris buffer. The fractions containing FGE
activity, eluting at 50-165 mM NaCl, of three runs were pooled (42
ml) and mixed with 2 ml of Concanavalin A-Sepharose (Amersham
Biosciences) that had been washed with 50 mM Hepes buffer, pH 7.4,
containing 0.5 M KCl, 1 mM MgCl.sub.2, 1 mM MnCl.sub.2, 1 mM
CaCl.sub.2, and 2.5 mM DTT. After incubation for 16 h at 4.degree.
C., the Concanavalin A-Sepharose was collected in a column and
washed with 6 ml of the same Hepes buffer. The bound material was
eluted by incubating the column for 1 h at room temperature with 6
ml 0.5 M a-methylmannoside in 50 mM Hepes, pH 7.4, 2.5 mM DTT. The
elution was repeated with 4 ml of the same eluent. The combined
eluates (10 ml) from Concanavalin A-Sepharose were adjusted to pH
8.0 with 0.5 M Tris/HCl, pH 9.0, and mixed with 2 ml of Affigel 10
(Bio-Rad Laboratories, Hercules, Calif.) that had been derivatized
with 10 mg of the scrambled peptide (PVSLPTRSCAALLTGR) (SEQ ID
NO:34) and washed with buffer A (50 mM Hepes, pH 8.0, containing
0.15 M potassium acetate, 0.125 M sucrose, 1 mM MgCl.sub.2, and 2.5
mM DTT). After incubation for 3 h at 4.degree. C. the affinity
matrix was collected in a column. The flow through and a wash
fraction with 4 ml of buffer A were collected, combined and mixed
with 2 ml of Affigel 10 that had been substituted with 10 mg of the
Ser69 peptide (PVSLSTPSRAALLTGR) (SEQ ID NO:35) and washed with
buffer A. After incubation overnight at 4.degree. C., the affinity
matrix was collected in a column, washed 3 times with 6 ml of
buffer B (buffer A containing 2 M NaCl and a mixture of the 20
proteinogenic amino acids, each at 50 mg/ml). The bound material
was eluted from the affinity matrix by incubating the Affigel twice
for 90 min each with 6 ml buffer B containing 25 mM Ser69 peptide.
An aliqout of the eluate was substituted with 1 mg/ml bovine serum
albumin, dialyzed against buffer A and analyzed for activity. The
remaining part of the activity (11.8 ml) was concentrated in a
Vivaspin 500 concentrator (Vivascience AG, Hannover, Germany), and
solubilized at 95.degree. C. in Laemmli SDS sample buffer. The
polypeptide composition of the starting material and preparations
obtained after the chromatographic steps were monitored by SDSPAGE
(15% acrylamide, 0.16% bisacrylamide) and staining with SYPRO Ruby
(Bio-Rad Laboratories).
Identification of FGE by Mass Spectrometry
[0301] For peptide mass fingerprint analysis the purified
polypeptides were in-gel digested with trypsin (Shevchenko et al.,
Anal. Chem., 1996, 68:850-855), desalted on C18 ZipTip and analyzed
by MALDI-TOF mass spectrometry using dihydrobenzoic acid as matrix
and two autolytic peptides from trypsin (m/z 842.51 and 2211.10) as
internal standards. For tandem mass spectrometry analysis selected
peptides were analyzed by MALDI-TOF post-source decay mass
spectrometry. Their corresponding doubly charged ions were isolated
and fragmented by offline nano-ESI ion trap mass spectrometry
(EsquireLC, Bruker Daltonics). The mass spectrometric data were
used by Mascot search algorithm for protein identification in the
NCBInr protein database and the NCBI EST nucleotide database.
Bioinformatics
[0302] Signal peptides and clevage sites were described with the
method of von Heijne (von Heijne, Nucleic Acids Res., 1986,
14:4683-90) implemented in EMBOSS (Rice et al., Trends in Genetics,
2000, 16:276-277). N-glycosylation sites were predicted using the
algorithm of Brunak (Gupta and Brunak, Pac. Symp. Biocomput., 2002,
310-22). Functional domains were detected by searching
PFAM-Hidden-Markov-Models (version 7.8) (Sonnhammer et al., Nucleic
Acids Res., 1998, 26:320-322). To search for FGE homologs, the
databases of the National Center for Biotechnology Information
(Wheeler et al., Nucleic Acids Res., 2002, 20:13-16) were queried
with BLAST (Altschul et al., Nucleic Acids Res., 1997,
25:3389-3402). Sequence similarities were computed using standard
tools from EMBOSS. Genomic loci organisation and synteny were
determined using the NCBI's human and mouse genome resources and
the Human-Mouse Homology Map also form NCBI, Bethesda, Md.).
Cloning of Human FGE cDNA
[0303] Total RNA, prepared from human fibroblasts using the
RNEASY.TM. Mini kit (Qiagen, Inc., Valencia, Calif.) was reverse
transcribed using the OMNISCRIPT RT.TM. kit (Qiagen, Inc.,
Valencia, Calif.) and either an oligo(dT) primer or the
FGE-specific primer 1199nc (CCAATGTAGGTCAGACACG) (SEQ ID NO:36).
The first strand cDNA was amplified by PCR using the forward primer
1c (ACATGGCCCGCGGGAC) (SEQ ID NO:37) and, as reverse primer, either
1199nc or 1182nc (CGACTGCTCCTTGGACTGG) (SEQ ID NO:38). The PCR
products were cloned directly into the pCR4-TOPOT.TM. vector
(Invitrogen Corporation, Carlsbad, Calif.). By sequencing multiple
of the cloned PCR products, which had been obtained from various
individuals and from independent RT and PCR reactions, the coding
sequence of the FGE cDNA was determined (SEQ ID NOs:1 and 3).
Mutation Detection, Genomic Sequencing, Site-Directed Mutagenesis
and Northern Blot Analysis
[0304] Standard protocols utilized in this study were essentially
as described in Liibke et al. (Nat. Gen., 2001, 28:73-76) and
Hansske et al. (J. Clin. Invest., 2002, 109:725-733). Northern
blots were hybridized with a cDNA probe covering the entire coding
region and a .beta.-actin cDNA probe as a control for RNA
loading.
Cell Lines and Cell Culture
[0305] The fibroblasts from MSD patients 1-6 were obtained from E.
Christenson (Rigshospitalet Copenhagen), M. Beck
(Universitatskinderklinik Mainz), A. KohlschUtter
(Universitatskrankenhaus Eppendorf, Hamburg), E. Zammarchi (Meyer
Hospital, University of Florence), K. Harzer (Institut fur
Hirnforschung, Universitat Tubingen), and A. Fensom (Guy's
Hospital, London), respectively. Human skin fibroblasts, HT-1080,
BHK21 and CHO cells were maintained at 37.degree. C. under 5% CO2
in Dulbecco's modified Eagle's medium containing 10% fetal calf
serum.
Transfection, Indirect Immunofluorescence, Western Blot Analysis
and Detection of FGE Activity
[0306] The FGE cDNA was equipped with a 5' EcoRI-site and either a
3' HA-, c-Myc or RGS-His.sub.6-tag sequence, followed by a
stop-codon and a HindIII site, by add-on PCR using Pfu polymerase
(Stratagene, La Jolla, Calif.) and the following primers:
GGAATTCGGGACAACATGGCTGCG (EcoRI) (SEQ ID NO:39), CCCAAGCTTATGC
GTAGTCAGGCACATCATACGGATAGTCCATGGTGGGCAGGC(HA) (SEQ ID NO:40),
CCCAAGCTTACAGGTCTTCTTCAGAAATCAGCTTTTGTTCGTCCATGGTGGGCAG GC (c-Myc)
(SEQ ID NO:41), CCCAAGCTTAGTGATGGTGATGGTGATGCGATC
CTCTGTCCATGGTGGGCAGGC (RGS-His.sub.6) (SEQ ID NO:42). The resulting
PCR products were cloned as EcoRI/HindIII fragments into pMPSVEH
(Artelt et al., Gene, 1988, 68:213-219). The plasmids obtained were
transiently transfected into HT-1080, BHK21 and CHO cells, grown on
cover slips, using EN-ECTENE.TM. (Qiagen) as transfection reagent.
48 h after transfection the cells were analyzed by indirect
immunofluorescence as described previously (Lubke et al., Nat.
Gen., 2001, 28:73-76; Hansske et al., J. Clin. Invest., 2002,
109:725-733), using monoclonal IgG1 antibodies against HA (Berkeley
Antibody Company, Richmond, Calif.), c-Myc (Santa Cruz
Biotechnology, Inc., Santa Cruz, Calif.) or RGS-His (Qiagen) as
primary antibodies. The endoplasmic reticulum marker protein
proteindisulfide isomerase (PDI) was detected with a monoclonal
antibody of different subtype (IgG2A, Stressgen Biotech., Victoria
BC, Canada). The primary antibodies were detected with
isotype-specific goat secondary antibodies coupled to CY2 or CY3,
respectively (Molecular Probes, Inc., Eugene, Oreg.).
Immunofluorescence images were obtained on a Leica TCS Sp2 AOBS
laser scan microscope. For Western blot analysis the same
monoclonal antibodies and a HRP-conjugated anti-mouse IgG as
secondary antibody were used. For determination of FGE activity,
the trypsinised cells were washed with phosphate buffered saline
containing a mixture of proteinase inhibitors (208 .mu.M
4-(2-aminoethyl)benzene sulfonyl fluoride hydrochloride, 0.16 AM
aprotinin, 4.2 .mu.M leupeptin, 7.2 .mu.M bestatin, 3 .mu.M
pepstatin A, 2.8 .mu.M E-64), solubilized in 10 mM Tris, pH 8.0,
containing 2.5 mM DTT, the proteinase inhibitors and 1% Triton
X-100, and cleared by centrifugation at 125,000 g for 1 h. The
supernatant was subjected to chromatography on a MonoQ PC 1.6/5
column using the conditions described above. Fractions eluting at
50-200 mM NaCl were pooled, lyophilised and reconstituted in one
tenth of the original pool volume prior determination of FGE
activity with peptide P23.
Retroviral Transduction
[0307] cDNAs of interest were cloned into the Moloney murine
leukemia virus based vector pLPCX and pLNCX2 (BD Biosciences
Clontech, Palo Alto, Calif.). The transfection of ecotropic FNX-Eco
cells (ATCC, Manassas, Va.) and the transduction of amphotropic
RETROPACK.TM. PT67 cells (BD Biosciences Clontech) and human
fibroblasts was performed as described (Lubke et al., Nat. Gen.,
2001, 28:73-76; Thiel et al., Biochem. J., 2002, 376, 195-201). For
some experiments pLPCX-transduced PT67 cells were selected with
puromycin prior determination of sulfatase activities.
Sulfatase Assays
[0308] Activity of ASA, STS and GalNAc6S were determined as
described in Rommerskirch and von Figura, Proc. Natl. Acad. Sci.,
USA, 1992, 89:2561-2565; Glossl and Kresse, Clin. Chim. Acta, 1978,
88:111-119.
Results
A Rapid Peptide Based Assay for FGE Activity
[0309] We had developed an assay for determining FGE activity in
microsome extracts using in vitro synthesized [.sup.35S] ASA
fragments as substrate. The fragments were added to the assay
mixture as ribosome-associated nascent chain complexes. The
quantitation of the product included tryptic digestion, separation
of the peptides by RP-HPLC and identification and quantitation of
the [.sup.35S]-labeled FGly containing tryptic peptide by a
combination of chemical derivatization to hydrazones, RP-HPLC
separation and liquid scintillation counting (Fey et al., J. Biol.
Chem., 2001, 276:47021-47028). For monitoring the enzyme activity
during purification, this cumbersome procedure needed to be
modified. A synthetic 16mer peptide corresponding to ASA residues
65-80 and containing the sequence motif required for FGly formation
inhibited the FGE activity in the in vitro assay. This suggested
that peptides such as ASA65-80 may serve as substrates for FGE. We
synthesized the 23mer peptide P23 (SEQ ID NO:33), which corresponds
to ASA residues 60-80 with an additional N-acetylated methionine
and a C-amidated serine residue to protect the N- and C-terminus,
respectively. The cysteine and the FGly containing forms of P23
could be identified and quantified by matrix-assisted laser
desorption/ionisation time of flight (MALDI-TOF) mass spectrometry.
The presence of the FGly residue in position 11 of P23 was verified
by MALDI-TOF post source decay mass spectrometry (see Peng et al.,
J. Mass Spec., 2003, 38:80-86). Incubation of P23 with extracts
from microsomes of bovine pancreas or bovine testis converted up to
95% of the peptide into a FGly containing derivative (FIG. 1).
Under standard conditions the reaction was proportional to the
amount of enzyme and time of incubation as long as less than 50% of
the substrate was consumed and the incubation period did not exceed
24 h. The k.sub.m for P23 was 13 nM. The effects of reduced and
oxidized glutathione, Ca.sup.2+ and pH were comparable to those
seen in the assay using ribosome-associated nascent chain complexes
as substrate (Fey et al., J. Biol. Chem., 2001,
276:47021-47028).
Purification of FGE
[0310] For purification of FGE the soluble fraction (reticuloplasm)
of bovine testis microsomes served as the starting material. The
specific activity of FGE was 10-20 times higher than that in
reticuloplasm from bovine pancreas microsomes (Fey et al., J. Biol.
Chem., 2001, 276:47021-47028). Purification of FGE was achieved by
a combination of four chromatographic steps. The first two steps
were chromatography on a MonoQ anion exchanger and on Concanavalin
A-Sepharose. At pH 8 the FGE activity bound to MonoQ and was eluted
at 50-165 mM NaCl with 60-90% recovery. When this fraction was
mixed with Concanavalin A-Sepharose, FGE was bound. 30-40% of the
starting activity could be eluted with 0.5 M a-methyl mannoside.
The two final purification steps were chromatography on affinity
matrices derivatized with 16mer peptides. The first affinity matrix
was Affigel 10 substituted with a variant of the ASA65-80 peptide,
in which residues Cys69, Pro71 and Arg73, critical for FGly
formation, were scrambled (scrambled peptide PVSLPTRSCAALLTGR--SEQ
ID NO:34). This peptide did not inhibit FGE activity when added at
10 mM concentration to the in vitro assay and, when immobilized to
Affigel 10, did not retain FGE activity. Chromatography on the
scrambled peptide affinity matrix removed peptide binding proteins
including chaperones of the endoplasmic reticulum. The second
affinity matrix was Affigel 10 substituted with a variant of the
ASA65-80 peptide, in which the Cys69 was replaced by a serine
(Ser69 peptide PVSLSTPSRAALLTGR-SEQ ID NO:35). The Ser69 peptide
affinity matrix efficiently bound FGE. The FGE activity could be
eluted with either 2 M KSCN or 25 mM Ser69 peptide with 20-40%
recovery. Prior to activity determination the KSCN or Ser69 peptide
had to be removed by dialysis. The substitution of Cys69 by serine
was crucial for the elution of active FGE. Affigel 10 substituted
with the wildtype ASA65-80 peptide bound FGE efficiently. However,
nearly no activity could be recovered in eluates with chaotropic
salts (KSCN, MgCl.sub.2), peptides (ASA65-80 or Ser69 peptide) or
buffers with low or high pH. In FIG. 2 the polypeptide pattern of
the starting material and of the active fractions obtained after
the four chromatographic steps of a typical purification is shown.
In the final fraction 5% of the starting FGE activity and 0.0006%
of the starting protein were recovered (8333-fold
purification).
The Purified 39.5 and 41.5 kDa Polypeptides are Encoded by a Single
Gene
[0311] The 39.5 and 41.5 kDa polypeptides in the purified FGE
preparation were subjected to peptide mass fingerprint analysis.
The mass spectra of the tryptic peptides of the two polypeptides
obtained by MALDI-TOF mass spectrometry were largely overlapping,
suggesting that the two proteins originate from the same gene.
Among the tryptic peptides of both polypeptides two abundant
peptides MH.sup.+ 1580.73, SQNTPDSSASNLGFR (SEQ ID NO:43), and
MH.sup.+ 2049.91, MVPIPAGVFTMGTDDPQIK--SEQ ID NO:44 plus two
methionine oxidations) were found, which matched to the protein
encoded by a cDNA with GenBank Acc. No. AK075459 (SEQ ID NO:4). The
amino acid sequence of the two peptides was confirmed by MALDI-TOF
post source decay spectra and by MS/MS analysis using offline
nano-electrospray ionisation (ESI) iontrap mass spectrometry. An
EST sequence of the bovine ortholog of the human cDNA covering the
C-terminal part of the FGE and matching the sequences of both
peptides provided additional sequence information for bovine
FGE.
Evolutionary Conservation and Domain Structure of FGE
[0312] The gene for human FGE is encoded by the cDNA of (SEQ ID
NOs:1 and/or 3) and located on chromosome 3p26. It spans--105 kb
and the coding sequence is distributed over 9 exons. Three
orthologs of the human FGE gene are found in mouse (87% identity),
Drosophila melanogaster (48% identity), and Anopheles gambiae (47%
identity). Orthologous EST sequences are found for 8 further
species including cow, pig, Xenopus laevis, Silurana tropicalis,
zebra fish, salmon and other fish species (for details see Example
2). The exon-intron structure between the human and the mouse gene
is conserved and the mouse gene on chromosome 6E2 is located within
a region syntenic to the human chromosome 3p26. The genomes of S.
cerevisiae and C. elegans lack FGE homologs. In prokaryotes 12
homologs of human FGE were found. The cDNA for human FGE is
predicted to encode a protein of 374 residues (FIG. 3 and SEQ ID
NO:2). The protein contains a cleavable signal sequence of 33
residues, which indicates translocation of FGE into the endoplasmic
reticulum, and contains a single N-glycosylation site at Asn141.
The binding of FGE to concanavalin A suggests that this
N-glycosylation site is utilized. Residues 87-367 of FGE are listed
in the PFAM protein motif database as a domain of unknown function
(PFAM: DUF323). Sequence comparison analysis of human FGE and its
eukaryotic orthologs identified in data bases indicates that this
domain is composed of three distinct subdomains.
[0313] The N-terminal subdomain (residues 91-154 in human FGE) has
a sequence identity of 46% and a similarity of 79% within the four
known eukaryotic FGE orthologs. In human FGE, this domain carries
the N-glycosylation site at Asn 141, which is conserved in the
other orthologs. The middle part of FGE (residues 179-308 in human
FGE) is represented by a tryptophan-rich subdomain (12 tryptophans
per 129 residues). The identity of the eukaryotic orthologs within
this subdomain is 57%, the similarity is 82%. The C-terminal
subdomain (residues 327-366 in human FGE) is the most highly
conserved sequence within the FGE family. The sequence identity of
the human C-terminal subdomain with the eukaryotic orthologs (3
full length sequences and 8 ESTs) is 85%, the similarity 97%.
Within the 40 residues of the subdomain 3 four cysteine residues
are fully conserved. Three of cysteins are also conserved in the
prokaryotic FGE orthologs. The 12 prokaryotic members of the
FGE-family (for details see Example 2) share the subdomain
structure with eukaryotic FGEs. The boundaries between the three
subdomains are more evident in the prokaryotic FGE family due to
non-conserved sequences of variable length separating the
subdomains from each other. The human and the mouse genome encode
two closely related homologs of FGE (SEQ ID NOs:43 and 44, GenBank
Acc. No. NM.sub.--015411, in man, and SEQ ID NOs:45 and 46, GenBank
Acc. No. AK076022, in mouse). The two paralogs are 86% identical.
Their genes are located on syntenic chromosome regions (7q11 in
human, 5G1 in mouse). Both paralogs share with the FGE orthologs
the subdomain structure and are 35% identical and 47% similar to
human FGE. In the third subdomain, which is 100% identical in both
homologs, the cysteine containing undecamer sequence of the
subdomain 3 is missing.
Expression, Subcellular Localization and Molecular Forms
[0314] A single transcript of 2.1 kb is detectable by Northern blot
analysis of total RNA from skin fibroblasts and poly A' RNA from
heart, brain, placenta, lung, liver, skeletal muscle, kidney and
pancreas. Relative to .beta.-actin RNA the abundance varies by one
order of magnitude and is highest in pancreas and kidney and lowest
in brain. Various eukaryotic cell lines stably or transiently
expressing the cDNA of human FGE or FGE derivatives C-terminally
extended by a HA-, Myc- or His.sub.6-tag were assayed for FGE
activity and subcellular localization of FGE. Transient expression
of tagged and non-tagged FGE increased the FGE activity
1.6-3.9-fold. Stable expression of FGE in PT67 cells increased the
activity of FGE about 100-fold. Detection of the tagged FGE form by
indirect immunofluorescence in BHK 21, CHO, and HT1080 cells showed
a colocalization of the variously tagged FGE forms with
proteindisulfide isomerase, a lumenal protein of the endoplasmic
reticulum. Western blot analysis of extracts from BHK 21 cells
transiently transfected with cDNA encoding tagged forms of FGE
showed a single immunoreactive band with an apparent size between
42 to 44 kDa.
The FGE Gene Carries Mutations in MSD
[0315] MSD is caused by a deficiency to generate FGly residues in
sulfatases (Schmidt, B., et al., Cell, 1995, 82:271-278). The FGE
gene is therefore a candidate gene for MSD. We amplified and
sequenced the FGE encoding cDNA of seven MSD patients and found ten
different mutations that were confirmed by sequencing the genomic
DNA (Table 1).
TABLE-US-00001 TABLE 1 Mutations in MSD patients Mutation Effect on
Protein Remarks Patient 1076C > A S359X Truncation of the
C-terminal 16 .sup. 1* residues IVS3 + 5-8 del Deletion of residues
In-frame deletion of exon 3 1, 2 149-173 979C > T R327X Loss of
subdomain 3 2 1045C > T R349W Substitution of a conserved
residue 3, 7 in subdomain 3 1046G > A R349Q Substitution of a
conserved residue 4 in subdomain 3 1006T > C C336R Substitution
of a conserved residue 4 in subdomain 3 836C > T A279V
Substitution of a conserved residue 5 in subdomain 2 243delC
frameshift and Loss of all three subdomains 5 truncation 661delG
frameshift and Loss of the C-terminal third of .sup. 6** truncation
FGE including subdomain 3 IVS6-1G > A Deletion of residues In =
frame deletion of exon 7 5 231-318 *Patient 1 is the MSD patient
Mo. in Schmidt, B., et al., Cell, 1995, 82:271-278 and Rommerskirch
and von Figura, Proc. Natl. Acad. Sci., USA, 1992, 89:2561-2565.
**Patient 6 is the MSD patient reported by Burk et al., J.
Pediatr., 1984, 104:574-578. The other patients represent
unpublished cases.
[0316] The first patient was heterozygous for a 1076C>A
substitution converting the codon for serine 359 into a stop codon
(S359X) and a mutation causing the deletion of the 25 residues
149-173 that are encoded by exon 3 and space the first and the
second domain of the protein. Genomic sequencing revealed a
deletion of nucleotides +5-8 of the third intron (IVS3+5-8 del)
thereby destroying the splice donor site of intron 3. The second
patient was heterozygous for the mutation causing the loss of exon
3 (IVS3+5-8 del) and a 979C>T substitution converting the codon
for arginine 327 into a stop codon (R327X). The truncated FGE
encoded by the 979C>T allele lacks most of subdomain 3. The
third patient was homozygous for a 1045C>T substitution
replacing the conserved arginine 349 in subdomain 3 by tryptophan
(R349W). The fourth patient was heterozygous for two missense
mutations replacing conserved residues in the FGE domain: a
1046>T substitution replacing arginine 349 by glutamine (R349Q)
and a 1006T>C substitution replacing cysteine 336 by arginine
(C336R). The fifth patient was heterozygous for a 836 C>T
substitution replacing the conserved alanine 279 by valine (A279V).
The second mutation is a single nucleotide deletion (243delC)
changing the sequence after proline 81 and causing a translation
stop after residue 139. The sixth patient was heterozygous for the
deletion of a single nucleotide (661delG) changing the amino acid
sequence after residue 220 and introducing a stop codon after
residue 266. The second mutation is a splice acceptor site mutation
of intron 6 (IVS6-1G>A) causing an in-frame deletion of exon 7
encoding residues 281-318. In the seventh patient the same
1045C>T substitution was found as in the third patient. In
addition we detected two polymorphisms in the coding region of 18
FGE alleles from controls and MSD patients. 22% carried a 188G>A
substitution, replacing serine 63 by asparagine (S63N) and 28% a
silent 1116C>T substitution.
Transduction of MSD Fibroblasts with Wild Type and Mutant FGE
cDNA
[0317] In order to confirm the deficiency of FGE as the cause of
the inactivity of sulfatases synthesized in MSD, we expressed the
FGE cDNA in MSD fibroblasts utilizing retroviral gene transfer. As
a control we transduced the retroviral vector without cDNA insert.
To monitor the complementation of the metabolic defect the activity
of ASA, steroid sulfatase (STS) and N-acetylgalactosamine
6-sulfatase (GalNAc6S) were measured in the transduced fibroblasts
prior or after selection. Transduction of the wild type FGE
partially restored the catalytic activity of the three sulfatases
in two MSD-cell lines (Table'2) and for STS in a third MSD cell
line. It should be noted that for ASA and GalNAc6S the restoration
was only partial after selection of the fibroblasts reaching 20 to
50% of normal activity. For STS the activity was found to be
restored to that in control fibroblasts after selection. Selection
increased the activity of ASA and STS by 50 to 80%, which is
compatible with the earlier observation that 15 to 50% of the
fibroblasts become transduced (Lubke et al., Nat. Gen., 2001,
28:73-76). The sulfatase activities in the MSD fibroblasts
transduced with the retroviral vector alone (Table 2) were
comparable to those in non-transduced MSD fibroblasts (not shown).
Transduction of FGE cDNA carrying the IVS3+5-8del mutation failed
to restore the sulfatase activities (Table 2).
TABLE-US-00002 TABLE 2 Complementation of MSD fibroblasts by
transduction of wild type or Sulfatase Fibroblasts FGE-insert ASA'
STS' GaINAc6S.sup.1 MSD 3.degree. -- 1.9 .+-. 0.2 <3 56.7 .+-.
32 FGE.sub.+ 7.9 13.5 n.d. FGE.sup.++ 12.2 .+-. 0.2 75.2 283 .+-.
42 FGE-IVS3 + 5-8del.sup.+ 1.8 <3 n.d. FGE-IVS3 + 5-8de1.sup.++
2.1 <3 98.5 MSD 4.degree. -- 1.1 .+-. 0.3 <3 n.d. FGE.sup.+
4.7 17.0 n.d. Control 58 .+-. 11 66 .+-. 31 828 .+-. 426
fibroblasts .sup.1The values give the ratio between ASA (mU/mg cell
protein), STS (AU/mg cell protein), Ga1NAc6S (AU/mg cell protein)
and that of f3-hexosaminidase (U/mg cell protein). For control
fibroblasts the mean and the variation of 6-11 cell lines is given.
Where indicated the range of two cultures transduced in parallel is
given for MSD fibroblasts. .degree.The number of MSD fibroblasts
refers to that of the patient in Table 1. .sup.+Activity
determination prior to selection. .sup.++Activity determination
after selection. n.d.: not determined
Discussion
FGE is a Highly Conserved Glycoprotein of the Endoplasmic
Reticulum.
[0318] Purification of FGE from bovine testis yielded two
polypeptides of 39.5 and 41.5 kDa which originate from the same
gene. The expression of three differently tagged versions of FGE in
three different eukaryotic cell lines as a single form suggests
that one of the two forms observed in the FGE preparation purified
from bovine testis may have been generated by limited proteolysis
during purification. The substitution of Cys69 in ASA65-80 peptide
by serine was critical for the purification of FGE by affinity
chromatography. FGE has a cleavable signal sequence that mediates
translocation across the membrane of the endoplasmic reticulum. The
greater part of the mature protein (275 residues out of 340)
defines a unique domain, which is likely to be composed of three
subdomains (see Example 2), for none of the three subdomains
homologs exist in proteins with known function. The recognition of
the linear FGly modification motif in newly synthesized sulfatase
polypeptides (Dierks et al., EMBO J., 1999, 18:2084-2091) could be
the function of a FGE subdomain. The catalytic domain could
catalyse the FGly formation in several ways. It has been proposed
that FGE abstracts electrons from the thiol group of the cysteine
and transfers them to an acceptor. The resulting thioaldehyde would
spontaneously hydrolyse to FGly and H.sub.2S (Schmidt, B., et al.,
Cell, 1995, 82:271-278). Alternatively FGE could act as a
mixed-function oxygenase (monooxygenase) introducing one atom of
O.sub.2 into the cysteine and the other in H.sub.2O with the help
of an electron donor such as FADH.sub.2. The resulting thioaldehyde
hydrate derivative of cysteine would spontaneously react to FGly
and H.sub.2S. Preliminary experiments with a partially purified FGE
preparation showed a critical dependence of the FGly formation on
molecular oxygen. This would suggest that FGE acts as a
mixed-function oxygenase. The particular high conservation of
subdomain 3 and the presence of three fully conserved cysteine
residues therein make this subdomain a likely candidate for the
catalytic site. It will be interesting to see whether the
structural elements mediating the recognition of the FGly motif and
the binding of an electron acceptor or electron donor correlate
with the domain structure of FGE.
[0319] Recombinant FGE is localized in the endoplasmic reticulum,
which is compatible with the proposed site of its action. FGly
residues are generated in newly synthesized sulfatases during or
shortly after their translocation into the endoplasmic reticulum
(Dierks et al., Proc. Natl. Acad. Sci. U.S.A., 1997,
94:11963-11968; Dierks et al., FEBS Lett., 1998, 423:61-65). FGE
itself does not contain an ER-retention signal of the KDEL (SEQ ID
NO:96) type. Its retention in the endoplasmic reticulum may
therefore be mediated by the interaction with other ER proteins.
Components of the translocation/N-glycosylation machinery are
attractive candidates for such interacting partners.
Mutations in FGE Cause MSD
[0320] We have shown that mutations in the gene encoding FGE cause
MSD. FGE also may interact with other components, and defects in
genes encoding the latter could equally well cause MSD. In seven
MSD patients we indeed found ten different mutations in the FGE
gene. All mutations have severe effects on the FGE protein by
replacing highly conserved residues in subdomain 3 (three
mutations) or subdomain 2 (one mutation) or C-terminal truncations
of various lengths (four mutations) or large inframe deletions (two
mutations). For two MSD-cell lines and one of the MSD mutations it
was shown that transduction of the wild type, but not of the mutant
FGE cDNA, partially restores the sulfatase activities. This clearly
identifies the FGE gene as the site of mutation and the disease
causing nature of the mutation. MSD is both clinically and
biochemically heterogenous. A rare neonatal form presenting at
birth and developing a hydrocephalus, a common form resembling
initially to an infantile metachromatic leukodystrophy and
subsequently developing ichthyosis- and mucopolysaccharidosis-like
features, and a less frequent mild form in which the clinical
features of a mucopolysaccharidosis prevail, have been
differentiated. Biochemically it is characteristic that a residual
activity of sulfatases can be detected, which for most cases in
cultured skin fibroblasts is below 10% of controls (Burch et al.,
Clin. Genet., 1986, 30:409-15; Basner et al., Pediatr. Res., 1979,
13:1316-1318). However, in some MSD cell lines the activity of
selected sulfatases can reach the normal range (Yutaka et al.,
Clin. Genet., 1981, 20:296-303). Furthermore, the residual activity
has been reported to be subject to variations depending on the cell
culture conditions and unknown factors. Biochemically, MSD has been
classified into two groups. In group I the residual activity of
sulfatases is below 15% including that of ASB. In group II the
residual activity of sulfatases is higher and particularly that of
ASB may reach values of up to 50-100% of control. All patients
reported here fall into group I except patient 5, which falls into
group II (ASB activity in the control range) of the biochemical
phenotype. Based on clinical criteria patients 1 and 6 are neonatal
cases, while patients 2-4 and 7 have the common and patient 5 the
mucopolysaccharidosis-like form of MSD.
[0321] The phenotypic heterogeneity suggests that the different
mutations in MSD patients are associated with different residual
activities of FGE. Preliminary data on PT67 cells stably expressing
FGE IVS3+5-8del indicate that the in-frame deletion of exon 3
abolishes FGE activity completely. The characterization of the
mutations in MSD, of the biochemical properties of the mutant FGE
and of the residual content of FGly in sulfatases using a recently
developed highly sensitive mass spectrometric method (Peng et al.,
J. Mass Spec., 2003, 38:80-86) will provide a better understanding
of the genotype-phenotype correlation in MSD.
Example 2
The Human FGE Gene Defines a New Gene Family Modifying Sulfatases
which is Conserved from Prokaryotes to Eukaryotes
Bioinformatics
[0322] Signal peptides and cleavage sites were described with the
method of von Heijne (Nucleic Acids Res., 1986, 14:4683)
implemented in EMBOSS (Rice et al., Trends in Genetics, 2000,
16:276-277), and the method of Nielsen et al. (Protein Engineering,
1997, 10:1-6). N-glycosylation sites were predicted using the
algorithm of Brunak (Gupta and Brunak, Pac. Symp. Biocomput., 2002,
310-22).
[0323] Functional domains were detected by searching
PFAM-Hidden-Markov-Models (version 7.8) (Sonnhammer et al., Nucleic
Acids Res., 1998, 26:320-322). Sequences from the PFAM DUF323 seed
were obtained from TrEMBL (Bairoch, A. and Apweiler, R., Nucleic
Acids Res., 2000, 28:45-48). Multiple alignments and phylogenetic
tree constructions were performed with Clustal W (Thompson, J., et
al., Nucleic Acids Res., 1994, 22:4673-4680). For phylogenetic tree
computation, gap positions were excluded and multiple substitutions
were corrected for. Tree bootstraping was performed to obtain
significant results. Trees were visualised using Njplot (Perriere,
G. and Gouy, M., Biochimie, 1996, 78:364-369). Alignments were
plotted using the pret-typlot command from EMBOSS.
[0324] To search for FGE homologs, the databases NR, NT and EST of
the National Center for Biotechnology Information (NCBI) (Wheeler
et al., Nucleic Acids Res., 2002, 20:13-16), were queried with
BLAST (Altschul et al., Nucleic Acids Res., 1997, 25:3389-3402).
For protein sequences, the search was performed using iterative
converging Psi-Blast against the current version of the NR database
using an expectation value cutoff of 10.sup.-40, and default
parameters. Convergence was reached after 5 iterations. For
nucleotide sequences, the search was performed with Psi-TBlastn:
using NR and the protein sequence of human FGE as input, a score
matrix for hFGE was built with iterative converging Psi-Blast. This
matrix was used as input for blastall to query the nucleotide
databases NT and EST. For both steps, an expectation value cutoff
of 10.sup.-20 was used.
[0325] Protein secondary structure prediction was done using
Psipred (Jones, D., J Mol. Biol., 1999, 292:1950-202; McGuffin, L.,
et al., Bioinformatics, 2000, 16:404-405).
[0326] Similarity scores of the subdomains were computed from
alignments using the cons algorithm form EMBOSS with default
parameters. The metaalignments were generated by aligning consensus
sequences of the FGE-family subgroups. Genomic loci organisation
and synteny were determined using the NCBI's human and mouse genome
resources at NCBI (Bethesda, Md.) and Softberry's (Mount Kisco,
N.Y.) Human-Mouse-Rat Synteny. Bacterial genome sequences were
downloaded from the NCBI-FTP-server. The NCBI microbial genome
annotation was used to obtain an overview of the genomic loci of
bacterial FGE genes.
Results and Discussion
Basic Features and Motifs of Human FGE and Related Proteins
[0327] The human FGE gene (SEQ ID NOs:1, 3) encodes the FGE protein
(SEQ ID NO:2) which is predicted to have 374 residues. A cleavage
signal between residues 22-33 (Heijne-Score of 15.29) and a
hydropathy-score (Kyte, J. and Doolittle, R., J Mol Biol., 1982,
157:105-132) of residues 17-29 between 1.7 and 3.3 indicate that
the 33 N-terminal residues are cleaved off after ER-translocation.
However with the algorithm of Nielsen et al. (Protein Engineering,
1997, 10:1-6), cleavage of the signal sequence is predicted after
residue 34. The protein has a single potential N-glycosylation site
at Asn 141.
[0328] A search with the FGE protein sequence against the protein
motif database PFAM (Sonnhammer et al., Nucleic Acids Res., 1998,
26:320-322) revealed that residues 87-367 of human FGE can be
classified as the protein domain DUF323 ("domain of unknown
function", PF03781) with a highly significant expectation value of
7:9*10.sup.-114. The PFAM-seed defining DUF323 consists of 25
protein sequences, of which the majority are hypothetical proteins
derived from sequencing data. To analyse the relationship between
human FGE and DUF323, a multiple alignment of FGE with the
sequences of the DUF323 seed was performed. Based on this, a
phylogenetic tree was constructed and bootstraped. Four of the
hypothetical sequences (TrEMBL-IDs Q9CK12, Q91761, 094632 and
Q9Y405) had such a strong divergence from the other members of the
seed that they prevented successful) bootstraping and had to be
removed from the set. FIG. 2 shows the bootstraped tree displaying
the relationship between human FGE and the remaining 21 DUF323 seed
proteins. The tree can be used to subdivide the seed members into
two categories: homologs closely related to human FGE and the
remaining, less related genes.
[0329] The topmost 7 proteins have a phylogenetic distance between
0.41 and 0.73 to human FGE. They only contain a single domain,
DUF323. The homology within this group extends over the whole amino
acid sequence, the greater part of which consists of the DUF323
domain. The DUF323 domain is strongly conserved within this group
of homologs, while the other 15 proteins of the seed are less
related to human FGE (phylogenetic distance between 1.14 and 1.93).
Their DUF323 domain diverges considerably from the highly conserved
DUF323-domain of the first group (cf. section "Subdomains of FGE
and mutations in the FGE gene"). Most of these 15 proteins are
hypothetical, six of them have been further investigated. One of
them, a serine/threonine kinase (TrEMBL: 084147) from C.
trachomatis contains other domains in addition to DUF323: an
ATP-binding domain and a kinase domain. The sequences from R.
sphaeroides (TrEMBL: Q9ALV8) and Pseudomonas sp. (TrEMBL: 052577)
encode. the protein NirV, a gene cotranscribed with the
copper-containing nitrite reductase nirK (Jain, R. and Shapleigh,
J., Microbiology, 2001, 147:2505-2515). CarC (TrEMBL: Q9XB56) is an
oxygenase involved in the synthesis of a .beta.-lactam antibiotic
from E. carotovora (McGowan, S., et al., Mol. Microbiol., 1996,
22:415-426; Khaleeli N, T. C., and Busby R W, Biochemistry, 2000,
39:8666-8673). Xy1R (TrEMBL: 031397) and BH0900 (TrEMBL: Q9KEF2)
are enhancer binding proteins involved in the regulation of pentose
utilisation (Rodionov, D., et al., FEMS Microbiol Lett., 2001,
205:305-314) in bacillaceae and clostridiaceae. The comparison of
FGE and DUF323 led to the establishment of a homology threshold
differentiating the FGE family from distant DUF323-containing
homologs with different functions. The latter include a
serine/threonine kinase and Xy1R, a transcription enhancer as well
as FGE, a FGly generating enzyme and CarC, an oxygenase. As
discussed in elsewhere herein, FGE might also exert its cysteine
modifying function as an oxygenase, suggesting that FGE and non-FGE
members of the DUF323 seed may share an oxygenase function.
Homologs of FGE
[0330] The presence of closely related homologs of human FGE in the
DUF323 seed directed us to search for homologs of human FGE in
NCBI's NR database (Wheeler et al., Nucleic Acids Res., 2002,
20:13-16). The threshold of the search was chosen in such a way
that all 6 homologs present in the DUF323 seed and other closely
related homologs were obtained without finding the other seed
members. This search led to the identification of three FGE
orthologs in eukaryotes, 12 orthologs in prokaryotes and two
paralogs in man and mouse (Table 3).
TABLE-US-00003 TABLE 3 The FGE gene family in eukaryotes and
prokaryotes SEQ ID NOs: NA, AA LENGTH SUB- [GI] SPECIES [AA] GROUP
1/3, 2 Homo sapiens 374 El 49, 50 Mus musculus 372f El [22122361]
51, 52 Drosophila melanogaster 336 El [20130397] 53, 54 Anopheles
gambiae 290 El [21289310] 47, 48 Mus musculus 308 E2 [26344956] 45,
46 Homo sapiens 301 E2 [24308053] 55, 56 Streptomyces coelicolor
A3(2) 314 PI [21225812] 57, 58 Corynebacterium efficiens YS-314 334
P1 [25028125] 59, 60 Novosphingobium aromaticivorans 338 P2
[23108562] 61, 62 Mesorhizobium loti 372 P2 [13474559] 63, 64
Burkholderia fungorum 416 P2 [22988809] 65, 66 Sinorhizobium
meliloti 303 P2 [16264068] 67, 68 Microscilla sp. 354 P2 [14518334]
69, 70 Pseudomonas putida KT2440 291 P2 [26990068] 71, 72 Ralstonia
metallidurans 259 P2 [22975289] 73, 74 Prochlorococcus marinus 291
P2 [23132010] 75, 76 Caulobacter crescentus CB 15 338 P2 [16125425]
77, 78 Mycobacterium tuberculosis Ht37Rv 299 P2 [15607852]
GI--GenBank protein identifier NA--nucleic acid AA--amino acids,
El--eukaryotic orthologs E2--eukaryotic paralogs P1--closely
related prokaryotic orthologs P2--other prokaryotic f--protein
sequence mispredicted in GenBank
[0331] Note that the mouse sequence GI 22122361 is predicted in
GenBank to encode a protein of 284 aa, although the cDNA sequence
NM 145937 encodes for a protein of 372 residues. This misprediction
is based on the omission of the first exon of the murine FGE gene.
All sequences found in the NR database are from higher eukaryotes
or prokaryotes. FGE-homologs were not detected in archaebacteriae
or plants. Searches with even lowered thresholds in the fully
sequenced genomes of C. elegans and S. cerevisiae and the related
ORF databases did not reveal any homologs. A search in the
eukaryotic sequences of the NT and EST nucleotide databases led to
the identification of 8 additional FGE orthologous ESTs with
3'-terminal cDNA sequence fragments showing a high degree of
conservation on the protein level which are not listed in the NR
database. These sequences do not encompass the full coding part of
the mRNAs and are all from higher eukaryotes (Table 4).
TABLE-US-00004 TABLE 4 FGE ortholog EST fragments in eukaryotes SEQ
ID NOs: NA [GB] SPECIES 80 Oncorhynchus mykiss [CA379852] 81 Danio
rerio [A1721440] 82 Oryzias latipes [BJ505402] 83 Xenopus laevis
[BJ054666] 84 Silurana tropicalis [AL892419] 85 Salmo salar
[CA064079] 86 Sus scrota [BF189614] 87 Bos Taurus [AV609121]
GB--GenBank Accession No. NA--nucleic acid
[0332] Multiple alignment and construction of a phylogenetic tree
(using ClustalW) of the coding sequences from the NR database
allowed the definition of four subgroups of homologs: eukaryotic
orthologs (human, mouse, mosquito and fruitfly FGE, eukaryotic
paralogs (human and mouse FGE paralog), prokaryotic orthologs
closely related to FGE (Streptomyces and Corynebacterium and other
prokaryotic orthologs (Caulobacter, Pseudomonas, Mycobacterium,
Prochlorococcus, Mesorhizobium, Sinorhizobium, Novosphingobium,
Ralstonia, Burkholderia, and Microscilla). The eukaryotic orthologs
show an overall identity to human FGE of 87% (mouse), 48%
(fruitfly) and 47% (anopheles). While FGE orthologs are found in
prokaryotes and higher eukaryotes, they are missing in the
completely sequenced genomes of lower eukaryotes phylogenetically
situated between S. cerevisiae and D. melanogaster. In addition,
FGE homologs are absent in the fully sequenced genomes of E. coli
and the pufferfish.
[0333] As discussed elsewhere herein, the FGE paralogs found in
human and mouse may have a minor FGly-generating activity and
contribute to the residual activities of sulfatases found in MSD
patients.
Subdomains of FGE
[0334] The members of the FGE gene family have three highly
conserved parts/domains (as described elsewhere herein). In
addition to the two non-conserved sequences separating the former,
they have non-conserved extensions at the N- and C-terminus. The
three conserved parts are considered to represent subdomains of the
DUF323 domain because they are spaced by non-conserved parts of
varying length. The length of the part spacing subdomains 1 and 2
varies between 22 and 29 residues and that spacing subdomains 2 and
3 between 7 to 38 amino acids. The N- and C-terminal non-conserved
parts show an even stronger variation in length (N-terminal: 0-90
AA, Cterminal: 0-28 AA). The sequence for the FGE gene from
Ralstonia metallidurans is probably incomplete as it lacks the
first subdomain.
[0335] To verify the plausibility of defining subdomains of DUF323,
we performed a secondary structure prediction of the human FGE
protein using Psipred. The hydrophobic ER-signal (residues 1-33) is
predicted to contain helix-structures confirming the signal
prediction of the von-Heijne algorithm. The N-terminal
non-conserved region (aa 34-89) and the spacing region between
subdomains 2 and 3 (aa 308-327) contain coiled sections. The region
spacing subdomains 1 and 2 contains a coil. The .alpha.-helix at aa
65/66 has a low prediction confidence and is probably a prediction
artefact. The subdomain boundaries are situated within coils and do
not interrupt .alpha.-helices or .beta.-strands. The first
subdomain is made up of several .beta.-strands and an
.alpha.-helix, the second subdomain contains two .beta.-strands and
four .alpha.-helices. The third subdomain has a a-helix region
flanked by a sheet at the beginning and the end of the subdomain.
In summary, the secondary structure is in agreement with the
proposed subdomain structure as the subdomain boundaries are
situated within coils and the subdomains contain structural
elements .alpha.-helices and (.beta.-strands).
[0336] It should be noted that none of the subdomains exists as an
isolated module in sequences listed in databases. Within each of
the four subgroups of the FGE family, the subdomains are highly
conserved, with the third subdomain showing the highest homology
(Table 5). This subdomain shows also the strongest homology across
the subgroups.
TABLE-US-00005 TABLE 5 Homology (% similarity) of the FGE family
subdomains Subdomain Subfamily Members 1 2 3 El 4 79 82 100 E2 2 90
94 100 P1 2 70 79 95 P2 10 59 79 80 El--eukaryotic orthologs
E2--eukaryotic paralogs P1--closely related prokaryotic orthologs
P2--other prokaryotic orthologs
[0337] The first subdomain of the FGE-family shows the weakest
homology across the subgroups. In the eukaryotic orthologs it
carries the N-glycosylation site: at residue Asn 141 in human, at
Asn 139 in the mouse and Asn 120 in the fruit fly. In anopheles, no
asparagine is found at the residue 130 homologous to D.
melanogaster Asn 120. However, a change of two nucleotides would
create an N-glycosylation site Asn 130 in anopheles. Therefore, the
sequence encompassing residue 130 needs to be resequenced. The
second subdomain is rich in tryptophans with 12 Trp in 129 residues
of human FGE. Ten of these tryptophans are conserved in the FGE
family.
[0338] High conservation of subdomain 3: subdomain 3 between
eukaryotic orthologs are 100% similar and 90% identical. The
importance of the third subdomain for the function of the protein
is underlined by the observation that this subdomain is a hot spot
for disease causing mutations in MSD patients. Seven of nine
mutations identified in six MSD patients described in Example 1 are
located in sequences that encode the 40 residues of subdomain 3.
The residues contain four cysteines, three of which are conserved
among the pro- and eukaryotic orthologs. The two eukaryotic
paralogs show the lowest homology to the other members of the
FGE-family, e.g. they lack two of the three conserved cysteines of
subdomain 3. Features conserved between subdomain 3 sequences of
orthologs and paralogs are the initial RVXXGG(A)S motif (SEQ ID
NO:79), a heptamer containing three arginines (residues 19-25 of
the subdomain consensus sequence) and the terminal GFR motif. A
comparison with the DUF323 domain of the 15 seed sequences that are
no close homologs of FGE shows marked sequence differences: the 15
seed sequences have a less conserved first and second subdomain,
although the overall subdomain structure is also visible. Subdomain
3, which is strongly conserved in the FGE family, is shorter and
has a significantly weaker homology to the eukaryotic subdomain 3
(similarity of about 20%) as compared to the prokaryotic FGE family
members (similarity of about 60%). Thus they lack all of the
conserved cysteine residues of subdomain 3. The only conserved
features are the initial RVXXGG(A)S motif (SEQ ID NO:79) and the
terminal GFR motif.
Genomic Organisation of the Human and Murine FGE Gene
[0339] The human FGE gene is located on chromosome 3p26. It
encompasses 105 kb and 9 exons for the translated sequence. The
murine FGE gene has a length of 80 Kb and is located on chromosome
6E2. The 9 exons of the murine FGE gene have nearly the same size
as the human exons (FIG. 3). Major differences between the human
and the mouse gene are the lower conservation of the 3'-UTR in exon
9 and the length of exon 9, which is 461 by longer in the murine
gene. Segment 6E2 of mouse chromosome 6 is highly syntenic to the
human chromosome segment 3p26. Towards the telomere, both the human
and the murine FGE loci are flanked by the genes coding for LMCD1,
KIAA0212, ITPR1, AXCAM, and IL5RA. In the centromeric direction,
both FGE loci are flanked by the loci of CAV3 and OXTR.
Genomic Organisation of the Prokaryotic FGE Genes
[0340] In prokaryotes the sulfatases are classified either as
cysteine- or serine-type sulfatases depending on the residue that
is converted to FGly in their active center (Miech, C., et al., J
Biol. Chem., 1998, 273:4835-4837; Dierks, T., et al., J Biol.
Chem., 1998, 273:25560-25564). In Klebsiella pneumoniae, E. coli
and Yersinia pestis, the serine-type sulfatases are part of an
operon with AtsB, which encodes a cytosolic protein containing
iron-sulfur cluster motifs and is critical for the generation of
FGly from serine residues (Marquordt, C., et al., J Biol. Chem.,
2003, 278:2212-2218; Szameit, C., et al., J Biol. Chem., 1999,
274:15375-15381).
[0341] It was therefore of interest to examine whether prokaryotic
FGE genes are localized in proximity to cysteine-type sulfatases
that are the substrates of FGE. Among the prokaryotic FGE genes
shown in Table 3, seven have fully sequenced genomes allowing a
neighbourhood analysis of the FGE loci. Indeed, in four of the 7
genomes (C. efficiens: PID 25028125, P. putida: PID 26990068, C.
crescentus: PID 16125425 and M. tuberculosis: PID 15607852) a
cysteine-type sulfatase is found in direct vicinity of FGE
compatible with a cotranscription of FGE and the sulfatase. In two
of them (C. efficiens and P. putida), FGE and the sulfatase have
even overlapping ORFs, strongly pointing to their coexpression.
Furthermore, the genomic neighbourhood of FGE and sulfatase genes
in four prokaryotes provides additional evidence for the assumption
that the bacterial FGEs are functional orthologs.
[0342] The remaining three organisms do contain cysteine-type
sulfatases (S. coelicolor: PID 24413927, M. loti: HD 13476324, S.
meliloti: PIDs 16262963, 16263377, 15964702), however, the genes
neighbouring FGE in these organisms neither contain a canonical
sulfatase signature (Dierks, T., et al., J Biol. Chem., 1998,
273:25560-25564) nor a domain that would indicate their function.
In these organisms the expression of FGE and cysteine-type
sulfatases is therefore likely to be regulated in trans.
Conclusions
[0343] The identification of human FGE whose deficiency causes the
autosomal-recessively transmitted lysosomal storage disease
Multiple Sulfatase Deficiency, allows the definition of a new gene
family which comprises FGE orthologs from prokaryotes and
eukaryotes as well as an FGE paralog in mouse and man. FGE is not
found in the fully sequenced genomes of E. coli, S. cerevisiae, C.
elegans and Fugu rubripes. In addition, there is a phylogenetic gap
between prokaryotes and higher eukaryotes with FGE lacking in any
species phylogenetically situated between prokaryotes and D.
melanogaster. However, some of these lower eukaryotes, e.g. C.
elegans, have cysteine-type sulfatase genes. This points to the
existence of a second FGly generating system acting on
cysteine-type sulfatases. This assumption is supported by the
observation that E. coli, which lacks FGE, can generate FGly in
cysteine-type sulfatases (Dierks, T., et al., J Biol. Chem., 1998,
273:25560-25564).
Example 3
FGE Expression Causes Significant Increases in Sulfatase Activity
in Cell Lines that Overexpress a Sulfatase
[0344] We wanted to examine the effects of FGE on cells
expressing/overexpressing a sulfatase. To this end, HT-1080 cells
expressing human sulfatases Iduronate 2-Sulfatase (I2S) or
N-Acetylgalactosamine 6-Sulfatase (GALNS) were transfected in
duplicate with either a FGE expression construct, pXMG.1.3 (Table 7
and FIG. 4) or a control plasmid, pXMG.1.2 (FGE in antisense
orientation incapable of producing functional FGE, Table 7). Media
samples were harvested 24, 48, and 72 hours following a 24 hour
post-electroporation medium change. The samples of medium were
tested for respective sulfatase activity by activity assay and
total sulfatase protein level estimated by ELISA specific for
either Iduronate 2-Sulfatase or N-Acetylgalactosamine
6-Sulfatase.
TABLE-US-00006 TABLE 6 Transfected Cell Lines Expressing Sulfatases
Used as Substrates for Transfection Cell Strain Plasmid Sulfatase
Expressed 36F pXFM4A.1 N-Acetylgalactosamine 6-Sulfatase 3006
pXI2S6 Iduronate 2-Sulfatase
TABLE-US-00007 TABLE 7 FGE and Control Plasmids Used to Transfect
Iduronate 2-Sulfatase and N-Acetylgalactosamine 6-Sulfatase
Expressing HT-1080 Cells Plasmid Configuration of Major DNA
Sequence Elements * pXMG.1.3 (FGE expression) >1.6 kb CMV
enhancer/promoter > 1.1 kb FGE cDNA > hGH3' untranslated
sequence < amp < DHFR cassette < Cdneo cassette (neomycin
phosphotransferase) pXMG.1.2 (control, >1.6 kb CMV
enhancer/promoter < 1.1 kb FGE cDNA < hGH3' FGE reverse
untranslated sequence < amp < DHFR cassette < Cdneo
cassette orientation) (neomycin phosphotransferase) * > denotes
orientation 5' to 3'
Experimental Procedures
Materials and Methods
[0345] Transfection of HT-1080 cells Producing Iduronate
2-Sulfatase and N-Acetylgalactosamine 6-Sulfatase
[0346] HT-1080 cells were harvested to obtain 9-12.times.10.sup.6
cells for each electroporation. Two plasmids were transfected in
duplicate: one to be tested (FGE) and a control; in this case the
control plasmid contained the FGE cDNA cloned in the reverse
orientation with respect to the CMV promoter. Cells were
centrifuged at approximately 1000 RPM for 5 minutes. Cells were
suspended in 1.times.PBS at 16.times.10.sup.6 cells/mL. To the
bottom of electroporation cuvette, 100 .mu.g of plasmid DNA was
added, 750 .mu.L of cell suspension (12.times.10.sup.6 cells) was
added to the DNA solution in the cuvette. The cells and DNA were
mixed gently with a plastic transfer pipette, being careful not to
create bubbles. The cells were electroporated at 450 V, 250 .mu.F
(BioRad Gene Pulser). The time constant was recorded.
[0347] The electroporated cells were allowed to sit undisturbed for
10-30 minutes. 1.25 mL of DMEM/10% calf serum was then added to
each cuvette, mixed, and all the cells transferred to a fresh T75
flask containing 20 mL DMEM/10. After 24 hours, the flask was
re-fed with 20 mL DMEM/10 to remove dead cells. 48-72 hours after
transfection, media samples were collected and the cells harvested
from duplicate T75 flasks.
Medium Preparation
[0348] 1 L DMEM/10 (contains: 23 ml of 2 mM L Glutamine, 115 mL
calf serum)
[0349] Cells were transfected in media without methotrexate (MTX).
24 hours later cells were re-fed with media containing the
appropriate amounts of MTX (36F=1.0 .mu.M MTX, 3006=0.1M MTX).
Medium was harvested and cells collected 24, 48, and 72 hours after
re-feed.
Activity Assays
Iduronate 2-Sulfatase (I2S).
[0350] NAPS Desalting columns (Amersham Pharmacia Biotech AB,
Uppsala, Sweden) were equilibrated with Dialysis Buffer (5 mM
sodium acetate, 5 mM tris, pH 7.0). I2S-containing sample was
applied to the column and allowed to enter the bed. The sample was
eluted in 1 mL of Dialysis Buffer. Desalted samples were further
diluted to approximately 100 ng/mL 12S in Reaction Buffer (5 mM
sodium acetate, 0.5 mg/L BSA, 0.1% Triton X-100, pH 4.5). 10 AL of
each 12S sample was added to the top row of a 96-well Fluormetric
Plate (Perkin Elmer, Norwalk, Conn.) and pre-incubated for 15
minutes at 37.degree. C. Substrate was prepared by dissolving
4-methyl-umbelliferyl sulfate (Fluka, Buchs, Switzerland) in
Substrate Buffer (5 mM sodium acetate, 0.5 mg/mL BSA, pH 4.5) at a
final concentration of 1.5 mg/mL. 100 .mu.L of Substrate was added
to each well containing 12S sample and the plate was incubated for
1 hour at 37.degree. C. in the dark. After the incubation 190 .mu.L
of Stop Buffer (332.5 mM glycine, 207.5 mM sodium carbonate, pH
10.7) was added to each well containing sample. Stock
4-methylumbelliferone (4-MUF, Sigma, St. Louis, Mo.) was prepared
as the product standard in reagent grade water to a final
concentration of 1 .mu.M. 150 .mu.L of 1 .mu.M 4-MUF Stock and 150
.mu.L Stop Buffer were added to one top row well in the plate. 150
.mu.L of Stop Buffer was added to every remaining well in the
96-well plate. Two fold serial dilutions were made from the top row
of each column down to the last row of the plate. The plate was
read on a Fusion Universal Microplate Analyzer (Packard, Meriden,
Conn.) with an excitation filter wavelength of 330 nm and an
emission filter wavelength of 440 nm. A standard curve of .mu.moles
of 4-MUF stock versus fluorescence was generated, and unknown
samples have their fluorescence extrapolated from this curve.
Results are reported as Units/mL where one Unit of activity was
equal to 1 .mu.mole of 4-MUF produced per minute at 37.degree.
C.
N-Acetylgalactosamine 6-Sulfatase (GALNS).
[0351] The GALNS activity assay makes use of the fluorescent
substrate, 4-methyl umbel li feryl-.beta.-D-galactopyranosi
de-6-sulfate (Toronto Research Chemicals Inc., Catalogue No.
M33448). The assay was comprised of two-steps. At the first step,
75 .mu.L of the 1.3 mM substrate prepared in reaction buffer (0.1M
sodium acetate, 0.1M sodium chloride, pH 4.3) was incubated for 4
hours at 37.degree. C. with 10 .mu.L of media/protein sample or its
corresponding dilutions. The reaction was stopped by the addition
of 54 of 2M monobasic sodium phosphate to inhibit the GALNS
activity. Following the addition of approximately 500 U of
.beta.-galactosidase from Aspergillus oryzae (Sigma, Catalogue No.
G5160), the reaction mixture was incubated at 37.degree. C. for an
additional hour to release the fluorescent moiety of the substrate.
The second reaction was stopped by the addition of 910 .mu.L of
stop solution (1% glycine, 1% sodium carbonate, pH 10.7). The
fluorescence of the resultant mixture was measured by using a
measurement wavelength of 359 nm and a reference wavelength of 445
nm with 4-methylumbelliferone (sodium salt from Sigma, Catalogue
No. M1508) serving as a reference standard. One unit of the
activity corresponds to nmoles of released 4-methylumbelliferone
per hour.
Immunoassays (ELISA)
Iduronate 2-Sulfatase (I2S).
[0352] A 96-well flat bottom plate was coated with a mouse
monoclonal anti-12S antibody diluted to 10 .mu.g/mL in 50 nM sodium
bicarbonate pH 9.6 for 1 hour at 37.degree. C. The mouse monoclonal
anti-I2S antibody was developed under contract by Maine
Biotechnology Services, Inc. (Portland, Me.) to a purified,
recombinantly-produced, full-length, human I2S polypeptide using
standard hybridoma-producing technology. The plate was washed 3
times with 1.times.PBS containing 0.1% Tween-20 and blocked for 1
hour with 2% BSA in wash buffer at 37.degree. C. Wash buffer with
2% BSA was used to dilute samples and standards. I2S standard was
diluted and used from 100 ng/mL to 1.56 ng/mL. After removal of the
blocking buffer, samples and standards were applied to the plate
and incubated for 1 hour at 37.degree. C. Detecting antibody,
horseradish peroxidase-conjugated mouse anti-I2S antibody, was
diluted to 0.15 .mu.g/mL in wash buffer with 2% BSA. The plate was
washed 3 times, detecting antibody added to the plate, and it was
incubated for 30 minutes at 37.degree. C. To develop the plate, TMB
substrate (Bio-Rad, Hercules, Calif.) was prepared. The plate was
washed 3 times, 100 .mu.L of substrate was added to each well and
it was incubated for 15 minutes at 37.degree. C. The reaction was
stopped with 2 N sulfuric acid (100 .mu.L/well) and the plate was
read on a microtiter plate reader at 450 nm, using 655 nm as the
reference wavelength.
N-Acetylgalactosamine 6-Sulfatase (GALNS).
[0353] Two mouse monoclonal anti-GALNS antibodies provided the
basis of the GALNS ELISA. The mouse monoclonal anti-GALNS
antibodies were also developed under contract by Maine
Biotechnology Services, Inc. (Portland, Me.) to a purified,
recombinantly-produced, full-length, human GALNS polypeptide using
standard hybridoma-producing technology. The first antibody, for
capture of GALNS was used to coat a F96 MaxiSorp Nunc-Immuno Plate
(Nalge Nunc, Catalogue No. 442404) in a coating buffer (50 mM
sodium bicarbonate, pH 9.6). After incubation for one hour at
37.degree. C. and washing with a wash buffer, the plate was blocked
with blocking buffer (PBS, 0.05% Tween-20, 2% BSA) for one hour at
37.degree. C. Experimental and control samples along with GALNS
standards were then loaded onto the plate and further incubated for
one hour at 37.degree. C. After washing with a wash buffer, the
second, detection antibody conjugated to HRP was applied in
blocking buffer followed by 30 minute incubation at 37.degree. C.
After washing the plate again, the Bio-Rad TMB substrate reagent
was added and incubated for 15 minutes. 2N sulfuric acid was then.
added to stop the reaction and results were scored
spectrophotometrically by using a Molecular Device plate reader at
450 nm wavelength.
Discussion
Effect of FGE on Sulfatase Activity
GALNS.
[0354] An approximately 50-fold increase in total GALNS activity
was observed over the control levels (FIG. 5). This level of
increased activity was observed with all three medium sampling time
points. Moreover, the GALNS activity was accumulated linearly over
time with a four-fold increase between 24 and 48 hours and a
two-fold increase between the 48 hour and 72 hour timepoints.
I2S.
[0355] Although of smaller absolute magnitude, a similar effect was
observed for total I2S activity where an approximately 5-fold
increase in total 12S activity was observed over the control
levels. This level of increased activity was sustained for the
duration of the experiment. 12S activity accumulated in the medium
linearly over time, similar to the results seen with GALNS
(2.3-fold between 24 and 48 hours, and 1.8-fold between 48 and 72
hours).
Effect of FGE on Sulfatase Specific Activity
GALNS.
[0356] Expression of FGE in 36F cells enhanced apparent specific
activity of GALNS (ratio of enzyme activity to total enzyme
estimated by ELISA) by 40-60 fold over the control levels (FIG. 6).
The increase in specific activity was sustained over the three time
points in the study and appeared to increase over the three days of
post-transfection accumulation.
I2S.
[0357] A similar effect was seen with I2S, where a 6-7-fold
increase in specific activity (3-5 U/mg) was observed over the
control values (0.5-0.7 U/mg).
[0358] The ELISA values for both GALNS (FIG. 7) and I2S were not
significantly affected by transfection of FGE. This indicates that
expression of FGE does not impair translational and secretory
pathways involved in sulfatase production.
[0359] In sum, all of these results for both sulfatases indicate
that FGE expression dramatically increases sulfatase specific
activity in cell lines that overexpress GALNS and I2S.
Co-Expression of FGE (SUMF1) and Other Sulfatase Genes
[0360] To test the effect of FGE (SUMF1) on additional sulfatase
activities in normal cells we overexpressed ARSA (SEQ ID NO:14),
ARSC (SEQ ID NO:18) and ARSE (SEQ ID NO:22) cDNAs in various cell
lines with and without co-transfection of the FGE (SUMF1) cDNA and
measured sulfatase activities. Overexpression of sulfatase cDNAs in
Cos-7 cells resulted in a moderate increase of sulfatase activity,
while a striking synergistic increase (20 to 50 fold) was observed
when both a sulfatase gene and the FGE (SUMF1) gene were
co-expressed. A similar, albeit lower, effect was observed in three
additional cell lines, HepG2, LE293, and U205. Simultaneous
overexpression of multiple sulfatase cDNAs resulted in a lower
increase of each specific sulfatase activity as compared to
overexpression of a single sulfatase, indicating the presence of
competition of the different sulfatases for the modification
machinery.
[0361] To test for functional conservation of the FGE (SUMF1) gene
during evolution we overexpressed ARSA, ARSC and ARSE cDNAs in
various cell lines with and without co-transfection of the MSD cDNA
and measured sulfatase activities. Both the murine and the
DroSophila FGE (SUMF1) genes were active on all three human
sulfatases, with the Drosophila FGE (SUMF1) being less efficient.
These data demonstrate a high degree of functional conservation of
FGE (SUMF1) during evolution implicating significant biological
importance to cellular function and survival. A similar and
consistent, albeit much weaker, effect was observed by using the
FGE2 (SUMF2) gene, suggesting that the protein encoded by this gene
also has a sulfatase modifying activity. These data demonstrate
that the amount of the FGE (SUMF1)-encoded protein is a limiting
factor for sulfatase activities, a finding with important
implications. for the large scale production of active sulfatases
to be utilized in enzyme replacement therapy.
Example 4
Identification of the Gene Mutated in MSD by Means of Functional
Complementation Using Microcell Mediated Chromosome Transfer
[0362] In a separate experiment using microcell mediated chromosome
transfer by means of functional complementation we confirmed that
the gene mutated in MSD is FGE. Our findings provide further
insight into a novel biological mechanism affecting an entire
family of proteins in distantly related organisms. In addition to
identifying the molecular basis of a rare genetic disease, our data
further confirms a powerful enhancing effect of the FGE gene
product on the activity of sulfatases. The latter finding has
direct clinical implications for the therapy of at least eight
human diseases caused by sulfatase deficiencies.
The Gene for MSD Maps to Chromosome 3p26
[0363] To identify the chromosomal location of the gene mutated in
MSD we attempted to rescue the deficient sulfatase enzymes by
functional complementation via microcell mediated chromosome
transfer. A panel of human/mouse hybrid cell lines, containing
individual normal human chromosomes tagged with the dominant
selectable marker HyTK, was used as the source of donor human
chromosomes and fused to an immortalized cell line from a patient
with MSD. All 22 human autosomes were transferred one by one to the
patient cell line and hybrids were selected in hygromycin.
Approximately 25 surviving colonies were picked in each of the 22
transfer experiments. These were grown separately and harvested for
subsequent enzymatic testing. ArylsulfataseA (ARSA) (SEQ ID NO:15),
ArylsulfataseB (ARSB) (SEQ ID NO:17), and ArylsulfataseC (ARSC)
(SEQ ID NO:19) activities were tested for each of the approximately
440 clones (20.times.22). This analysis clearly indicated that
sulfatase activities of several clones deriving from the chromosome
3 transfer was significantly higher compared to that of all the
other clones. A striking variability was observed when analyzing
the activities of each individual clone from the chromosome 3
transfer. To verify whether each clone had an intact human
chromosome 3 from the donor cell line, we used a panel of 23
chromosome 3 polymorphic genetic markers, evenly distributed along
the length of the chromosome and previously selected on the basis
of having different alleles between the donor and the patient cell
lines. This allowed us to examine for the presence of the donor
chromosome and to identify possible loss of specific regions due to
incidental chromosomal breakage. Each clone having high enzymatic
activity retained the entire chromosome 3 from the donor cell line,
whereas clones with low activities appeared to have lost the entire
chromosome on the basis of the absence of chromosome 3 alleles from
the donor cell line. The latter clones probably retained a small
region of the donor chromosome containing the selectable marker
gene that enabled them to survive in hygromycin containing medium.
These data indicate that a normal human chromosome 3 was able to
complement the defect observed in the MSD patient cell line.
[0364] To determine the specific chromosomal region containing the
gene responsible for the complementing activity we used Neo-tagged
chromosome 3 hybrids which were found to have lost various portions
of the chromosome. In addition, we performed irradiated
microcell-mediated chromosome transfer of HyTK-tagged human
chromosomes 3. One hundred and fifteen chromosome 3 irradiated
hybrids were tested for sulfatase activities and genotyped using a
panel of 31 polymorphic microsatellite markers spanning the entire
chromosome. All clones displaying high enzymatic activities
appeared to have retained chromosome 3p26. A higher resolution
analysis using additional markers from this region mapped the
putative location for the complementing gene between markers
D3S3630 and D3S2397.
Identification of the Gene Mutated in MSD
[0365] We investigated genes from the 3p26 genomic region for
mutations in MSD patients. Each exon including splice junctions
were PCR-amplified and analyzed by direct sequencing. Mutation
analysis was performed on twelve unrelated affected individuals;
five previously described MSD patients and seven unpublished cases.
Several mutations were identified from our MSD cohort in the
expressed sequence tag (EST) AK075459 (SEQ ID NOs:4,5),
corresponding to a gene of unknown function, strongly suggesting
that this was the gene involved in MSD. Each mutation was found to
be absent in 100 control individuals, thus excluding the presence
of a sequence polymorphism. Additional confirmatory mutation
analysis was performed on reverse transcribed patients' RNAs,
particularly in those cases in which genomic DNA analysis revealed
the presence of a mutation in or near a splice site, possibly
affecting splicing. Frameshift, nonsense, splicing, and missense
mutations were also identified, suggesting that the disease is
caused by a loss of function mechanism, as anticipated for a
recessive disorder. This is also consistent with the observation
that almost all missense mutations affect amino acids that are
highly conserved throughout evolution (see below).
TABLE-US-00008 TABLE 8 Additional MSD Mutations identified
nucleotide amino acid Case reference phenotype exon change change
1. BA426 Conary et al, 1988 moderate 3 463T > C S155P 3 463T
> C S155P 2. BA428 Burch et al, 1986 severe neonatal 5 661delG
frameshift 3. BA431 Zenger et al, 1989 moderate 1 2T > G M R 2
276delC frameshift 4. BA799 Burk et al, 1981 mild-moderate 3 463T
> C S155P 3 463T > C S155P 5. BA806 unpublished several
neonatal 9 1045T > C R349W 6. BA807 Schmidt et al, 1995 unknown
3 c519 + 4delGTAA ex 3 skipping 9 1076C > A S359X 7. BA809
Couchot et al, 1974 mild-moderate 1 1A > G M V 9 1042G > C
A348P 8. BA810 unpublished severe 8 1006T > C C336R 9 1046G >
A R349Q 9. BA811 unpublished several neonatal 3 c519 + 4delGTAA ex
3 skipping 8 979C > T 327X 10. BA815 unpublished moderate 5 c
603 - 6delC ex 6 skipping 6 836C > T A279V 11. BA919 unpublished
mild-moderate 9 1033C > T R345C 9 1033C > T R345C 12. BA920
unpublished moderate 5 653G > A C218Y 9 1033C > T R345C
indicates data missing or illegible when filed
[0366] Mutations were identified in each MSD patient tested, thus
excluding locus heterogeneity. No obvious correlation was observed
between the types of mutations identified and the severity of the
phenotype reported in the patients, suggesting that clinical
variability is not caused by allelic heterogeneity. In three
instances different patients (case 1 and 4, case 6 and 9, and case
11 and 12 in Table 6) were found to carry the same mutation. Two of
these patients (case 11 and 12) originate from the same town in
Sicily, suggesting the presence of a founder effect that was indeed
confirmed by haplotype analysis. Surprisingly, most patients were
found to be compound heterozygotes, carrying different allelic
mutations, while only a few were homozygous. Albeit consistent with
the absence of consanguinity reported by the parents, this was a
somehow unexpected finding for a very rare recessive disorder such
as MSD.
The FGE Gene and Protein
[0367] The consensus cDNA sequence of the human FGE (also used
interchangeably herein as SUMF1) cDNA (SEQ ID NO:1) was assembled
from several expressed sequence tag (EST) clones and partly from
the corresponding genomic sequence. The gene contains nine exons
and spans approximately 105 kb (see Example 1). Sequence comparison
also identified the presence of a FGE gene paralog located on human
chromosome 7 that we designated FGE2 (also used interchangeably
herein as SUMF2) (SEQ ID NOs: 45, 46).
Functional Complementation of Sulfatase Deficiencies
[0368] Fibroblasts from two patients (case 1 and 12 in Table 8)
with MSD in whom we identified mutations of the FGE (SUMF1) gene
(cell lines BA426 and BA920) were infected with HSV viruses
containing the wild type and two mutated forms of the FGE (SUMF1)
cDNA (R327X and .DELTA.ex3). ARSA, ARSB, and ARSC activities were
tested 72 hrs after infection. Expression of the wild type FGE
(SUMF1) cDNA resulted in functional complementation of all three
activities, while mutant FGE (SUMF1) cDNAs did not (Table 9). These
data provide conclusive evidence for the identity of FGE (SUMF1) as
the MSD gene and they prove the functional relevance of the
mutations found in patients. The disease-associated mutations
result in sulfatase deficiency, thus demonstrating that FGE (SUMF1)
is an essential factor for sulfatase activity.
TABLE-US-00009 TABLE 9 Functional complementation of sulfatase
deficiencies Recipient MSD cell line construct ARSA.sup.(1)
ARSB.sup.(1) ARSC.sup.(1) BA426 HSV amplicon 24.0 22.5 0.15
SUMF1-Aex3 42.0 23.8 0.29 SUMF1-R327X 33.6 24.2 0.16 SUMF1 119.5
(4.9.times.) 37.8 (1.7.times.) 0.62(4.1 BA920 HSV amplicon 16.6
11.3 0.15 SUMF1-Aex3 17.2 14.4 0.07 SUMF1-R327X 36.0 13.5 0.13
SUMF1 66.5 (4.0.times.) 21.6 (1.9.times.) 0.42(2.8 Control range
123.7-394.6 50.6-60.7 1.80-1.58 .sup.(1)enzymatic activities are
expressed as nmoles 4-methylumbelliferone iberated mg
protein.sup.-1 3 hrs. MSD cell lines BA426 and BA920 were infected
with the HSV amplicon alone, and with constructs carrying either
mutant or wild-type SUMF1 cDNAs. The increase of single
arylsulfatase activities in fibroblasts infected with the wild-type
SUMF1 gene, as compared to those of cells infected with the vector
alone, is indicated in parentheses. Activities measured in
uninfected control fibroblasts are indicated.
Molecular Basis of MSD
[0369] Based on the hypothesis that the disease gene should be able
to complement the enzymatic deficiency in a patient cell line, we
performed microcell-mediated chromosome transfer to an immortalized
cell line from a patient with MSD. This technique has been
successfully used for the identification of genes whose predicted
function could be assessed in cell lines (e.g. by measuring
enzymatic activity or by detecting morphologic features). To
address the problem of stochastic variability of enzyme activity we
measured the activities of three different sulfatases (ARSA, ARSB
and ARSC) in the complementation assay. The results of chromosome
transfer clearly indicated mapping of the complementing gene to
chromosome 3. Subregional mapping was achieved by generating a
radiation hybrid panel for chromosome 3. Individual hybrid clones
were characterized both at the genomic level, by typing 31
microsatellite markers displaying different alleles between donor
and recipient cell lines, and at the functional level by testing
sulfatase activities. The analysis of 130 such hybrids resulted in
the mapping of the complementing region to chromosome 3p26.
[0370] Once the critical genomic region was defined, the FGE
(SUMF1) gene was also identified by mutation analysis in patients'
DNA. Mutations were found in all patients tested, proving that a
single gene is involved in MSD. The mutations found were of
different types, the majority (e.g. splice site, start site,
nonsense, frameshift) putatively result in a loss function of the
encoded protein, as expected for a recessive disease. Most missense
mutations affect codons corresponding to amino acids that have been
highly conserved during evolution, suggesting that also these
mutations cause a loss of function. No correlations could be drawn
between the type. of mutation and the severity of the phenotype,
indicating that the latter is due to unrelated factors.
Unexpectedly for a rare genetic disease, many patients were found
to be compound heterozygotes, carrying two different mutations.
However, a founder effect was identified for one mutation
originating from a small town in Sicily.
FGE (SUMF1) Gene Function
[0371] The identity of the FGE (SUMF1) gene as the "complementing
factor" was demonstrated definitively by rescuing the enzymatic
deficiency of four different sulfatases upon expression of
exogenous FGE (SUMF1) cDNA, inserted into a viral vector, in two
different patient cell lines. In each case a consistent, albeit
partial, restoration of all sulfatase activities tested was
observed, as compared to control patient cell lines transfected
with empty vectors. On average, the increase of enzyme activities
ranged between 1.7 to 4.9 fold and reached approximately half of
the levels observed in normal cell lines. Enzyme activity
correlates with the number of virus particles used in each
experiment and with the efficiency of the infection as tested by
marker protein (GFP) analysis. In the same experiments vectors
containing FGE (SUMF1) cDNAs carrying two of the mutations found in
the patients, R327X and .DELTA.ex3, were used and no significant
increase of enzyme activity was observed, thus demonstrating the
functional relevance of these mutations.
[0372] As mentioned elsewhere herein, Schmidt et al. first
discovered that sulfatases undergo a post-translational
modification of a highly conserved cysteine, that is found at the
active site of most sulfatases, to C.alpha.-formylglycine. They
also showed that this modification was defective in MSD (Schmidt,
B., et al., Cell, 1995, 82:271-278). Our mutational and functional
data provide strong evidence that FGE (SUMF1) is responsible for
this modification.
[0373] The FGE (SUMF1) gene shows an extremely high degree of
sequence conservation across all distantly related species
analyzed, from bacteria to man. We provide evidence that that the
Drosophila homologue of the human FGE (SUMF1) gene is able to
activate overexpressed human sulfatases, proving that the observed
high level of sequence similarity of the FGE (SUMF1) genes of
distantly related species correlates with a striking functional
conservation. A notable exception is yeast, which appears to lack
the FGE (SUMF1) gene as well as any sulfatase encoding genes,
indicating that sulfatase function is not required by this organism
and suggesting the presence of a reciprocal influence on the
evolution of FGE (SUMF1) and sulfatase genes.
[0374] Interestingly, there are two homologous genes, FGE (SUMF1)
and FGE2 (SUMF2), in the genomes of all vertebrates analyzed,
including humans. As evident from the phylogenetic tree, the FGE2
(SUMF2) gene appears to have evolved independently from the FGE
(SUMF1) gene. In our assays the FGE2 (SUMF2) gene is also able to
activate sulfatases, however it does it in a much less efficient
manner compared to the FGE (SUMF1) gene. This may account for the
residual sulfatase activity found in MSD patients and suggests that
a complete sulfatase deficiency would be lethal. At the moment we
cannot rule out the possibility that the FGE2 (SUMF2) gene has an
additional, yet unknown, function.
Impact on the Therapy of Diseases Due to Sulfatase Deficiencies
[0375] A strong increase, up to 50 fold, of sulfatase activities
was observed in cells overexpressing FGE (SUMF1) cDNA together with
either ARSA, ARSC, or ARSE cDNAs, compared to cells overexpressing
single sulfatases alone. In all cell lines a significant synergic
effect was found, indicating that FGE (SUMF1) is a limiting factor
for sulfatase activity. However, variability was observed among
different sulfatases, possibly due to different affinity of the FGE
(SUMF1)-encoded protein with the various sulfatases. Variability
was also observed between different cell lines which may have
different levels of endogenous formylglycine generating enzyme.
Consistent with these observations, we found that the expression of
the MSD gene varies among different tissues, with significantly
high levels in kidney. and liver. This may have important
implications as tissues with low FGE (SUMF1) gene expression levels
may be less capable of effectively modifying exogenously delivered
sulfatase proteins (see below). Together these data suggest that
the function of the FGE (SUMF1) gene has evolved to achieve a dual
regulatory system, with each sulfatase being controlled by both an
individual mechanism, responsible for the mRNA levels of each
structural sulfatase gene, and a common mechanism shared by all
sulfatases. In addition, FGE2 (SUMF2) provides partial redundancy
for sulfatase modification.
[0376] These data have profound implications for the mass
production of active sulfatases to be utilized in enzyme
replacement therapy. Enzyme replacement studies have been reported
on animal models of sulfatase deficiencies, such as a feline model
of mucopolysaccharidosis VI, and proved to be effective in
preventing and curing several symptoms. Therapeutic trials in
humans are currently being performed for two congenital disorders
due to sulfatase deficiencies, MPSII (Hunter syndrome) and MPSVI
(Maroteaux-Lamy syndrome) and will soon be extended to a large
number of patients.
Example 5
Enzyme Replacement Therapy with FGE-Activated GALNS for Morquio
Disease MPS IVA
[0377] The primary cause of skeletal pathology in Morquio patients
is keratan sulfate (KS) accumulation in epiphyseal disk (growth
plate) chondrocytes due to deficiency of the lysosomal sulfatase,
GALNS. The primary objective of in vivo research studies was to
determine whether intravenously (IV) administered FGE-activated
GALNS was able to penetrate chondrocytes of the growth plate as
well as other appropriate cell types in normal mice.
Notwithstanding a general lack of skeletal abnormalities, a GALNS
deficient mouse model (Morquio Knock-In--MKI, S. Tomatsu, St. Louis
University, MO) was also used to demonstrate in vivo biochemical
activity of repeatedly administered FGE-activated GALNS. The lack
of skeletal pathology in mouse models reflects the fact that
skeletal KS is either greatly reduced or absent in rodents (Venn G,
& Mason R M., Biochem J., 1985, 228:443-450). These mice did,
however, demonstrate detectable accumulation of GAG and other
cellular abnormalities in various organs and tissues. Therefore,
the overall objective of the studies was to demonstrate that
FGE-activated GALNS penetrates into the growth plate
(biodistribution study) and show functional GALNS enzyme activity
directed towards removal of accumulated GAG in affected tissues
(pharmacodynamic study).
[0378] The results of these studies demonstrated that IV injected
FGE-activated GALNS was internalized by chondrocytes of the growth
plate, albeit at relatively low levels compared to other tissues.
In addition, FGE-activated GALNS injection over the course of 16
weeks in MKI mice effectively cleared accumulated GAG and reduced
lysosomal biomarker staining in all soft tissues examined. In sum,
the experiments successfully demonstrated GALNS delivery to growth
plate chondrocytes and demonstrated biochemical activity in terms
of GAG clearance in multiple tissues.
Biodistribution Study
[0379] Four-week-old ICR (normal) mice were given a single IV
injection of 5 mg/kg FGE-activated GALNS. Liver, femur (bone),
heart, kidney and spleen were collected two hours after injection
and prepared for histological examination. A monoclonal anti-human
GALNS antibody was used to detect the presence of injected GALNS in
the various tissues. GALNS was detected in all tissues examined as
compared to the vehicle controls. Moreover, GALNS was readily
observed in all tissues examined using a horseradish-peroxidase
reporter system, with the exception of bone. Demonstration of GALNS
uptake in the growth plate required the use of a more sensitive
fluorescein-isothiocyanate (FITC) reporter system and indicates
that although GALNS penetrates the growth plate, it is less readily
available to growth plate chondrocytes than to cells of soft
tissues. Notwithstanding the requirement of a more sensitive
fluorescent detection method, GALNS delivery to bone growth plate
chondrocytes was observed in all growth plate sections examined as
compared to the vehicle controls.
Pharmacodynamic Study in MKI Mice
[0380] Four-week-old MKI or wild-type mice were given weekly IV
injections (n=8 per group) through 20 weeks of age. Each weekly
injection consisted of either 2 mg/kg FGE-activated GALNS or
vehicle control (no injection for wild-type mice). All mice were
sacrificed for histological examination at 20 weeks of age and
stained using the following methods: hematoxylin and eosin for
cellular morphology, alcian blue for detection of GAGs.
[0381] Clearance of accumulated GAG was demonstrated by reduced or
absent alcian blue staining in all soft tissues examined (liver,
heart, kidney and spleen). This was observed only in the GALNS
injected mice. Although the growth plate in the MKI mice functioned
normally as evidenced by normal skeletal morphology, there were
more subtle cellular abnormalities observed (including
vacuolization of chondrocytes without apparent pathological
effect). The vacuolized chondrocytes of the hypertrophic and
proliferating zones of the growth plate were unaffected by GALNS
administration. This was in contrast to the chondrocytes in the
calcification zone of the growth plate where a reduction of
vacuolization was observed in GALNS injected mice. The
vacuolization of chondrocytes and accumulation of presumed non-KS
GAG in the growth plate in MKI mice was, in general, surprising and
unexpected due to the known lack of KS in the growth plate of mice.
These particular observations likely reflect the fact that, in the
knock-in mice, high levels of mutant GALNS are present (as opposed
to knock-out mice where there is no residual mutant GALNS, no
growth plate chondrocyte vacuolization and no GAG
accumulation--Tomatsu S. et al., Human Molecular Genetics, 2003,
12:3349-3358). The vacuolization phenomenon in the growth plate may
be indicative of a secondary effect on a subset of cells expressing
mutant GALNS. Nonetheless, enzyme injection over the course of 16
weeks demonstrated strong evidence of multiple tissue FGE-activated
GALNS delivery and in vivo enzymatic activity.
DETAILED DESCRIPTION OF THE DRAWINGS
[0382] FIG. 1: MALDI-TOF mass spectra of P23 after incubation in
the absence (A) or presence (B) of a soluble extract from bovine
testis microsomes. 6 pmol of P23 were incubated under standard
conditions for 10 min at 37.degree. C. in the absence or presence
of 1 .mu.l microsomal extract. The samples were prepared for
MALDI-TOF mass spectrometry as described in Experimental
Procedures. The monoisotopic masses MH.sup.+ of P23 (2526.28) and
its FGly derivative (2508.29) are indicated.
[0383] FIG. 2: Phylogenetic tree derived from an alignment of human
FGE and 21 proteins of the PFAM-DUF323 seed. The numbers at the
branches indicate phylogenetic distance. The proteins are
designated by their TrEMBL ID number and the species name.
hFGE-human FGE. Upper right: scale of the phylogenetic distances. A
asterisk indicates that the gene has been further investigated. The
top seven genes are part of the FGE gene family.
[0384] FIG. 3: Organisation of the human and murine FGE gene locus.
Exons are shown to scale as dark boxes (human locus) and bright
boxes (murine locus). The bar in the lower right corner shows the
scale. The lines between the exons show the introns (not to scale).
The numbers above the intron lines indicate the size of the introns
in kilobases.
[0385] FIG. 4: Diagram showing a map of FGE Expression Plasmid
pXMG.1.3
[0386] FIG. 5: Bar graph depicting N-Acetylgalactosamine
6-Sulfatase Activity in 36F Cells Transiently Transfected with FGE
Expression Plasmid. Cells were transfected with either a control
plasmid, pXMG.1.2, with the FGE cDNA in the reverse orientation, or
a FGE expression plasmid, pXMG.1.3 in media without methotrexate
(MTX). 24 hours later cells were re-fed with media containing 1.0
.mu.M MTX. Medium was harvested and cells collected 24, 48, and 72
hours after re-feed. N-Acetylgalactosamine 6-Sulfatase activity was
determined by activity assay. Each value shown is the average of
two separate transfections with standard deviations indicated by
error bars.
[0387] FIG. 6: Bar graph depicting N-Acetylgalactosamine
6-Sulfatase Specific Activity in 36F Cells Transiently Transfected
with FGE Expression Plasmid. Cells were transfected with either a
control plasmid, pXMG.1.2, with the FGE cDNA in the reverse
orientation, or a FGE expression plasmid, pXMG.1.3 in media without
methotrexate (MTX). 24 hours later cells were re-fed with media
containing 1.0 .mu.M MTX. Medium was harvested and cells collected
24, 48, and 72 hours after re-feed. N-Acetylgalactosamine
6-Sulfatase specific activity was determined by activity assay and
ELISA and is represented as a ratio of N-Acetylgalactosamine
6-Sulfatase activity per mg of ELISA-reactive N-Acetylgalactosamine
6-Sulfatase. Each value shown is the average of two separate
transfections.
[0388] FIG. 7: Bar graph depicting N-Acetylgalactosamine
6-Sulfatase Production in 36F Cells Transiently Transfected with
FGE Expression Plasmid. Cells were transfected with either a
control plasmid, pXMG.1.2, with the FGE cDNA in the reverse
orientation, or a FGE expression plasmid, pXMG.1.3 in media without
methotrexate (MTX). 24 hours later cells were re-fed with media
containing 1.0 .mu.M MTX. Medium was harvested and cells collected
24, 48, and 72 hours after re-feed. N-Acetylgalactosamine
6-Sulfatase total protein was determined by ELISA. Each value shown
is the average of two separate transfections with standard
deviations indicated by error bars.
[0389] FIG. 8: Graph depicting Iduronate 2-Sulfatase Activity in
3006 Cells Transiently Transfected with FGE Expression Plasmid.
Cells were transfected with either a control plasmid, pXMG.1.2,
with the FGE cDNA in the reverse orientation, or a FGE expression
plasmid, pXMG.1.3 in media without methotrexate (MTX). 24 hours
later cells were re-fed with media containing 0.1 .mu.M MTX. Medium
was harvested and cells collected 24, 48, and 72 hours after
re-feed. Iduronate 2-Sulfatase activity was determined by activity
assay. Each value shown is the average of two separate
transfections.
[0390] FIG. 9: Depicts a kit embodying features of the present
invention.
EQUIVALENTS
[0391] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0392] All references disclosed herein are incorporated by
reference in their entirety. What is claimed is presented below and
is followed by a Sequence Listing.
Sequence CWU 1
1
9611180DNAHomo sapiensCDS(20)..(1141) 1acatggcccg cgggacaac atg gct
gcg ccc gca cta ggg ctg gtg tgt gga 52 Met Ala Ala Pro Ala Leu Gly
Leu Val Cys Gly 1 5 10cgt tgc cct gag ctg ggt ctc gtc ctc ttg ctg
ctg ctg ctc tcg ctg 100Arg Cys Pro Glu Leu Gly Leu Val Leu Leu Leu
Leu Leu Leu Ser Leu 15 20 25ctg tgt gga gcg gca ggg agc cag gag gcc
ggg acc ggt gcg ggc gcg 148Leu Cys Gly Ala Ala Gly Ser Gln Glu Ala
Gly Thr Gly Ala Gly Ala 30 35 40ggg tcc ctt gcg ggt tct tgc ggc tgc
ggc acg ccc cag cgg cct ggc 196Gly Ser Leu Ala Gly Ser Cys Gly Cys
Gly Thr Pro Gln Arg Pro Gly 45 50 55gcc cat ggc agt tcg gca gcc gct
cac cga tac tcg cgg gag gct aac 244Ala His Gly Ser Ser Ala Ala Ala
His Arg Tyr Ser Arg Glu Ala Asn60 65 70 75gct ccg ggc ccc gta ccc
gga gag cgg caa ctc gcg cac tca aag atg 292Ala Pro Gly Pro Val Pro
Gly Glu Arg Gln Leu Ala His Ser Lys Met 80 85 90gtc ccc atc cct gct
gga gta ttt aca atg ggc aca gat gat cct cag 340Val Pro Ile Pro Ala
Gly Val Phe Thr Met Gly Thr Asp Asp Pro Gln 95 100 105ata aag cag
gat ggg gaa gca cct gcg agg aga gtt act att gat gcc 388Ile Lys Gln
Asp Gly Glu Ala Pro Ala Arg Arg Val Thr Ile Asp Ala 110 115 120ttt
tac atg gat gcc tat gaa gtc agt aat act gaa ttt gag aag ttt 436Phe
Tyr Met Asp Ala Tyr Glu Val Ser Asn Thr Glu Phe Glu Lys Phe 125 130
135gtg aac tca act ggc tat ttg aca gag gct gag aag ttt ggc gac tcc
484Val Asn Ser Thr Gly Tyr Leu Thr Glu Ala Glu Lys Phe Gly Asp
Ser140 145 150 155ttt gtc ttt gaa ggc atg ttg agt gag caa gtg aag
acc aat att caa 532Phe Val Phe Glu Gly Met Leu Ser Glu Gln Val Lys
Thr Asn Ile Gln 160 165 170cag gca gtt gca gct gct ccc tgg tgg tta
cct gtg aaa ggc gct aac 580Gln Ala Val Ala Ala Ala Pro Trp Trp Leu
Pro Val Lys Gly Ala Asn 175 180 185tgg aga cac cca gaa ggg cct gac
tct act att ctg cac agg ccg gat 628Trp Arg His Pro Glu Gly Pro Asp
Ser Thr Ile Leu His Arg Pro Asp 190 195 200cat cca gtt ctc cat gtg
tcc tgg aat gat gcg gtt gcc tac tgc act 676His Pro Val Leu His Val
Ser Trp Asn Asp Ala Val Ala Tyr Cys Thr 205 210 215tgg gca ggg aag
cgg ctg ccc acg gaa gct gag tgg gaa tac agc tgt 724Trp Ala Gly Lys
Arg Leu Pro Thr Glu Ala Glu Trp Glu Tyr Ser Cys220 225 230 235cga
gga ggc ctg cat aat aga ctt ttc ccc tgg ggc aac aaa ctg cag 772Arg
Gly Gly Leu His Asn Arg Leu Phe Pro Trp Gly Asn Lys Leu Gln 240 245
250ccc aaa ggc cag cat tat gcc aac att tgg cag ggc gag ttt ccg gtg
820Pro Lys Gly Gln His Tyr Ala Asn Ile Trp Gln Gly Glu Phe Pro Val
255 260 265acc aac act ggt gag gat ggc ttc caa gga act gcg cct gtt
gat gcc 868Thr Asn Thr Gly Glu Asp Gly Phe Gln Gly Thr Ala Pro Val
Asp Ala 270 275 280ttc cct ccc aat ggt tat ggc tta tac aac ata gtg
ggg aac gca tgg 916Phe Pro Pro Asn Gly Tyr Gly Leu Tyr Asn Ile Val
Gly Asn Ala Trp 285 290 295gaa tgg act tca gac tgg tgg act gtt cat
cat tct gtt gaa gaa acg 964Glu Trp Thr Ser Asp Trp Trp Thr Val His
His Ser Val Glu Glu Thr300 305 310 315ctt aac cca aaa ggt ccc cct
tct ggg aaa gac cga gtg aag aaa ggt 1012Leu Asn Pro Lys Gly Pro Pro
Ser Gly Lys Asp Arg Val Lys Lys Gly 320 325 330gga tcc tac atg tgc
cat agg tct tat tgt tac agg tat cgc tgt gct 1060Gly Ser Tyr Met Cys
His Arg Ser Tyr Cys Tyr Arg Tyr Arg Cys Ala 335 340 345gct cgg agc
cag aac aca cct gat agc tct gct tcg aat ctg gga ttc 1108Ala Arg Ser
Gln Asn Thr Pro Asp Ser Ser Ala Ser Asn Leu Gly Phe 350 355 360cgc
tgt gca gcc gac cgc ctg ccc acc atg gac tgacaaccaa gggtagtctt
1161Arg Cys Ala Ala Asp Arg Leu Pro Thr Met Asp 365 370ccccagtcca
aggagcagt 11802374PRTHomo sapiens 2Met Ala Ala Pro Ala Leu Gly Leu
Val Cys Gly Arg Cys Pro Glu Leu1 5 10 15Gly Leu Val Leu Leu Leu Leu
Leu Leu Ser Leu Leu Cys Gly Ala Ala 20 25 30Gly Ser Gln Glu Ala Gly
Thr Gly Ala Gly Ala Gly Ser Leu Ala Gly 35 40 45Ser Cys Gly Cys Gly
Thr Pro Gln Arg Pro Gly Ala His Gly Ser Ser 50 55 60Ala Ala Ala His
Arg Tyr Ser Arg Glu Ala Asn Ala Pro Gly Pro Val65 70 75 80Pro Gly
Glu Arg Gln Leu Ala His Ser Lys Met Val Pro Ile Pro Ala 85 90 95Gly
Val Phe Thr Met Gly Thr Asp Asp Pro Gln Ile Lys Gln Asp Gly 100 105
110Glu Ala Pro Ala Arg Arg Val Thr Ile Asp Ala Phe Tyr Met Asp Ala
115 120 125Tyr Glu Val Ser Asn Thr Glu Phe Glu Lys Phe Val Asn Ser
Thr Gly 130 135 140Tyr Leu Thr Glu Ala Glu Lys Phe Gly Asp Ser Phe
Val Phe Glu Gly145 150 155 160Met Leu Ser Glu Gln Val Lys Thr Asn
Ile Gln Gln Ala Val Ala Ala 165 170 175Ala Pro Trp Trp Leu Pro Val
Lys Gly Ala Asn Trp Arg His Pro Glu 180 185 190Gly Pro Asp Ser Thr
Ile Leu His Arg Pro Asp His Pro Val Leu His 195 200 205Val Ser Trp
Asn Asp Ala Val Ala Tyr Cys Thr Trp Ala Gly Lys Arg 210 215 220Leu
Pro Thr Glu Ala Glu Trp Glu Tyr Ser Cys Arg Gly Gly Leu His225 230
235 240Asn Arg Leu Phe Pro Trp Gly Asn Lys Leu Gln Pro Lys Gly Gln
His 245 250 255Tyr Ala Asn Ile Trp Gln Gly Glu Phe Pro Val Thr Asn
Thr Gly Glu 260 265 270Asp Gly Phe Gln Gly Thr Ala Pro Val Asp Ala
Phe Pro Pro Asn Gly 275 280 285Tyr Gly Leu Tyr Asn Ile Val Gly Asn
Ala Trp Glu Trp Thr Ser Asp 290 295 300Trp Trp Thr Val His His Ser
Val Glu Glu Thr Leu Asn Pro Lys Gly305 310 315 320Pro Pro Ser Gly
Lys Asp Arg Val Lys Lys Gly Gly Ser Tyr Met Cys 325 330 335His Arg
Ser Tyr Cys Tyr Arg Tyr Arg Cys Ala Ala Arg Ser Gln Asn 340 345
350Thr Pro Asp Ser Ser Ala Ser Asn Leu Gly Phe Arg Cys Ala Ala Asp
355 360 365Arg Leu Pro Thr Met Asp 37031122DNAHomo sapiens
3atggctgcgc ccgcactagg gctggtgtgt ggacgttgcc ctgagctggg tctcgtcctc
60ttgctgctgc tgctctcgct gctgtgtgga gcggcaggga gccaggaggc cgggaccggt
120gcgggcgcgg ggtcccttgc gggttcttgc ggctgcggca cgccccagcg
gcctggcgcc 180catggcagtt cggcagccgc tcaccgatac tcgcgggagg
ctaacgctcc gggccccgta 240cccggagagc ggcaactcgc gcactcaaag
atggtcccca tccctgctgg agtatttaca 300atgggcacag atgatcctca
gataaagcag gatggggaag cacctgcgag gagagttact 360attgatgcct
tttacatgga tgcctatgaa gtcagtaata ctgaatttga gaagtttgtg
420aactcaactg gctatttgac agaggctgag aagtttggcg actcctttgt
ctttgaaggc 480atgttgagtg agcaagtgaa gaccaatatt caacaggcag
ttgcagctgc tccctggtgg 540ttacctgtga aaggcgctaa ctggagacac
ccagaagggc ctgactctac tattctgcac 600aggccggatc atccagttct
ccatgtgtcc tggaatgatg cggttgccta ctgcacttgg 660gcagggaagc
ggctgcccac ggaagctgag tgggaataca gctgtcgagg aggcctgcat
720aatagacttt tcccctgggg caacaaactg cagcccaaag gccagcatta
tgccaacatt 780tggcagggcg agtttccggt gaccaacact ggtgaggatg
gcttccaagg aactgcgcct 840gttgatgcct tccctcccaa tggttatggc
ttatacaaca tagtggggaa cgcatgggaa 900tggacttcag actggtggac
tgttcatcat tctgttgaag aaacgcttaa cccaaaaggt 960cccccttctg
ggaaagaccg agtgaagaaa ggtggatcct acatgtgcca taggtcttat
1020tgttacaggt atcgctgtgc tgctcggagc cagaacacac ctgatagctc
tgcttcgaat 1080ctgggattcc gctgtgcagc cgaccgcctg cccaccatgg ac
112242130DNAHomo sapiens 4acatggcccg cgggacaaca tggctgcgcc
cgcactaggg ctggtgtgtg gacgttgccc 60tgagctgggt ctcgtcctct tgctgctgct
gctctcgctg ctgtgtggag cggcagggag 120ccaggaggcc gggaccggtg
cgggcgcggg gtcccttgcg ggttcttgcg gctgcggcac 180gccccagcgg
cctggcgccc atggcagttc ggcagccgct caccgatact cgcgggaggc
240taacgctccg ggccccgtac ccggagagcg gcaactcgcg cactcaaaga
tggtccccat 300ccctgctgga gtatttacaa tgggcacaga tgatcctcag
ataaagcagg atggggaagc 360acctgcgagg agagttacta ttgatgccct
ttacatggat gcctatgaag tcagtaatac 420tgaatttgag aagtttgtga
actcaactgg ctatttgaca gaggctgaga agtttggcga 480ctcctttgtc
tttgaaggca tgttgagtga gcaagtgaag accaatattc aacaggcagt
540tgcagctgct ccctggtggt tacctgtgaa aggcgctaac tggagacacc
cagaagggcc 600tgactctact attctgcaca ggccggatca tccagttctc
catgtgtcct ggaatgatgc 660ggttgcctac tgcacttggg cagggaagcg
gctgcccacg gaagctgagt gggaatacag 720ctgtcgagga ggcctgcata
atagactttt cccctggggc aacaaactgc agcccaaagg 780ccagcattat
gccaacattt ggcagggcga ttttccggtg accaacactg gtgaggatgg
840cttccaagga actgcgcctg ttgatgcctt ccctcccaat ggttatggct
tatacaacat 900agtggggaac gcatgggaat ggacttcaga ctggtggact
gttcatcatt ctgttgaaga 960aacgcttaac ccaaaaggtc ccccttctgg
gaaagaccga gtgaagaaag gtggatccta 1020catgtgccat aggtcttatt
gttacaggta tcgctgtgct gctcggagcc agaacacacc 1080tgatagctct
gcttcgaatc tgggattccg ctgtgcagcc gaccgcctgc ccaccatgga
1140ctgacaacca agggtagtct tccccagtcc aaggagcagt cgtgtctgac
ctacattggg 1200ctttcctcag aactttgaac gatcccatgc aaagaattcc
caccctgagg tgggttacat 1260acctgcccaa tggccaaagg aaccgccttg
tgagaccaaa ttgctgacct gggtcagtgc 1320atgtgcttta tggtgtggtg
catctttgga gatcatcacc atattttact tttgagagtc 1380tttaaagagg
aaggggagtg gagggaaccc tgagctaggc ttcaggaggc ccgcatccta
1440cgcaggctct gccacagggg ttagacccca ggtccgacgc ttgaccttcc
tgggcctcaa 1500gtgccctccc ctatcaaatg aaggaatgga cagcatgacc
tctgggtgtc tctccaactc 1560accagttcta aaaagggtat cagattctat
tgtgacttca tagaatttat gatagattat 1620tttttagcta ttttttccat
gtgtgaacct tgagtgatac taatcatgta aagtaagagt 1680tctcttatgt
attatgttcg gaagaggggt gtggtgactc ctttatattc gtactgcact
1740ttgtttttcc aaggaaatca gtgtctttta cgttgttatg atgaatccca
catggggccg 1800gtgatggtat gctgaagttc agccgttgaa cacataggaa
tgtctgtggg gtgactctac 1860tgtgctttat cttttaacat taagtgcctt
tggttcagag gggcagtcat aagctctgtt 1920tccccctctc cccaaagcct
tcagcgaacg tgaaatgtgc gctaaacggg gaaacctgtt 1980taattctaga
tatagggaaa aaggaacgag gaccttgaat gagctatatt cagggtatcc
2040ggtattttgt aatagggaat aggaaacctt gttggctgtg gaatatccga
tgctttgaat 2100catgcactgt gttgaataaa cgtatctgct 21305374PRTHomo
sapiens 5Met Ala Ala Pro Ala Leu Gly Leu Val Cys Gly Arg Cys Pro
Glu Leu1 5 10 15Gly Leu Val Leu Leu Leu Leu Leu Leu Ser Leu Leu Cys
Gly Ala Ala 20 25 30Gly Ser Gln Glu Ala Gly Thr Gly Ala Gly Ala Gly
Ser Leu Ala Gly 35 40 45Ser Cys Gly Cys Gly Thr Pro Gln Arg Pro Gly
Ala His Gly Ser Ser 50 55 60Ala Ala Ala His Arg Tyr Ser Arg Glu Ala
Asn Ala Pro Gly Pro Val65 70 75 80Pro Gly Glu Arg Gln Leu Ala His
Ser Lys Met Val Pro Ile Pro Ala 85 90 95Gly Val Phe Thr Met Gly Thr
Asp Asp Pro Gln Ile Lys Gln Asp Gly 100 105 110Glu Ala Pro Ala Arg
Arg Val Thr Ile Asp Ala Leu Tyr Met Asp Ala 115 120 125Tyr Glu Val
Ser Asn Thr Glu Phe Glu Lys Phe Val Asn Ser Thr Gly 130 135 140Tyr
Leu Thr Glu Ala Glu Lys Phe Gly Asp Ser Phe Val Phe Glu Gly145 150
155 160Met Leu Ser Glu Gln Val Lys Thr Asn Ile Gln Gln Ala Val Ala
Ala 165 170 175Ala Pro Trp Trp Leu Pro Val Lys Gly Ala Asn Trp Arg
His Pro Glu 180 185 190Gly Pro Asp Ser Thr Ile Leu His Arg Pro Asp
His Pro Val Leu His 195 200 205Val Ser Trp Asn Asp Ala Val Ala Tyr
Cys Thr Trp Ala Gly Lys Arg 210 215 220Leu Pro Thr Glu Ala Glu Trp
Glu Tyr Ser Cys Arg Gly Gly Leu His225 230 235 240Asn Arg Leu Phe
Pro Trp Gly Asn Lys Leu Gln Pro Lys Gly Gln His 245 250 255Tyr Ala
Asn Ile Trp Gln Gly Asp Phe Pro Val Thr Asn Thr Gly Glu 260 265
270Asp Gly Phe Gln Gly Thr Ala Pro Val Asp Ala Phe Pro Pro Asn Gly
275 280 285Tyr Gly Leu Tyr Asn Ile Val Gly Asn Ala Trp Glu Trp Thr
Ser Asp 290 295 300Trp Trp Thr Val His His Ser Val Glu Glu Thr Leu
Asn Pro Lys Gly305 310 315 320Pro Pro Ser Gly Lys Asp Arg Val Lys
Lys Gly Gly Ser Tyr Met Cys 325 330 335His Arg Ser Tyr Cys Tyr Arg
Tyr Arg Cys Ala Ala Arg Ser Gln Asn 340 345 350Thr Pro Asp Ser Ser
Ala Ser Asn Leu Gly Phe Arg Cys Ala Ala Asp 355 360 365Arg Leu Pro
Thr Met Asp 37062297DNAHomo sapiens 6cggctgtgtt gcgcagtctt
catgggttcc cgacgaggag gtctctgtgg ctgcggcggc 60tgctaactgc gccacctgct
gcagcctgtc cccgccgctc tgaagcggcc gcgtcgaagc 120cgaaatgccg
ccaccccgga ccggccgagg ccttctctgg ctgggtctgg ttctgagctc
180cgtctgcgtc gccctcggat ccgaaacgca ggccaactcg accacagatg
ctctgaacgt 240tcttctcatc atcgtggatg acctgcgccc ctccctgggc
tgttatgggg ataagctggt 300gaggtcccca aatattgacc aactggcatc
ccacagcctc ctcttccaga atgcctttgc 360gcagcaagca gtgtgcgccc
cgagccgcgt ttctttcctc actggcagga gacctgacac 420cacccgcctg
tacgacttca actcctactg gagggtgcac gctggaaact tctccaccat
480cccccagtac ttcaaggaga atggctatgt gaccatgtcg gtgggaaaag
tctttcaccc 540tgggatatct tctaaccata ccgatgattc tccgtatagc
tggtcttttc caccttatca 600tccttcctct gagaagtatg aaaacactaa
gacatgtcga gggccagatg gagaactcca 660tgccaacctg ctttgccctg
tggatgtgct ggatgttccc gagggcacct tgcctgacaa 720acagagcact
gagcaagcca tacagttgtt ggaaaagatg aaaacgtcag ccagtccttt
780cttcctggcc gttgggtatc ataagccaca catccccttc agatacccca
aggaatttca 840gaagttgtat cccttggaga acatcaccct ggcccccgat
cccgaggtcc ctgatggcct 900accccctgtg gcctacaacc cctggatgga
catcaggcaa cgggaagacg tccaagcctt 960aaacatcagt gtgccgtatg
gtccaattcc tgtggacttt cagcggaaaa tccgccagag 1020ctactttgcc
tctgtgtcat atttggatac acaggtcggc cgcctcttga gtgctttgga
1080cgatcttcag ctggccaaca gcaccatcat tgcatttacc tcggatcatg
ggtgggctct 1140aggtgaacat ggagaatggg ccaaatacag caattttgat
gttgctaccc atgttcccct 1200gatattctat gttcctggaa ggacggcttc
acttccggag gcaggcgaga agcttttccc 1260ttacctcgac ccttttgatt
ccgcctcaca gttgatggag ccaggcaggc aatccatgga 1320ccttgtggaa
cttgtgtctc tttttcccac gctggctgga cttgcaggac tgcaggttcc
1380acctcgctgc cccgttcctt catttcacgt tgagctgtgc agagaaggca
agaaccttct 1440gaagcatttt cgattccgtg acttggaaga ggatccgtac
ctccctggta atccccgtga 1500actgattgcc tatagccagt atccccggcc
ttcagacatc cctcagtgga attctgacaa 1560gccgagttta aaagatataa
agatcatggg ctattccata cgcaccatag actataggta 1620tactgtgtgg
gttggcttca atcctgatga atttctagct aacttttctg acatccatgc
1680aggggaactg tattttgtgg attctgaccc attgcaggat cacaatatgt
ataatgattc 1740ccaaggtgga gatcttttcc agttgttgat gccttgagtt
ttgccaacca tggatggcaa 1800atgtgatgtg ctcccttcca gctggtgaga
ggaggagtta gagctggtcg ttttgtgatt 1860acccataata ttggaagcag
cctgagggct agttaatcca aacatgcatc aacaatttgg 1920cctgagaata
tgtaacagcc aaaccttttc gtttagtctt tattaaaatt tataattggt
1980aattggacca gttttttttt taatttccct ctttttaaaa cagttacggc
ttatttactg 2040aataaataca aagcaaacaa actcaagtta tgtcatacct
ttggatacga agaccataca 2100taataaccaa acataacatt atacacaaag
aatactttca ttatttgtgg aatttagtgc 2160atttcaaaaa gtaatcatat
atcaaactag gcaccacact aagttcctga ttattttgtt 2220tataatttaa
taatatatct tatgagccct atatattcaa aatattatgt taacatgtaa
2280tccatgtttc tttttcc 22977550PRTHomo sapiens 7Met Pro Pro Pro Arg
Thr Gly Arg Gly Leu Leu Trp Leu Gly Leu Val1 5 10 15Leu Ser Ser Val
Cys Val Ala Leu Gly Ser Glu Thr Gln Ala Asn Ser 20 25 30Thr Thr Asp
Ala Leu Asn Val Leu Leu Ile Ile Val Asp Asp Leu Arg 35 40 45Pro Ser
Leu Gly Cys Tyr Gly Asp Lys Leu Val Arg Ser Pro Asn Ile 50 55 60Asp
Gln Leu Ala Ser His Ser Leu Leu Phe Gln Asn Ala Phe Ala Gln65 70 75
80Gln Ala Val Cys Ala Pro Ser Arg Val Ser Phe Leu Thr Gly Arg Arg
85 90 95Pro Asp Thr Thr Arg Leu Tyr Asp Phe Asn Ser Tyr Trp Arg Val
His 100 105 110Ala Gly Asn Phe Ser Thr Ile Pro Gln Tyr Phe Lys Glu
Asn Gly Tyr 115 120 125Val Thr Met Ser Val Gly Lys Val Phe His Pro
Gly Ile Ser Ser Asn 130 135 140His Thr Asp Asp Ser Pro Tyr Ser Trp
Ser Phe Pro Pro Tyr His Pro145 150 155 160Ser Ser
Glu Lys Tyr Glu Asn Thr Lys Thr Cys Arg Gly Pro Asp Gly 165 170
175Glu Leu His Ala Asn Leu Leu Cys Pro Val Asp Val Leu Asp Val Pro
180 185 190Glu Gly Thr Leu Pro Asp Lys Gln Ser Thr Glu Gln Ala Ile
Gln Leu 195 200 205Leu Glu Lys Met Lys Thr Ser Ala Ser Pro Phe Phe
Leu Ala Val Gly 210 215 220Tyr His Lys Pro His Ile Pro Phe Arg Tyr
Pro Lys Glu Phe Gln Lys225 230 235 240Leu Tyr Pro Leu Glu Asn Ile
Thr Leu Ala Pro Asp Pro Glu Val Pro 245 250 255Asp Gly Leu Pro Pro
Val Ala Tyr Asn Pro Trp Met Asp Ile Arg Gln 260 265 270Arg Glu Asp
Val Gln Ala Leu Asn Ile Ser Val Pro Tyr Gly Pro Ile 275 280 285Pro
Val Asp Phe Gln Arg Lys Ile Arg Gln Ser Tyr Phe Ala Ser Val 290 295
300Ser Tyr Leu Asp Thr Gln Val Gly Arg Leu Leu Ser Ala Leu Asp
Asp305 310 315 320Leu Gln Leu Ala Asn Ser Thr Ile Ile Ala Phe Thr
Ser Asp His Gly 325 330 335Trp Ala Leu Gly Glu His Gly Glu Trp Ala
Lys Tyr Ser Asn Phe Asp 340 345 350Val Ala Thr His Val Pro Leu Ile
Phe Tyr Val Pro Gly Arg Thr Ala 355 360 365Ser Leu Pro Glu Ala Gly
Glu Lys Leu Phe Pro Tyr Leu Asp Pro Phe 370 375 380Asp Ser Ala Ser
Gln Leu Met Glu Pro Gly Arg Gln Ser Met Asp Leu385 390 395 400Val
Glu Leu Val Ser Leu Phe Pro Thr Leu Ala Gly Leu Ala Gly Leu 405 410
415Gln Val Pro Pro Arg Cys Pro Val Pro Ser Phe His Val Glu Leu Cys
420 425 430Arg Glu Gly Lys Asn Leu Leu Lys His Phe Arg Phe Arg Asp
Leu Glu 435 440 445Glu Asp Pro Tyr Leu Pro Gly Asn Pro Arg Glu Leu
Ile Ala Tyr Ser 450 455 460Gln Tyr Pro Arg Pro Ser Asp Ile Pro Gln
Trp Asn Ser Asp Lys Pro465 470 475 480Ser Leu Lys Asp Ile Lys Ile
Met Gly Tyr Ser Ile Arg Thr Ile Asp 485 490 495Tyr Arg Tyr Thr Val
Trp Val Gly Phe Asn Pro Asp Glu Phe Leu Ala 500 505 510Asn Phe Ser
Asp Ile His Ala Gly Glu Leu Tyr Phe Val Asp Ser Asp 515 520 525Pro
Leu Gln Asp His Asn Met Tyr Asn Asp Ser Gln Gly Gly Asp Leu 530 535
540Phe Gln Leu Leu Met Pro545 55082657DNAHomo sapiens 8gaattccggg
ccatgagctg ccccgtgccc gcctgctgcg cgctgctgct agtcctgggg 60ctctgccggg
cgcgtccccg gaacgcactg ctgctcctcg cggatgacgg aggctttgag
120agtggcgcgt acaacaacag cgccatcgcc accccgcacc tggacgcctt
ggcccgccgc 180agcctcctct ttcgcaatgc cttcacctcg gtcagcagct
gctctcccag ccgcgccagc 240ctcctcactg gcctgcccca gcatcagaat
gggatgtacg ggctgcacca ggacgtgcac 300cacttcaact ccttcgacaa
ggtgcggagc ctgccgctgc tgctcagcca agctggtgtg 360cgcacaggca
tcatcgggaa gaagcacgtg gggccggaga ccgtgtaccc gtttgacttt
420gcgtacacgg aggagaatgg ctccgtcctc caggtggggc ggaacatcac
tagaattaag 480ctgctcgtcc ggaaattcct gcagactcag gatgaccggc
ctttcttcct ctacgtcgcc 540ttccacgacc cccaccgctg tgggcactcc
cagccccagt acggaacctt ctgtgagaag 600tttggcaacg gagagagcgg
catgggtcgt atcccagact ggacccccca ggcctacgac 660ccactggacg
tgctggtgcc ttacttcgtc cccaacaccc cggcagcccg agccgacctg
720gccgctcagt acaccaccgt cggccgcatg gaccaaggag ttggactggt
gctccaggag 780ctgcgtgacg ccggtgtcct gaacgacaca ctggtgatct
tcacgtccga caacgggatc 840cccttcccca gcggcaggac caacctgtac
tggccgggca ctgctgaacc cttactggtg 900tcatccccgg agcacccaaa
acgctggggc caagtcagcg aggcctacgt gagcctccta 960gacctcacgc
ccaccatctt ggattggttc tcgatcccgt accccagcta cgccatcttt
1020ggctcgaaga ccatccacct cactggccgg tccctcctgc cggcgctgga
ggccgagccc 1080ctctgggcca ccgtctttgg cagccagagc caccacgagg
tcaccatgtc ctaccccatg 1140cgctccgtgc agcaccggca cttccgcctc
gtgcacaacc tcaacttcaa gatgcccttt 1200cccatcgacc aggacttcta
cgtctcaccc accttccagg acctcctgaa ccgcaccaca 1260gctggtcagc
ccacgggctg gtacaaggac ctccgtcatt actactaccg ggcgcgctgg
1320gagctctacg accggagccg ggacccccac gagacccaga acctggccac
cgacccgcgc 1380tttgctcagc ttctggagat gcttcgggac cagctggcca
agtggcagtg ggagacccac 1440gacccctggg tgtgcgcccc cgacggcgtc
ctggaggaga agctctctcc ccagtgccag 1500cccctccaca atgagctgtg
accatcccag gaggcctgtg cacacatccc aggcatgtcc 1560cagacacatc
ccacacgtgt ccgtgtggcc ggccagcctg gggagtagtg gcaacagccc
1620ttccgtccac actcccatcc aaggagggtt cttccttcct gtggggtcac
tcttgccatt 1680gcctggaggg ggaccagagc atgtgaccag agcatgtgcc
cagcccctcc accaccaggg 1740gcactgccgt catggcaggg gacacagttg
tccttgtgtc tgaaccatgt cccagcacgg 1800gaattctaga catacgtggt
ctgcggacag ggcagcgccc ccagcccatg acaagggagt 1860cttgttttct
ggcttggttt ggggacctgc aaatgggagg cctgaggccc tcttcaggct
1920ttggcagcca cagatacttc tgaacccttc acagagagca ggcaggggct
tcggtgccgc 1980gtgggcagta cgcaggtccc accgacactc acctgggagc
acggcgcctg gctcttacca 2040gcgtctggcc tagaggaagc ctttgagcga
cctttgggca ggtttctgct tcttctgttt 2100tgcccatggt caagtccctg
ttccccaggc aggtttcagc tgattggcag caggctccct 2160gagtgatgag
cttgaacctg tggtgtttct gggcagaagc ttatcttttt tgagagtgtc
2220cgaagatgaa ggcatggcga tgcccgtcct ctggcttggg ttaattcttc
ggtgacactg 2280gcattgctgg gtggtgatgc ccgtcctctg gcttgggtta
attcttcggt gacactggcg 2340ttgctgggtg gcaatgcccg tcctctggct
tgggttaatt cttcggtgac actggcgttg 2400ctgggtggcg atgcccgtcc
tctggcttgg gttaattctt ggatgacgtc ggcgttgctg 2460ggagaatgtg
ccgttcctgc cctgcctcca cccacctcgg gagcagaagc ccggcctgga
2520cacccctcgg cctggacacc cctcgaagga gagggcgctt ccttgagtag
gtgggctccc 2580cttgcccttc cctccctatc actccatact ggggtgggct
ggaggaggcc acaggccagc 2640tattgtaaaa gcttttt 26579502PRTHomo
sapiens 9Met Ser Cys Pro Val Pro Ala Cys Cys Ala Leu Leu Leu Val
Leu Gly1 5 10 15Leu Cys Arg Ala Arg Pro Arg Asn Ala Leu Leu Leu Leu
Ala Asp Asp 20 25 30Gly Gly Phe Glu Ser Gly Ala Tyr Asn Asn Ser Ala
Ile Ala Thr Pro 35 40 45His Leu Asp Ala Leu Ala Arg Arg Ser Leu Leu
Phe Arg Asn Ala Phe 50 55 60Thr Ser Val Ser Ser Cys Ser Pro Ser Arg
Ala Ser Leu Leu Thr Gly65 70 75 80Leu Pro Gln His Gln Asn Gly Met
Tyr Gly Leu His Gln Asp Val His 85 90 95His Phe Asn Ser Phe Asp Lys
Val Arg Ser Leu Pro Leu Leu Leu Ser 100 105 110Gln Ala Gly Val Arg
Thr Gly Ile Ile Gly Lys Lys His Val Gly Pro 115 120 125Glu Thr Val
Tyr Pro Phe Asp Phe Ala Tyr Thr Glu Glu Asn Gly Ser 130 135 140Val
Leu Gln Val Gly Arg Asn Ile Thr Arg Ile Lys Leu Leu Val Arg145 150
155 160Lys Phe Leu Gln Thr Gln Asp Asp Arg Pro Phe Phe Leu Tyr Val
Ala 165 170 175Phe His Asp Pro His Arg Cys Gly His Ser Gln Pro Gln
Tyr Gly Thr 180 185 190Phe Cys Glu Lys Phe Gly Asn Gly Glu Ser Gly
Met Gly Arg Ile Pro 195 200 205Asp Trp Thr Pro Gln Ala Tyr Asp Pro
Leu Asp Val Leu Val Pro Tyr 210 215 220Phe Val Pro Asn Thr Pro Ala
Ala Arg Ala Asp Leu Ala Ala Gln Tyr225 230 235 240Thr Thr Val Gly
Arg Met Asp Gln Gly Val Gly Leu Val Leu Gln Glu 245 250 255Leu Arg
Asp Ala Gly Val Leu Asn Asp Thr Leu Val Ile Phe Thr Ser 260 265
270Asp Asn Gly Ile Pro Phe Pro Ser Gly Arg Thr Asn Leu Tyr Trp Pro
275 280 285Gly Thr Ala Glu Pro Leu Leu Val Ser Ser Pro Glu His Pro
Lys Arg 290 295 300Trp Gly Gln Val Ser Glu Ala Tyr Val Ser Leu Leu
Asp Leu Thr Pro305 310 315 320Thr Ile Leu Asp Trp Phe Ser Ile Pro
Tyr Pro Ser Tyr Ala Ile Phe 325 330 335Gly Ser Lys Thr Ile His Leu
Thr Gly Arg Ser Leu Leu Pro Ala Leu 340 345 350Glu Ala Glu Pro Leu
Trp Ala Thr Val Phe Gly Ser Gln Ser His His 355 360 365Glu Val Thr
Met Ser Tyr Pro Met Arg Ser Val Gln His Arg His Phe 370 375 380Arg
Leu Val His Asn Leu Asn Phe Lys Met Pro Phe Pro Ile Asp Gln385 390
395 400Asp Phe Tyr Val Ser Pro Thr Phe Gln Asp Leu Leu Asn Arg Thr
Thr 405 410 415Ala Gly Gln Pro Thr Gly Trp Tyr Lys Asp Leu Arg His
Tyr Tyr Tyr 420 425 430Arg Ala Arg Trp Glu Leu Tyr Asp Arg Ser Arg
Asp Pro His Glu Thr 435 440 445Gln Asn Leu Ala Thr Asp Pro Arg Phe
Ala Gln Leu Leu Glu Met Leu 450 455 460Arg Asp Gln Leu Ala Lys Trp
Gln Trp Glu Thr His Asp Pro Trp Val465 470 475 480Cys Ala Pro Asp
Gly Val Leu Glu Glu Lys Leu Ser Pro Gln Cys Gln 485 490 495Pro Leu
His Asn Glu Leu 500101014DNAHomo sapiens 10cgtgcctgta atcccagcag
ctactcactc aggaggctga ggcaggagaa tctcttgaac 60ccggaaggca gaggttgcag
tgagccaaga tcgcgccact gaactccagc ctgggtgaca 120gagtgagact
gtctcagaac agcaacaaca aaatgcccgc tgctgctggg tccagaagag
180cttgaataac tgcatgttct ttttctcaat tttcatttcc cagaactggg
cacctccggg 240ctgtgaaaag ttagggaagt gtctgacacc tccagaatcc
attcccaaga agtgcctctg 300gtcccactag cacctgcgca gactcaggcc
aggcctagaa tctccagttg gccctgcaag 360tgcctggagg aaggatggct
ctggcctcgg tcctccccca accctgccca agccagacag 420acagcacctg
cagacgcagg gggactgcac aattccacct gcccaggacc tgaccctggc
480gtgtgcttgg ccctcctcct cgcccacggc gcctcagatt tcaggaccct
cctcctcgcc 540cacggcgcct cagacctcag gaccctgccg tctcacgcct
ttgtgaaccc caaatatctg 600agaccagtct cagtttattt tgccaaggtt
aaggatgcac ctgtgacagc ctcaggaggt 660cctgacaaca ggtgcccgag
gtggctgggg atacagtttg cctttataca tcttagggag 720acacaagatc
agtatgtgta tggcgtacat tggttcagtc agccttccac tgaatacacg
780attgagtctg gcccagtgaa tccgcatttt tatgtaaaca gtaagggaac
ggggcaatca 840tataagcgtt tgtctcaggg gagccccaga gggatgactt
ccagttccgt ctgtcctttg 900tccacaagga atttccctgg gcgctaatta
tgagggaggc gtgtagcttc ttatcattgt 960agctatgtta tttagaaata
aaacgggagg caggtttgcc taattcccag gttg 101411522PRTHomo sapiens
11Met Ala Ala Val Val Ala Ala Thr Arg Trp Trp Gln Leu Leu Leu Val1
5 10 15Leu Ser Ala Ala Gly Met Gly Ala Ser Gly Ala Pro Gln Pro Pro
Asn 20 25 30Ile Leu Leu Leu Leu Met Asp Asp Met Gly Trp Gly Asp Leu
Gly Val 35 40 45Tyr Gly Glu Pro Ser Arg Glu Thr Pro Asn Leu Asp Arg
Met Ala Ala 50 55 60Glu Gly Leu Leu Phe Pro Asn Phe Tyr Ser Ala Asn
Pro Leu Cys Ser65 70 75 80Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg
Leu Pro Ile Arg Asn Gly 85 90 95Phe Tyr Thr Thr Asn Ala His Ala Arg
Asn Ala Tyr Thr Pro Gln Glu 100 105 110Ile Val Gly Gly Ile Pro Asp
Ser Glu Gln Leu Leu Pro Glu Leu Leu 115 120 125Lys Lys Ala Gly Tyr
Val Ser Lys Ile Val Gly Lys Trp His Leu Gly 130 135 140His Arg Pro
Gln Phe His Pro Leu Lys His Gly Phe Asp Glu Trp Phe145 150 155
160Gly Ser Pro Asn Cys His Phe Gly Pro Tyr Asp Asn Lys Ala Arg Pro
165 170 175Asn Ile Pro Val Tyr Arg Asp Trp Glu Met Val Gly Arg Tyr
Tyr Glu 180 185 190Glu Phe Pro Ile Asn Leu Lys Thr Gly Glu Ala Asn
Leu Thr Gln Ile 195 200 205Tyr Leu Gln Glu Ala Leu Asp Phe Ile Lys
Arg Gln Ala Arg His His 210 215 220Pro Phe Phe Leu Tyr Trp Ala Val
Asp Ala Thr His Ala Pro Val Tyr225 230 235 240Ala Ser Lys Pro Phe
Leu Gly Thr Ser Gln Arg Gly Arg Tyr Gly Asp 245 250 255Ala Val Arg
Glu Ile Asp Asp Ser Ile Gly Lys Ile Leu Glu Leu Leu 260 265 270Gln
Asp Leu His Val Ala Asp Asn Thr Phe Val Phe Phe Thr Ser Asp 275 280
285Asn Gly Ala Ala Leu Ile Ser Ala Pro Glu Gln Gly Gly Ser Asn Gly
290 295 300Pro Phe Leu Cys Gly Lys Gln Thr Thr Phe Glu Gly Gly Met
Arg Glu305 310 315 320Pro Ala Leu Ala Trp Trp Pro Gly His Val Thr
Ala Gly Gln Val Ser 325 330 335His Gln Leu Gly Ser Ile Met Asp Leu
Phe Thr Thr Ser Leu Ala Leu 340 345 350Ala Gly Leu Thr Pro Pro Ser
Asp Arg Ala Ile Asp Gly Leu Asn Leu 355 360 365Leu Pro Thr Leu Leu
Gln Gly Arg Leu Met Asp Arg Pro Ile Phe Tyr 370 375 380Tyr Arg Gly
Asp Thr Leu Met Ala Ala Thr Leu Gly Gln His Lys Ala385 390 395
400His Phe Trp Thr Trp Thr Asn Ser Trp Glu Asn Phe Arg Gln Gly Ile
405 410 415Asp Phe Cys Pro Gly Gln Asn Val Ser Gly Val Thr Thr His
Asn Leu 420 425 430Glu Asp His Thr Lys Leu Pro Leu Ile Phe His Leu
Gly Arg Asp Pro 435 440 445Gly Glu Arg Phe Pro Leu Ser Phe Ala Ser
Ala Glu Tyr Gln Glu Ala 450 455 460Leu Ser Arg Ile Thr Ser Val Val
Gln Gln His Gln Glu Ala Leu Val465 470 475 480Pro Ala Gln Pro Gln
Leu Asn Val Cys Asn Trp Ala Val Met Asn Trp 485 490 495Ala Pro Pro
Gly Cys Glu Lys Leu Gly Lys Cys Leu Thr Pro Pro Glu 500 505 510Ser
Ile Pro Lys Lys Cys Leu Trp Ser His 515 520122379DNAHomo sapiens
12ggaattccgg tcggcctctc gcccttcagc tacctgtgcg tccctccgtc ccgtcccgtc
60ccggggtcac cccggagcct gtccgctatg cggctcctgc ctctagcccc aggtcggctc
120cggcggggca gcccccgcca cctgccctcc tgcagcccag cgctgctact
gctggtgctg 180ggcggctgcc tgggggtctt cggggtggct gcgggaaccc
ggaggcccaa cgtggtgctg 240ctcctcacgg acgaccagga cgaagtgctc
ggcggcatga caccactaaa gaaaaccaaa 300gctctcatcg gagagatggg
gatgactttt tccagtgctt atgtgccaag tgctctctgc 360tgccccagca
gagccagtat cctgacagga aagtacccac ataatcatca cgttgtgaac
420aacactctgg aggggaactg cagtagtaag tcctggcaga agatccaaga
accaaatact 480ttcccagcaa ttctcagatc aatgtgtggt tatcagacct
tttttgcagg gaaatattta 540aatgagtacg gagccccaga tgcaggtgga
ctagaacacg ttcctctggg ttggagttac 600tggtatgcct tggaaaagaa
ttctaagtat tataattaca ccctgtctat caatgggaag 660gcacggaagc
atggtgaaaa ctatagtgtg gactacctga cagatgtttt ggctaatgtc
720tccttggact ttctggacta caagtccaac tttgagccct tcttcatgat
gatcgccact 780ccagcgcctc attcgccttg gacagctgca cctcagtacc
agaaggcttt ccagaatgtc 840tttgcaccaa gaaacaagaa cttcaacatc
catggaacga acaagcactg gttaattagg 900caagccaaga ctccaatgac
taattcttca atacagtttt tagataatgc atttaggaaa 960aggtggcaaa
ctctcctctc agttgatgac cttgtggaga aactggtcaa gaggctggag
1020ttcactgggg agctcaacaa cacttacatc ttctatacct cagacaatgg
ctatcacaca 1080ggacagtttt ccttgccaat agacaagaga cagctgtatg
agtttgatat caaagttcca 1140ctgttggttc gaggacctgg gatcaaacca
aatcagacaa gcaagatgct ggttgccaac 1200attgacttgg gtcctactat
tttggacatt gctggctacg acctaaataa gacacagatg 1260gatgggatgt
ccttattgcc cattttgaga ggtgccagta acttgacctg gcgatcagat
1320gtcctggtgg aataccaagg agaaggccgt aacgtcactg acccaacatg
cccttccctg 1380agtcctggcg tatctcaatg cttcccagac tgtgtatgtg
aagatgctta taacaatacc 1440tatgcctgtg tgaggacaat gtcagcattg
tggaatttgc agtattgcga gtttgatgac 1500caggaggtgt ttgtagaagt
ctataatctg actgcagacc cagaccagat cactaacatt 1560gctaaaacca
tagacccaga gcttttagga aagatgaact atcggttaat gatgttacag
1620tcctgttctg ggccaacctg tcgcactcca ggggtttttg accccggata
caggtttgac 1680ccccgtctca tgttcagcaa tcgcggcagt gtcaggactc
gaagattttc caaacatctt 1740ctgtagcgac ctcacacagc ctctgcagat
ggatccctgc acgcctcttt ctgatgaagt 1800gattgtagta ggtgtctgta
gctagtcttc aagaccacac ctggaagagt ttctgggctg 1860gctttaagtc
ctgtttgaaa aagcaaccca gtcagctgac ttcctcgtgc aatgtgttaa
1920actgtgaact ctgcccatgt gtcaggagtg gctgtctctg gtctcttcct
ttagctgaca 1980aggacactcc tgaggtcttt gttctcactg tatttttttt
atcctggggc cacagttctt 2040gattattcct cttgtggtta aagactgaat
ttgtaaaccc attcagataa atggcagtac 2100tttaggacac acacaaacac
acagatacac cttttgatat gtaagcttga cctaaagtca 2160aaggacctgt
gtagcatttc agattgagca cttcactatc aaaaatacta acatcacatg
2220gcttgaagag taaccatcag agctgaatca tccaagtaag aacaagtacc
attgttgatt 2280gataagtaga gatacatttt ttatgatgtt catcacagtg
tggtaaggtt gcaaattcaa 2340aacatgtcac ccaagctctg ttcatgtttt
tgtgaattc 237913552PRTHomo sapiens 13Met Arg Leu Leu Pro Leu Ala
Pro Gly Arg Leu Arg Arg Gly Ser Pro1 5 10 15Arg His Leu Pro Ser Cys
Ser Pro Ala Leu Leu Leu Leu Val Leu Gly 20 25 30Gly Cys Leu Gly Val
Phe Gly
Val Ala Ala Gly Thr Arg Arg Pro Asn 35 40 45Val Val Leu Leu Leu Thr
Asp Asp Gln Asp Glu Val Leu Gly Gly Met 50 55 60Thr Pro Leu Lys Lys
Thr Lys Ala Leu Ile Gly Glu Met Gly Met Thr65 70 75 80Phe Ser Ser
Ala Tyr Val Pro Ser Ala Leu Cys Cys Pro Ser Arg Ala 85 90 95Ser Ile
Leu Thr Gly Lys Tyr Pro His Asn His His Val Val Asn Asn 100 105
110Thr Leu Glu Gly Asn Cys Ser Ser Lys Ser Trp Gln Lys Ile Gln Glu
115 120 125Pro Asn Thr Phe Pro Ala Ile Leu Arg Ser Met Cys Gly Tyr
Gln Thr 130 135 140Phe Phe Ala Gly Lys Tyr Leu Asn Glu Tyr Gly Ala
Pro Asp Ala Gly145 150 155 160Gly Leu Glu His Val Pro Leu Gly Trp
Ser Tyr Trp Tyr Ala Leu Glu 165 170 175Lys Asn Ser Lys Tyr Tyr Asn
Tyr Thr Leu Ser Ile Asn Gly Lys Ala 180 185 190Arg Lys His Gly Glu
Asn Tyr Ser Val Asp Tyr Leu Thr Asp Val Leu 195 200 205Ala Asn Val
Ser Leu Asp Phe Leu Asp Tyr Lys Ser Asn Phe Glu Pro 210 215 220Phe
Phe Met Met Ile Ala Thr Pro Ala Pro His Ser Pro Trp Thr Ala225 230
235 240Ala Pro Gln Tyr Gln Lys Ala Phe Gln Asn Val Phe Ala Pro Arg
Asn 245 250 255Lys Asn Phe Asn Ile His Gly Thr Asn Lys His Trp Leu
Ile Arg Gln 260 265 270Ala Lys Thr Pro Met Thr Asn Ser Ser Ile Gln
Phe Leu Asp Asn Ala 275 280 285Phe Arg Lys Arg Trp Gln Thr Leu Leu
Ser Val Asp Asp Leu Val Glu 290 295 300Lys Leu Val Lys Arg Leu Glu
Phe Thr Gly Glu Leu Asn Asn Thr Tyr305 310 315 320Ile Phe Tyr Thr
Ser Asp Asn Gly Tyr His Thr Gly Gln Phe Ser Leu 325 330 335Pro Ile
Asp Lys Arg Gln Leu Tyr Glu Phe Asp Ile Lys Val Pro Leu 340 345
350Leu Val Arg Gly Pro Gly Ile Lys Pro Asn Gln Thr Ser Lys Met Leu
355 360 365Val Ala Asn Ile Asp Leu Gly Pro Thr Ile Leu Asp Ile Ala
Gly Tyr 370 375 380Asp Leu Asn Lys Thr Gln Met Asp Gly Met Ser Leu
Leu Pro Ile Leu385 390 395 400Arg Gly Ala Ser Asn Leu Thr Trp Arg
Ser Asp Val Leu Val Glu Tyr 405 410 415Gln Gly Glu Gly Arg Asn Val
Thr Asp Pro Thr Cys Pro Ser Leu Ser 420 425 430Pro Gly Val Ser Gln
Cys Phe Pro Asp Cys Val Cys Glu Asp Ala Tyr 435 440 445Asn Asn Thr
Tyr Ala Cys Val Arg Thr Met Ser Ala Leu Trp Asn Leu 450 455 460Gln
Tyr Cys Glu Phe Asp Asp Gln Glu Val Phe Val Glu Val Tyr Asn465 470
475 480Leu Thr Ala Asp Pro Asp Gln Ile Thr Asn Ile Ala Lys Thr Ile
Asp 485 490 495Pro Glu Leu Leu Gly Lys Met Asn Tyr Arg Leu Met Met
Leu Gln Ser 500 505 510Cys Ser Gly Pro Thr Cys Arg Thr Pro Gly Val
Phe Asp Pro Gly Tyr 515 520 525Arg Phe Asp Pro Arg Leu Met Phe Ser
Asn Arg Gly Ser Val Arg Thr 530 535 540Arg Arg Phe Ser Lys His Leu
Leu545 550142022DNAHomo sapiens 14ccggtaccgg ctcctcctgg gctccctcta
gcgccttccc cccggcccga ctgcctggtc 60agcgccaagt gacttacgcc cccgaccctg
agcccggacc gctaggcgag gaggatcaga 120tctccgctcg agaatctgaa
ggtgccctgg tcctggagga gttccgtccc agccctgcgg 180tctcccggta
ctgctcgccc cggccctctg gagcttcagg aggcggccgt cagggtcggg
240gagtatttgg gtccggggtc tcagggaagg gcggcgcctg ggtctgcggt
atcggaaaga 300gcctgctgga gccaagtagc cctccctctc ttgggacaga
cccctcggtc ccatgtccat 360gggggcaccg cggtccctcc tcctggccct
ggctgctggc ctggccgttg cccgtccgcc 420caacatcgtg ctgatctttg
ccgacgacct cggctatggg gacctgggct gctatgggca 480ccccagctct
accactccca acctggacca gctggcggcg ggagggctgc ggttcacaga
540cttctacgtg cctgtgtctc tgtgcacacc ctctagggcc gccctcctga
ccggccggct 600cccggttcgg atgggcatgt accctggcgt cctggtgccc
agctcccggg ggggcctgcc 660cctggaggag gtgaccgtgg ccgaagtcct
ggctgcccga ggctacctca caggaatggc 720cggcaagtgg caccttgggg
tggggcctga gggggccttc ctgccccccc atcagggctt 780ccatcgattt
ctaggcatcc cgtactccca cgaccagggc ccctgccaga acctgacctg
840cttcccgccg gccactcctt gcgacggtgg ctgtgaccag ggcctggtcc
ccatcccact 900gttggccaac ctgtccgtgg aggcgcagcc cccctggctg
cccggactag aggcccgcta 960catggctttc gcccatgacc tcatggccga
cgcccagcgc caggatcgcc ccttcttcct 1020gtactatgcc tctcaccaca
cccactaccc tcagttcagt gggcagagct ttgcagagcg 1080ttcaggccgc
gggccatttg gggactccct gatggagctg gatgcagctg tggggaccct
1140gatgacagcc ataggggacc tggggctgct tgaagagacg ctggtcatct
tcactgcaga 1200caatggacct gagaccatgc gtatgtcccg aggcggctgc
tccggtctct tgcggtgtgg 1260aaagggaacg acctacgagg gcggtgtccg
agagcctgcc ttggccttct ggccaggtca 1320tatcgctccc ggcgtgaccc
acgagctggc cagctccctg gacctgctgc ctaccctggc 1380agccctggct
ggggccccac tgcccaatgt caccttggat ggctttgacc tcagccccct
1440gctgctgggc acaggcaaga gccctcggca gtctctcttc ttctacccgt
cctacccaga 1500cgaggtccgt ggggtttttg ctgtgcggac tggaaagtac
aaggctcact tcttcaccca 1560gggctctgcc cacagtgata ccactgcaga
ccctgcctgc cacgcctcca gctctctgac 1620tgctcatgag cccccgctgc
tctatgacct gtccaaggac cctggtgaga actacaacct 1680gctggggggt
gtggccgggg ccaccccaga ggtgctgcaa gccctgaaac agcttcagct
1740gctcaaggcc cagttagacg cagctgtgac cttcggcccc agccaggtgg
cccggggcga 1800ggaccccgcc ctgcagatct gctgtcatcc tggctgcacc
ccccgcccag cttgctgcca 1860ttgcccagat ccccatgcct gagggcccct
cggctggcct gggcatgtga tggctcctca 1920ctgggagcct gtgggggagg
ctcaggtgtc tggagggggt ttgtgcctga taacgtaata 1980acaccagtgg
agacttgcac atctgaaaaa aaaaaaaaaa aa 202215507PRTHomo sapiens 15Met
Gly Ala Pro Arg Ser Leu Leu Leu Ala Leu Ala Ala Gly Leu Ala1 5 10
15Val Ala Arg Pro Pro Asn Ile Val Leu Ile Phe Ala Asp Asp Leu Gly
20 25 30Tyr Gly Asp Leu Gly Cys Tyr Gly His Pro Ser Ser Thr Thr Pro
Asn 35 40 45Leu Asp Gln Leu Ala Ala Gly Gly Leu Arg Phe Thr Asp Phe
Tyr Val 50 55 60Pro Val Ser Leu Cys Thr Pro Ser Arg Ala Ala Leu Leu
Thr Gly Arg65 70 75 80Leu Pro Val Arg Met Gly Met Tyr Pro Gly Val
Leu Val Pro Ser Ser 85 90 95Arg Gly Gly Leu Pro Leu Glu Glu Val Thr
Val Ala Glu Val Leu Ala 100 105 110Ala Arg Gly Tyr Leu Thr Gly Met
Ala Gly Lys Trp His Leu Gly Val 115 120 125Gly Pro Glu Gly Ala Phe
Leu Pro Pro His Gln Gly Phe His Arg Phe 130 135 140Leu Gly Ile Pro
Tyr Ser His Asp Gln Gly Pro Cys Gln Asn Leu Thr145 150 155 160Cys
Phe Pro Pro Ala Thr Pro Cys Asp Gly Gly Cys Asp Gln Gly Leu 165 170
175Val Pro Ile Pro Leu Leu Ala Asn Leu Ser Val Glu Ala Gln Pro Pro
180 185 190Trp Leu Pro Gly Leu Glu Ala Arg Tyr Met Ala Phe Ala His
Asp Leu 195 200 205Met Ala Asp Ala Gln Arg Gln Asp Arg Pro Phe Phe
Leu Tyr Tyr Ala 210 215 220Ser His His Thr His Tyr Pro Gln Phe Ser
Gly Gln Ser Phe Ala Glu225 230 235 240Arg Ser Gly Arg Gly Pro Phe
Gly Asp Ser Leu Met Glu Leu Asp Ala 245 250 255Ala Val Gly Thr Leu
Met Thr Ala Ile Gly Asp Leu Gly Leu Leu Glu 260 265 270Glu Thr Leu
Val Ile Phe Thr Ala Asp Asn Gly Pro Glu Thr Met Arg 275 280 285Met
Ser Arg Gly Gly Cys Ser Gly Leu Leu Arg Cys Gly Lys Gly Thr 290 295
300Thr Tyr Glu Gly Gly Val Arg Glu Pro Ala Leu Ala Phe Trp Pro
Gly305 310 315 320His Ile Ala Pro Gly Val Thr His Glu Leu Ala Ser
Ser Leu Asp Leu 325 330 335Leu Pro Thr Leu Ala Ala Leu Ala Gly Ala
Pro Leu Pro Asn Val Thr 340 345 350Leu Asp Gly Phe Asp Leu Ser Pro
Leu Leu Leu Gly Thr Gly Lys Ser 355 360 365Pro Arg Gln Ser Leu Phe
Phe Tyr Pro Ser Tyr Pro Asp Glu Val Arg 370 375 380Gly Val Phe Ala
Val Arg Thr Gly Lys Tyr Lys Ala His Phe Phe Thr385 390 395 400Gln
Gly Ser Ala His Ser Asp Thr Thr Ala Asp Pro Ala Cys His Ala 405 410
415Ser Ser Ser Leu Thr Ala His Glu Pro Pro Leu Leu Tyr Asp Leu Ser
420 425 430Lys Asp Pro Gly Glu Asn Tyr Asn Leu Leu Gly Gly Val Ala
Gly Ala 435 440 445Thr Pro Glu Val Leu Gln Ala Leu Lys Gln Leu Gln
Leu Leu Lys Ala 450 455 460Gln Leu Asp Ala Ala Val Thr Phe Gly Pro
Ser Gln Val Ala Arg Gly465 470 475 480Glu Asp Pro Ala Leu Gln Ile
Cys Cys His Pro Gly Cys Thr Pro Arg 485 490 495Pro Ala Cys Cys His
Cys Pro Asp Pro His Ala 500 505162228DNAHomo sapiens 16acaaggatgg
gtccgcgcgg cgcggcgagc ttgccccgag gccccggacc tcggcggctg 60ctcctccccg
tcgtcctccc gctgctgctg ctgctgttgt tggcgccgcc gggctcgggc
120gccggggcca gccggccgcc ccacctggtc ttcttgctgg cagacgacct
aggctggaac 180gacgtcggct tccacggctc ccgcatccgc acgccgcacc
tggacgcgct ggcggccggc 240ggggtgctcc tggacaacta ctacacgcag
ccgctgtgca cgccgtcgcg gagccagctg 300ctcactggcc gctaccagat
ccgtacaggt ttacagcacc aaataatctg gccctgtcag 360cccagctgtg
ttcctctgga tgaaaaactc ctgccccagc tcctaaaaga agcaggttat
420actacccata tggtcggaaa atggcacctg ggaatgtacc ggaaagaatg
ccttccaacc 480cgccgaggat ttgataccta ctttggatat ctcctgggta
gtgaagatta ttattcccat 540gaacgctgta cattaattga cgctctgaat
gtcacacgat gtgctcttga ttttcgagat 600ggcgaagaag ttgcaacagg
atataaaaat atgtattcaa caaacatatt caccaaaagg 660gctatagccc
tcataactaa ccatccacca gagaagcctc tgtttctcta ccttgctctc
720cagtctgtgc atgagcccct tcaggtccct gaggaatact tgaagccata
tgactttatc 780caagacaaga acaggcatca ctatgcagga atggtgtccc
ttatggatga agcagtagga 840aatgtcactg cagctttaaa aagcagtggg
ctctggaaca acacggtgtt catcttttct 900acagataacg gagggcagac
tttggcaggg ggtaataact ggccccttcg aggaagaaaa 960tggagcctgt
gggaaggagg cgtccgaggg gtgggctttg tggcaagccc cttgctgaag
1020cagaagggcg tgaagaaccg ggagctcatc cacatctctg actggctgcc
aacactcgtg 1080aagctggcca ggggacacac caatggcaca aagcctctgg
atggcttcga cgtgtggaaa 1140accatcagtg aaggaagccc atcccccaga
attgagctgc tgcataatat tgacccaaac 1200ttcgtggact cttcaccgtg
tcccaggaac agcatggctc cagcaaagga tgactcttct 1260cttccagaat
attcagcctt taacacatct gtccatgctg caattagaca tggaaattgg
1320aaactcctca cgggctaccc aggctgtggt tactggttcc ctccaccgtc
tcaatacaat 1380gtttctgaga taccctcatc agacccacca accaagaccc
tctggctctt tgatattgat 1440cgggaccctg aagaaagaca tgacctgtcc
agagaatatc ctcacatcgt cacaaagctc 1500ctgtcccgcc tacagttcta
ccataaacac tcagtccccg tgtacttccc tgcacaggac 1560ccccgctgtg
atcccaaggc cactggggtg tggggccctt ggatgtagga tttcagggag
1620gctagaaaac ctttcaattg gaagttggac ctcaggcctt ttctcacgac
tcttgtctca 1680tttgttatcc caacctgggt tcacttggcc cttctcttgc
tcttaaacca caccgaggtg 1740tctaatttca acccctaatg catttaagaa
gctgataaaa tctgcaacac tcctgctgtt 1800ggctggagca tgtgtctaga
ggtgggggtg gctgggttta tccccctttc ctaagccttg 1860ggacagctgg
gaacttaact tgaaatagga agttctcact gaatcctgga ggctggaaca
1920gctggctctt ttagactcac aagtcagacg ttcgattccc ctctgccaat
agccagtttt 1980attggagtga atcacatttc ttacgcaaat gaagggagca
gacagtgatt aatggttctg 2040ttggccaagg cttctccctg tcggtgaagg
atcatgttca ggcactccaa gtgaaccacc 2100cctcttggtt caccccttac
tcacttatct catcacagag cataaggccc attttgttgt 2160tcaggtcaac
agcaaaatgg cctgcaccat gactgtggct tttaaaataa agaaatgtgt 2220ttttatcg
222817533PRTHomo sapiens 17Met Gly Pro Arg Gly Ala Ala Ser Leu Pro
Arg Gly Pro Gly Pro Arg1 5 10 15Arg Leu Leu Leu Pro Val Val Leu Pro
Leu Leu Leu Leu Leu Leu Leu 20 25 30Ala Pro Pro Gly Ser Gly Ala Gly
Ala Ser Arg Pro Pro His Leu Val 35 40 45Phe Leu Leu Ala Asp Asp Leu
Gly Trp Asn Asp Val Gly Phe His Gly 50 55 60Ser Arg Ile Arg Thr Pro
His Leu Asp Ala Leu Ala Ala Gly Gly Val65 70 75 80Leu Leu Asp Asn
Tyr Tyr Thr Gln Pro Leu Cys Thr Pro Ser Arg Ser 85 90 95Gln Leu Leu
Thr Gly Arg Tyr Gln Ile Arg Thr Gly Leu Gln His Gln 100 105 110Ile
Ile Trp Pro Cys Gln Pro Ser Cys Val Pro Leu Asp Glu Lys Leu 115 120
125Leu Pro Gln Leu Leu Lys Glu Ala Gly Tyr Thr Thr His Met Val Gly
130 135 140Lys Trp His Leu Gly Met Tyr Arg Lys Glu Cys Leu Pro Thr
Arg Arg145 150 155 160Gly Phe Asp Thr Tyr Phe Gly Tyr Leu Leu Gly
Ser Glu Asp Tyr Tyr 165 170 175Ser His Glu Arg Cys Thr Leu Ile Asp
Ala Leu Asn Val Thr Arg Cys 180 185 190Ala Leu Asp Phe Arg Asp Gly
Glu Glu Val Ala Thr Gly Tyr Lys Asn 195 200 205Met Tyr Ser Thr Asn
Ile Phe Thr Lys Arg Ala Ile Ala Leu Ile Thr 210 215 220Asn His Pro
Pro Glu Lys Pro Leu Phe Leu Tyr Leu Ala Leu Gln Ser225 230 235
240Val His Glu Pro Leu Gln Val Pro Glu Glu Tyr Leu Lys Pro Tyr Asp
245 250 255Phe Ile Gln Asp Lys Asn Arg His His Tyr Ala Gly Met Val
Ser Leu 260 265 270Met Asp Glu Ala Val Gly Asn Val Thr Ala Ala Leu
Lys Ser Ser Gly 275 280 285Leu Trp Asn Asn Thr Val Phe Ile Phe Ser
Thr Asp Asn Gly Gly Gln 290 295 300Thr Leu Ala Gly Gly Asn Asn Trp
Pro Leu Arg Gly Arg Lys Trp Ser305 310 315 320Leu Trp Glu Gly Gly
Val Arg Gly Val Gly Phe Val Ala Ser Pro Leu 325 330 335Leu Lys Gln
Lys Gly Val Lys Asn Arg Glu Leu Ile His Ile Ser Asp 340 345 350Trp
Leu Pro Thr Leu Val Lys Leu Ala Arg Gly His Thr Asn Gly Thr 355 360
365Lys Pro Leu Asp Gly Phe Asp Val Trp Lys Thr Ile Ser Glu Gly Ser
370 375 380Pro Ser Pro Arg Ile Glu Leu Leu His Asn Ile Asp Pro Asn
Phe Val385 390 395 400Asp Ser Ser Pro Cys Pro Arg Asn Ser Met Ala
Pro Ala Lys Asp Asp 405 410 415Ser Ser Leu Pro Glu Tyr Ser Ala Phe
Asn Thr Ser Val His Ala Ala 420 425 430Ile Arg His Gly Asn Trp Lys
Leu Leu Thr Gly Tyr Pro Gly Cys Gly 435 440 445Tyr Trp Phe Pro Pro
Pro Ser Gln Tyr Asn Val Ser Glu Ile Pro Ser 450 455 460Ser Asp Pro
Pro Thr Lys Thr Leu Trp Leu Phe Asp Ile Asp Arg Asp465 470 475
480Pro Glu Glu Arg His Asp Leu Ser Arg Glu Tyr Pro His Ile Val Thr
485 490 495Lys Leu Leu Ser Arg Leu Gln Phe Tyr His Lys His Ser Val
Pro Val 500 505 510Tyr Phe Pro Ala Gln Asp Pro Arg Cys Asp Pro Lys
Ala Thr Gly Val 515 520 525Trp Gly Pro Trp Met 530182401DNAHomo
sapiens 18gcctccagca gctgacggga cccagctgta gtgaggttgc agtgattgag
taggattggc 60ctgcttcaaa gcagaggttt ctcatgggaa tatgcttatt aaactcccac
tggtgcagaa 120accatgaaca gaggatgaac aagtgaagtt gcaatctcct
ccatcacagc tcagttcccc 180aacaacagga tcacaagctg gagatgcctt
taaggaagat gaagatccct ttcctcctac 240tgttctttct gtgggaagcc
gagagccacg cagcatcaag gccgaacatc atcctggtga 300tggctgacga
cctcggcatt ggagatcctg ggtgctatgg gaacaaaact atcaggactc
360ccaatatcga ccggttggcc agtgggggag tgaaactcac tcagcacctg
gcagcatcac 420cgctgtgcac accaagcagg gcagccttca tgactggccg
gtaccctgtc cgatcaggaa 480tggcatcttg gtcccgcact ggagttttcc
tcttcacagc ctcttcggga ggacttccca 540ccgatgagat tacctttgct
aagcttctga aggatcaagg ttattcaaca gcactgatag 600ggaaatggca
ccttgggatg agctgtcaca gcaagactga cttctgtcac caccctttac
660atcacggctt caattatttc tatgggatct ctttgaccaa tctgagagac
tgcaagcccg 720gagagggcag tgtcttcacc acgggcttca agaggctggt
cttcctcccc ctgcagatcg 780tcggggtcac cctccttacc cttgctgcac
tcaattgtct ggggctactc cacgtgcctc 840taggcgtttt tttcagcctt
ctcttcctag cagccctaat cctgaccctt ttcttgggct 900tccttcatta
cttccggccc ctgaactgct tcatgatgag gaactacgag atcattcagc
960agcccatgtc ctatgacaat ctcacccaga ggctaacggt ggaggcggcc
cagttcatac 1020agcggaacac tgagactccg ttcctgcttg tcttgtccta
cctccacgtg cacacagccc 1080tgttctccag caaagacttt gctggcaaaa
gtcaacacgg agtctacggg gatgctgttg 1140aggaaatgga ctggagtgtg
gggcagatct tgaaccttct ggatgagctg agattggcta
1200atgataccct catctacttc acatcggacc agggagcaca tgtagaggag
gtgtcttcca 1260aaggagaaat tcatggcgga agtaatggga tctataaagg
aggaaaagca aacaactggg 1320aaggaggtat ccgggttcca ggcatccttc
gttggcccag ggtgatacag gctggccaga 1380agattgatga gcccactagc
aacatggaca tatttcctac agtagccaag ctggctggag 1440ctcccttgcc
tgaggacagg atcattgatg gacgtgatct gatgcccctg cttgaaggaa
1500aaagccaacg ctccgatcat gagtttctct tccattactg caacgcctac
ttaaatgctg 1560tgcgctggca ccctcagaac agcacatcca tctggaaggc
ctttttcttc acccccaact 1620tcaaccccgt gggttccaac ggatgctttg
ccacacacgt gtgcttctgt ttcgggagtt 1680atgtcaccca tcacgaccca
cctttactct ttgatatttc caaagatccc agagagagaa 1740acccactaac
tccagcatcc gagccccggt tttatgaaat cctcaaagtc atgcaggaag
1800ctgcggacag acacacccag accctgccag aggtgcccga tcagttttca
tggaacaact 1860ttctttggaa gccctggctt cagctgtgct gtccttccac
cggcctgtct tgccagtgtg 1920atagagaaaa acaggataag agactgagcc
gctagcagcg cctggggacc agacagacgc 1980atgtggcaaa gctcaccatc
ttcactacaa acacgcctga gagtggcact ggggaaacat 2040aactccatct
acaccttgga tttggactga ttctccattt tatcacctga aggcttgggc
2100cagagctcaa cagctactca actggagggg tgagggggat aaggtctgta
gtatacagac 2160aggaagatgg taggtttatg ccttctgtgg ccagagtctt
ggactcatgg aaatagaatg 2220aatagagggg cattcacaag gcacaccagt
gcaagcagat gacaaaaagg tgcagaaggc 2280aatcttaaaa cagaaaggtg
caggaggtac cttaactcac ccctcagcaa atacctatgt 2340caacagtata
agttaccatt tactctataa tctgcagtga tgcaataacc agcataataa 2400a
240119583PRTHomo sapiens 19Met Pro Leu Arg Lys Met Lys Ile Pro Phe
Leu Leu Leu Phe Phe Leu1 5 10 15Trp Glu Ala Glu Ser His Ala Ala Ser
Arg Pro Asn Ile Ile Leu Val 20 25 30Met Ala Asp Asp Leu Gly Ile Gly
Asp Pro Gly Cys Tyr Gly Asn Lys 35 40 45Thr Ile Arg Thr Pro Asn Ile
Asp Arg Leu Ala Ser Gly Gly Val Lys 50 55 60Leu Thr Gln His Leu Ala
Ala Ser Pro Leu Cys Thr Pro Ser Arg Ala65 70 75 80Ala Phe Met Thr
Gly Arg Tyr Pro Val Arg Ser Gly Met Ala Ser Trp 85 90 95Ser Arg Thr
Gly Val Phe Leu Phe Thr Ala Ser Ser Gly Gly Leu Pro 100 105 110Thr
Asp Glu Ile Thr Phe Ala Lys Leu Leu Lys Asp Gln Gly Tyr Ser 115 120
125Thr Ala Leu Ile Gly Lys Trp His Leu Gly Met Ser Cys His Ser Lys
130 135 140Thr Asp Phe Cys His His Pro Leu His His Gly Phe Asn Tyr
Phe Tyr145 150 155 160Gly Ile Ser Leu Thr Asn Leu Arg Asp Cys Lys
Pro Gly Glu Gly Ser 165 170 175Val Phe Thr Thr Gly Phe Lys Arg Leu
Val Phe Leu Pro Leu Gln Ile 180 185 190Val Gly Val Thr Leu Leu Thr
Leu Ala Ala Leu Asn Cys Leu Gly Leu 195 200 205Leu His Val Pro Leu
Gly Val Phe Phe Ser Leu Leu Phe Leu Ala Ala 210 215 220Leu Ile Leu
Thr Leu Phe Leu Gly Phe Leu His Tyr Phe Arg Pro Leu225 230 235
240Asn Cys Phe Met Met Arg Asn Tyr Glu Ile Ile Gln Gln Pro Met Ser
245 250 255Tyr Asp Asn Leu Thr Gln Arg Leu Thr Val Glu Ala Ala Gln
Phe Ile 260 265 270Gln Arg Asn Thr Glu Thr Pro Phe Leu Leu Val Leu
Ser Tyr Leu His 275 280 285Val His Thr Ala Leu Phe Ser Ser Lys Asp
Phe Ala Gly Lys Ser Gln 290 295 300His Gly Val Tyr Gly Asp Ala Val
Glu Glu Met Asp Trp Ser Val Gly305 310 315 320Gln Ile Leu Asn Leu
Leu Asp Glu Leu Arg Leu Ala Asn Asp Thr Leu 325 330 335Ile Tyr Phe
Thr Ser Asp Gln Gly Ala His Val Glu Glu Val Ser Ser 340 345 350Lys
Gly Glu Ile His Gly Gly Ser Asn Gly Ile Tyr Lys Gly Gly Lys 355 360
365Ala Asn Asn Trp Glu Gly Gly Ile Arg Val Pro Gly Ile Leu Arg Trp
370 375 380Pro Arg Val Ile Gln Ala Gly Gln Lys Ile Asp Glu Pro Thr
Ser Asn385 390 395 400Met Asp Ile Phe Pro Thr Val Ala Lys Leu Ala
Gly Ala Pro Leu Pro 405 410 415Glu Asp Arg Ile Ile Asp Gly Arg Asp
Leu Met Pro Leu Leu Glu Gly 420 425 430Lys Ser Gln Arg Ser Asp His
Glu Phe Leu Phe His Tyr Cys Asn Ala 435 440 445Tyr Leu Asn Ala Val
Arg Trp His Pro Gln Asn Ser Thr Ser Ile Trp 450 455 460Lys Ala Phe
Phe Phe Thr Pro Asn Phe Asn Pro Val Gly Ser Asn Gly465 470 475
480Cys Phe Ala Thr His Val Cys Phe Cys Phe Gly Ser Tyr Val Thr His
485 490 495His Asp Pro Pro Leu Leu Phe Asp Ile Ser Lys Asp Pro Arg
Glu Arg 500 505 510Asn Pro Leu Thr Pro Ala Ser Glu Pro Arg Phe Tyr
Glu Ile Leu Lys 515 520 525Val Met Gln Glu Ala Ala Asp Arg His Thr
Gln Thr Leu Pro Glu Val 530 535 540Pro Asp Gln Phe Ser Trp Asn Asn
Phe Leu Trp Lys Pro Trp Leu Gln545 550 555 560Leu Cys Cys Pro Ser
Thr Gly Leu Ser Cys Gln Cys Asp Arg Glu Lys 565 570 575Gln Asp Lys
Arg Leu Ser Arg 580201945DNAHomo sapiens 20ggaagccttg gcactagcgg
cgcccgggcg cggagtgcgc agggcaaggt cctgcgctct 60gggccagcgc tcggccatgc
gatccgccgc gcggagggga cgcgccgcgc ccgccgccag 120ggactctttg
ccggtgctac tgtttttatg cttgcttctg aagacgtgtg aacctaaaac
180tgcaaatgcc tttaaaccaa atatcctact gatcatggcg gatgatctag
gcactgggga 240tctcggttgc tacgggaaca atacactgag aacgccgaat
attgaccagc ttgcagagga 300aggtgtgagg ctcactcagc acctggcggc
cgccccgctc tgcaccccaa gccgagctgc 360attcctcaca gggagacatt
ccttcagatc aggcatggac gccagcaatg gataccgggc 420ccttcagtgg
aacgcaggct caggtggact ccctgagaac gaaaccactt ttgcaagaat
480cttgcagcag catggctatg caaccggcct cataggaaaa tggcaccagg
gtgtgaattg 540tgcatcccgc ggggatcact gccaccaccc cctgaaccac
ggatttgact atttctacgg 600catgcccttc acgctcacaa acgactgtga
cccaggcagg ccccccgaag tggacgccgc 660cctgagggcg cagctctggg
gttacaccca gttcctggcg ctggggattc tcaccctggc 720tgccggccag
acctgcggtt tcttctctgt ctccgcgaga gcagtcaccg gcatggccgg
780cgtgggctgc ctgtttttca tctcttggta ctcctccttc gggtttgtgc
gacgctggaa 840ctgtatcctg atgagaaacc atgacgtcac ggagcaaccc
atggttctgg agaaaacagc 900gagtcttatg ctaaaggaag ctgtttccta
tattgaaaga cacaagcatg ggccatttct 960cctcttcctt tctttgctgc
atgtgcacat tccccttgtg accacgagtg cattcctggg 1020gaaaagtcag
catggcttat atggtgataa tgtggaggag atggactggc tcataggtaa
1080ggttcttaat gccatcgaag acaatggttt aaagaactca acattcacgt
atttcacctc 1140tgaccatgga ggacatttag aggcaagaga tggacacagc
cagttagggg gatggaacgg 1200aatttacaaa ggtgggaagg gcatgggagg
atgggaaggt gggatccgag tgcccgggat 1260cttccactgg ccgggggtgc
tcccggccgg ccgagtgatt ggagagccca cgagcctgat 1320ggacgtgttc
cctactgtgg tccagctggt gggtggcgag gtgccccagg acagggtgat
1380tgatggccac agcctggtac ccttgctgca gggagctgag gcacgctcgg
cacatgagtt 1440cctgtttcat tactgtgggc agcatcttca cgcagcacgc
tggcaccaga aggacagtgg 1500aagcgtctgg aaggttcatt acacgacccc
gcagttccac cccgaggagc ggggcctgct 1560aacggccgag gcgtctgccc
atgctgaatg gggaggcgtg acccatcaca gacccccttt 1620gctctttgac
ctctccaggg acccctccga ggcacggccc ctgacccccg actccgagcc
1680cctgtaccac gccgtgatag caagggtagg tgccgcggtg tcggagcatc
ggcagaccct 1740gagtcctgtg ccccagcagt tttccatgag caacatcctg
tggaagccgt ggctgcagcc 1800gtgctgcgga catttcccgt tctgttcatg
ccacgaggat ggggatggca ccccctgaat 1860gccaggactg tgagagagga
tccaggagag cctgactgcg ttgcaaacaa aattctccaa 1920gcttggttct
atcttcagtc cggaa 194521593PRTHomo sapiens 21Met Arg Ser Ala Ala Arg
Arg Gly Arg Ala Ala Pro Ala Ala Arg Asp1 5 10 15Ser Leu Pro Val Leu
Leu Phe Leu Cys Leu Leu Leu Lys Thr Cys Glu 20 25 30Pro Lys Thr Ala
Asn Ala Phe Lys Pro Asn Ile Leu Leu Ile Met Ala 35 40 45Asp Asp Leu
Gly Thr Gly Asp Leu Gly Cys Tyr Gly Asn Asn Thr Leu 50 55 60Arg Thr
Pro Asn Ile Asp Gln Leu Ala Glu Glu Gly Val Arg Leu Thr65 70 75
80Gln His Leu Ala Ala Ala Pro Leu Cys Thr Pro Ser Arg Ala Ala Phe
85 90 95Leu Thr Gly Arg His Ser Phe Arg Ser Gly Met Asp Ala Ser Asn
Gly 100 105 110Tyr Arg Ala Leu Gln Trp Asn Ala Gly Ser Gly Gly Leu
Pro Glu Asn 115 120 125Glu Thr Thr Phe Ala Arg Ile Leu Gln Gln His
Gly Tyr Ala Thr Gly 130 135 140Leu Ile Gly Lys Trp His Gln Gly Val
Asn Cys Ala Ser Arg Gly Asp145 150 155 160His Cys His His Pro Leu
Asn His Gly Phe Asp Tyr Phe Tyr Gly Met 165 170 175Pro Phe Thr Leu
Thr Asn Asp Cys Asp Pro Gly Arg Pro Pro Glu Val 180 185 190Asp Ala
Ala Leu Arg Ala Gln Leu Trp Gly Tyr Thr Gln Phe Leu Ala 195 200
205Leu Gly Ile Leu Thr Leu Ala Ala Gly Gln Thr Cys Gly Phe Phe Ser
210 215 220Val Ser Ala Arg Ala Val Thr Gly Met Ala Gly Val Gly Cys
Leu Phe225 230 235 240Phe Ile Ser Trp Tyr Ser Ser Phe Gly Phe Val
Arg Arg Trp Asn Cys 245 250 255Ile Leu Met Arg Asn His Asp Val Thr
Glu Gln Pro Met Val Leu Glu 260 265 270Lys Thr Ala Ser Leu Met Leu
Lys Glu Ala Val Ser Tyr Ile Glu Arg 275 280 285His Lys His Gly Pro
Phe Leu Leu Phe Leu Ser Leu Leu His Val His 290 295 300Ile Pro Leu
Val Thr Thr Ser Ala Phe Leu Gly Lys Ser Gln His Gly305 310 315
320Leu Tyr Gly Asp Asn Val Glu Glu Met Asp Trp Leu Ile Gly Lys Val
325 330 335Leu Asn Ala Ile Glu Asp Asn Gly Leu Lys Asn Ser Thr Phe
Thr Tyr 340 345 350Phe Thr Ser Asp His Gly Gly His Leu Glu Ala Arg
Asp Gly His Ser 355 360 365Gln Leu Gly Gly Trp Asn Gly Ile Tyr Lys
Gly Gly Lys Gly Met Gly 370 375 380Gly Trp Glu Gly Gly Ile Arg Val
Pro Gly Ile Phe His Trp Pro Gly385 390 395 400Val Leu Pro Ala Gly
Arg Val Ile Gly Glu Pro Thr Ser Leu Met Asp 405 410 415Val Phe Pro
Thr Val Val Gln Leu Val Gly Gly Glu Val Pro Gln Asp 420 425 430Arg
Val Ile Asp Gly His Ser Leu Val Pro Leu Leu Gln Gly Ala Glu 435 440
445Ala Arg Ser Ala His Glu Phe Leu Phe His Tyr Cys Gly Gln His Leu
450 455 460His Ala Ala Arg Trp His Gln Lys Asp Ser Gly Ser Val Trp
Lys Val465 470 475 480His Tyr Thr Thr Pro Gln Phe His Pro Glu Glu
Arg Gly Leu Leu Thr 485 490 495Ala Glu Ala Ser Ala His Ala Glu Trp
Gly Gly Val Thr His His Arg 500 505 510Pro Pro Leu Leu Phe Asp Leu
Ser Arg Asp Pro Ser Glu Ala Arg Pro 515 520 525Leu Thr Pro Asp Ser
Glu Pro Leu Tyr His Ala Val Ile Ala Arg Val 530 535 540Gly Ala Ala
Val Ser Glu His Arg Gln Thr Leu Ser Pro Val Pro Gln545 550 555
560Gln Phe Ser Met Ser Asn Ile Leu Trp Lys Pro Trp Leu Gln Pro Cys
565 570 575Cys Gly His Phe Pro Phe Cys Ser Cys His Glu Asp Gly Asp
Gly Thr 580 585 590Pro221858DNAHomo sapiens 22ccttcctctt cttgatcggg
gattcaggaa ggagcccagg agcagaggaa gtagagagag 60agacaacatg ttacatctgc
accattcttg tttgtgtttc aggagctggc tgccagcgat 120gctcgctgta
ctgctaagtt tggcaccatc agcttccagc gacatttccg cctcccgacc
180gaacatcctt cttctgatgg cggacgacct tggcattggg gacattggct
gctatggcaa 240caacaccatg aggactccga atattgaccg ccttgcagag
gacggcgtga agctgaccca 300acacatctct gccgcatctt tgtgcacccc
aagcagagcc gccttcctca cgggcagata 360ccctgtgcga tcagggatgg
tttccagcat tggttaccgt gttcttcagt ggaccggagc 420atctggaggt
cttccaacaa atgagacaac ttttgcaaaa atactgaaag agaaaggcta
480tgccactgga ctcattggaa aatggcatct gggtctcaac tgtgagtcag
ccagtgatca 540ttgccaccac cctctccatc atggctttga gcatttctac
ggaatgcctt tctccttgat 600gggtgattgc gcccgctggg aactctcaga
gaagcgtgtc aacctggaac aaaaactcaa 660cttcctcttc caagtcctgg
ccttggttgc cctcacactg gtagcaggga agctcacaca 720cctgataccc
gtctcgtgga tgccggtcat ctggtcagcc ctttcggccg tcctcctcct
780cgcaagctcc tattttgtgg gtgctctgat tgtccatgcc gattgctttc
tgatgagaaa 840ccacaccatc acggagcagc ccatgtgctt ccaaagaacg
acacccctta ttctgcagga 900ggttgcgtcc tttctcaaaa ggaataagca
tgggcctttc ctcctctttg tttcctttct 960acacgttcac atccctctta
tcactatgga gaacttcctc gggaagagtc tccacgggct 1020gtatggggac
aacgtagagg agatggactg gatggtagga cggatccttg acactttgga
1080cgtggagggt ttgagcaaca gcaccctcat ttattttacg tcggatcacg
gcggttccct 1140agagaatcaa cttggaaaca cccagtatgg tggctggaat
ggaatttata aaggtgggaa 1200gggcatggga ggatgggaag gtgggatccg
cgtgcccggg atcttccgct ggcccggggt 1260gctcccggcc ggccgagtga
ttggcgagcc cacgagtctg atggacgtgt tccccaccgt 1320ggtccggctg
gcgggcggcg aggtgcccca ggacagagtg attgacggcc aagaccttct
1380gcccttgctc ctggggacag cccaacactc agaccacgag ttcctgatgc
attattgtga 1440gaggtttctg cacgcagcca ggtggcatca acgggacaga
ggaacaatgt ggaaagtcca 1500ctttgtgacg cctgtgttcc agccagaggg
agccggtgcc tgctatggaa gaaaggtctg 1560cccgtgcttt ggggaaaaag
tagtccacca cgatccacct ttgctctttg acctctcaag 1620agacccttct
gagacccaca tcctcacacc agcctcagag cccgtgttct atcaggtgat
1680ggaacgagtc cagcaggcgg tgtgggaaca ccagcggaca ctcagcccag
ttcctctgca 1740gctggacagg ctgggcaaca tctggagacc gtggctgcag
ccctgctgtg gcccgttccc 1800cctctgctgg tgccttaggg aagatgaccc
acaataaatg tctgcagtga aaagctgg 185823589PRTHomo sapiens 23Met Leu
His Leu His His Ser Cys Leu Cys Phe Arg Ser Trp Leu Pro1 5 10 15Ala
Met Leu Ala Val Leu Leu Ser Leu Ala Pro Ser Ala Ser Ser Asp 20 25
30Ile Ser Ala Ser Arg Pro Asn Ile Leu Leu Leu Met Ala Asp Asp Leu
35 40 45Gly Ile Gly Asp Ile Gly Cys Tyr Gly Asn Asn Thr Met Arg Thr
Pro 50 55 60Asn Ile Asp Arg Leu Ala Glu Asp Gly Val Lys Leu Thr Gln
His Ile65 70 75 80Ser Ala Ala Ser Leu Cys Thr Pro Ser Arg Ala Ala
Phe Leu Thr Gly 85 90 95Arg Tyr Pro Val Arg Ser Gly Met Val Ser Ser
Ile Gly Tyr Arg Val 100 105 110Leu Gln Trp Thr Gly Ala Ser Gly Gly
Leu Pro Thr Asn Glu Thr Thr 115 120 125Phe Ala Lys Ile Leu Lys Glu
Lys Gly Tyr Ala Thr Gly Leu Ile Gly 130 135 140Lys Trp His Leu Gly
Leu Asn Cys Glu Ser Ala Ser Asp His Cys His145 150 155 160His Pro
Leu His His Gly Phe Glu His Phe Tyr Gly Met Pro Phe Ser 165 170
175Leu Met Gly Asp Cys Ala Arg Trp Glu Leu Ser Glu Lys Arg Val Asn
180 185 190Leu Glu Gln Lys Leu Asn Phe Leu Phe Gln Val Leu Ala Leu
Val Ala 195 200 205Leu Thr Leu Val Ala Gly Lys Leu Thr His Leu Ile
Pro Val Ser Trp 210 215 220Met Pro Val Ile Trp Ser Ala Leu Ser Ala
Val Leu Leu Leu Ala Ser225 230 235 240Ser Tyr Phe Val Gly Ala Leu
Ile Val His Ala Asp Cys Phe Leu Met 245 250 255Arg Asn His Thr Ile
Thr Glu Gln Pro Met Cys Phe Gln Arg Thr Thr 260 265 270Pro Leu Ile
Leu Gln Glu Val Ala Ser Phe Leu Lys Arg Asn Lys His 275 280 285Gly
Pro Phe Leu Leu Phe Val Ser Phe Leu His Val His Ile Pro Leu 290 295
300Ile Thr Met Glu Asn Phe Leu Gly Lys Ser Leu His Gly Leu Tyr
Gly305 310 315 320Asp Asn Val Glu Glu Met Asp Trp Met Val Gly Arg
Ile Leu Asp Thr 325 330 335Leu Asp Val Glu Gly Leu Ser Asn Ser Thr
Leu Ile Tyr Phe Thr Ser 340 345 350Asp His Gly Gly Ser Leu Glu Asn
Gln Leu Gly Asn Thr Gln Tyr Gly 355 360 365Gly Trp Asn Gly Ile Tyr
Lys Gly Gly Lys Gly Met Gly Gly Trp Glu 370 375 380Gly Gly Ile Arg
Val Pro Gly Ile Phe Arg Trp Pro Gly Val Leu Pro385 390 395 400Ala
Gly Arg Val Ile Gly Glu Pro Thr Ser Leu Met Asp Val Phe Pro 405 410
415Thr Val Val Arg Leu Ala Gly Gly Glu Val Pro Gln Asp Arg Val Ile
420 425 430Asp Gly Gln Asp Leu Leu Pro Leu Leu Leu Gly Thr Ala Gln
His Ser 435 440
445Asp His Glu Phe Leu Met His Tyr Cys Glu Arg Phe Leu His Ala Ala
450 455 460Arg Trp His Gln Arg Asp Arg Gly Thr Met Trp Lys Val His
Phe Val465 470 475 480Thr Pro Val Phe Gln Pro Glu Gly Ala Gly Ala
Cys Tyr Gly Arg Lys 485 490 495Val Cys Pro Cys Phe Gly Glu Lys Val
Val His His Asp Pro Pro Leu 500 505 510Leu Phe Asp Leu Ser Arg Asp
Pro Ser Glu Thr His Ile Leu Thr Pro 515 520 525Ala Ser Glu Pro Val
Phe Tyr Gln Val Met Glu Arg Val Gln Gln Ala 530 535 540Val Trp Glu
His Gln Arg Thr Leu Ser Pro Val Pro Leu Gln Leu Asp545 550 555
560Arg Leu Gly Asn Ile Trp Arg Pro Trp Leu Gln Pro Cys Cys Gly Pro
565 570 575Phe Pro Leu Cys Trp Cys Leu Arg Glu Asp Asp Pro Gln 580
585241996DNAHomo sapiens 24gggttctgct cctagacatt agagagataa
tacggctgat agacaacaag aaggtattcc 60aagctgcaca atgaggccca ggagaccgtt
ggtcttcatg tctttggtgt gtgcactctt 120gaacacatgg ccagggcaca
cagggtgcat gacgacaagg cctaatattg tcctaatcat 180ggttgatgac
ctgggtattg gagatctggg ctgctacggc aatgacacca tgaggacgcc
240tcacatcgac cgccttgcca gggaaggcgt gcgactgact cagcacatct
ctgccgcctc 300cctctgcagc ccaagccggt ccgcgttctt gacgggaaga
taccccatcc gatcaggtat 360ggtttctagt ggtaatagac gtgtcatcca
aaatcttgca gtccccgcag gcctccctct 420taatgagaca acacttgcag
ccttgctaaa gaagcaagga tacagcacgg ggcttatagg 480caaatggcac
caaggcttga actgcgactc ccgaagtgac cagtgccacc atccatataa
540ttatgggttt gactactact atggcatgcc gttcactctc gttgacagct
gctggccgga 600cccctctcgt aacacggaat tagcctttga gagtcagctc
tggctctgtg tgcagctagt 660tgccattgcc atcctcaccc taacctttgg
gaagctgagc ggctgggtct ctgttccctg 720gctcctgatc ttctccatga
ttctgtttat tttcctcttg ggctatgctt ggttctccag 780ccacacgtcc
cctttatact gggactgcct cctcatgcgg gggcacgaga tcacggagca
840gcccatgaag gctgaacgag ctggatccat tatggtgaag gaagcgattt
cctttttaga 900aaggcacagt aaggaaactt tccttctctt tttctccttt
cttcacgtgc acacacctct 960ccccaccacg gacgatttca ctggcaccag
caagcatggc ttgtatgggg ataatgtgga 1020agagatggac tccatggtgg
gcaagattct tgatgctatc gatgattttg gcctaaggaa 1080caacaccctt
gtctacttta catcagatca cggagggcat ttggaagcta ggcgagggca
1140tgcccaactt ggtggatgga atggaatata caaaggtgga aaaggcatgg
ggggctggga 1200aggtggaatc cgcgtcccag gaattgtccg atggcctgga
aaggtaccag ctggacggtt 1260gattaaggaa cctacaagtt taatggatat
tttaccaact gtcgcatcag tgtcaggagg 1320aagtctccct caggacaggg
tcattgacgg ccgagacctc atgcccttgc tgcagggcaa 1380cgtcaggcac
tcggagcatg aatttctttt ccactactgt ggctcctacc tgcacgccgt
1440gcggtggatc cccaaggacg acagtgggtc agtttggaag gctcactatg
tgaccccggt 1500attccagcca ccagcttctg gtggctgcta tgtcacctca
ttatgcagat gtttcggaga 1560acaggttacc taccacaacc cccctctgct
cttcgatctc tccagggacc cctcagagtc 1620cacacccctg acacctgcca
cagagcccct ctatgatttt gtgattaaaa aggtggccaa 1680cgccctgaag
gaacaccagg aaaccatcgt gcctgtgacc taccaactct cagaactgaa
1740tcagggcagg acgtggctga agccttgctg tggggtgttc ccattttgtc
tgtgtgacaa 1800ggaagaggaa gtctctcagc ctcggggtcc taacgagaag
agataattac aatcaggcta 1860ccagaggaag cctttggtcc taacgagaag
agataattac aatcaggcta ccaaaggaag 1920cactaacttt ggtgctttca
agttggcaag gagtgcattt aatagtcaat aaattcatct 1980accattccag attatt
199625591PRTHomo sapiens 25Met Arg Pro Arg Arg Pro Leu Val Phe Met
Ser Leu Val Cys Ala Leu1 5 10 15Leu Asn Thr Trp Pro Gly His Thr Gly
Cys Met Thr Thr Arg Pro Asn 20 25 30Ile Val Leu Ile Met Val Asp Asp
Leu Gly Ile Gly Asp Leu Gly Cys 35 40 45Tyr Gly Asn Asp Thr Met Arg
Thr Pro His Ile Asp Arg Leu Ala Arg 50 55 60Glu Gly Val Arg Leu Thr
Gln His Ile Ser Ala Ala Ser Leu Cys Ser65 70 75 80Pro Ser Arg Ser
Ala Phe Leu Thr Gly Arg Tyr Pro Ile Arg Ser Gly 85 90 95Met Val Ser
Ser Gly Asn Arg Arg Val Ile Gln Asn Leu Ala Val Pro 100 105 110Ala
Gly Leu Pro Leu Asn Glu Thr Thr Leu Ala Ala Leu Leu Lys Lys 115 120
125Gln Gly Tyr Ser Thr Gly Leu Ile Gly Lys Trp His Gln Gly Leu Asn
130 135 140Cys Asp Ser Arg Ser Asp Gln Cys His His Pro Tyr Asn Tyr
Gly Phe145 150 155 160Asp Tyr Tyr Tyr Gly Met Pro Phe Thr Leu Val
Asp Ser Cys Trp Pro 165 170 175Asp Pro Ser Arg Asn Thr Glu Leu Ala
Phe Glu Ser Gln Leu Trp Leu 180 185 190Cys Val Gln Leu Val Ala Ile
Ala Ile Leu Thr Leu Thr Phe Gly Lys 195 200 205Leu Ser Gly Trp Val
Ser Val Pro Trp Leu Leu Ile Phe Ser Met Ile 210 215 220Leu Phe Ile
Phe Leu Leu Gly Tyr Ala Trp Phe Ser Ser His Thr Ser225 230 235
240Pro Leu Tyr Trp Asp Cys Leu Leu Met Arg Gly His Glu Ile Thr Glu
245 250 255Gln Pro Met Lys Ala Glu Arg Ala Gly Ser Ile Met Val Lys
Glu Ala 260 265 270Ile Ser Phe Leu Glu Arg His Ser Lys Glu Thr Phe
Leu Leu Phe Phe 275 280 285Ser Phe Leu His Val His Thr Pro Leu Pro
Thr Thr Asp Asp Phe Thr 290 295 300Gly Thr Ser Lys His Gly Leu Tyr
Gly Asp Asn Val Glu Glu Met Asp305 310 315 320Ser Met Val Gly Lys
Ile Leu Asp Ala Ile Asp Asp Phe Gly Leu Arg 325 330 335Asn Asn Thr
Leu Val Tyr Phe Thr Ser Asp His Gly Gly His Leu Glu 340 345 350Ala
Arg Arg Gly His Ala Gln Leu Gly Gly Trp Asn Gly Ile Tyr Lys 355 360
365Gly Gly Lys Gly Met Gly Gly Trp Glu Gly Gly Ile Arg Val Pro Gly
370 375 380Ile Val Arg Trp Pro Gly Lys Val Pro Ala Gly Arg Leu Ile
Lys Glu385 390 395 400Pro Thr Ser Leu Met Asp Ile Leu Pro Thr Val
Ala Ser Val Ser Gly 405 410 415Gly Ser Leu Pro Gln Asp Arg Val Ile
Asp Gly Arg Asp Leu Met Pro 420 425 430Leu Leu Gln Gly Asn Val Arg
His Ser Glu His Glu Phe Leu Phe His 435 440 445Tyr Cys Gly Ser Tyr
Leu His Ala Val Arg Trp Ile Pro Lys Asp Asp 450 455 460Ser Gly Ser
Val Trp Lys Ala His Tyr Val Thr Pro Val Phe Gln Pro465 470 475
480Pro Ala Ser Gly Gly Cys Tyr Val Thr Ser Leu Cys Arg Cys Phe Gly
485 490 495Glu Gln Val Thr Tyr His Asn Pro Pro Leu Leu Phe Asp Leu
Ser Arg 500 505 510Asp Pro Ser Glu Ser Thr Pro Leu Thr Pro Ala Thr
Glu Pro Leu Tyr 515 520 525Asp Phe Val Ile Lys Lys Val Ala Asn Ala
Leu Lys Glu His Gln Glu 530 535 540Thr Ile Val Pro Val Thr Tyr Gln
Leu Ser Glu Leu Asn Gln Gly Arg545 550 555 560Thr Trp Leu Lys Pro
Cys Cys Gly Val Phe Pro Phe Cys Leu Cys Asp 565 570 575Lys Glu Glu
Glu Val Ser Gln Pro Arg Gly Pro Asn Glu Lys Arg 580 585
590261578DNAHomo sapiens 26atgggctggc tttttctaaa ggttttgttg
gcgggagtga gtttctcagg atttctttat 60cctcttgtgg atttttgcat cagtgggaaa
acaagaggac agaagccaaa ctttgtgatt 120attttggccg atgacatggg
gtggggtgac ctgggagcaa actgggcaga aacaaaggac 180actgccaacc
ttgataagat ggcttcggag ggaatgaggt ttgtggattt ccatgcagct
240gcctccacct gctcaccctc ccgggcttcc ttgctcaccg gccggcttgg
ccttcgcaat 300ggagtcacac gcaactttgc agtcacttct gtgggaggcc
ttccgctcaa cgagaccacc 360ttggcagagg tgctgcagca ggcgggttac
gtcactggga taataggcaa atggcatctt 420ggacaccacg gctcttatca
ccccaacttc cgtggttttg attactactt tggaatccca 480tatagccatg
atatgggctg tactgatact ccaggctaca accaccctcc ttgtccagcg
540tgtccacagg gtgatggacc atcaaggaac cttcaaagag actgttacac
tgacgtggcc 600ctccctcttt atgaaaacct caacattgtg gagcagccgg
tgaacttgag cagccttgcc 660cagaagtatg ctgagaaagc aacccagttc
atccagcgtg caagcaccag cgggaggccc 720ttcctgctct atgtggctct
ggcccacatg cacgtgccct tacctgtgac tcagctacca 780gcagcgccac
ggggcagaag cctgtatggt gcagggctct gggagatgga cagtctggtg
840ggccagatca aggacaaagt tgaccacaca gtgaaggaaa acacattcct
ctggtttaca 900ggagacaatg gcccgtgggc tcagaagtgt gagctagcgg
gcagtgtggg tcccttcact 960ggattttggc aaactcgtca agggggaagt
ccagccaagc agacgacctg ggaaggaggg 1020caccgggtcc cagcactggc
ttactggcct ggcagagttc cagttaatgt caccagcact 1080gccttgttaa
gcgtgctgga catttttcca actgtggtag ccctggccca ggccagctta
1140cctcaaggac ggcgctttga tggtgtggac gtctccgagg tgctctttgg
ccggtcacag 1200cctgggcaca gggtgctgtt ccaccccaac agcggggcag
ctggagagtt tggagccctg 1260cagactgtcc gcctggagcg ttacaaggcc
ttctacatta ccggtggagc cagggcgtgt 1320gatgggagca cggggcctga
gctgcagcat aagtttcctc tgattttcaa cctggaagac 1380gataccgcag
aagctgtgcc cctagaaaga ggtggtgcgg agtaccaggc tgtgctgccc
1440gaggtcagaa aggttcttgc agacgtcctc caagacattg ccaacgacaa
catctccagc 1500gcagattaca ctcaggaccc ttcagtaact ccctgctgta
atccctacca aattgcctgc 1560cgctgtcaag ccgcataa 157827525PRTHomo
sapiens 27Met Gly Trp Leu Phe Leu Lys Val Leu Leu Ala Gly Val Ser
Phe Ser1 5 10 15Gly Phe Leu Tyr Pro Leu Val Asp Phe Cys Ile Ser Gly
Lys Thr Arg 20 25 30Gly Gln Lys Pro Asn Phe Val Ile Ile Leu Ala Asp
Asp Met Gly Trp 35 40 45Gly Asp Leu Gly Ala Asn Trp Ala Glu Thr Lys
Asp Thr Ala Asn Leu 50 55 60Asp Lys Met Ala Ser Glu Gly Met Arg Phe
Val Asp Phe His Ala Ala65 70 75 80Ala Ser Thr Cys Ser Pro Ser Arg
Ala Ser Leu Leu Thr Gly Arg Leu 85 90 95Gly Leu Arg Asn Gly Val Thr
Arg Asn Phe Ala Val Thr Ser Val Gly 100 105 110Gly Leu Pro Leu Asn
Glu Thr Thr Leu Ala Glu Val Leu Gln Gln Ala 115 120 125Gly Tyr Val
Thr Gly Ile Ile Gly Lys Trp His Leu Gly His His Gly 130 135 140Ser
Tyr His Pro Asn Phe Arg Gly Phe Asp Tyr Tyr Phe Gly Ile Pro145 150
155 160Tyr Ser His Asp Met Gly Cys Thr Asp Thr Pro Gly Tyr Asn His
Pro 165 170 175Pro Cys Pro Ala Cys Pro Gln Gly Asp Gly Pro Ser Arg
Asn Leu Gln 180 185 190Arg Asp Cys Tyr Thr Asp Val Ala Leu Pro Leu
Tyr Glu Asn Leu Asn 195 200 205Ile Val Glu Gln Pro Val Asn Leu Ser
Ser Leu Ala Gln Lys Tyr Ala 210 215 220Glu Lys Ala Thr Gln Phe Ile
Gln Arg Ala Ser Thr Ser Gly Arg Pro225 230 235 240Phe Leu Leu Tyr
Val Ala Leu Ala His Met His Val Pro Leu Pro Val 245 250 255Thr Gln
Leu Pro Ala Ala Pro Arg Gly Arg Ser Leu Tyr Gly Ala Gly 260 265
270Leu Trp Glu Met Asp Ser Leu Val Gly Gln Ile Lys Asp Lys Val Asp
275 280 285His Thr Val Lys Glu Asn Thr Phe Leu Trp Phe Thr Gly Asp
Asn Gly 290 295 300Pro Trp Ala Gln Lys Cys Glu Leu Ala Gly Ser Val
Gly Pro Phe Thr305 310 315 320Gly Phe Trp Gln Thr Arg Gln Gly Gly
Ser Pro Ala Lys Gln Thr Thr 325 330 335Trp Glu Gly Gly His Arg Val
Pro Ala Leu Ala Tyr Trp Pro Gly Arg 340 345 350Val Pro Val Asn Val
Thr Ser Thr Ala Leu Leu Ser Val Leu Asp Ile 355 360 365Phe Pro Thr
Val Val Ala Leu Ala Gln Ala Ser Leu Pro Gln Gly Arg 370 375 380Arg
Phe Asp Gly Val Asp Val Ser Glu Val Leu Phe Gly Arg Ser Gln385 390
395 400Pro Gly His Arg Val Leu Phe His Pro Asn Ser Gly Ala Ala Gly
Glu 405 410 415Phe Gly Ala Leu Gln Thr Val Arg Leu Glu Arg Tyr Lys
Ala Phe Tyr 420 425 430Ile Thr Gly Gly Ala Arg Ala Cys Asp Gly Ser
Thr Gly Pro Glu Leu 435 440 445Gln His Lys Phe Pro Leu Ile Phe Asn
Leu Glu Asp Asp Thr Ala Glu 450 455 460Ala Val Pro Leu Glu Arg Gly
Gly Ala Glu Tyr Gln Ala Val Leu Pro465 470 475 480Glu Val Arg Lys
Val Leu Ala Asp Val Leu Gln Asp Ile Ala Asn Asp 485 490 495Asn Ile
Ser Ser Ala Asp Tyr Thr Gln Asp Pro Ser Val Thr Pro Cys 500 505
510Cys Asn Pro Tyr Gln Ile Ala Cys Arg Cys Gln Ala Ala 515 520
525284669DNAHomo sapiens 28cgcagaccgt cgctaatgaa tcttggggcc
ggtgtcgggc cggggcggct tgatcggcaa 60ctaggaaacc ccaggcgcag aggccaggag
cgagggcagc gaggatcaga ggccaggcct 120tcccggctgc cggcgctcct
cggaggtcag ggcagatgag gaacatgact ctcccccttc 180ggaggaggaa
ggaagtcccg ctgccacctt atctctgctc ctctgcctcc tccctgttcc
240cagagctttt tctctagaga agattttgaa ggcggctttt gtgctgacgg
ccacccacca 300tcatctaaag aagataaact tggcaaatga catgcaggtt
cttcaaggca gaataattgc 360agaaaatctt caaaggaccc tatctgcaga
tgttctgaat acctctgaga atagagattg 420attattcaac caggatacct
aattcaagaa ctccagaaat caggagacgg agacattttg 480tcagttttgc
aacattggac caaatacaat gaagtattct tgctgtgctc tggttttggc
540tgtcctgggc acagaattgc tgggaagcct ctgttcgact gtcagatccc
cgaggttcag 600aggacggata cagcaggaac gaaaaaacat ccgacccaac
attattcttg tgcttaccga 660tgatcaagat gtggagctgg ggtccctgca
agtcatgaac aaaacgagaa agattatgga 720acatgggggg gccaccttca
tcaatgcctt tgtgactaca cccatgtgct gcccgtcacg 780gtcctccatg
ctcaccggga agtatgtgca caatcacaat gtctacacca acaacgagaa
840ctgctcttcc ccctcgtggc aggccatgca tgagcctcgg acttttgctg
tatatcttaa 900caacactggc tacagaacag ccttttttgg aaaatacctc
aatgaatata atggcagcta 960catcccccct gggtggcgag aatggcttgg
attaatcaag aattctcgct tctataatta 1020cactgtttgt cgcaatggca
tcaaagaaaa gcatggattt gattatgcaa aggactactt 1080cacagactta
atcactaacg agagcattaa ttacttcaaa atgtctaaga gaatgtatcc
1140ccataggccc gttatgatgg tgatcagcca cgctgcgccc cacggccccg
aggactcagc 1200cccacagttt tctaaactgt accccaatgc ttcccaacac
ataactccta gttataacta 1260tgcaccaaat atggataaac actggattat
gcagtacaca ggaccaatgc tgcccatcca 1320catggaattt acaaacattc
tacagcgcaa aaggctccag actttgatgt cagtggatga 1380ttctgtggag
aggctgtata acatgctcgt ggagacgggg gagctggaga atacttacat
1440catttacacc gccgaccatg gttaccatat tgggcagttt ggactggtca
aggggaaatc 1500catgccatat gactttgata ttcgtgtgcc tttttttatt
cgtggtccaa gtgtagaacc 1560aggatcaata gtcccacaga tcgttctcaa
cattgacttg gcccccacga tcctggatat 1620tgctgggctc gacacacctc
ctgatgtgga cggcaagtct gtcctcaaac ttctggaccc 1680agaaaagcca
ggtaacaggt ttcgaacaaa caagaaggcc aaaatttggc gtgatacatt
1740cctagtggaa agaggcaaat ttctacgtaa gaaggaagaa tccagcaaga
atatccaaca 1800gtcaaatcac ttgcccaaat atgaacgggt caaagaacta
tgccagcagg ccaggtacca 1860gacagcctgt gaacaaccgg ggcagaagtg
gcaatgcatt gaggatacat ctggcaagct 1920tcgaattcac aagtgtaaag
gacccagtga cctgctcaca gtccggcaga gcacgcggaa 1980cctctacgct
cgcggcttcc atgacaaaga caaagagtgc agttgtaggg agtctggtta
2040ccgtgccagc agaagccaaa gaaagagtca acggcaattc ttgagaaacc
aggggactcc 2100aaagtacaag cccagatttg tccatactcg gcagacacgt
tccttgtccg tcgaatttga 2160aggtgaaata tatgacataa atctggaaga
agaagaagaa ttgcaagtgt tgcaaccaag 2220aaacattgct aagcgtcatg
atgaaggcca caaggggcca agagatctcc aggcttccag 2280tggtggcaac
aggggcagga tgctggcaga tagcagcaac gccgtgggcc cacctaccac
2340tgtccgagtg acacacaagt gttttattct tcccaatgac tctatccatt
gtgagagaga 2400actgtaccaa tcggccagag cgtggaagga ccataaggca
tacattgaca aagagattga 2460agctctgcaa gataaaatta agaatttaag
agaagtgaga ggacatctga agagaaggaa 2520gcctgaggaa tgtagctgca
gtaaacaaag ctattacaat aaagagaaag gtgtaaaaaa 2580gcaagagaaa
ttaaagagcc atcttcaccc attcaaggag gctgctcagg aagtagatag
2640caaactgcaa cttttcaagg agaacaaccg taggaggaag aaggagagga
aggagaagag 2700acggcagagg aagggggaag agtgcagcct gcctggcctc
acttgcttca cgcatgacaa 2760caaccactgg cagacagccc cgttctggaa
cctgggatct ttctgtgctt gcacgagttc 2820taacaataac acctactggt
gtttgcgtac agttaatgag acgcataatt ttcttttctg 2880tgagtttgct
actggctttt tggagtattt tgatatgaat acagatcctt atcagctcac
2940aaatacagtg cacacggtag aacgaggcat tttgaatcag ctacacgtac
aactaatgga 3000gctcagaagc tgtcaaggat ataagcagtg caacccaaga
cctaagaatc ttgatgttgg 3060aaataaagat ggaggaagct atgacctaca
cagaggacag ttatgggatg gatgggaagg 3120ttaatcagcc ccgtctcact
gcagacatca actggcaagg cctagaggag ctacacagtg 3180tgaatgaaaa
catctatgag tacagacaaa actacagact tagtctggtg gactggacta
3240attacttgaa ggatttagat agagtatttg cactgctgaa gagtcactat
gagcaaaata 3300aaacaaataa gactcaaact gctcaaagtg acgggttctt
ggttgtctct gctgagcacg 3360ctgtgtcaat ggagatggcc tctgctgact
cagatgaaga cccaaggcat aaggttggga 3420aaacacctca tttgaccttg
ccagctgacc ttcaaaccct gcatttgaac cgaccaacat 3480taagtccaga
gagtaaactt gaatggaata acgacattcc agaagttaat catttgaatt
3540ctgaacactg gagaaaaacc gaaaaatgga cggggcatga agagactaat
catctggaaa 3600ccgatttcag tggcgatggc atgacagagc tagagctcgg
gcccagcccc aggctgcagc 3660ccattcacag gcacccgaaa gaacttcccc
agtatggtgg tcctggaaag gacatttttg 3720aagatcaact atatcttcct
gtgcattccg atggaatttc agttcatcag atgttcacca 3780tggccaccgc
agaacaccga agtaattcca gcatagcggg gaagatgttg accaaggtgg
3840agaagaatca cgaaaaggag aagtcacagc acctagaagg cagcacctcc
tcttcactct 3900cctctgatta gatgaaactg ttaccttacc ctaaacacag
tatttctttt taactttttt 3960atttgtaaac taataaaggt aatcacagcc
accaacattc caagctaccc tgggtacctt 4020tgtgcagtag aagctagtga
gcatgtgagc aagcggtgtg cacacggaga ctcatcgtta 4080taatttacta
tctgccaaga gtagaaagaa aggctgggga tatttgggtt ggcttggttt
4140tgattttttg cttgtttgtt tgttttgtac taaaacagta ttatcttttg
aatatcgtag 4200ggacataagt atatacatgt tatccaatca agatggctag
aatggtgcct ttctgagtgt 4260ctaaaacttg acacccctgg taaatctttc
aacacacttc cactgcctgc gtaatgaagt 4320tttgattcat ttttaaccac
tggaattttt caatgccgtc attttcagtt agatgatttt 4380gcactttgag
attaaaatgc catgtctatt tgattagtct tattttttta tttttacagg
4440cttatcagtc tcactgttgg ctgtcattgt gacaaagtca aataaacccc
caaggacgac 4500acacagtatg gatcacatat tgtttgacat taagcttttg
ccagaaaatg ttgcatgtgt 4560tttacctcga cttgctaaaa tcgattagca
gaaaggcatg gctaataatg ttggtggtga 4620aaataaataa ataagtaaat
gaaaaaaaaa aaaaaaaaaa aaaaaaaaa 466929871PRTHomo sapiens 29Met Lys
Tyr Ser Cys Cys Ala Leu Val Leu Ala Val Leu Gly Thr Glu1 5 10 15Leu
Leu Gly Ser Leu Cys Ser Thr Val Arg Ser Pro Arg Phe Arg Gly 20 25
30Arg Ile Gln Gln Glu Arg Lys Asn Ile Arg Pro Asn Ile Ile Leu Val
35 40 45Leu Thr Asp Asp Gln Asp Val Glu Leu Gly Ser Leu Gln Val Met
Asn 50 55 60Lys Thr Arg Lys Ile Met Glu His Gly Gly Ala Thr Phe Ile
Asn Ala65 70 75 80Phe Val Thr Thr Pro Met Cys Cys Pro Ser Arg Ser
Ser Met Leu Thr 85 90 95Gly Lys Tyr Val His Asn His Asn Val Tyr Thr
Asn Asn Glu Asn Cys 100 105 110Ser Ser Pro Ser Trp Gln Ala Met His
Glu Pro Arg Thr Phe Ala Val 115 120 125Tyr Leu Asn Asn Thr Gly Tyr
Arg Thr Ala Phe Phe Gly Lys Tyr Leu 130 135 140Asn Glu Tyr Asn Gly
Ser Tyr Ile Pro Pro Gly Trp Arg Glu Trp Leu145 150 155 160Gly Leu
Ile Lys Asn Ser Arg Phe Tyr Asn Tyr Thr Val Cys Arg Asn 165 170
175Gly Ile Lys Glu Lys His Gly Phe Asp Tyr Ala Lys Asp Tyr Phe Thr
180 185 190Asp Leu Ile Thr Asn Glu Ser Ile Asn Tyr Phe Lys Met Ser
Lys Arg 195 200 205Met Tyr Pro His Arg Pro Val Met Met Val Ile Ser
His Ala Ala Pro 210 215 220His Gly Pro Glu Asp Ser Ala Pro Gln Phe
Ser Lys Leu Tyr Pro Asn225 230 235 240Ala Ser Gln His Ile Thr Pro
Ser Tyr Asn Tyr Ala Pro Asn Met Asp 245 250 255Lys His Trp Ile Met
Gln Tyr Thr Gly Pro Met Leu Pro Ile His Met 260 265 270Glu Phe Thr
Asn Ile Leu Gln Arg Lys Arg Leu Gln Thr Leu Met Ser 275 280 285Val
Asp Asp Ser Val Glu Arg Leu Tyr Asn Met Leu Val Glu Thr Gly 290 295
300Glu Leu Glu Asn Thr Tyr Ile Ile Tyr Thr Ala Asp His Gly Tyr
His305 310 315 320Ile Gly Gln Phe Gly Leu Val Lys Gly Lys Ser Met
Pro Tyr Asp Phe 325 330 335Asp Ile Arg Val Pro Phe Phe Ile Arg Gly
Pro Ser Val Glu Pro Gly 340 345 350Ser Ile Val Pro Gln Ile Val Leu
Asn Ile Asp Leu Ala Pro Thr Ile 355 360 365Leu Asp Ile Ala Gly Leu
Asp Thr Pro Pro Asp Val Asp Gly Lys Ser 370 375 380Val Leu Lys Leu
Leu Asp Pro Glu Lys Pro Gly Asn Arg Phe Arg Thr385 390 395 400Asn
Lys Lys Ala Lys Ile Trp Arg Asp Thr Phe Leu Val Glu Arg Gly 405 410
415Lys Phe Leu Arg Lys Lys Glu Glu Ser Ser Lys Asn Ile Gln Gln Ser
420 425 430Asn His Leu Pro Lys Tyr Glu Arg Val Lys Glu Leu Cys Gln
Gln Ala 435 440 445Arg Tyr Gln Thr Ala Cys Glu Gln Pro Gly Gln Lys
Trp Gln Cys Ile 450 455 460Glu Asp Thr Ser Gly Lys Leu Arg Ile His
Lys Cys Lys Gly Pro Ser465 470 475 480Asp Leu Leu Thr Val Arg Gln
Ser Thr Arg Asn Leu Tyr Ala Arg Gly 485 490 495Phe His Asp Lys Asp
Lys Glu Cys Ser Cys Arg Glu Ser Gly Tyr Arg 500 505 510Ala Ser Arg
Ser Gln Arg Lys Ser Gln Arg Gln Phe Leu Arg Asn Gln 515 520 525Gly
Thr Pro Lys Tyr Lys Pro Arg Phe Val His Thr Arg Gln Thr Arg 530 535
540Ser Leu Ser Val Glu Phe Glu Gly Glu Ile Tyr Asp Ile Asn Leu
Glu545 550 555 560Glu Glu Glu Glu Leu Gln Val Leu Gln Pro Arg Asn
Ile Ala Lys Arg 565 570 575His Asp Glu Gly His Lys Gly Pro Arg Asp
Leu Gln Ala Ser Ser Gly 580 585 590Gly Asn Arg Gly Arg Met Leu Ala
Asp Ser Ser Asn Ala Val Gly Pro 595 600 605Pro Thr Thr Val Arg Val
Thr His Lys Cys Phe Ile Leu Pro Asn Asp 610 615 620Ser Ile His Cys
Glu Arg Glu Leu Tyr Gln Ser Ala Arg Ala Trp Lys625 630 635 640Asp
His Lys Ala Tyr Ile Asp Lys Glu Ile Glu Ala Leu Gln Asp Lys 645 650
655Ile Lys Asn Leu Arg Glu Val Arg Gly His Leu Lys Arg Arg Lys Pro
660 665 670Glu Glu Cys Ser Cys Ser Lys Gln Ser Tyr Tyr Asn Lys Glu
Lys Gly 675 680 685Val Lys Lys Gln Glu Lys Leu Lys Ser His Leu His
Pro Phe Lys Glu 690 695 700Ala Ala Gln Glu Val Asp Ser Lys Leu Gln
Leu Phe Lys Glu Asn Asn705 710 715 720Arg Arg Arg Lys Lys Glu Arg
Lys Glu Lys Arg Arg Gln Arg Lys Gly 725 730 735Glu Glu Cys Ser Leu
Pro Gly Leu Thr Cys Phe Thr His Asp Asn Asn 740 745 750His Trp Gln
Thr Ala Pro Phe Trp Asn Leu Gly Ser Phe Cys Ala Cys 755 760 765Thr
Ser Ser Asn Asn Asn Thr Tyr Trp Cys Leu Arg Thr Val Asn Glu 770 775
780Thr His Asn Phe Leu Phe Cys Glu Phe Ala Thr Gly Phe Leu Glu
Tyr785 790 795 800Phe Asp Met Asn Thr Asp Pro Tyr Gln Leu Thr Asn
Thr Val His Thr 805 810 815Val Glu Arg Gly Ile Leu Asn Gln Leu His
Val Gln Leu Met Glu Leu 820 825 830Arg Ser Cys Gln Gly Tyr Lys Gln
Cys Asn Pro Arg Pro Lys Asn Leu 835 840 845Asp Val Gly Asn Lys Asp
Gly Gly Ser Tyr Asp Leu His Arg Gly Gln 850 855 860Leu Trp Asp Gly
Trp Glu Gly865 870304279DNAHomo sapiens 30gggccatttc tggacaacag
ctgctatttt cacttgagcc caagttaatt tctcggggag 60ttctcgggcg cgcacaggca
gctcggtttg ccctgcgatt gagctgcggg tcgcggccgg 120cgccggcctc
tccaatggca aatgtgtgtg gctggaggcg agcgcgaggc tttcggcaaa
180ggcagtcgag tgtttgcaga ccggggcgag tcctgtgaaa gcagataaaa
gaaaacattt 240attaacgtgt cattacgagg ggagcgcccg gccggggctg
tcgcactccc cgcggaacat 300ttggctccct ccagctccta gagaggagaa
gaagaaagcg gaaaagaggc agattcacgt 360cgtttccagc caagtggacc
tgatcgatgg ccctcctgaa tttatcacga tatttgattt 420attagcgatg
ccccctggtt tgtgtgttac gcacacacac gtgcacacaa ggctctggct
480cgcttccctc cctcgtttcc agctcctggg cgaatcccac atctgtttca
actctccgcc 540gagggcgagc aggagcgaga gtgtgtcgaa tctgcgagtg
aagagggacg agggaaaaga 600aacaaagcca cagacgcaac ttgagactcc
cgcatcccaa aagaagcacc agatcagcaa 660aaaaagaaga tgggcccccc
gagcctcgtg ctgtgcttgc tgtccgcaac tgtgttctcc 720ctgctgggtg
gaagctcggc cttcctgtcg caccaccgcc tgaaaggcag gtttcagagg
780gaccgcagga acatccgccc caacatcatc ctggtgctga cggacgacca
ggatgtggag 840ctgggttcca tgcaggtgat gaacaagacc cggcgcatca
tggagcaggg cggggcgcac 900ttcatcaacg ccttcgtgac cacacccatg
tgctgcccct cacgctcctc catcctcacc 960ggcaagtacg tccacaacca
caacacctac accaacaatg agaactgctc ctcgccctcc 1020tggcaggcac
agcacgagag ccgcaccttt gccgtgtacc tcaatagcac tggctaccgg
1080acagctttct tcgggaagta tcttaatgaa tacaacggct cctacgtgcc
acccggctgg 1140aaggagtggg tcggactcct taaaaactcc cgcttttata
actacacgct gtgtcggaac 1200ggggtgaaag agaagcacgg ctccgactac
tccaaggatt acctcacaga cctcatcacc 1260aatgacagcg tgagcttctt
ccgcacgtcc aagaagatgt acccgcacag gccagtcctc 1320atggtcatca
gccatgcagc cccccacggc cctgaggatt cagccccaca atattcacgc
1380ctcttcccaa acgcatctca gcacatcacg ccgagctaca actacgcgcc
caacccggac 1440aaacactgga tcatgcgcta cacggggccc atgaagccca
tccacatgga attcaccaac 1500atgctccagc ggaagcgctt gcagaccctc
atgtcggtgg acgactccat ggagacgatt 1560tacaacatgc tggttgagac
gggcgagctg gacaacacgt acatcgtata caccgccgac 1620cacggttacc
acatcggcca gtttggcctg gtgaaaggga aatccatgcc atatgagttt
1680gacatcaggg tcccgttcta cgtgaggggc cccaacgtgg aagccggctg
tctgaatccc 1740cacatcgtcc tcaacattga cctggccccc accatcctgg
acattgcagg cctggacata 1800cctgcggata tggacgggaa atccatcctc
aagctgctgg acacggagcg gccggtgaat 1860cggtttcact tgaaaaagaa
gatgagggtc tggcgggact ccttcttggt ggagagaggc 1920aagctgctac
acaagagaga caatgacaag gtggacgccc aggaggagaa ctttctgccc
1980aagtaccagc gtgtgaagga cctgtgtcag cgtgctgagt accagacggc
gtgtgagcag 2040ctgggacaga agtggcagtg tgtggaggac gccacgggga
agctgaagct gcataagtgc 2100aagggcccca tgcggctggg cggcagcaga
gccctctcca acctcgtgcc caagtactac 2160gggcagggca gcgaggcctg
cacctgtgac agcggggact acaagctcag cctggccgga 2220cgccggaaaa
aactcttcaa gaagaagtac aaggccagct atgtccgcag tcgctccatc
2280cgctcagtgg ccatcgaggt ggacggcagg gtgtaccacg taggcctggg
tgatgccgcc 2340cagccccgaa acctcaccaa gcggcactgg ccaggggccc
ctgaggacca agatgacaag 2400gatggtgggg acttcagtgg cactggaggc
cttcccgact actcagccgc caaccccatt 2460aaagtgacac atcggtgcta
catcctagag aacgacacag tccagtgtga cctggacctg 2520tacaagtccc
tgcaggcctg gaaagaccac aagctgcaca tcgaccacga gattgaaacc
2580ctgcagaaca aaattaagaa cctgagggaa gtccgaggtc acctgaagaa
aaagcggcca 2640gaagaatgtg actgtcacaa aatcagctac cacacccagc
acaaaggccg cctcaagcac 2700agaggctcca gtctgcatcc tttcaggaag
ggcctgcaag agaaggacaa ggtgtggctg 2760ttgcgggagc agaagcgcaa
gaagaaactc cgcaagctgc tcaagcgcct gcagaacaac 2820gacacgtgca
gcatgccagg cctcacgtgc ttcacccacg acaaccagca ctggcagacg
2880gcgcctttct ggacactggg gcctttctgt gcctgcacca gcgccaacaa
taacacgtac 2940tggtgcatga ggaccatcaa tgagactcac aatttcctct
tctgtgaatt tgcaactggc 3000ttcctagagt actttgatct caacacagac
ccctaccagc tgatgaatgc agtgaacaca 3060ctggacaggg atgtcctcaa
ccagctacac gtacagctca tggagctgag gagctgcaag 3120ggttacaagc
agtgtaaccc ccggactcga aacatggacc tgggacttaa agatggagga
3180agctatgagc aatacaggca gtttcagcgt cgaaagtggc cagaaatgaa
gagaccttct 3240tccaaatcac tgggacaact gtgggaaggc tgggaaggtt
aagaaacaac agaggtggac 3300ctccaaaaac atagaggcat cacctgactg
cacaggcaat gaaaaaccat gtgggtgatt 3360tccagcagac ctgtgctatt
ggccaggagg cctgagaaag caagcacgca ctctcagtca 3420acatgacaga
ttctggagga taaccagcag gagcagagat aacttcagga agtccatttt
3480tgcccctgct tttgctttgg attatacctc accagctgca caaaatgcat
tttttcgtat 3540caaaaagtca ccactaaccc tcccccagaa gctcacaaag
gaaaacggag agagcgagcg 3600agagagattt ccttggaaat ttctcccaag
ggcgaaagtc attggaattt ttaaatcata 3660ggggaaaagc agtcctgttc
taaatcctct tattcttttg gtttgtcaca aagaaggaac 3720taagaagcag
gacagaggca acgtggagag gctgaaaaca gtgcagagac gtttgacaat
3780gagtcagtag cacaaaagag atgacattta cctagcatat aaaccctggt
tgcctctgaa 3840gaaactgcct tcattgtata tatgtgacta tttacatgta
atcaacatgg gaacttttag 3900gggaacctaa taagaaatcc caattttcag
gagtggtggt gtcaataaac gctctgtggc 3960cagtgtaaaa gaaaaaaaaa
aaaaattgtg gacatttctg ttcctgtcca gataccattt 4020ctcctagtat
ttctttgtta tgtcccagaa ctgatgtttt ttttttaagg tactgaaaag
4080aaatgaagtt gatgtatgtc ccaagttttg atgaaactgt atttgtaaaa
aaaattttgt 4140agtttaagta ttgtcataca gtgttcaaaa ccccagccaa
tgaccagcag ttggtatgaa 4200gaacctttga cattttgtaa aaggccattt
cttggggaaa aaaaaaaaaa aaaaaaaaaa 4260aaaaaaaaaa aaaaaaaaa
427931870PRTHomo sapiens 31Met Gly Pro Pro Ser Leu Val Leu Cys Leu
Leu Ser Ala Thr Val Phe1 5 10 15Ser Leu Leu Gly Gly Ser Ser Ala Phe
Leu Ser His His Arg Leu Lys 20 25 30Gly Arg Phe Gln Arg Asp Arg Arg
Asn Ile Arg Pro Asn Ile Ile Leu 35 40 45Val Leu Thr Asp Asp Gln Asp
Val Glu Leu Gly Ser Met Gln Val Met 50 55 60Asn Lys Thr Arg Arg Ile
Met Glu Gln Gly Gly Ala His Phe Ile Asn65 70 75 80Ala Phe Val Thr
Thr Pro Met Cys Cys Pro Ser Arg Ser Ser Ile Leu 85 90 95Thr Gly Lys
Tyr Val His Asn His Asn Thr Tyr Thr Asn Asn Glu Asn 100 105 110Cys
Ser Ser Pro Ser Trp Gln Ala Gln His Glu Ser Arg Thr Phe Ala 115 120
125Val Tyr Leu Asn Ser Thr Gly Tyr Arg Thr Ala Phe Phe Gly Lys Tyr
130 135 140Leu Asn Glu Tyr Asn Gly Ser Tyr Val Pro Pro Gly Trp Lys
Glu Trp145 150 155 160Val Gly Leu Leu Lys Asn Ser Arg Phe Tyr Asn
Tyr Thr Leu Cys Arg 165 170 175Asn Gly Val Lys Glu Lys His Gly Ser
Asp Tyr Ser Lys Asp Tyr Leu 180 185 190Thr Asp Leu Ile Thr Asn Asp
Ser Val Ser Phe Phe Arg Thr Ser Lys 195 200 205Lys Met Tyr Pro His
Arg Pro Val Leu Met Val Ile Ser His Ala Ala 210 215 220Pro His Gly
Pro Glu Asp Ser Ala Pro Gln Tyr Ser Arg Leu Phe Pro225 230 235
240Asn Ala Ser Gln His Ile Thr Pro Ser Tyr Asn Tyr Ala Pro Asn Pro
245 250 255Asp Lys His Trp Ile Met Arg Tyr Thr Gly Pro Met Lys Pro
Ile His 260 265 270Met Glu Phe Thr Asn Met Leu Gln Arg Lys Arg Leu
Gln Thr Leu Met 275 280 285Ser Val Asp Asp Ser Met Glu Thr Ile Tyr
Asn Met Leu Val Glu Thr 290 295 300Gly Glu Leu Asp Asn Thr Tyr Ile
Val Tyr Thr Ala Asp His Gly Tyr305 310 315 320His Ile Gly Gln Phe
Gly Leu Val Lys Gly Lys Ser Met Pro Tyr Glu 325 330 335Phe Asp Ile
Arg Val Pro Phe Tyr Val Arg Gly Pro Asn Val Glu Ala 340 345 350Gly
Cys Leu Asn Pro His Ile Val Leu Asn Ile Asp Leu Ala Pro Thr 355 360
365Ile Leu Asp Ile Ala Gly Leu Asp Ile Pro Ala Asp Met Asp Gly Lys
370 375 380Ser Ile Leu Lys Leu Leu Asp Thr Glu Arg Pro Val Asn Arg
Phe His385 390 395 400Leu Lys Lys Lys Met Arg Val Trp Arg Asp Ser
Phe Leu Val Glu Arg 405 410 415Gly Lys Leu Leu His Lys Arg Asp Asn
Asp Lys Val Asp Ala Gln Glu 420 425 430Glu Asn Phe Leu Pro Lys Tyr
Gln Arg Val Lys Asp Leu Cys Gln Arg 435 440 445Ala Glu Tyr Gln Thr
Ala Cys Glu Gln Leu Gly Gln Lys Trp Gln Cys 450 455 460Val Glu Asp
Ala Thr Gly Lys Leu Lys Leu His Lys Cys Lys Gly Pro465 470 475
480Met Arg Leu Gly Gly Ser Arg Ala Leu Ser Asn Leu Val Pro Lys Tyr
485 490 495Tyr Gly Gln Gly Ser Glu Ala Cys Thr Cys Asp Ser Gly Asp
Tyr Lys 500 505 510Leu Ser Leu Ala Gly Arg Arg Lys Lys Leu Phe Lys
Lys Lys Tyr Lys 515 520 525Ala Ser Tyr Val Arg Ser Arg Ser Ile Arg
Ser Val Ala Ile Glu Val 530 535 540Asp Gly Arg Val Tyr His Val Gly
Leu Gly Asp Ala Ala Gln Pro Arg545 550 555 560Asn Leu Thr Lys Arg
His Trp Pro Gly Ala Pro Glu Asp Gln Asp Asp 565 570 575Lys Asp Gly
Gly Asp Phe Ser Gly Thr Gly Gly Leu Pro Asp Tyr Ser 580 585 590Ala
Ala Asn Pro Ile Lys Val Thr His Arg Cys Tyr Ile Leu Glu Asn 595 600
605Asp Thr Val Gln Cys Asp Leu Asp Leu Tyr Lys Ser Leu Gln Ala Trp
610 615 620Lys Asp His Lys Leu His Ile Asp His Glu Ile Glu Thr Leu
Gln Asn625 630 635 640Lys Ile Lys Asn Leu Arg Glu Val Arg Gly His
Leu Lys Lys Lys Arg 645 650 655Pro Glu Glu Cys Asp Cys His Lys Ile
Ser Tyr His Thr Gln His Lys 660 665 670Gly Arg Leu Lys His Arg Gly
Ser Ser Leu His Pro Phe Arg Lys Gly 675 680 685Leu Gln Glu Lys Asp
Lys Val Trp Leu Leu Arg Glu Gln Lys Arg Lys 690 695 700Lys Lys Leu
Arg Lys Leu Leu Lys Arg Leu Gln Asn Asn Asp Thr Cys705
710 715 720Ser Met Pro Gly Leu Thr Cys Phe Thr His Asp Asn Gln His
Trp Gln 725 730 735Thr Ala Pro Phe Trp Thr Leu Gly Pro Phe Cys Ala
Cys Thr Ser Ala 740 745 750Asn Asn Asn Thr Tyr Trp Cys Met Arg Thr
Ile Asn Glu Thr His Asn 755 760 765Phe Leu Phe Cys Glu Phe Ala Thr
Gly Phe Leu Glu Tyr Phe Asp Leu 770 775 780Asn Thr Asp Pro Tyr Gln
Leu Met Asn Ala Val Asn Thr Leu Asp Arg785 790 795 800Asp Val Leu
Asn Gln Leu His Val Gln Leu Met Glu Leu Arg Ser Cys 805 810 815Lys
Gly Tyr Lys Gln Cys Asn Pro Arg Thr Arg Asn Met Asp Leu Gly 820 825
830Leu Lys Asp Gly Gly Ser Tyr Glu Gln Tyr Arg Gln Phe Gln Arg Arg
835 840 845Lys Trp Pro Glu Met Lys Arg Pro Ser Ser Lys Ser Leu Gly
Gln Leu 850 855 860Trp Glu Gly Trp Glu Gly865 870326PRTArtificial
SequenceConsensus sequence 32Xaa Xaa Xaa Pro Ser Arg1
53323PRTArtificial SequenceSequence derived from human
Arylsulfatase A 33Met Thr Asp Phe Tyr Val Pro Val Ser Leu Cys Thr
Pro Ser Arg Ala1 5 10 15Ala Leu Leu Thr Gly Arg Ser
203416PRTArtificial Sequencea variant of the ASA65-80 peptide, in
which residues Cys69, Pro71 and Arg73, critical for FGly formation,
were scrambled 34Pro Val Ser Leu Pro Thr Arg Ser Cys Ala Ala Leu
Leu Thr Gly Arg1 5 10 153516PRTArtificial Sequencea variant of the
ASA65-80 peptide, in which the Cys69 was replaced by a Serine 35Pro
Val Ser Leu Ser Thr Pro Ser Arg Ala Ala Leu Leu Thr Gly Arg1 5 10
153619DNAArtificial Sequencehuman FGE-specific PCR primer
36ccaatgtagg tcagacacg 193716DNAArtificial Sequencehuman
FGE-specific PCR primer 37acatggcccg cgggac 163819DNAArtificial
Sequencehuman FGE-specific PCR primer 38cgactgctcc ttggactgg
193924DNAArtificial Sequencehuman FGE-specific PCR primer
39ggaattcggg acaacatggc tgcg 244054DNAArtificial
SequenceHA-specific primer 40cccaagctta tgcgtagtca ggcacatcat
acggatagtc catggtgggc aggc 544157DNAArtificial Sequencec-myc
-specific primer 41cccaagctta caggtcttct tcagaaatca gcttttgttc
gtccatggtg ggcaggc 574254DNAArtificial SequenceRGS-His6 - specific
primer 42cccaagctta gtgatggtga tggtgatgcg atcctctgtc catggtgggc
aggc 544315PRTArtificial Sequencetryptic oligopeptide from a human
FGE preparation 43Ser Gln Asn Thr Pro Asp Ser Ser Ala Ser Asn Leu
Gly Phe Arg1 5 10 154419PRTArtificial Sequencetryptic oligopeptide
from a human FGE preparation 44Met Val Pro Ile Pro Ala Gly Val Phe
Thr Met Gly Thr Asp Asp Pro1 5 10 15Gln Ile Lys45906DNAHomo sapiens
45atggcccggc atgggttacc gctgctgccc ctgctgtcgc tcctggtcgg cgcgtggctc
60aagctaggaa atggacaggc tactagcatg gtccaactgc agggtgggag attcctgatg
120ggaacaaatt ctccagacag cagagatggt gaagggcctg tgcgggaggc
gacagtgaaa 180ccctttgcca tcgacatatt tcctgtcacc aacaaagatt
tcagggattt tgtcagggag 240aaaaagtatc ggacagaagc tgagatgttt
ggatggagct ttgtctttga ggactttgtc 300tctgatgagc tgagaaacaa
agccacccag ccaatgaagt ctgtactctg gtggcttcca 360gtggaaaagg
cattttggag gcagcctgca ggtcctggct ctggcatccg agagagactg
420gagcacccag tgttacacgt gagctggaat gacgcccgtg cctactgtgc
ttggcgggga 480aaacgactgc ccacggagga agagtgggag tttgccgccc
gagggggctt gaagggtcaa 540gtttacccat gggggaactg gttccagcca
aaccgcacca acctgtggca gggaaagttc 600cccaagggag acaaagctga
ggatggcttc catggagtct ccccagtgaa tgctttcccc 660gcccagaaca
actacgggct ctatgacctc ctggggaacg tgtgggagtg gacagcatca
720ccgtaccagg ctgctgagca ggacatgcgc gtcctccggg gggcatcctg
gatcgacaca 780gctgatggct ctgccaatca ccgggcccgg gtcaccacca
ggatgggcaa cactccagat 840tcagcctcag acaacctcgg tttccgctgt
gctgcagacg caggccggcc gccaggggag 900ctgtaa 90646301PRTHomo sapiens
46Met Ala Arg His Gly Leu Pro Leu Leu Pro Leu Leu Ser Leu Leu Val1
5 10 15Gly Ala Trp Leu Lys Leu Gly Asn Gly Gln Ala Thr Ser Met Val
Gln 20 25 30Leu Gln Gly Gly Arg Phe Leu Met Gly Thr Asn Ser Pro Asp
Ser Arg 35 40 45Asp Gly Glu Gly Pro Val Arg Glu Ala Thr Val Lys Pro
Phe Ala Ile 50 55 60Asp Ile Phe Pro Val Thr Asn Lys Asp Phe Arg Asp
Phe Val Arg Glu65 70 75 80Lys Lys Tyr Arg Thr Glu Ala Glu Met Phe
Gly Trp Ser Phe Val Phe 85 90 95Glu Asp Phe Val Ser Asp Glu Leu Arg
Asn Lys Ala Thr Gln Pro Met 100 105 110Lys Ser Val Leu Trp Trp Leu
Pro Val Glu Lys Ala Phe Trp Arg Gln 115 120 125Pro Ala Gly Pro Gly
Ser Gly Ile Arg Glu Arg Leu Glu His Pro Val 130 135 140Leu His Val
Ser Trp Asn Asp Ala Arg Ala Tyr Cys Ala Trp Arg Gly145 150 155
160Lys Arg Leu Pro Thr Glu Glu Glu Trp Glu Phe Ala Ala Arg Gly Gly
165 170 175Leu Lys Gly Gln Val Tyr Pro Trp Gly Asn Trp Phe Gln Pro
Asn Arg 180 185 190Thr Asn Leu Trp Gln Gly Lys Phe Pro Lys Gly Asp
Lys Ala Glu Asp 195 200 205Gly Phe His Gly Val Ser Pro Val Asn Ala
Phe Pro Ala Gln Asn Asn 210 215 220Tyr Gly Leu Tyr Asp Leu Leu Gly
Asn Val Trp Glu Trp Thr Ala Ser225 230 235 240Pro Tyr Gln Ala Ala
Glu Gln Asp Met Arg Val Leu Arg Gly Ala Ser 245 250 255Trp Ile Asp
Thr Ala Asp Gly Ser Ala Asn His Arg Ala Arg Val Thr 260 265 270Thr
Arg Met Gly Asn Thr Pro Asp Ser Ala Ser Asp Asn Leu Gly Phe 275 280
285Arg Cys Ala Ala Asp Ala Gly Arg Pro Pro Gly Glu Leu 290 295
30047927DNAMus musculus 47atgcgctctg agttctggtt ccccagcatg
ggttccttgc tccctccggt gttgctgctg 60aggctcctgt cctgccccag gcttcagcta
ggacatgccc aggatcctgc catggtgcat 120ctgccaggtg gccggtttct
gatggggaca gacgctccag atggcagaga cggtgaaggg 180cctgcccggg
aagtgacagt aaaacccttt gccatcgaca tatttccagt caccaataaa
240gacttcaggg agtttgtcag ggagaagaag taccagactg aagccgaggc
attcgggtgg 300agcttcgtct ttgaggattt tgtctcccct gagctcagaa
agcaagaaaa tctgatgccg 360gctgttcact ggtggcagcc agtgccaaag
gcattttgga ggcagcctgc aggtcccggc 420tctggcatcc gagagaaact
ggagcttccc gtggtacacg tgagctggaa cgacgctggt 480gcttactgcg
catggcgggg gagacgcttg cccacagaag aggagtggga gtttgcagcc
540cgagggggct tgaagggtca ggtttatcca tgggggaacc ggttccagcc
aaaccgcacc 600aacttatggc agggaaagtt ccccaaaggt gacaaagctg
aagatggttt tcatggactg 660tcaccagtga acgctttccc cccacagaac
aactacggac tgtatgacct catgggcaat 720gtgtgggagt ggacagcgtc
cacataccaa cctgctggcc aggacatgcg tgtcctccgg 780ggggcatcat
ggatcgacac cgcagacggc tctgctaatc acagggctcg ggtcaccacc
840aggatgggaa acactccaga ctcagcctca gacaacctgg gcttccgctg
cgcctccagt 900gcaggccgac cgaaggagga cctgtga 92748308PRTMus musculus
48Met Arg Ser Glu Phe Trp Phe Pro Ser Met Gly Ser Leu Leu Pro Pro1
5 10 15Val Leu Leu Leu Arg Leu Leu Ser Cys Pro Arg Leu Gln Leu Gly
His 20 25 30Ala Gln Asp Pro Ala Met Val His Leu Pro Gly Gly Arg Phe
Leu Met 35 40 45Gly Thr Asp Ala Pro Asp Gly Arg Asp Gly Glu Gly Pro
Ala Arg Glu 50 55 60Val Thr Val Lys Pro Phe Ala Ile Asp Ile Phe Pro
Val Thr Asn Lys65 70 75 80Asp Phe Arg Glu Phe Val Arg Glu Lys Lys
Tyr Gln Thr Glu Ala Glu 85 90 95Ala Phe Gly Trp Ser Phe Val Phe Glu
Asp Phe Val Ser Pro Glu Leu 100 105 110Arg Lys Gln Glu Asn Leu Met
Pro Ala Val His Trp Trp Gln Pro Val 115 120 125Pro Lys Ala Phe Trp
Arg Gln Pro Ala Gly Pro Gly Ser Gly Ile Arg 130 135 140Glu Lys Leu
Glu Leu Pro Val Val His Val Ser Trp Asn Asp Ala Gly145 150 155
160Ala Tyr Cys Ala Trp Arg Gly Arg Arg Leu Pro Thr Glu Glu Glu Trp
165 170 175Glu Phe Ala Ala Arg Gly Gly Leu Lys Gly Gln Val Tyr Pro
Trp Gly 180 185 190Asn Arg Phe Gln Pro Asn Arg Thr Asn Leu Trp Gln
Gly Lys Phe Pro 195 200 205Lys Gly Asp Lys Ala Glu Asp Gly Phe His
Gly Leu Ser Pro Val Asn 210 215 220Ala Phe Pro Pro Gln Asn Asn Tyr
Gly Leu Tyr Asp Leu Met Gly Asn225 230 235 240Val Trp Glu Trp Thr
Ala Ser Thr Tyr Gln Pro Ala Gly Gln Asp Met 245 250 255Arg Val Leu
Arg Gly Ala Ser Trp Ile Asp Thr Ala Asp Gly Ser Ala 260 265 270Asn
His Arg Ala Arg Val Thr Thr Arg Met Gly Asn Thr Pro Asp Ser 275 280
285Ala Ser Asp Asn Leu Gly Phe Arg Cys Ala Ser Ser Ala Gly Arg Pro
290 295 300Lys Glu Asp Leu30549855DNAMus musculus 49atggtcccca
ttcctgctgg agtattcaca atgggcactg atgatcctca gatcaggcag 60gatggagaag
cccctgccag gagagtcact gttgatggct tttacatgga cgcctatgaa
120gtcagcaatg cggattttga gaagtttgtg aactcgactg gctatttgac
agaggctgag 180aagtttggag actctttcgt ctttgaaggc atgttgagcg
agcaagtgaa aacgcatatc 240caccaggcag ttgcagctgc tccatggtgg
ttgcctgtca agggagctaa ttggagacac 300ccagagggtc cggactccag
tattctgcac aggtcaaatc atccggttct ccatgtttcc 360tggaacgatg
ctgttgccta ctgcacatgg gcgggcaaga ggttgcctac tgaggcagag
420tgggaataca gctgtagagg aggcctgcag aacaggcttt tcccctgggg
caacaaactg 480cagcccaaag gacagcatta tgccaacatc tggcagggca
agtttcctgt gagcaacact 540ggcgaggatg gcttccaagg aactgccccc
gttgatgcct ttcctcccaa tggctatggc 600ttatacaaca tagtggggaa
tgtgtgggag tggacctcag actggtggac tgttcaccat 660tctgttgagg
aaacgttcaa cccaaagggt cccacttctg ggaaagaccg agtgaagaag
720ggtggatcct acatgtgcca taagtcctat tgctataggt accgctgtgc
agctcgaagc 780cagaacacac cagatagctc tgcatccaac ctgggattcc
gatgtgcagc cgaccacctg 840cccaccgcag actga 85550284PRTMus musculus
50Met Val Pro Ile Pro Ala Gly Val Phe Thr Met Gly Thr Asp Asp Pro1
5 10 15Gln Ile Arg Gln Asp Gly Glu Ala Pro Ala Arg Arg Val Thr Val
Asp 20 25 30Gly Phe Tyr Met Asp Ala Tyr Glu Val Ser Asn Ala Asp Phe
Glu Lys 35 40 45Phe Val Asn Ser Thr Gly Tyr Leu Thr Glu Ala Glu Lys
Phe Gly Asp 50 55 60Ser Phe Val Phe Glu Gly Met Leu Ser Glu Gln Val
Lys Thr His Ile65 70 75 80His Gln Ala Val Ala Ala Ala Pro Trp Trp
Leu Pro Val Lys Gly Ala 85 90 95Asn Trp Arg His Pro Glu Gly Pro Asp
Ser Ser Ile Leu His Arg Ser 100 105 110Asn His Pro Val Leu His Val
Ser Trp Asn Asp Ala Val Ala Tyr Cys 115 120 125Thr Trp Ala Gly Lys
Arg Leu Pro Thr Glu Ala Glu Trp Glu Tyr Ser 130 135 140Cys Arg Gly
Gly Leu Gln Asn Arg Leu Phe Pro Trp Gly Asn Lys Leu145 150 155
160Gln Pro Lys Gly Gln His Tyr Ala Asn Ile Trp Gln Gly Lys Phe Pro
165 170 175Val Ser Asn Thr Gly Glu Asp Gly Phe Gln Gly Thr Ala Pro
Val Asp 180 185 190Ala Phe Pro Pro Asn Gly Tyr Gly Leu Tyr Asn Ile
Val Gly Asn Val 195 200 205Trp Glu Trp Thr Ser Asp Trp Trp Thr Val
His His Ser Val Glu Glu 210 215 220Thr Phe Asn Pro Lys Gly Pro Thr
Ser Gly Lys Asp Arg Val Lys Lys225 230 235 240Gly Gly Ser Tyr Met
Cys His Lys Ser Tyr Cys Tyr Arg Tyr Arg Cys 245 250 255Ala Ala Arg
Ser Gln Asn Thr Pro Asp Ser Ser Ala Ser Asn Leu Gly 260 265 270Phe
Arg Cys Ala Ala Asp His Leu Pro Thr Ala Asp 275
280511011DNADrosophila melanogaster 51atgacaacaa ttatattagt
cctctttatt tggatagttt tattcaatga cgtatccagc 60gactgtggct gccaaaagct
cgaccggaag gccccggata tgccgtccat ttccggacaa 120gtgtgccagc
aacgagcaca gggtgcacac agccactacc gggattacta tggcgaactg
180gagccaaata ttgcggacat gtcactgctt ccgggaggca cggtttacat
gggtactgac 240aaaccgcact ttccggccga ccgcgaggct ccggaacggc
aggtgaagct gaatgacttc 300tacatcgaca agtatgaggt ttccaacgaa
gcctttgcga agtttgttct gcacactaac 360tacaccacgg aggctgagcg
atatggcgac agttttctgt ttaagagcct tttgagccca 420ttggagcaga
agaacctaga ggacttccga gtggcgagcg ctgtctggtg gtacaaagtg
480gccggcgtga actggcgaca tccaaatggc gtggacagcg atatagacca
cttaggccga 540cacccggtag tgcacgtatc gtggcgcgac gctgtggagt
actgtaagtg ggccggcaag 600cggttgccca gcgaggcgga gtgggaggcg
gcttgcaggg gcggcaagga gcgcaaactg 660tttccctggg gcaacaagct
gatgccaagg aatgaacatt ggctgaacat ctggcaggga 720gactttcccg
atggcaacct ggctgaagat gggtttgagt acaccagccc cgtggatgcc
780ttccgacaga atatttacga cctgcacaac atggtgggca acgtctggga
gtggacggca 840gatctgtggg acgtaaatga cgttagcgat aatccaaatc
gggtcaagaa gggcggttct 900tatctgtgtc acaagtccta ctgctacagg
tacaggtgcg cggcacgctc gcagaacaca 960gaagacagtt cagccggtaa
cctgggtttt cggtgcgcca agaatgcgtg a 101152336PRTDrosophila
melanogaster 52Met Thr Thr Ile Ile Leu Val Leu Phe Ile Trp Ile Val
Leu Phe Asn1 5 10 15Asp Val Ser Ser Asp Cys Gly Cys Gln Lys Leu Asp
Arg Lys Ala Pro 20 25 30Asp Met Pro Ser Ile Ser Gly Gln Val Cys Gln
Gln Arg Ala Gln Gly 35 40 45Ala His Ser His Tyr Arg Asp Tyr Tyr Gly
Glu Leu Glu Pro Asn Ile 50 55 60Ala Asp Met Ser Leu Leu Pro Gly Gly
Thr Val Tyr Met Gly Thr Asp65 70 75 80Lys Pro His Phe Pro Ala Asp
Arg Glu Ala Pro Glu Arg Gln Val Lys 85 90 95Leu Asn Asp Phe Tyr Ile
Asp Lys Tyr Glu Val Ser Asn Glu Ala Phe 100 105 110Ala Lys Phe Val
Leu His Thr Asn Tyr Thr Thr Glu Ala Glu Arg Tyr 115 120 125Gly Asp
Ser Phe Leu Phe Lys Ser Leu Leu Ser Pro Leu Glu Gln Lys 130 135
140Asn Leu Glu Asp Phe Arg Val Ala Ser Ala Val Trp Trp Tyr Lys
Val145 150 155 160Ala Gly Val Asn Trp Arg His Pro Asn Gly Val Asp
Ser Asp Ile Asp 165 170 175His Leu Gly Arg His Pro Val Val His Val
Ser Trp Arg Asp Ala Val 180 185 190Glu Tyr Cys Lys Trp Ala Gly Lys
Arg Leu Pro Ser Glu Ala Glu Trp 195 200 205Glu Ala Ala Cys Arg Gly
Gly Lys Glu Arg Lys Leu Phe Pro Trp Gly 210 215 220Asn Lys Leu Met
Pro Arg Asn Glu His Trp Leu Asn Ile Trp Gln Gly225 230 235 240Asp
Phe Pro Asp Gly Asn Leu Ala Glu Asp Gly Phe Glu Tyr Thr Ser 245 250
255Pro Val Asp Ala Phe Arg Gln Asn Ile Tyr Asp Leu His Asn Met Val
260 265 270Gly Asn Val Trp Glu Trp Thr Ala Asp Leu Trp Asp Val Asn
Asp Val 275 280 285Ser Asp Asn Pro Asn Arg Val Lys Lys Gly Gly Ser
Tyr Leu Cys His 290 295 300Lys Ser Tyr Cys Tyr Arg Tyr Arg Cys Ala
Ala Arg Ser Gln Asn Thr305 310 315 320Glu Asp Ser Ser Ala Gly Asn
Leu Gly Phe Arg Cys Ala Lys Asn Ala 325 330 33553870DNAAnopheles
gambiae 53ccggagagct tgctcgatct ggtggaacat tccaagcggt tcgaagacat
gagccttatc 60ccaggaggtg aatatgtaat cggcacaaat gaacctatct tcgtcaagga
tcgcgaatca 120ccggcccggc ccgcgacgat ccgcgacttt tacctcgacc
agtacgaagt ctccaacgca 180cagttcaagg cattcgtcga ccagacgggc
tacgtcacgg aggcggaaaa gtttggcgac 240agcttcgtct tccagcagct
gctcagcgaa ccggtgcgcc agcagtacga agatttccgc 300gtggcggcgg
cgccctggtg gtacaaggta cgtggagcct cctggcagca tccggaaggt
360gatgtgtcac gtgatataag cgaccgattg gaccatccgg tggtgcacgt
gtcctggaac 420gatgcggtcg cgtactgcgc ctggaaaggg aagcgcctgc
cgacggaagc ggaatgggaa 480gcggcctgcc ggggcggtcg caagcagaag
ctgttcccct ggggtaacaa gctgatgccg 540aaggagcagc acatgatgaa
catatggcag ggcgagttcc cggacagcaa tctgaaggag 600gatggctacg
agaccacctg cccggtgacg tccttccgcc agaacccgtt cgagctgtac
660aacatcgttg gcaacgtgtg ggagtggacg gcggatcttt gggacgcgaa
ggatgcggcc 720atcgagcgca agccgggcag cgatccaccg aatcgggtga
aaaagggtgg ctcatacctg 780tgtcacgaat cgtactgcta tcgctatcgc
tgtgcggctc gatcgcagaa caccgaggac 840agttcggcgg gcaatctggg
cttccggtgc 87054290PRTAnopheles gambiae 54Pro Glu Ser Leu Leu Asp
Leu Val Glu His Ser Lys Arg Phe Glu Asp1 5 10 15Met Ser Leu Ile Pro
Gly Gly Glu Tyr Val Ile Gly Thr Asn Glu Pro 20 25 30Ile Phe Val Lys
Asp Arg Glu Ser Pro Ala Arg Pro Ala Thr Ile Arg 35 40 45Asp Phe Tyr
Leu Asp Gln Tyr Glu Val Ser Asn Ala Gln Phe Lys Ala 50 55 60Phe Val
Asp Gln Thr Gly Tyr Val Thr Glu Ala Glu Lys Phe Gly Asp65 70 75
80Ser Phe Val Phe Gln Gln Leu Leu Ser Glu Pro Val Arg Gln Gln Tyr
85 90 95Glu Asp Phe Arg Val Ala Ala Ala Pro Trp Trp Tyr Lys Val Arg
Gly 100 105 110Ala Ser Trp Gln His Pro Glu Gly Asp Val Ser Arg Asp
Ile Ser Asp 115 120 125Arg Leu Asp His Pro Val Val His Val Ser Trp
Asn Asp Ala Val Ala 130 135 140Tyr Cys Ala Trp Lys Gly Lys Arg Leu
Pro Thr Glu Ala Glu Trp Glu145 150 155 160Ala Ala Cys Arg Gly Gly
Arg Lys Gln Lys Leu Phe Pro Trp Gly Asn 165 170 175Lys Leu Met Pro
Lys Glu Gln His Met Met Asn Ile Trp Gln Gly Glu 180 185 190Phe Pro
Asp Ser Asn Leu Lys Glu Asp Gly Tyr Glu Thr Thr Cys Pro 195 200
205Val Thr Ser Phe Arg Gln Asn Pro Phe Glu Leu Tyr Asn Ile Val Gly
210 215 220Asn Val Trp Glu Trp Thr Ala Asp Leu Trp Asp Ala Lys Asp
Ala Ala225 230 235 240Ile Glu Arg Lys Pro Gly Ser Asp Pro Pro Asn
Arg Val Lys Lys Gly 245 250 255Gly Ser Tyr Leu Cys His Glu Ser Tyr
Cys Tyr Arg Tyr Arg Cys Ala 260 265 270Ala Arg Ser Gln Asn Thr Glu
Asp Ser Ser Ala Gly Asn Leu Gly Phe 275 280 285Arg Cys
29055945DNAStreptomyces coelicolor 55gtggccgtgg ccgccccgtc
ccccgcggcc gccgcggagc cggggcccgc cgcccgtccg 60cgctcgaccc gcggacaggt
gcgcctgccg ggcggtgagt tcgcgatggg ggacgccttc 120ggggagggat
atccggccga cggcgagaca cccgtgcaca cggtgcgcct gcggcccttc
180cacatcgacg agaccgccgt caccaacgcc cggttcgccg ccttcgtcaa
ggcgaccggc 240catgtgaccg acgccgaacg cttcggctcc tcggccgtct
tccacctggt cgtcgccgcc 300ccggacgccg acgtcctcgg cagcgccgcc
ggcgccccct ggtggatcaa cgtgcggggc 360gcccactggc gccgccccga
gggcgcccgc tccgacatca ccggccggcc gaaccatccg 420gtcgtccacg
tctcctggaa cgatgccacc gcctacgcgc ggtgggccgg caagcgcctg
480cccaccgagg ccgaatggga gtacgccgcc cgcgggggac tggccggccg
ccgctacgcc 540tggggcgacg agctgacccc gggcggccgg tggcgctgca
acatctggca gggccgcttc 600ccgcacgtca acacggccga ggacgggcac
ctgagcaccg caccggtcaa gtcctaccgg 660cccaacggcc acggcctgtg
gaacaccgcg ggcaacgtgt gggaatggtg ctccgactgg 720ttctcgccca
cctactacgc cgaatcaccc accgtcgacc cgcacggccc cgggaccggg
780gcggcacggg tgctgcgcgg cggctcctac ctgtgccacg actcctactg
caaccgctac 840cgggtcgccg cccgctcctc caacaccccg gactcctcgt
ccggcaacct cggattccgc 900tgcgccaacg acgcggacct cacgtccgga
tcagccgctg agtga 94556314PRTStreptomyces coelicolor 56Met Ala Val
Ala Ala Pro Ser Pro Ala Ala Ala Ala Glu Pro Gly Pro1 5 10 15Ala Ala
Arg Pro Arg Ser Thr Arg Gly Gln Val Arg Leu Pro Gly Gly 20 25 30Glu
Phe Ala Met Gly Asp Ala Phe Gly Glu Gly Tyr Pro Ala Asp Gly 35 40
45Glu Thr Pro Val His Thr Val Arg Leu Arg Pro Phe His Ile Asp Glu
50 55 60Thr Ala Val Thr Asn Ala Arg Phe Ala Ala Phe Val Lys Ala Thr
Gly65 70 75 80His Val Thr Asp Ala Glu Arg Phe Gly Ser Ser Ala Val
Phe His Leu 85 90 95Val Val Ala Ala Pro Asp Ala Asp Val Leu Gly Ser
Ala Ala Gly Ala 100 105 110Pro Trp Trp Ile Asn Val Arg Gly Ala His
Trp Arg Arg Pro Glu Gly 115 120 125Ala Arg Ser Asp Ile Thr Gly Arg
Pro Asn His Pro Val Val His Val 130 135 140Ser Trp Asn Asp Ala Thr
Ala Tyr Ala Arg Trp Ala Gly Lys Arg Leu145 150 155 160Pro Thr Glu
Ala Glu Trp Glu Tyr Ala Ala Arg Gly Gly Leu Ala Gly 165 170 175Arg
Arg Tyr Ala Trp Gly Asp Glu Leu Thr Pro Gly Gly Arg Trp Arg 180 185
190Cys Asn Ile Trp Gln Gly Arg Phe Pro His Val Asn Thr Ala Glu Asp
195 200 205Gly His Leu Ser Thr Ala Pro Val Lys Ser Tyr Arg Pro Asn
Gly His 210 215 220Gly Leu Trp Asn Thr Ala Gly Asn Val Trp Glu Trp
Cys Ser Asp Trp225 230 235 240Phe Ser Pro Thr Tyr Tyr Ala Glu Ser
Pro Thr Val Asp Pro His Gly 245 250 255Pro Gly Thr Gly Ala Ala Arg
Val Leu Arg Gly Gly Ser Tyr Leu Cys 260 265 270His Asp Ser Tyr Cys
Asn Arg Tyr Arg Val Ala Ala Arg Ser Ser Asn 275 280 285Thr Pro Asp
Ser Ser Ser Gly Asn Leu Gly Phe Arg Cys Ala Asn Asp 290 295 300Ala
Asp Leu Thr Ser Gly Ser Ala Ala Glu305 310571005DNACorynebacterium
efficiens 57gtggttcgcc atcgactggg ccaccggccc tgcacactga ggattacgtc
catgagtaac 60tgctgctccc cgtcaagcgc acaatggcgt accactaccc gggatttatc
agatcctgtc 120aatcccacca ctccatgcaa cccggaacaa tcccgcgatg
ctgtgacact gccgggtgga 180gctttccaca tgggcgatca tcacggggag
gggtacccgg cggacgggga ggggccagta 240catgaggttc acctcgcccc
cttcggcatt aatgtcacca cggtcacgaa tgccgagttc 300ggacgattta
ttgaagccac agggtatacg acgacagcgg aacgctacgg tgtctcggct
360gtattctacg cagcgttcca agggcaacgc gctgacattc ttcgccaggt
tcccggcgtg 420ccctggtggc tggcggtcaa gggtgcgaac tggcagcgtc
ccaacggccc cggatccacc 480ctggacgggc ttgaggacca ccccgtcgtt
cacgtttcct gggatgatgc cgttgcctac 540tgcacctggg ctggcggtcg
tctgcccacc gaagccgagt gggaatacgc cgcccggggt 600ggactgcagg
gcgcacgata tgcctggggg gataacctcg ccctagacgg gaggtggaac
660tgcaatatct ggcagggggg cttccccatg gagaacaccg ccgcggatgg
ttacctcacc 720actgcaccgg tgaagaccta cacgcccaat ggatacggtc
tgtggcagat ggcagggaat 780gtatgggaat ggtgccagga ctggtttgat
gcggagtact actcccgtgc ttcctccatc 840aacccgcggg gaccggatac
cggtgcgcgc cgggtgatgc gcggaggctc gtatctctgc 900catgattcct
actgcaacag ataccgggtg gccgcccgca attcgaacac cccggattcc
960acctcgggga ataccggttt ccggtgcgtt ttcgatagtc cttga
100558334PRTCorynebacterium efficiens 58Met Val Arg His Arg Leu Gly
His Arg Pro Cys Thr Leu Arg Ile Thr1 5 10 15Ser Met Ser Asn Cys Cys
Ser Pro Ser Ser Ala Gln Trp Arg Thr Thr 20 25 30Thr Arg Asp Leu Ser
Asp Pro Val Asn Pro Thr Thr Pro Cys Asn Pro 35 40 45Glu Gln Ser Arg
Asp Ala Val Thr Leu Pro Gly Gly Ala Phe His Met 50 55 60Gly Asp His
His Gly Glu Gly Tyr Pro Ala Asp Gly Glu Gly Pro Val65 70 75 80His
Glu Val His Leu Ala Pro Phe Gly Ile Asn Val Thr Thr Val Thr 85 90
95Asn Ala Glu Phe Gly Arg Phe Ile Glu Ala Thr Gly Tyr Thr Thr Thr
100 105 110Ala Glu Arg Tyr Gly Val Ser Ala Val Phe Tyr Ala Ala Phe
Gln Gly 115 120 125Gln Arg Ala Asp Ile Leu Arg Gln Val Pro Gly Val
Pro Trp Trp Leu 130 135 140Ala Val Lys Gly Ala Asn Trp Gln Arg Pro
Asn Gly Pro Gly Ser Thr145 150 155 160Leu Asp Gly Leu Glu Asp His
Pro Val Val His Val Ser Trp Asp Asp 165 170 175Ala Val Ala Tyr Cys
Thr Trp Ala Gly Gly Arg Leu Pro Thr Glu Ala 180 185 190Glu Trp Glu
Tyr Ala Ala Arg Gly Gly Leu Gln Gly Ala Arg Tyr Ala 195 200 205Trp
Gly Asp Asn Leu Ala Leu Asp Gly Arg Trp Asn Cys Asn Ile Trp 210 215
220Gln Gly Gly Phe Pro Met Glu Asn Thr Ala Ala Asp Gly Tyr Leu
Thr225 230 235 240Thr Ala Pro Val Lys Thr Tyr Thr Pro Asn Gly Tyr
Gly Leu Trp Gln 245 250 255Met Ala Gly Asn Val Trp Glu Trp Cys Gln
Asp Trp Phe Asp Ala Glu 260 265 270Tyr Tyr Ser Arg Ala Ser Ser Ile
Asn Pro Arg Gly Pro Asp Thr Gly 275 280 285Ala Arg Arg Val Met Arg
Gly Gly Ser Tyr Leu Cys His Asp Ser Tyr 290 295 300Cys Asn Arg Tyr
Arg Val Ala Ala Arg Asn Ser Asn Thr Pro Asp Ser305 310 315 320Thr
Ser Gly Asn Thr Gly Phe Arg Cys Val Phe Asp Ser Pro 325
330591017DNANovosphingobium aromaticivorans 59atggcgcaac cattccgatc
gacggcggcc agtcgtacaa gtattgaacg ccatctcgaa 60cccaattgca ggagcacgtc
gcgaatggtc gaacgccccg gcatgcgcct gatcgaaggc 120ggcactttca
ccatgggctc ggaagccttc tacccggagg aagcgccgct tcgccgggtg
180aaggtagaca gcttctggat cgatgaagcg ccggtgacga acgcacagtt
cgccgcattc 240gtggaggcca cgggatacgt cactgtggcc gagatcgagc
cggatcccaa ggactacccc 300ggcatgctcc cgggcatgga ccgcgcggga
tcgctggtgt tccagaaaac agcagggccg 360gtcgacatgg cggatgcgtc
caactggtgg cactttacct ttggcgcctg ctggaagcat 420ccacttggac
cgggcagttc catcgatggg atcgaggacc atcccgtcgt tcacgtcgcc
480tatgccgatg ccgaggccta tgccaaatgg gcgggcaagg atctgccgac
cgaagccgag 540ttcgaatatg ctgcgcgcgg cgggttggac ggttccgaat
tttcctgggg agacgaactc 600gcacctgaag gccggatgat ggccaactac
tggcaaggcc tgtttccctt cgccaaccag 660tgcctcgatg gctgggaacg
gacatcgccc gtccgcaact tcccgcccaa cggctatggt 720ctttacgaca
tgatcgggaa cacgtgggag tggacctgcg attggtgggc cgacaagccg
780ctgactccgc aaaggaaatc ggcatgctgc gcgatcagca atccgcgcgg
cggcaagctc 840aaggacagct tcgacccgtc gcaacccgca atgcgcatcg
gccggaaggt cataaagggc 900ggttcgcacc tgtgtgcggc caattactgc
cagcgctatc gccccgcagc acgccatcct 960gaaatggttg ataccgcgac
gacgcacatc ggcttcaggt gtgtggtgcg gccctga
101760338PRTNovosphingobium aromaticivorans 60Met Ala Gln Pro Phe
Arg Ser Thr Ala Ala Ser Arg Thr Ser Ile Glu1 5 10 15Arg His Leu Glu
Pro Asn Cys Arg Ser Thr Ser Arg Met Val Glu Arg 20 25 30Pro Gly Met
Arg Leu Ile Glu Gly Gly Thr Phe Thr Met Gly Ser Glu 35 40 45Ala Phe
Tyr Pro Glu Glu Ala Pro Leu Arg Arg Val Lys Val Asp Ser 50 55 60Phe
Trp Ile Asp Glu Ala Pro Val Thr Asn Ala Gln Phe Ala Ala Phe65 70 75
80Val Glu Ala Thr Gly Tyr Val Thr Val Ala Glu Ile Glu Pro Asp Pro
85 90 95Lys Asp Tyr Pro Gly Met Leu Pro Gly Met Asp Arg Ala Gly Ser
Leu 100 105 110Val Phe Gln Lys Thr Ala Gly Pro Val Asp Met Ala Asp
Ala Ser Asn 115 120 125Trp Trp His Phe Thr Phe Gly Ala Cys Trp Lys
His Pro Leu Gly Pro 130 135 140Gly Ser Ser Ile Asp Gly Ile Glu Asp
His Pro Val Val His Val Ala145 150 155 160Tyr Ala Asp Ala Glu Ala
Tyr Ala Lys Trp Ala Gly Lys Asp Leu Pro 165 170 175Thr Glu Ala Glu
Phe Glu Tyr Ala Ala Arg Gly Gly Leu Asp Gly Ser 180 185 190Glu Phe
Ser Trp Gly Asp Glu Leu Ala Pro Glu Gly Arg Met Met Ala 195 200
205Asn Tyr Trp Gln Gly Leu Phe Pro Phe Ala Asn Gln Cys Leu Asp Gly
210 215 220Trp Glu Arg Thr Ser Pro Val Arg Asn Phe Pro Pro Asn Gly
Tyr Gly225 230 235 240Leu Tyr Asp Met Ile Gly Asn Thr Trp Glu Trp
Thr Cys Asp Trp Trp 245 250 255Ala Asp Lys Pro Leu Thr Pro Gln Arg
Lys Ser Ala Cys Cys Ala Ile 260 265 270Ser Asn Pro Arg Gly Gly Lys
Leu Lys Asp Ser Phe Asp Pro Ser Gln 275 280 285Pro Ala Met Arg Ile
Gly Arg Lys Val Ile Lys Gly Gly Ser His Leu 290 295 300Cys Ala Ala
Asn Tyr Cys Gln Arg Tyr Arg Pro Ala Ala Arg His Pro305 310 315
320Glu Met Val Asp Thr Ala Thr Thr His Ile Gly Phe Arg Cys Val Val
325 330 335Arg Pro611119DNAMesorhizobium loti 61atgggcccac
gaggtcgagg tcaaaaaccg catgaaaggc gacgcggtca tgttcgacat 60tgccgggaag
ttctagccga tagcgggtgg gcggctgatg gagatgagca cgccgtgtca
120tttcgggatc tttcgatgaa cgcccctgcc gaagtcttcg agcgcgctgc
agccgaacgg 180tcgtaccccg gaatggtctg gatccccggc ggtaccttcc
tgatgggctc agacaaccac 240tatccggagg aggcaccggc ccaccgggtc
agggtcgacg gcttctggat ggacaaattc 300accgtctcca accgcgactt
cgaacgcttc gttgcggcga caggacatgt cactcttgcc 360gagaaacccg
ccaatcccga cgactatccc ggtgccttac ccgatctgct ggctccgtcc
420tcgatgatgt tcaggaagcc ggccggccct gtcgaccttg gcaatcacta
caattggtgg 480gtctatgtcc gcggcgccaa ctggcgccat ccacgcgggc
cggcaagtac aatcaagaag 540gttgcagatc atccggtcgt gcatgtggcc
tacgaggatg tcgtggccta tgccaactgg 600gcaggcaagg aacttcccac
cgaggccgag tgggaattcg cggcgcgagg cggcctcgat 660gccgccgaat
acgtctgggg caacgagctt acgccggccg ggaagcacat ggccaacatc
720tggcaaggag actttcccta ccggaatact gtcgacgacg gttacgaata
tacggcccca 780gtaggctcgt tcccggccaa cgactacggt ctctacgaca
tggccggcaa tgtctggcaa 840tggacgaccg actggtacca ggaccacaag
gcgatcgaca gcccgtgctg caccgctgtc 900aatccgcgtg gcggccatcg
cgaagcgagc tatgacaccc ggctacctga cgttaagatc 960cctcgcaagg
tcaccaaggg tggctcccat ctgtgcgcgc cgaactactg tcggcgctac
1020cggcccgcgg cgcgaatggc gcaacccgtc gacactgcaa tctcccatct
cggctttcgc 1080tgcatcgtgc gaaggaaaat ggaattgaac gcgcagtaa
111962372PRTMesorhizobium loti 62Met Gly Pro Arg Gly Arg Gly Gln
Lys Pro His Glu Arg Arg Arg Gly1 5 10 15His Val Arg His Cys Arg Glu
Val Leu Ala Asp Ser Gly Trp Ala Ala 20 25 30Asp Gly Asp Glu His Ala
Val Ser Phe Arg Asp Leu Ser Met Asn Ala 35 40 45Pro Ala Glu Val Phe
Glu Arg Ala Ala Ala Glu Arg Ser Tyr Pro Gly 50 55 60Met Val Trp Ile
Pro Gly Gly Thr Phe Leu Met Gly Ser Asp Asn His65 70 75 80Tyr Pro
Glu Glu Ala Pro Ala His Arg Val Arg Val Asp Gly Phe Trp 85 90 95Met
Asp Lys Phe Thr Val Ser Asn Arg Asp Phe Glu Arg Phe Val Ala 100 105
110Ala Thr Gly His Val Thr Leu Ala Glu Lys Pro Ala Asn Pro Asp Asp
115 120 125Tyr Pro Gly Ala Leu Pro Asp Leu Leu Ala Pro Ser Ser Met
Met Phe 130 135 140Arg Lys Pro Ala Gly Pro Val Asp Leu Gly Asn His
Tyr Asn Trp Trp145 150 155 160Val Tyr Val Arg Gly Ala Asn Trp Arg
His Pro Arg Gly Pro Ala Ser 165 170 175Thr Ile Lys Lys Val Ala Asp
His Pro Val Val His Val Ala Tyr Glu 180 185 190Asp Val Val Ala Tyr
Ala Asn Trp Ala Gly Lys Glu Leu Pro Thr Glu 195 200 205Ala Glu Trp
Glu Phe Ala Ala Arg Gly Gly Leu Asp Ala Ala Glu Tyr 210 215 220Val
Trp Gly Asn Glu Leu Thr Pro Ala Gly Lys His Met Ala Asn Ile225 230
235 240Trp Gln Gly Asp Phe Pro Tyr Arg Asn Thr Val Asp Asp Gly Tyr
Glu 245 250 255Tyr Thr Ala Pro Val Gly Ser Phe Pro Ala Asn Asp Tyr
Gly Leu Tyr 260 265 270Asp Met Ala Gly Asn Val Trp Gln Trp Thr Thr
Asp Trp Tyr Gln Asp 275 280 285His Lys Ala Ile Asp Ser Pro Cys Cys
Thr Ala Val Asn Pro Arg Gly 290 295 300Gly His Arg Glu Ala Ser Tyr
Asp Thr Arg Leu Pro Asp Val Lys Ile305 310 315 320Pro Arg Lys Val
Thr Lys Gly Gly Ser His Leu Cys Ala Pro Asn Tyr 325 330 335Cys Arg
Arg Tyr Arg Pro Ala Ala Arg Met Ala Gln Pro Val Asp Thr 340 345
350Ala Ile Ser His Leu Gly Phe Arg Cys Ile Val Arg Arg Lys Met Glu
355 360 365Leu Asn Ala Gln 370631251DNABurkholderia fungorum
63atgaagagtg aaagagatcg agagcccgca aagtcgtccc gctcgaacgg gtcggtcgca
60gcaacccaaa cgcgcgccgg tcgcgtgcgc aaactaatgt tgtggggcgc cctgctcgtc
120atactgcccg cctgtgtcgg cgccgcggtc agttgggcct tcacgccgca
cgcacccgct 180cacccgcaaa tcgttttcgg cgacggcacg catggtccgc
tcggcatggc gtgggtgccc 240ggcggccagt tcctcatggg cagcgacgcc
aaacaggcgc aaccgaacga acgccccgcg 300cacaaggtca aggtgcacgg
cttctggatg gaccgccatc acgtgaccaa cgccgaattc 360cgccgcttcg
tcgaagcgac cggctacgtc accacggccg agaagaaacc cgactgggag
420accctgaaag tccagttgcc gcccggcacg ccgcgcccgc ccgagagcgc
gatggtggcg 480ggtgcaatgg tgttcgtcgg caccagccgt cccgtgccgc
tagacgacta ttcgcagtgg
540tggcgctatg tgcctggcgc taactggcgt catccagccg ggcctgagag
caacatcatc 600ggtaaagatg atcaccccgt ggttcaagtg tcctacgaag
atgcgcaggc ttatgcgaaa 660tgggccggca agcgtctgcc gaccgaagcc
gaatgggaat tcgccgcgcg cggcggcctc 720gaacaggcca cgtatgcgtg
gggcgatcag ttctctccca acggcaaaca gatggccaac 780gtctggcagg
gccagcagcc gcagtctttc cccgttgtca acccgaaagc gggtggcgcg
840ctcggtacaa gtccggtggg tactttcccg gccaacggct acggcctttc
cgacatgacc 900ggcaacgcct ggcagtgggt tgccgactgg tatcgcgcgg
atcagttcag gcgtgaggcg 960gtaagcacca gcgcgatcga caatccggtg
ggcccgagcg agtcgtggga ccccgcagac 1020cagggcgtgc ccgtcaacgc
gcccaagcgt gtcacacgcg gcggttcgtt cctctgcaac 1080gaaatctatt
gcctgagcta ccggcccagc gcgagacgcg gcaccgatcc ctacaacagc
1140atgtcgcatc tgggcttccg gctggtgatg gacgaagaca cctggaaaga
agccggtgct 1200cgccaggctt cggcgaaagc tgccggcgcg cctggaaccc
ctggcggcta g 125164416PRTBurkholderia fungorum 64Met Lys Ser Glu
Arg Asp Arg Glu Pro Ala Lys Ser Ser Arg Ser Asn1 5 10 15Gly Ser Val
Ala Ala Thr Gln Thr Arg Ala Gly Arg Val Arg Lys Leu 20 25 30Met Leu
Trp Gly Ala Leu Leu Val Ile Leu Pro Ala Cys Val Gly Ala 35 40 45Ala
Val Ser Trp Ala Phe Thr Pro His Ala Pro Ala His Pro Gln Ile 50 55
60Val Phe Gly Asp Gly Thr His Gly Pro Leu Gly Met Ala Trp Val Pro65
70 75 80Gly Gly Gln Phe Leu Met Gly Ser Asp Ala Lys Gln Ala Gln Pro
Asn 85 90 95Glu Arg Pro Ala His Lys Val Lys Val His Gly Phe Trp Met
Asp Arg 100 105 110His His Val Thr Asn Ala Glu Phe Arg Arg Phe Val
Glu Ala Thr Gly 115 120 125Tyr Val Thr Thr Ala Glu Lys Lys Pro Asp
Trp Glu Thr Leu Lys Val 130 135 140Gln Leu Pro Pro Gly Thr Pro Arg
Pro Pro Glu Ser Ala Met Val Ala145 150 155 160Gly Ala Met Val Phe
Val Gly Thr Ser Arg Pro Val Pro Leu Asp Asp 165 170 175Tyr Ser Gln
Trp Trp Arg Tyr Val Pro Gly Ala Asn Trp Arg His Pro 180 185 190Ala
Gly Pro Glu Ser Asn Ile Ile Gly Lys Asp Asp His Pro Val Val 195 200
205Gln Val Ser Tyr Glu Asp Ala Gln Ala Tyr Ala Lys Trp Ala Gly Lys
210 215 220Arg Leu Pro Thr Glu Ala Glu Trp Glu Phe Ala Ala Arg Gly
Gly Leu225 230 235 240Glu Gln Ala Thr Tyr Ala Trp Gly Asp Gln Phe
Ser Pro Asn Gly Lys 245 250 255Gln Met Ala Asn Val Trp Gln Gly Gln
Gln Pro Gln Ser Phe Pro Val 260 265 270Val Asn Pro Lys Ala Gly Gly
Ala Leu Gly Thr Ser Pro Val Gly Thr 275 280 285Phe Pro Ala Asn Gly
Tyr Gly Leu Ser Asp Met Thr Gly Asn Ala Trp 290 295 300Gln Trp Val
Ala Asp Trp Tyr Arg Ala Asp Gln Phe Arg Arg Glu Ala305 310 315
320Val Ser Thr Ser Ala Ile Asp Asn Pro Val Gly Pro Ser Glu Ser Trp
325 330 335Asp Pro Ala Asp Gln Gly Val Pro Val Asn Ala Pro Lys Arg
Val Thr 340 345 350Arg Gly Gly Ser Phe Leu Cys Asn Glu Ile Tyr Cys
Leu Ser Tyr Arg 355 360 365Pro Ser Ala Arg Arg Gly Thr Asp Pro Tyr
Asn Ser Met Ser His Leu 370 375 380Gly Phe Arg Leu Val Met Asp Glu
Asp Thr Trp Lys Glu Ala Gly Ala385 390 395 400Arg Gln Ala Ser Ala
Lys Ala Ala Gly Ala Pro Gly Thr Pro Gly Gly 405 410
41565912DNASinorhizobium meliloti 65atggtctggg ttcccggagc
gaccttcatg atggggtcga acgaccatta cccggaggaa 60gcgcccgtgc atccggtaac
cgtcgacgga ttctggatcg atgtgacacc ggtaacgaac 120cgccagtttc
tcgaattcgt aaatgcgacg gggcatgtga ccttcgcgga aagaaagccg
180cgcgccgaag actatccggg cgctccgcca tccaatctaa gggccggttc
gctcgtcttc 240acacccccga agcgaccgct gcagggaacg gatatatcgc
agtggtggat attcacgctg 300ggtgccaact ggcggcaccc gctcgggcgc
aagagcagca tcggagcgat tctggatcat 360ccggtcgtcc atgtcgctta
cagcgacgca aaggcctatg ccgaatgggc cggcaaggac 420ctcccgaccg
agaccgagtg ggagctggcg gcccgcggcg gcctcgatgg ggctgaattt
480tcctggggcg gcgagcttgc gccgggcgga aatcacatgg ccaatacttg
gcagggaagt 540tttccggtcg agaattctat ggacgatggt ttcgcgcgaa
catcgccggt cagattttac 600ccgccgaacg gctacggcct ctacgacatg
atcggcaatg tgtgggagtg gaccacggat 660tactggtccg tgcgccaccc
ggaagcggcc gccaagcctt gctgcattcc gagcaatccc 720cgcaatgccg
atgccgatgc gagtatcgat ccggcggcga gcgtgaaagt tccgcgccgg
780gtgctcaagg gtggatcgca tctctgcgcg ccgaactact gccggcggta
ccgccctgcg 840gcgaggcacg cccaggaaat cgacacgacg accagccatg
tcggtttccg atgtgtcagg 900cgcgttcgat aa 91266303PRTSinorhizobium
meliloti 66Met Val Trp Val Pro Gly Ala Thr Phe Met Met Gly Ser Asn
Asp His1 5 10 15Tyr Pro Glu Glu Ala Pro Val His Pro Val Thr Val Asp
Gly Phe Trp 20 25 30Ile Asp Val Thr Pro Val Thr Asn Arg Gln Phe Leu
Glu Phe Val Asn 35 40 45Ala Thr Gly His Val Thr Phe Ala Glu Arg Lys
Pro Arg Ala Glu Asp 50 55 60Tyr Pro Gly Ala Pro Pro Ser Asn Leu Arg
Ala Gly Ser Leu Val Phe65 70 75 80Thr Pro Pro Lys Arg Pro Leu Gln
Gly Thr Asp Ile Ser Gln Trp Trp 85 90 95Ile Phe Thr Leu Gly Ala Asn
Trp Arg His Pro Leu Gly Arg Lys Ser 100 105 110Ser Ile Gly Ala Ile
Leu Asp His Pro Val Val His Val Ala Tyr Ser 115 120 125Asp Ala Lys
Ala Tyr Ala Glu Trp Ala Gly Lys Asp Leu Pro Thr Glu 130 135 140Thr
Glu Trp Glu Leu Ala Ala Arg Gly Gly Leu Asp Gly Ala Glu Phe145 150
155 160Ser Trp Gly Gly Glu Leu Ala Pro Gly Gly Asn His Met Ala Asn
Thr 165 170 175Trp Gln Gly Ser Phe Pro Val Glu Asn Ser Met Asp Asp
Gly Phe Ala 180 185 190Arg Thr Ser Pro Val Arg Phe Tyr Pro Pro Asn
Gly Tyr Gly Leu Tyr 195 200 205Asp Met Ile Gly Asn Val Trp Glu Trp
Thr Thr Asp Tyr Trp Ser Val 210 215 220Arg His Pro Glu Ala Ala Ala
Lys Pro Cys Cys Ile Pro Ser Asn Pro225 230 235 240Arg Asn Ala Asp
Ala Asp Ala Ser Ile Asp Pro Ala Ala Ser Val Lys 245 250 255Val Pro
Arg Arg Val Leu Lys Gly Gly Ser His Leu Cys Ala Pro Asn 260 265
270Tyr Cys Arg Arg Tyr Arg Pro Ala Ala Arg His Ala Gln Glu Ile Asp
275 280 285Thr Thr Thr Ser His Val Gly Phe Arg Cys Val Arg Arg Val
Arg 290 295 300671065DNAMicroscilla sp. 67atgaaataca tttttttagt
tcttttctta tgggccttga cccgatgtac cggaaagtat 60gaggacaaga gagtggaaac
tgatacttcc agaccaaaag ccgaagcgtc agatataaaa 120gttcccgaag
gaatggctta tattcccgcg ggccagtaca tgatgggagg taaatcagac
180caggcttata aggatgaata tccccgccat aacgtgaagg tttcggcttt
ttatatggac 240cttacagaag tgaccaatgc ggagtttaag cggtttgtag
acgaaacggg ctacgtgacc 300attgctgaga aagatattga ctgggaagag
ttaaagtctc aggtgccaca gggtaccccg 360aagcctcctg attctgtgct
tcaggcaggt tcactggttt tcaagcagac agatgaaccc 420gtttctctcc
aggattattc acagtggtgg gaatggacta tcggagccaa ctggcgaaat
480ccggagggtc caggtagtac gattgaggat cgtatggatc atccggtggt
acacgtttcc 540tttgaagatg tccaagcgta tgcggattgg gccggtaagc
gcctgcctac tgaggcagaa 600tgggaatggg ccgccatggg aggccaaaat
gacgtgaaat atccatgggg aaatgaatcg 660gtcgaacaag catccgataa
agcaaacttt tggcagggga attttccaca tcaaaactat 720gccctcgatg
gattcgaacg caccgcccct gtacgctcct tcccagcgaa tgggtacggc
780ctatatgata tggctggcaa tgtgtgggaa tggtgccagg ataagtatga
tgtcaatgct 840tatgaaagct ataagcaaaa aggactgaca gaagacccca
cgggttctga gcactacaac 900gaccctaggg aaccgtatac tcctaagcat
gtgatcagag ggggttcttt cctatgcaat 960gacagctact gtagtgggta
tcgtgtttca cgtcgtatga gttccagtag agattcaggt 1020tttaatcata
cgggattcag gtgtgtgaaa gatgtaaatg gatag 106568354PRTMicroscilla sp.
68Met Lys Tyr Ile Phe Leu Val Leu Phe Leu Trp Ala Leu Thr Arg Cys1
5 10 15Thr Gly Lys Tyr Glu Asp Lys Arg Val Glu Thr Asp Thr Ser Arg
Pro 20 25 30Lys Ala Glu Ala Ser Asp Ile Lys Val Pro Glu Gly Met Ala
Tyr Ile 35 40 45Pro Ala Gly Gln Tyr Met Met Gly Gly Lys Ser Asp Gln
Ala Tyr Lys 50 55 60Asp Glu Tyr Pro Arg His Asn Val Lys Val Ser Ala
Phe Tyr Met Asp65 70 75 80Leu Thr Glu Val Thr Asn Ala Glu Phe Lys
Arg Phe Val Asp Glu Thr 85 90 95Gly Tyr Val Thr Ile Ala Glu Lys Asp
Ile Asp Trp Glu Glu Leu Lys 100 105 110Ser Gln Val Pro Gln Gly Thr
Pro Lys Pro Pro Asp Ser Val Leu Gln 115 120 125Ala Gly Ser Leu Val
Phe Lys Gln Thr Asp Glu Pro Val Ser Leu Gln 130 135 140Asp Tyr Ser
Gln Trp Trp Glu Trp Thr Ile Gly Ala Asn Trp Arg Asn145 150 155
160Pro Glu Gly Pro Gly Ser Thr Ile Glu Asp Arg Met Asp His Pro Val
165 170 175Val His Val Ser Phe Glu Asp Val Gln Ala Tyr Ala Asp Trp
Ala Gly 180 185 190Lys Arg Leu Pro Thr Glu Ala Glu Trp Glu Trp Ala
Ala Met Gly Gly 195 200 205Gln Asn Asp Val Lys Tyr Pro Trp Gly Asn
Glu Ser Val Glu Gln Ala 210 215 220Ser Asp Lys Ala Asn Phe Trp Gln
Gly Asn Phe Pro His Gln Asn Tyr225 230 235 240Ala Leu Asp Gly Phe
Glu Arg Thr Ala Pro Val Arg Ser Phe Pro Ala 245 250 255Asn Gly Tyr
Gly Leu Tyr Asp Met Ala Gly Asn Val Trp Glu Trp Cys 260 265 270Gln
Asp Lys Tyr Asp Val Asn Ala Tyr Glu Ser Tyr Lys Gln Lys Gly 275 280
285Leu Thr Glu Asp Pro Thr Gly Ser Glu His Tyr Asn Asp Pro Arg Glu
290 295 300Pro Tyr Thr Pro Lys His Val Ile Arg Gly Gly Ser Phe Leu
Cys Asn305 310 315 320Asp Ser Tyr Cys Ser Gly Tyr Arg Val Ser Arg
Arg Met Ser Ser Ser 325 330 335Arg Asp Ser Gly Phe Asn His Thr Gly
Phe Arg Cys Val Lys Asp Val 340 345 350Asn Gly69876DNAPseudomonas
putida KT2440 69atggtgcacg tgccgggcgg cgagttcagc tttggttcaa
gccgctttta cgacgaagaa 60ggcccgcctc accccgccaa ggtgtccggc ttctggattg
acgtgcatcc ggtcaccaac 120gcccagttcg cgcgcttcgt caaggccacg
gggtatgtca cccatgccga gcgcggtacc 180cgtgtcgagg acgaccctgc
cctgcccgac gcgctgcgga taccgggtgc gatggtgttt 240catcagggtg
cggacgtgct cggccccggc tggcagttcg tgcccggcgc caactggcga
300cacccgcaag ggccgggcag cagcctggcc gggctggaca accatccggt
ggtgcagatc 360gccctggaag atgcccaggc ctatgcccgc tgggcaggcc
gcgaactgcc cagcgaggcg 420cagctggaat acgccatgcg cggcggcctg
accgatgccg acttcagctg gggtaccacc 480gagcagccca agggcaagct
catggccaat acctggcagg gtcagttccc ttatcgcaat 540gcggcgaagg
atggttttac cggtacatcg cccgtgggtt gcttcccggc caacggcttt
600ggcctgttcg atgccggcgg caatgtctgg gagctgactc gcacgggcta
tcggccaggc 660catgacgcac agcgcgacgc caagctcgac ccctcaggcc
cggccctgag tgacagcttc 720gacccggcag accccggcgt gccggtggcg
gtaatcaaag gcggctcgca cctgtgttcg 780gcggaccgct gcatgcgcta
ccgcccctcg gcacgccagc cgcagccggt gttcatgacg 840acctcgcacg
tgggtttcag aacgattcgg caatga 87670291PRTPseudomonas putida KT2440
70Met Val His Val Pro Gly Gly Glu Phe Ser Phe Gly Ser Ser Arg Phe1
5 10 15Tyr Asp Glu Glu Gly Pro Pro His Pro Ala Lys Val Ser Gly Phe
Trp 20 25 30Ile Asp Val His Pro Val Thr Asn Ala Gln Phe Ala Arg Phe
Val Lys 35 40 45Ala Thr Gly Tyr Val Thr His Ala Glu Arg Gly Thr Arg
Val Glu Asp 50 55 60Asp Pro Ala Leu Pro Asp Ala Leu Arg Ile Pro Gly
Ala Met Val Phe65 70 75 80His Gln Gly Ala Asp Val Leu Gly Pro Gly
Trp Gln Phe Val Pro Gly 85 90 95Ala Asn Trp Arg His Pro Gln Gly Pro
Gly Ser Ser Leu Ala Gly Leu 100 105 110Asp Asn His Pro Val Val Gln
Ile Ala Leu Glu Asp Ala Gln Ala Tyr 115 120 125Ala Arg Trp Ala Gly
Arg Glu Leu Pro Ser Glu Ala Gln Leu Glu Tyr 130 135 140Ala Met Arg
Gly Gly Leu Thr Asp Ala Asp Phe Ser Trp Gly Thr Thr145 150 155
160Glu Gln Pro Lys Gly Lys Leu Met Ala Asn Thr Trp Gln Gly Gln Phe
165 170 175Pro Tyr Arg Asn Ala Ala Lys Asp Gly Phe Thr Gly Thr Ser
Pro Val 180 185 190Gly Cys Phe Pro Ala Asn Gly Phe Gly Leu Phe Asp
Ala Gly Gly Asn 195 200 205Val Trp Glu Leu Thr Arg Thr Gly Tyr Arg
Pro Gly His Asp Ala Gln 210 215 220Arg Asp Ala Lys Leu Asp Pro Ser
Gly Pro Ala Leu Ser Asp Ser Phe225 230 235 240Asp Pro Ala Asp Pro
Gly Val Pro Val Ala Val Ile Lys Gly Gly Ser 245 250 255His Leu Cys
Ser Ala Asp Arg Cys Met Arg Tyr Arg Pro Ser Ala Arg 260 265 270Gln
Pro Gln Pro Val Phe Met Thr Thr Ser His Val Gly Phe Arg Thr 275 280
285Ile Arg Gln 29071780DNARalstonia metallidurans 71atggtcgcgg
gcgggatggt gttcgtcggc accaacagcc cggtgccgct gcgcgaatac 60tggcgctggt
ggcgcttcgt acctggcgcg gactggcgtc acccgaccgg cccgggcagt
120tccatcgaag gcaaggacaa tcatcccgtc gtgcaggtct cgtatgaaga
cgcgcaggcg 180tacgccaagt gggccggcaa gcgtctgccc accgaggccg
agtgggagtt tgccgcccgt 240ggcggcctgg agcaggccac ctacgcctgg
ggtgacaagt tcgcgccgga tggccggcag 300atggcgaatg tctggcaggg
ccagcaggtg cagccgttcc cggtggtcag cgccaaggcg 360ggcggcgcgg
ctggcaccag tgctgtcggc acgttcccgg gcaatggcta tgggctctat
420gacatgaccg gcaacgcctg gcagtgggtg gccgactggt atcgcgcgga
ccagttccgc 480cgcgaagcca cggtggcggc agtgctgcag aatccgaccg
gcccggccga ttcgtgggac 540ccgaccgaac ctggcgtgcc ggtgtcggcg
cccaagcggg tcacgcgcgg tggctcgttc 600ctctgcaacg aggacttctg
cctcagctac cgcccgagtg cccggcgcgg taccgacccg 660tacaccagca
tgtcgcacct aggcttccgg ctcgtgatgg atgacgcccg ttgggcagaa
720gttcgcaagc agccagccgt ggcaatggcc gcgggcgggc agcagaacgt
gcagaaataa 78072259PRTRalstonia metallidurans 72Met Val Ala Gly Gly
Met Val Phe Val Gly Thr Asn Ser Pro Val Pro1 5 10 15Leu Arg Glu Tyr
Trp Arg Trp Trp Arg Phe Val Pro Gly Ala Asp Trp 20 25 30Arg His Pro
Thr Gly Pro Gly Ser Ser Ile Glu Gly Lys Asp Asn His 35 40 45Pro Val
Val Gln Val Ser Tyr Glu Asp Ala Gln Ala Tyr Ala Lys Trp 50 55 60Ala
Gly Lys Arg Leu Pro Thr Glu Ala Glu Trp Glu Phe Ala Ala Arg65 70 75
80Gly Gly Leu Glu Gln Ala Thr Tyr Ala Trp Gly Asp Lys Phe Ala Pro
85 90 95Asp Gly Arg Gln Met Ala Asn Val Trp Gln Gly Gln Gln Val Gln
Pro 100 105 110Phe Pro Val Val Ser Ala Lys Ala Gly Gly Ala Ala Gly
Thr Ser Ala 115 120 125Val Gly Thr Phe Pro Gly Asn Gly Tyr Gly Leu
Tyr Asp Met Thr Gly 130 135 140Asn Ala Trp Gln Trp Val Ala Asp Trp
Tyr Arg Ala Asp Gln Phe Arg145 150 155 160Arg Glu Ala Thr Val Ala
Ala Val Leu Gln Asn Pro Thr Gly Pro Ala 165 170 175Asp Ser Trp Asp
Pro Thr Glu Pro Gly Val Pro Val Ser Ala Pro Lys 180 185 190Arg Val
Thr Arg Gly Gly Ser Phe Leu Cys Asn Glu Asp Phe Cys Leu 195 200
205Ser Tyr Arg Pro Ser Ala Arg Arg Gly Thr Asp Pro Tyr Thr Ser Met
210 215 220Ser His Leu Gly Phe Arg Leu Val Met Asp Asp Ala Arg Trp
Ala Glu225 230 235 240Val Arg Lys Gln Pro Ala Val Ala Met Ala Ala
Gly Gly Gln Gln Asn 245 250 255Val Gln Lys73876DNAProchlorococcus
marinus 73gtgaccacat ctttgccagt agagatggta accatccccg cagggctcta
tcgagttggc 60tgtgatcgct gctatccgga tggttcagtt cgctgctatc cggaggaaac
acccgcgcga 120gaagtgcagc ttgactcatt ccagatcgac gtagggccag
tcaccaatgc ccagttccga 180gctttcgtta gcgccacgca gcatctcaca
gtctcggagc taccacctga tccaacgctc 240tatcccgatc tagcgcccga
ggaacgcatc cctgaatcag ttgtctttca accgcctcca 300gcaacggtgg
atcgcagcaa acccttgagc tggtggaccc tcatggctgg ggctgattgg
360cgtcatcccc aaggacccga aagcacgatc gatggccttg atgatcaccc
tgtcgtgcat 420gtcgcctatg ccgacgccat cgcctatgcc cattgggctg
gcaagcgtct cccctctgct 480gaagagtggg
aagtagccgc ccgcgggggt cttgtcgatg cccaatacgc ctgggggaat
540gaactcactc ccaataaccg ctggatggcg aacatctggc aaggtccttt
cccttggcac 600aacgaggagc tagacggctg gttctggacc tcgcccgttg
gcagctttcc tgccaacggc 660tatggactct tggatgtttg cggcaatgtg
tgggaatgga ccaactctgt ttatcccgtg 720gcgtcaggcc accaggaacg
gcgaactatc aaaggcggat cgtttctctg cgcagataat 780tactgcgtac
gttatcgacc ctctgcacta caaggccaga cagtagacac tgccacctgt
840cacatgggct ttcgctgtgc aaaaggaggg ccttga
87674291PRTProchlorococcus marinus 74Met Thr Thr Ser Leu Pro Val
Glu Met Val Thr Ile Pro Ala Gly Leu1 5 10 15Tyr Arg Val Gly Cys Asp
Arg Cys Tyr Pro Asp Gly Ser Val Arg Cys 20 25 30Tyr Pro Glu Glu Thr
Pro Ala Arg Glu Val Gln Leu Asp Ser Phe Gln 35 40 45Ile Asp Val Gly
Pro Val Thr Asn Ala Gln Phe Arg Ala Phe Val Ser 50 55 60Ala Thr Gln
His Leu Thr Val Ser Glu Leu Pro Pro Asp Pro Thr Leu65 70 75 80Tyr
Pro Asp Leu Ala Pro Glu Glu Arg Ile Pro Glu Ser Val Val Phe 85 90
95Gln Pro Pro Pro Ala Thr Val Asp Arg Ser Lys Pro Leu Ser Trp Trp
100 105 110Thr Leu Met Ala Gly Ala Asp Trp Arg His Pro Gln Gly Pro
Glu Ser 115 120 125Thr Ile Asp Gly Leu Asp Asp His Pro Val Val His
Val Ala Tyr Ala 130 135 140Asp Ala Ile Ala Tyr Ala His Trp Ala Gly
Lys Arg Leu Pro Ser Ala145 150 155 160Glu Glu Trp Glu Val Ala Ala
Arg Gly Gly Leu Val Asp Ala Gln Tyr 165 170 175Ala Trp Gly Asn Glu
Leu Thr Pro Asn Asn Arg Trp Met Ala Asn Ile 180 185 190Trp Gln Gly
Pro Phe Pro Trp His Asn Glu Glu Leu Asp Gly Trp Phe 195 200 205Trp
Thr Ser Pro Val Gly Ser Phe Pro Ala Asn Gly Tyr Gly Leu Leu 210 215
220Asp Val Cys Gly Asn Val Trp Glu Trp Thr Asn Ser Val Tyr Pro
Val225 230 235 240Ala Ser Gly His Gln Glu Arg Arg Thr Ile Lys Gly
Gly Ser Phe Leu 245 250 255Cys Ala Asp Asn Tyr Cys Val Arg Tyr Arg
Pro Ser Ala Leu Gln Gly 260 265 270Gln Thr Val Asp Thr Ala Thr Cys
His Met Gly Phe Arg Cys Ala Lys 275 280 285Gly Gly Pro
290751017DNACaulobacter crescentus CB15 75ttgggaaaac tgacggcgct
tcccgtcctg atgcttctgg cgctggccgg ctgcggccag 60ccggcgccca aggcttgcct
ggcggacctg ccggttccag atccccagaa ccgcacggcg 120ggtatggttc
ggctggcggg cggcgacttc cagatgggcg ctgcgccgct gcgtccggag
180gagggaccgc cccagacggt cacggtcccg ccgttctgga tcgatcagac
agaggtcacc 240aacgccgcct tcgcgcggtt cgtcgaggcc acgggttatc
gcaccgtggc cgagcgaccg 300ctcgaccccg cgcgctacgc ccacgtaccg
gcggcgcagc ggcgtccggc ctcgctcgtc 360ttcgtggggg cgaagggggc
gaggtcggac gatccttccc aatggtggca ggtgatcccc 420ggcgccgact
ggcggcatcc cgaaggtccc ggctcgaaca tccggggcag ggacgcctgg
480ccggtggtgc atatcgcgtg ggaggacgcc atggcctacg cccgctggct
gggccgtgac 540ctgcccacag aggccgaatg ggagtacgcc gcgcgcggcg
ggctggttgg caagcgctac 600acctggggcg accaggctca ggatcctgca
aagccgcgcg ccaatacttg gcaaggcgtg 660ttcccggccc aggaccttgg
caatgacggc ttcaaggcca agcccgcgcc ggtcggctgc 720ttcccgccca
acggctatgg cctgcgcgac atggccggca atgtctggga gtggacccgc
780gactggttca agccgggcct ggatccggtc agcgtcctcg aaaccggcgg
gccgcccgag 840gcccgcgcgc tggatcccga ggacccgaac acgcccaagc
acgtcgtgaa gggcggttcg 900ttcctgtgcg ccgacgacta ctgcttccgc
tatcgacctg cggcgcgaac gccggggccg 960ccggacagcg gcgcatcgca
tgtcggtttc cgcaccgtgc tccgcgccga gcgctga 101776338PRTCaulobacter
crescentus CB15 76Met Gly Lys Leu Thr Ala Leu Pro Val Leu Met Leu
Leu Ala Leu Ala1 5 10 15Gly Cys Gly Gln Pro Ala Pro Lys Ala Cys Leu
Ala Asp Leu Pro Val 20 25 30Pro Asp Pro Gln Asn Arg Thr Ala Gly Met
Val Arg Leu Ala Gly Gly 35 40 45Asp Phe Gln Met Gly Ala Ala Pro Leu
Arg Pro Glu Glu Gly Pro Pro 50 55 60Gln Thr Val Thr Val Pro Pro Phe
Trp Ile Asp Gln Thr Glu Val Thr65 70 75 80Asn Ala Ala Phe Ala Arg
Phe Val Glu Ala Thr Gly Tyr Arg Thr Val 85 90 95Ala Glu Arg Pro Leu
Asp Pro Ala Arg Tyr Ala His Val Pro Ala Ala 100 105 110Gln Arg Arg
Pro Ala Ser Leu Val Phe Val Gly Ala Lys Gly Ala Arg 115 120 125Ser
Asp Asp Pro Ser Gln Trp Trp Gln Val Ile Pro Gly Ala Asp Trp 130 135
140Arg His Pro Glu Gly Pro Gly Ser Asn Ile Arg Gly Arg Asp Ala
Trp145 150 155 160Pro Val Val His Ile Ala Trp Glu Asp Ala Met Ala
Tyr Ala Arg Trp 165 170 175Leu Gly Arg Asp Leu Pro Thr Glu Ala Glu
Trp Glu Tyr Ala Ala Arg 180 185 190Gly Gly Leu Val Gly Lys Arg Tyr
Thr Trp Gly Asp Gln Ala Gln Asp 195 200 205Pro Ala Lys Pro Arg Ala
Asn Thr Trp Gln Gly Val Phe Pro Ala Gln 210 215 220Asp Leu Gly Asn
Asp Gly Phe Lys Ala Lys Pro Ala Pro Val Gly Cys225 230 235 240Phe
Pro Pro Asn Gly Tyr Gly Leu Arg Asp Met Ala Gly Asn Val Trp 245 250
255Glu Trp Thr Arg Asp Trp Phe Lys Pro Gly Leu Asp Pro Val Ser Val
260 265 270Leu Glu Thr Gly Gly Pro Pro Glu Ala Arg Ala Leu Asp Pro
Glu Asp 275 280 285Pro Asn Thr Pro Lys His Val Val Lys Gly Gly Ser
Phe Leu Cys Ala 290 295 300Asp Asp Tyr Cys Phe Arg Tyr Arg Pro Ala
Ala Arg Thr Pro Gly Pro305 310 315 320Pro Asp Ser Gly Ala Ser His
Val Gly Phe Arg Thr Val Leu Arg Ala 325 330 335Glu
Arg77900DNAMycobacterium tuberculosis H37Rv 77gtgctgaccg agttggttga
cctgcccggc ggatcgttcc gcatgggctc gacgcgcttc 60taccccgaag aagcgccgat
tcataccgtg accgtgcgcg cctttgcggt agagcgacac 120ccggtgacca
acgcgcaatt tgccgaattc gtctccgcga caggctatgt gacggttgca
180gaacaacccc ttgaccccgg gctctaccca ggagtggacg cagcagacct
gtgtcccggt 240gcgatggtgt tttgtccgac ggccgggccg gtcgacctgc
gtgactggcg gcaatggtgg 300gactgggtac ctggcgcctg ctggcgccat
ccgtttggcc gggacagcga tatcgccgac 360cgagccggcc acccggtcgt
acaggtggcc tatccggacg ccgtggccta cgcacgatgg 420gctggtcgac
gcctaccgac cgaggccgag tgggagtacg cggcccgtgg cggaaccacg
480gcaacctatg cgtggggcga ccaggagaag ccggggggca tgctcatggc
gaacacctgg 540cagggccggt ttccttaccg caacgacggt gcattgggct
gggtgggaac ctccccggtg 600ggcaggtttc cggccaacgg gtttggcttg
ctcgacatga tcggaaacgt ttgggagtgg 660accaccaccg agttctatcc
acaccatcgc atcgatccac cctcgacggc ctgctgcgca 720ccggtcaagc
tcgctacagc cgccgacccg acgatcagcc agaccctcaa gggcggctcg
780cacctgtgcg cgccggagta ctgccaccgc taccgcccgg cggcgcgctc
gccgcagtcg 840caggacaccg cgaccaccca tatcgggttc cggtgcgtgg
ccgacccggt gtccgggtag 90078299PRTMycobacterium tuberculosis H37Rv
78Met Leu Thr Glu Leu Val Asp Leu Pro Gly Gly Ser Phe Arg Met Gly1
5 10 15Ser Thr Arg Phe Tyr Pro Glu Glu Ala Pro Ile His Thr Val Thr
Val 20 25 30Arg Ala Phe Ala Val Glu Arg His Pro Val Thr Asn Ala Gln
Phe Ala 35 40 45Glu Phe Val Ser Ala Thr Gly Tyr Val Thr Val Ala Glu
Gln Pro Leu 50 55 60Asp Pro Gly Leu Tyr Pro Gly Val Asp Ala Ala Asp
Leu Cys Pro Gly65 70 75 80Ala Met Val Phe Cys Pro Thr Ala Gly Pro
Val Asp Leu Arg Asp Trp 85 90 95Arg Gln Trp Trp Asp Trp Val Pro Gly
Ala Cys Trp Arg His Pro Phe 100 105 110Gly Arg Asp Ser Asp Ile Ala
Asp Arg Ala Gly His Pro Val Val Gln 115 120 125Val Ala Tyr Pro Asp
Ala Val Ala Tyr Ala Arg Trp Ala Gly Arg Arg 130 135 140Leu Pro Thr
Glu Ala Glu Trp Glu Tyr Ala Ala Arg Gly Gly Thr Thr145 150 155
160Ala Thr Tyr Ala Trp Gly Asp Gln Glu Lys Pro Gly Gly Met Leu Met
165 170 175Ala Asn Thr Trp Gln Gly Arg Phe Pro Tyr Arg Asn Asp Gly
Ala Leu 180 185 190Gly Trp Val Gly Thr Ser Pro Val Gly Arg Phe Pro
Ala Asn Gly Phe 195 200 205Gly Leu Leu Asp Met Ile Gly Asn Val Trp
Glu Trp Thr Thr Thr Glu 210 215 220Phe Tyr Pro His His Arg Ile Asp
Pro Pro Ser Thr Ala Cys Cys Ala225 230 235 240Pro Val Lys Leu Ala
Thr Ala Ala Asp Pro Thr Ile Ser Gln Thr Leu 245 250 255Lys Gly Gly
Ser His Leu Cys Ala Pro Glu Tyr Cys His Arg Tyr Arg 260 265 270Pro
Ala Ala Arg Ser Pro Gln Ser Gln Asp Thr Ala Thr Thr His Ile 275 280
285Gly Phe Arg Cys Val Ala Asp Pro Val Ser Gly 290
295797PRTArtificial Sequenceconserved domain in prokaryotes and
prokaryotes 79Arg Val Xaa Xaa Gly Xaa Ser1 580630DNAOncorhynchus
mykiss 80tcaggtggct gctgccccct ggtggttgcc tgtcagagga gcagactgga
ggcaccctga 60gggccccgac tccagcatca cagacaggct ggaccaccct gtgctgcatg
tgtcatggca 120ggacgctgtg gcctactgct cctgggccta caagagacta
cccacagagg ctgagtggga 180gtacgcctgc agagggggcc tacaggagag
actttacccg tgggggaaca aactgaaacc 240taaaggacag cactacgcca
acctctggca gggaaagttc cccacacaca actcagaaga 300ggacgggtac
actaaaacct caccagtgaa gtcatttcct gcaaatggct atggcctgta
360caacatggta gggaatgcat gggagtggac atctgactgg tggactgtac
accacaccac 420agatgaacag cacaacccgg caggtccacc atcaggcaca
gaccgagtga agaaaggagg 480ctcctacatg tgccataagt catactgtta
caggtacagg tgtgcagcac ggagtcagaa 540cacccctgac agctctgcct
ctaacctagg gttccgctgt gtctcccagg agcagccgta 600acctttcacc
ctcgaccctg acatgggtag 63081655DNADanio reriomisc_feature590n is a,
c, g, or t 81caaatggttt tatttacata aaaaaatcct cttagtttga agtgtaagac
agtgagatta 60gtgatgtttg aggttatgga tcaacatcag aggcgcagcg gaagcccaag
ttcgaggctg 120aactgtccgg tgtgttctga ctgcgagcgg cacacctgta
tctgtagcag taagacttgt 180ggcacatgta ggatcctcct ttcttgactc
tgtctgtccc tgattctggt ccctttgggt 240taaacttgtc ttctgcagtg
tgatgcacag tccaccagtc tgccgtccac tcccacgcat 300ttcccaccat
gtcatacagg ccaaagccat tgggaggaaa agacatcacc ggggatgtgt
360tggcatagcc gtcctctgca gtgttgtgat tagggaaatc tccctgccac
aggttagcat 420agtgctgccc tcttggcatt aatttatttc cccatgggta
catcctgtcc tgtagtcctc 480ctctacaggc caactcccat tcagcttctg
taggaagtct gcgtttggcc cattgacagt 540acgcccgtgc atcatcccat
gaaacatgca gagcagggtg attcattctn gtgtgtatgg 600ttgaatctgg
tcctttctgg tgtctncagt ctgcaccttt cactggtgac cacca
65582773DNAOryzias latipesmisc_feature690n is a, c, g, or t
82tctccttttt tccataaata acattagagt ccttacattc tgcctttaca tacattgtca
60gagacagtac aaaaaatctg cctttgtaaa attagagtta caaaaatata ttttagattt
120gacttcttca gaattgtcgg tggcagcaaa agaatcggat tgatctcatg
acaagagcgt 180gagccagaag ttcttggatc aaactgattt ggttctgtca
tcgtttctgt tcagcagcac 240agcgaaaacc aagattggaa gcggagctgt
ctggagtgtt ttggcttcga gcagcacatc 300tgtacctgta acaataagac
ttgtggcaca tgtacgagcc tcctttcttc accttatctg 360tgcctgacgg
aggacccgtt gggttgtgct gatggtctgt tgtgtggtgc acgctccacc
420agtctgaggt ccactcccat gcgttcccca ccatgtcata cagaccaaaa
gcattgcctg 480ggaaggacat caccggggag gttttagtgt agccatcctc
tgcagagttg tgtgctggga 540attccccctg ccagaggttg gcgtaatgct
gtccctttgg gtttagcttg tttccccagg 600ggtagagtct gtccttcagg
ccgcccctgc aggcaacctc ccactctgcc tcagtgggaa 660gtctcttgtt
gacccaggag cagtaagccn aggcatcatt cccagaaacc tgaacgacgg
720atgatccatc ctgtctgtga tgttggagtc tggancttca gggtgcttcc agt
77383566DNAXenopus laevismisc_feature6n is a, c, g, or t
83atatgnaact aaaggtaatg taattggaat gatggatttc acaaggnctg agagttccct
60attgctcctg cttgtcgtgt nacaggtcac ggagccggcg ccacacagcg aaatcccagg
120ttggaggccg agctgtcggg tgtattctga cttcgagcag cacagcgata
cctgtagcaa 180taggactcat ggcacatgta ggagcctcct ttcttcactc
tatcatttcc cgtagaaggt 240cctttcgggt tgtgaacctc atctgctgta
tgatgagtgt cccaccaatc agatgtccac 300tcccaagcat ttcccaccat
gttatataga ccataaccat tggctgggaa agcagttaca 360ggtgaagtct
gcacataacc atcctctcca gtgttttggg ttggaaaatc cccctgccag
420acattcgcat aatgttgtcc ctttggttcc agcttgttcc cccatggaaa
aatcctgttc 480tcaagtcccc cgcggcaggc gtattcccac tcagcttcag
ttggaaggcg tttacctgcc 540caggtgcaga aagcagaagc atcatt
56684647DNASilurana tropicalis 84gccgcttttt tttttttttt tttttttttt
catcacaaaa ataattttat taataaaata 60ggattttgtg ttcattctta ttatgaagga
caaggaatgt cattgaaatt tttgttttca 120caaggtcttg ggagttcctt
cctgctcagg tcattttgca gtggtcacgg agccgacgcc 180acgcagcgga
atcccaggtt agaggccgag ctgtcaggtg tattctgact tcgagcagca
240cagcgatacc tgtagcagta ggactcatgg cacatgtatg agcctccttt
tttcaccttg 300tcttttcccg taaaaggacc tttcgggttg taagtctcat
ctgctgtatg atgagtgtcc 360caccaatcgg atgtccactc ccaagcattt
cccaccatgt tatataggct ataaccattg 420gctgggaaag cggttacagg
tgaagtctgc acatagccgt cctctccagt gttttgggtt 480ggaaattccc
cctgccagac attcgcataa tgttctccct ttggttccag cttgttcccc
540cacggaaaaa gcctgttctc aagtccccca cgggaggcat attcccactc
agcttctgtc 600ggaaggcgct tacccgccca ggtgcagaag gcagaagcat cgttcca
64785636DNASalmo salar 85atagacattt tttaaatatt ttacaacaaa
atatattcca taaatatcca catgtcatgc 60ggtaatcctg catttcatga agaacactga
catcactggc tgtatgaaga ggtgcacttg 120atttgtttcg cctggcgggc
aagataggca gagttagcac cctagactag agccaatggc 180gaatggtaca
aaaagggaaa agtcagacta cccatgtcag ggtcaagggt aaaaggttac
240ggctgctcct gggagacaca gcggaaccct aggttagagg cagagctgtc
aggggtgttc 300tgactccgtg ctgcacacct gtacctgtaa cagtatgact
tatggcacat gtaggagcct 360cctttcttca ctcggtctgt gcctgatggt
ggacctgccg ggttgtgccg ttcatctgtg 420gtgtggtgta cagtccacca
gtcagatgtc cactcccatg cattccctac catgttgtac 480aggccatagc
catttgcagg aaatgacttc actggtgagg ttttggtgta cccgtcctct
540tctgagttgt gtgtggggaa ctttccctgc cagaggttgg cgtagtgctg
tcctttaggt 600ttcagtttgt tcccccacgg gtaaagtctg tcctgt
63686415DNASus scrofa 86agtttcctgt gaccaacacc ggagaggatg gcttccgagg
aactgcgcct gttgatgcct 60ttcctcccaa tggttatggc ctttacaata tagtagggaa
cgcctgggaa tggacctcag 120actggtggac cattcaccat gctgctgaag
aaacaattaa cccatcaagt tcttcctgct 180gcaccgaata acagagccgc
cactacgtga tgaaagcaga gaaaggcccc ccttctggga 240aagaccgggt
gaagaaaggg ggatcctata tgtgccataa gtcctactgc tacaggtacc
300gctgtgctgc tcgaagccag aacacgccgg acagctcggc ttcaaatctg
gggttccgct 360gtgcagctga ccaccagccc accacaggct gagtcaggaa
gagtcttccc gaatc 41587595DNABos taurus 87ccacgcgtcc gggggcaaca
aactgcagcc gaaaggccag cattatagcc aacatcttgg 60caaggcgagt ttcctgtgac
caacaccggg gaggacggct tccgagggac cgcgcctgtt 120gacgcctttc
ctcccaatgg ttattggctt atacaatata gtagggaacg cctgggagtg
180gacttcagac tggtggactg ttcaccattc tgctgaagaa acgattaacc
caaaaggccc 240cccttctggg aaagaccggg tgaagaaagg tggatcctac
atgtgccata aatcctattg 300ctacaggtat cgctgtgctg ctcgaagcca
gaacacaccc gacagctctg cttcgaatct 360gggattccgt tgtgcagctg
accacctgcc caccacaggc taagagccaa aaagagcctt 420cccgaacccg
agaagtcgtg tctactctgc acgcggcttc cctcagaagg ctgaacaacc
480tgctgtgaag aattcccacc ccaaggtggg ttacatacct tgcccagtgg
ccaaaggacc 540tatggcaaga ccaaattgct gagctgatca gcatgtgcgc
tttattgggg gatgg 595881611DNAHomo sapiensCDS(1)..(1608) 88atg cta
ctg ctg tgg gtg tcg gtg gtc gca gcc ttg gcg ctg gcg gta 48Met Leu
Leu Leu Trp Val Ser Val Val Ala Ala Leu Ala Leu Ala Val1 5 10 15ctg
gcc ccc gga gca ggg gag cag agg cgg aga gca gcc aaa gcg ccc 96Leu
Ala Pro Gly Ala Gly Glu Gln Arg Arg Arg Ala Ala Lys Ala Pro 20 25
30aat gtg gtg ctg gtc gtg agc gac tcc ttc gat gga agg tta aca ttt
144Asn Val Val Leu Val Val Ser Asp Ser Phe Asp Gly Arg Leu Thr Phe
35 40 45cat cca gga agt cag gta gtg aaa ctt cct ttt atc aac ttt atg
aag 192His Pro Gly Ser Gln Val Val Lys Leu Pro Phe Ile Asn Phe Met
Lys 50 55 60aca cgt ggg act tcc ttt ctg aat gcc tac aca aac tct cca
att tgt 240Thr Arg Gly Thr Ser Phe Leu Asn Ala Tyr Thr Asn Ser Pro
Ile Cys65 70 75 80tgc cca tca cgc gca gca atg tgg agt ggc ctc ttc
act cac tta aca 288Cys Pro Ser Arg Ala Ala Met Trp Ser Gly Leu Phe
Thr His Leu Thr 85 90 95gaa tct tgg aat aat ttt aag ggt cta gat cca
aat tat aca aca tgg 336Glu Ser Trp Asn Asn Phe Lys Gly Leu Asp Pro
Asn Tyr Thr Thr Trp 100 105 110atg gat gtc atg gag agg cat ggc tac
cga aca cag aaa ttt ggg aaa 384Met Asp Val Met Glu Arg His Gly Tyr
Arg Thr Gln Lys Phe Gly Lys 115 120 125ctg gac tat act tca gga cat
cac tcc att agt aat cgt gtg gaa gcg 432Leu Asp Tyr Thr Ser Gly His
His Ser Ile Ser Asn Arg Val Glu Ala 130 135 140tgg aca aga gat
gtt
gct ttc tta ctc aga caa gaa ggc agg ccc atg 480Trp Thr Arg Asp Val
Ala Phe Leu Leu Arg Gln Glu Gly Arg Pro Met145 150 155 160gtt aat
ctt atc cgt aac agg act aaa gtc aga gtg atg gaa agg gat 528Val Asn
Leu Ile Arg Asn Arg Thr Lys Val Arg Val Met Glu Arg Asp 165 170
175tgg cag aat aca gac aaa gca gta aac tgg tta aga aag gaa gca att
576Trp Gln Asn Thr Asp Lys Ala Val Asn Trp Leu Arg Lys Glu Ala Ile
180 185 190aat tac act gaa cca ttt gtt att tac ttg gga tta aat tta
cca cac 624Asn Tyr Thr Glu Pro Phe Val Ile Tyr Leu Gly Leu Asn Leu
Pro His 195 200 205cct tac cct tca cca tct tct gga gaa aat ttt gga
tct tca aca ttt 672Pro Tyr Pro Ser Pro Ser Ser Gly Glu Asn Phe Gly
Ser Ser Thr Phe 210 215 220cac aca tct ctt tat tgg ctt gaa aaa gtg
tct cat gat gcc atc aaa 720His Thr Ser Leu Tyr Trp Leu Glu Lys Val
Ser His Asp Ala Ile Lys225 230 235 240atc cca aag tgg tca cct ttg
tca gaa atg cac cct gta gat tat tac 768Ile Pro Lys Trp Ser Pro Leu
Ser Glu Met His Pro Val Asp Tyr Tyr 245 250 255tct tct tat aca aaa
aac tgc act gga aga ttt aca aaa aaa gaa att 816Ser Ser Tyr Thr Lys
Asn Cys Thr Gly Arg Phe Thr Lys Lys Glu Ile 260 265 270aag aat att
aga gca ttt tat tat gct atg tgt gct gag aca gat gcc 864Lys Asn Ile
Arg Ala Phe Tyr Tyr Ala Met Cys Ala Glu Thr Asp Ala 275 280 285atg
ctt ggt gaa att att ttg gcc ctt cat caa tta gat ctt ctt cag 912Met
Leu Gly Glu Ile Ile Leu Ala Leu His Gln Leu Asp Leu Leu Gln 290 295
300aaa act att gtc ata tac tcc tca gac cat gga gag ctg gcc atg gaa
960Lys Thr Ile Val Ile Tyr Ser Ser Asp His Gly Glu Leu Ala Met
Glu305 310 315 320cat cga cag ttt tat aaa atg agc atg tac gag gct
agt gca cat gtt 1008His Arg Gln Phe Tyr Lys Met Ser Met Tyr Glu Ala
Ser Ala His Val 325 330 335ccg ctt ttg atg atg gga cca gga att aaa
gcc ggc cta caa gta tca 1056Pro Leu Leu Met Met Gly Pro Gly Ile Lys
Ala Gly Leu Gln Val Ser 340 345 350aat gtg gtt tct ctt gtg gat att
tac cct acc atg ctt gat att gct 1104Asn Val Val Ser Leu Val Asp Ile
Tyr Pro Thr Met Leu Asp Ile Ala 355 360 365gga att cct ctg cct cag
aac ctg agt gga tac tct ttg ttg ccg tta 1152Gly Ile Pro Leu Pro Gln
Asn Leu Ser Gly Tyr Ser Leu Leu Pro Leu 370 375 380tca tca gaa aca
ttt aag aat gaa cat aaa gtc aaa aac ctg cat cca 1200Ser Ser Glu Thr
Phe Lys Asn Glu His Lys Val Lys Asn Leu His Pro385 390 395 400ccc
tgg att ctg agt gaa ttc cat gga tgt aat gtg aat gcc tcc acc 1248Pro
Trp Ile Leu Ser Glu Phe His Gly Cys Asn Val Asn Ala Ser Thr 405 410
415tac atg ctt cga act aac cac tgg aaa tat ata gcc tat tcg gat ggt
1296Tyr Met Leu Arg Thr Asn His Trp Lys Tyr Ile Ala Tyr Ser Asp Gly
420 425 430gca tca ata ttg cct caa ctc ttt gat ctt tcc tcg gat cca
gat gaa 1344Ala Ser Ile Leu Pro Gln Leu Phe Asp Leu Ser Ser Asp Pro
Asp Glu 435 440 445tta aca aat gtt gct gta aaa ttt cca gaa att act
tat tct ttg gat 1392Leu Thr Asn Val Ala Val Lys Phe Pro Glu Ile Thr
Tyr Ser Leu Asp 450 455 460cag aag ctt cat tcc att ata aac tac cct
aaa gtt tct gct tct gtc 1440Gln Lys Leu His Ser Ile Ile Asn Tyr Pro
Lys Val Ser Ala Ser Val465 470 475 480cac cag tat aat aaa gag cag
ttt atc aag tgg aaa caa agt ata gga 1488His Gln Tyr Asn Lys Glu Gln
Phe Ile Lys Trp Lys Gln Ser Ile Gly 485 490 495cag aat tat tca aac
gtt ata gca aat ctt agg tgg cac caa gac tgg 1536Gln Asn Tyr Ser Asn
Val Ile Ala Asn Leu Arg Trp His Gln Asp Trp 500 505 510cag aag gaa
cca agg aag tat gaa aat gca att gat cag tgg ctt aaa 1584Gln Lys Glu
Pro Arg Lys Tyr Glu Asn Ala Ile Asp Gln Trp Leu Lys 515 520 525acc
cat atg aat cca aga gca gtt tga 1611Thr His Met Asn Pro Arg Ala Val
530 53589536PRTHomo sapiens 89Met Leu Leu Leu Trp Val Ser Val Val
Ala Ala Leu Ala Leu Ala Val1 5 10 15Leu Ala Pro Gly Ala Gly Glu Gln
Arg Arg Arg Ala Ala Lys Ala Pro 20 25 30Asn Val Val Leu Val Val Ser
Asp Ser Phe Asp Gly Arg Leu Thr Phe 35 40 45His Pro Gly Ser Gln Val
Val Lys Leu Pro Phe Ile Asn Phe Met Lys 50 55 60Thr Arg Gly Thr Ser
Phe Leu Asn Ala Tyr Thr Asn Ser Pro Ile Cys65 70 75 80Cys Pro Ser
Arg Ala Ala Met Trp Ser Gly Leu Phe Thr His Leu Thr 85 90 95Glu Ser
Trp Asn Asn Phe Lys Gly Leu Asp Pro Asn Tyr Thr Thr Trp 100 105
110Met Asp Val Met Glu Arg His Gly Tyr Arg Thr Gln Lys Phe Gly Lys
115 120 125Leu Asp Tyr Thr Ser Gly His His Ser Ile Ser Asn Arg Val
Glu Ala 130 135 140Trp Thr Arg Asp Val Ala Phe Leu Leu Arg Gln Glu
Gly Arg Pro Met145 150 155 160Val Asn Leu Ile Arg Asn Arg Thr Lys
Val Arg Val Met Glu Arg Asp 165 170 175Trp Gln Asn Thr Asp Lys Ala
Val Asn Trp Leu Arg Lys Glu Ala Ile 180 185 190Asn Tyr Thr Glu Pro
Phe Val Ile Tyr Leu Gly Leu Asn Leu Pro His 195 200 205Pro Tyr Pro
Ser Pro Ser Ser Gly Glu Asn Phe Gly Ser Ser Thr Phe 210 215 220His
Thr Ser Leu Tyr Trp Leu Glu Lys Val Ser His Asp Ala Ile Lys225 230
235 240Ile Pro Lys Trp Ser Pro Leu Ser Glu Met His Pro Val Asp Tyr
Tyr 245 250 255Ser Ser Tyr Thr Lys Asn Cys Thr Gly Arg Phe Thr Lys
Lys Glu Ile 260 265 270Lys Asn Ile Arg Ala Phe Tyr Tyr Ala Met Cys
Ala Glu Thr Asp Ala 275 280 285Met Leu Gly Glu Ile Ile Leu Ala Leu
His Gln Leu Asp Leu Leu Gln 290 295 300Lys Thr Ile Val Ile Tyr Ser
Ser Asp His Gly Glu Leu Ala Met Glu305 310 315 320His Arg Gln Phe
Tyr Lys Met Ser Met Tyr Glu Ala Ser Ala His Val 325 330 335Pro Leu
Leu Met Met Gly Pro Gly Ile Lys Ala Gly Leu Gln Val Ser 340 345
350Asn Val Val Ser Leu Val Asp Ile Tyr Pro Thr Met Leu Asp Ile Ala
355 360 365Gly Ile Pro Leu Pro Gln Asn Leu Ser Gly Tyr Ser Leu Leu
Pro Leu 370 375 380Ser Ser Glu Thr Phe Lys Asn Glu His Lys Val Lys
Asn Leu His Pro385 390 395 400Pro Trp Ile Leu Ser Glu Phe His Gly
Cys Asn Val Asn Ala Ser Thr 405 410 415Tyr Met Leu Arg Thr Asn His
Trp Lys Tyr Ile Ala Tyr Ser Asp Gly 420 425 430Ala Ser Ile Leu Pro
Gln Leu Phe Asp Leu Ser Ser Asp Pro Asp Glu 435 440 445Leu Thr Asn
Val Ala Val Lys Phe Pro Glu Ile Thr Tyr Ser Leu Asp 450 455 460Gln
Lys Leu His Ser Ile Ile Asn Tyr Pro Lys Val Ser Ala Ser Val465 470
475 480His Gln Tyr Asn Lys Glu Gln Phe Ile Lys Trp Lys Gln Ser Ile
Gly 485 490 495Gln Asn Tyr Ser Asn Val Ile Ala Asn Leu Arg Trp His
Gln Asp Trp 500 505 510Gln Lys Glu Pro Arg Lys Tyr Glu Asn Ala Ile
Asp Gln Trp Leu Lys 515 520 525Thr His Met Asn Pro Arg Ala Val 530
535901722DNAHomo sapiensCDS(1)..(1719) 90atg ggg gcg ctg gca gga
ttc tgg atc ctc tgc ctc ctc act tat ggt 48Met Gly Ala Leu Ala Gly
Phe Trp Ile Leu Cys Leu Leu Thr Tyr Gly1 5 10 15tac ctg tcc tgg ggc
cag gcc tta gaa gag gag gaa gaa ggg gcc tta 96Tyr Leu Ser Trp Gly
Gln Ala Leu Glu Glu Glu Glu Glu Gly Ala Leu 20 25 30cta gct caa gct
gga gag aaa cta gag ccc agc aca act tcc acc tcc 144Leu Ala Gln Ala
Gly Glu Lys Leu Glu Pro Ser Thr Thr Ser Thr Ser 35 40 45cag ccc cat
ctc att ttc atc cta gcg gat gat cag gga ttt aga gat 192Gln Pro His
Leu Ile Phe Ile Leu Ala Asp Asp Gln Gly Phe Arg Asp 50 55 60gtg ggt
tac cac gga tct gag att aaa aca cct act ctt gac aag ctc 240Val Gly
Tyr His Gly Ser Glu Ile Lys Thr Pro Thr Leu Asp Lys Leu65 70 75
80gct gcc gaa gga gtt aaa ctg gag aac tac tat gtc cag cct att tgc
288Ala Ala Glu Gly Val Lys Leu Glu Asn Tyr Tyr Val Gln Pro Ile Cys
85 90 95aca cca tcc agg agt cag ttt att act gga aag tat cag ata cac
acc 336Thr Pro Ser Arg Ser Gln Phe Ile Thr Gly Lys Tyr Gln Ile His
Thr 100 105 110gga ctt caa cat tct atc ata aga cct acc caa ccc aac
tgt tta cct 384Gly Leu Gln His Ser Ile Ile Arg Pro Thr Gln Pro Asn
Cys Leu Pro 115 120 125ctg gac aat gcc acc cta cct cag aaa ctg aag
gag gtt gga tat tca 432Leu Asp Asn Ala Thr Leu Pro Gln Lys Leu Lys
Glu Val Gly Tyr Ser 130 135 140acg cat atg gtc gga aaa tgg cac ttg
ggt ttt tac aga aaa gaa tgc 480Thr His Met Val Gly Lys Trp His Leu
Gly Phe Tyr Arg Lys Glu Cys145 150 155 160atg ccc acc aga aga gga
ttt gat acc ttt ttt ggt tcc ctt ttg gga 528Met Pro Thr Arg Arg Gly
Phe Asp Thr Phe Phe Gly Ser Leu Leu Gly 165 170 175agt ggg gat tac
tat aca cac tac aaa tgt gac agt cct ggg atg tgt 576Ser Gly Asp Tyr
Tyr Thr His Tyr Lys Cys Asp Ser Pro Gly Met Cys 180 185 190ggc tat
gac ttg tat gaa aac gac aat gct gcc tgg gac tat gac aat 624Gly Tyr
Asp Leu Tyr Glu Asn Asp Asn Ala Ala Trp Asp Tyr Asp Asn 195 200
205ggc ata tac tcc aca cag atg tac act cag aga gta cag caa atc tta
672Gly Ile Tyr Ser Thr Gln Met Tyr Thr Gln Arg Val Gln Gln Ile Leu
210 215 220gct tcc cat aac ccc aca aag cct ata ttt tta tat att gcc
tat caa 720Ala Ser His Asn Pro Thr Lys Pro Ile Phe Leu Tyr Ile Ala
Tyr Gln225 230 235 240gct gtt cat tca cca ctg caa gct cct ggc agg
tat ttc gaa cac tac 768Ala Val His Ser Pro Leu Gln Ala Pro Gly Arg
Tyr Phe Glu His Tyr 245 250 255cga tcc att atc aac ata aac agg agg
aga tat gct gcc atg ctt tcc 816Arg Ser Ile Ile Asn Ile Asn Arg Arg
Arg Tyr Ala Ala Met Leu Ser 260 265 270tgc tta gat gaa gca atc aac
aac gtg aca ttg gct cta aag act tat 864Cys Leu Asp Glu Ala Ile Asn
Asn Val Thr Leu Ala Leu Lys Thr Tyr 275 280 285ggt ttc tat aac aac
agc att atc att tac tct tca gat aat ggt ggc 912Gly Phe Tyr Asn Asn
Ser Ile Ile Ile Tyr Ser Ser Asp Asn Gly Gly 290 295 300cag cct acg
gca gga ggg agt aac tgg cct ctc aga ggt agc aaa gga 960Gln Pro Thr
Ala Gly Gly Ser Asn Trp Pro Leu Arg Gly Ser Lys Gly305 310 315
320aca tat tgg gaa gga ggg atc cgg gct gta ggc ttt gtg cat agc cca
1008Thr Tyr Trp Glu Gly Gly Ile Arg Ala Val Gly Phe Val His Ser Pro
325 330 335ctt ctg aaa aac aag gga aca gtg tgt aag gaa ctt gtg cac
atc act 1056Leu Leu Lys Asn Lys Gly Thr Val Cys Lys Glu Leu Val His
Ile Thr 340 345 350gac tgg tac ccc act ctc att tca ctg gct gaa gga
cag att gat gag 1104Asp Trp Tyr Pro Thr Leu Ile Ser Leu Ala Glu Gly
Gln Ile Asp Glu 355 360 365gac att caa cta gat ggc tat gat atc tgg
gag acc ata agt gag ggt 1152Asp Ile Gln Leu Asp Gly Tyr Asp Ile Trp
Glu Thr Ile Ser Glu Gly 370 375 380ctt cgc tca ccc cga gta gat att
ttg cat aac att gac ccc ata tac 1200Leu Arg Ser Pro Arg Val Asp Ile
Leu His Asn Ile Asp Pro Ile Tyr385 390 395 400acc aag gca aaa aat
ggc tcc tgg gca gca ggc tat ggg atc tgg aac 1248Thr Lys Ala Lys Asn
Gly Ser Trp Ala Ala Gly Tyr Gly Ile Trp Asn 405 410 415act gca atc
cag tca gcc atc aga gtg cag cac tgg aaa ttg ctt aca 1296Thr Ala Ile
Gln Ser Ala Ile Arg Val Gln His Trp Lys Leu Leu Thr 420 425 430gga
aat cct ggc tac agc gac tgg gtc ccc cct cag tct ttc agc aac 1344Gly
Asn Pro Gly Tyr Ser Asp Trp Val Pro Pro Gln Ser Phe Ser Asn 435 440
445ctg gga ccg aac cgg tgg cac aat gaa cgg atc acc ttg tca act ggc
1392Leu Gly Pro Asn Arg Trp His Asn Glu Arg Ile Thr Leu Ser Thr Gly
450 455 460aaa agt gta tgg ctt ttc aac atc aca gcc gac cca tat gag
agg gtg 1440Lys Ser Val Trp Leu Phe Asn Ile Thr Ala Asp Pro Tyr Glu
Arg Val465 470 475 480gac cta tct aac agg tat cca gga atc gtg aag
aag ctc cta cgg agg 1488Asp Leu Ser Asn Arg Tyr Pro Gly Ile Val Lys
Lys Leu Leu Arg Arg 485 490 495ctc tca cag ttc aac aaa act gca gtg
ccg gtc agg tat ccc ccc aaa 1536Leu Ser Gln Phe Asn Lys Thr Ala Val
Pro Val Arg Tyr Pro Pro Lys 500 505 510gac ccc aga agt aac cct agg
ctc aat gga ggg gtc tgg gga cca tgg 1584Asp Pro Arg Ser Asn Pro Arg
Leu Asn Gly Gly Val Trp Gly Pro Trp 515 520 525tat aaa gag gaa acc
aag aaa aag aag cca agc aaa aat cag gct gag 1632Tyr Lys Glu Glu Thr
Lys Lys Lys Lys Pro Ser Lys Asn Gln Ala Glu 530 535 540aaa aag caa
aag aaa agc aaa aaa aag aag aag aaa cag cag aaa gca 1680Lys Lys Gln
Lys Lys Ser Lys Lys Lys Lys Lys Lys Gln Gln Lys Ala545 550 555
560gtc tca ggt tca act tgc cat tca ggt gtt act tgt gga taa 1722Val
Ser Gly Ser Thr Cys His Ser Gly Val Thr Cys Gly 565 57091573PRTHomo
sapiens 91Met Gly Ala Leu Ala Gly Phe Trp Ile Leu Cys Leu Leu Thr
Tyr Gly1 5 10 15Tyr Leu Ser Trp Gly Gln Ala Leu Glu Glu Glu Glu Glu
Gly Ala Leu 20 25 30Leu Ala Gln Ala Gly Glu Lys Leu Glu Pro Ser Thr
Thr Ser Thr Ser 35 40 45Gln Pro His Leu Ile Phe Ile Leu Ala Asp Asp
Gln Gly Phe Arg Asp 50 55 60Val Gly Tyr His Gly Ser Glu Ile Lys Thr
Pro Thr Leu Asp Lys Leu65 70 75 80Ala Ala Glu Gly Val Lys Leu Glu
Asn Tyr Tyr Val Gln Pro Ile Cys 85 90 95Thr Pro Ser Arg Ser Gln Phe
Ile Thr Gly Lys Tyr Gln Ile His Thr 100 105 110Gly Leu Gln His Ser
Ile Ile Arg Pro Thr Gln Pro Asn Cys Leu Pro 115 120 125Leu Asp Asn
Ala Thr Leu Pro Gln Lys Leu Lys Glu Val Gly Tyr Ser 130 135 140Thr
His Met Val Gly Lys Trp His Leu Gly Phe Tyr Arg Lys Glu Cys145 150
155 160Met Pro Thr Arg Arg Gly Phe Asp Thr Phe Phe Gly Ser Leu Leu
Gly 165 170 175Ser Gly Asp Tyr Tyr Thr His Tyr Lys Cys Asp Ser Pro
Gly Met Cys 180 185 190Gly Tyr Asp Leu Tyr Glu Asn Asp Asn Ala Ala
Trp Asp Tyr Asp Asn 195 200 205Gly Ile Tyr Ser Thr Gln Met Tyr Thr
Gln Arg Val Gln Gln Ile Leu 210 215 220Ala Ser His Asn Pro Thr Lys
Pro Ile Phe Leu Tyr Ile Ala Tyr Gln225 230 235 240Ala Val His Ser
Pro Leu Gln Ala Pro Gly Arg Tyr Phe Glu His Tyr 245 250 255Arg Ser
Ile Ile Asn Ile Asn Arg Arg Arg Tyr Ala Ala Met Leu Ser 260 265
270Cys Leu Asp Glu Ala Ile Asn Asn Val Thr Leu Ala Leu Lys Thr Tyr
275 280 285Gly Phe Tyr Asn Asn Ser Ile Ile Ile Tyr Ser Ser Asp Asn
Gly Gly 290 295 300Gln Pro Thr Ala Gly Gly Ser Asn Trp Pro Leu Arg
Gly Ser Lys Gly305 310 315 320Thr Tyr Trp Glu Gly Gly Ile Arg Ala
Val Gly Phe Val His Ser Pro 325 330 335Leu Leu Lys Asn Lys Gly Thr
Val Cys Lys Glu Leu Val His Ile Thr 340 345 350Asp Trp Tyr Pro Thr
Leu Ile Ser Leu Ala Glu Gly Gln Ile Asp Glu 355 360 365Asp Ile Gln
Leu Asp Gly Tyr Asp Ile Trp Glu Thr Ile Ser
Glu Gly 370 375 380Leu Arg Ser Pro Arg Val Asp Ile Leu His Asn Ile
Asp Pro Ile Tyr385 390 395 400Thr Lys Ala Lys Asn Gly Ser Trp Ala
Ala Gly Tyr Gly Ile Trp Asn 405 410 415Thr Ala Ile Gln Ser Ala Ile
Arg Val Gln His Trp Lys Leu Leu Thr 420 425 430Gly Asn Pro Gly Tyr
Ser Asp Trp Val Pro Pro Gln Ser Phe Ser Asn 435 440 445Leu Gly Pro
Asn Arg Trp His Asn Glu Arg Ile Thr Leu Ser Thr Gly 450 455 460Lys
Ser Val Trp Leu Phe Asn Ile Thr Ala Asp Pro Tyr Glu Arg Val465 470
475 480Asp Leu Ser Asn Arg Tyr Pro Gly Ile Val Lys Lys Leu Leu Arg
Arg 485 490 495Leu Ser Gln Phe Asn Lys Thr Ala Val Pro Val Arg Tyr
Pro Pro Lys 500 505 510Asp Pro Arg Ser Asn Pro Arg Leu Asn Gly Gly
Val Trp Gly Pro Trp 515 520 525Tyr Lys Glu Glu Thr Lys Lys Lys Lys
Pro Ser Lys Asn Gln Ala Glu 530 535 540Lys Lys Gln Lys Lys Ser Lys
Lys Lys Lys Lys Lys Gln Gln Lys Ala545 550 555 560Val Ser Gly Ser
Thr Cys His Ser Gly Val Thr Cys Gly 565 570921710DNAHomo
sapiensCDS(1)..(1707) 92atg cac acc ctc act ggc ttc tcc ctg gtc agc
ctg ctc agc ttc ggc 48Met His Thr Leu Thr Gly Phe Ser Leu Val Ser
Leu Leu Ser Phe Gly1 5 10 15tac ctg tcc tgg gac tgg gcc aag ccg agc
ttc gtg gcc gac ggg ccc 96Tyr Leu Ser Trp Asp Trp Ala Lys Pro Ser
Phe Val Ala Asp Gly Pro 20 25 30ggg gag gct ggc gag cag ccc tcg gcc
gct ccg ccc cag cct ccc cac 144Gly Glu Ala Gly Glu Gln Pro Ser Ala
Ala Pro Pro Gln Pro Pro His 35 40 45atc atc ttc atc ctc acg gac gac
caa ggc tac cac gac gtg ggc tac 192Ile Ile Phe Ile Leu Thr Asp Asp
Gln Gly Tyr His Asp Val Gly Tyr 50 55 60cat ggt tca gat atc gag acc
cct acg ctg gac agg ctg gcg gcc aag 240His Gly Ser Asp Ile Glu Thr
Pro Thr Leu Asp Arg Leu Ala Ala Lys65 70 75 80ggg gtc aag ttg gag
aat tat tac atc cag ccc atc tgc acg cct tcg 288Gly Val Lys Leu Glu
Asn Tyr Tyr Ile Gln Pro Ile Cys Thr Pro Ser 85 90 95cgg agc cag ctc
ctc act ggc agg tac cag atc cac aca gga ctc cag 336Arg Ser Gln Leu
Leu Thr Gly Arg Tyr Gln Ile His Thr Gly Leu Gln 100 105 110cat tcc
atc atc cgc cca cag cag ccc aac tgc ctg ccc ctg gac cag 384His Ser
Ile Ile Arg Pro Gln Gln Pro Asn Cys Leu Pro Leu Asp Gln 115 120
125gtg aca ctg cca cag aag ctg cag gag gca ggt tat tcc acc cat atg
432Val Thr Leu Pro Gln Lys Leu Gln Glu Ala Gly Tyr Ser Thr His Met
130 135 140gtg ggc aag tgg cac ctg ggc ttc tac cgg aag gag tgt ctg
ccc acc 480Val Gly Lys Trp His Leu Gly Phe Tyr Arg Lys Glu Cys Leu
Pro Thr145 150 155 160cgt cgg ggc ttc gac acc ttc ctg ggc tcg ctc
acg ggc aat gtg gac 528Arg Arg Gly Phe Asp Thr Phe Leu Gly Ser Leu
Thr Gly Asn Val Asp 165 170 175tat tac acc tat gac aac tgt gat ggc
cca ggc gtg tgc ggc ttc gac 576Tyr Tyr Thr Tyr Asp Asn Cys Asp Gly
Pro Gly Val Cys Gly Phe Asp 180 185 190ctg cac gag ggt gag aat gtg
gcc tgg ggg ctc agc ggc cag tac tcc 624Leu His Glu Gly Glu Asn Val
Ala Trp Gly Leu Ser Gly Gln Tyr Ser 195 200 205act atg ctt tac gcc
cag cgc gcc agc cat atc ctg gcc agc cac agc 672Thr Met Leu Tyr Ala
Gln Arg Ala Ser His Ile Leu Ala Ser His Ser 210 215 220cct cag cgt
ccc ctc ttc ctc tat gtg gcc ttc cag gca gta cac aca 720Pro Gln Arg
Pro Leu Phe Leu Tyr Val Ala Phe Gln Ala Val His Thr225 230 235
240ccc ctg cag tcc cct cgt gag tac ctg tac cgc tac cgc acc atg ggc
768Pro Leu Gln Ser Pro Arg Glu Tyr Leu Tyr Arg Tyr Arg Thr Met Gly
245 250 255aat gtg gcc cgg cgg aag tac gcg gcc atg gtg acc tgc atg
gat gag 816Asn Val Ala Arg Arg Lys Tyr Ala Ala Met Val Thr Cys Met
Asp Glu 260 265 270gct gtg cgc aac atc acc tgg gcc ctc aag cgc tac
ggt ttc tac aac 864Ala Val Arg Asn Ile Thr Trp Ala Leu Lys Arg Tyr
Gly Phe Tyr Asn 275 280 285aac agt gtc atc atc ttc tcc agt gac aat
ggt ggc cag act ttc tcg 912Asn Ser Val Ile Ile Phe Ser Ser Asp Asn
Gly Gly Gln Thr Phe Ser 290 295 300ggg ggc agc aac tgg ccg ctc cga
gga cgc aag ggc act tat tgg gaa 960Gly Gly Ser Asn Trp Pro Leu Arg
Gly Arg Lys Gly Thr Tyr Trp Glu305 310 315 320ggt ggc gtg cgg ggc
cta ggc ttt gtc cac agt ccc ctg ctc aag cga 1008Gly Gly Val Arg Gly
Leu Gly Phe Val His Ser Pro Leu Leu Lys Arg 325 330 335aag caa cgg
aca agc cgg gca ctg atg cac atc act gac tgg tac ccg 1056Lys Gln Arg
Thr Ser Arg Ala Leu Met His Ile Thr Asp Trp Tyr Pro 340 345 350acc
ctg gtg ggt ctg gca ggt ggt acc acc tca gca gcc gat ggg cta 1104Thr
Leu Val Gly Leu Ala Gly Gly Thr Thr Ser Ala Ala Asp Gly Leu 355 360
365gat ggc tac gac gtg tgg ccg gcc atc agc gag ggc cgg gcc tca cca
1152Asp Gly Tyr Asp Val Trp Pro Ala Ile Ser Glu Gly Arg Ala Ser Pro
370 375 380cgc acg gag atc ctg cac aac att gac cca ctc tac aac cat
gcc cag 1200Arg Thr Glu Ile Leu His Asn Ile Asp Pro Leu Tyr Asn His
Ala Gln385 390 395 400cat ggc tcc ctg gag ggc ggc ttt ggc atc tgg
aac acc gcc gtg cag 1248His Gly Ser Leu Glu Gly Gly Phe Gly Ile Trp
Asn Thr Ala Val Gln 405 410 415gct gcc atc cgc gtg ggt gag tgg aag
ctg ctg aca gga gac ccc ggc 1296Ala Ala Ile Arg Val Gly Glu Trp Lys
Leu Leu Thr Gly Asp Pro Gly 420 425 430tat ggc gat tgg atc cca ccg
cag aca ctg gcc acc ttc ccg ggt agc 1344Tyr Gly Asp Trp Ile Pro Pro
Gln Thr Leu Ala Thr Phe Pro Gly Ser 435 440 445tgg tgg aac ctg gaa
cga atg gcc agt gtc cgc cag gcc gtg tgg ctc 1392Trp Trp Asn Leu Glu
Arg Met Ala Ser Val Arg Gln Ala Val Trp Leu 450 455 460ttc aac atc
agt gct gac cct tat gaa cgg gag gac ctg gct ggc cag 1440Phe Asn Ile
Ser Ala Asp Pro Tyr Glu Arg Glu Asp Leu Ala Gly Gln465 470 475
480cgg cct gat gtg gtc cgc acc ctg ctg gct cgc ctg gcc gaa tat aac
1488Arg Pro Asp Val Val Arg Thr Leu Leu Ala Arg Leu Ala Glu Tyr Asn
485 490 495cgc aca gcc atc ccg gta cgc tac cca gct gag aac ccc cgg
gct cat 1536Arg Thr Ala Ile Pro Val Arg Tyr Pro Ala Glu Asn Pro Arg
Ala His 500 505 510cct gac ttt aat ggg ggt gct tgg ggg ccc tgg gcc
agt gat gag gaa 1584Pro Asp Phe Asn Gly Gly Ala Trp Gly Pro Trp Ala
Ser Asp Glu Glu 515 520 525gag gag gaa gag gaa ggg agg gct cga agc
ttc tcc cgg ggt cgt cgc 1632Glu Glu Glu Glu Glu Gly Arg Ala Arg Ser
Phe Ser Arg Gly Arg Arg 530 535 540aag aaa aaa tgc aag att tgc aag
ctt cga tcc ttt ttc cgt aaa ctc 1680Lys Lys Lys Cys Lys Ile Cys Lys
Leu Arg Ser Phe Phe Arg Lys Leu545 550 555 560aac acc agg cta atg
tcc caa cgg atc tga 1710Asn Thr Arg Leu Met Ser Gln Arg Ile
56593569PRTHomo sapiens 93Met His Thr Leu Thr Gly Phe Ser Leu Val
Ser Leu Leu Ser Phe Gly1 5 10 15Tyr Leu Ser Trp Asp Trp Ala Lys Pro
Ser Phe Val Ala Asp Gly Pro 20 25 30Gly Glu Ala Gly Glu Gln Pro Ser
Ala Ala Pro Pro Gln Pro Pro His 35 40 45Ile Ile Phe Ile Leu Thr Asp
Asp Gln Gly Tyr His Asp Val Gly Tyr 50 55 60His Gly Ser Asp Ile Glu
Thr Pro Thr Leu Asp Arg Leu Ala Ala Lys65 70 75 80Gly Val Lys Leu
Glu Asn Tyr Tyr Ile Gln Pro Ile Cys Thr Pro Ser 85 90 95Arg Ser Gln
Leu Leu Thr Gly Arg Tyr Gln Ile His Thr Gly Leu Gln 100 105 110His
Ser Ile Ile Arg Pro Gln Gln Pro Asn Cys Leu Pro Leu Asp Gln 115 120
125Val Thr Leu Pro Gln Lys Leu Gln Glu Ala Gly Tyr Ser Thr His Met
130 135 140Val Gly Lys Trp His Leu Gly Phe Tyr Arg Lys Glu Cys Leu
Pro Thr145 150 155 160Arg Arg Gly Phe Asp Thr Phe Leu Gly Ser Leu
Thr Gly Asn Val Asp 165 170 175Tyr Tyr Thr Tyr Asp Asn Cys Asp Gly
Pro Gly Val Cys Gly Phe Asp 180 185 190Leu His Glu Gly Glu Asn Val
Ala Trp Gly Leu Ser Gly Gln Tyr Ser 195 200 205Thr Met Leu Tyr Ala
Gln Arg Ala Ser His Ile Leu Ala Ser His Ser 210 215 220Pro Gln Arg
Pro Leu Phe Leu Tyr Val Ala Phe Gln Ala Val His Thr225 230 235
240Pro Leu Gln Ser Pro Arg Glu Tyr Leu Tyr Arg Tyr Arg Thr Met Gly
245 250 255Asn Val Ala Arg Arg Lys Tyr Ala Ala Met Val Thr Cys Met
Asp Glu 260 265 270Ala Val Arg Asn Ile Thr Trp Ala Leu Lys Arg Tyr
Gly Phe Tyr Asn 275 280 285Asn Ser Val Ile Ile Phe Ser Ser Asp Asn
Gly Gly Gln Thr Phe Ser 290 295 300Gly Gly Ser Asn Trp Pro Leu Arg
Gly Arg Lys Gly Thr Tyr Trp Glu305 310 315 320Gly Gly Val Arg Gly
Leu Gly Phe Val His Ser Pro Leu Leu Lys Arg 325 330 335Lys Gln Arg
Thr Ser Arg Ala Leu Met His Ile Thr Asp Trp Tyr Pro 340 345 350Thr
Leu Val Gly Leu Ala Gly Gly Thr Thr Ser Ala Ala Asp Gly Leu 355 360
365Asp Gly Tyr Asp Val Trp Pro Ala Ile Ser Glu Gly Arg Ala Ser Pro
370 375 380Arg Thr Glu Ile Leu His Asn Ile Asp Pro Leu Tyr Asn His
Ala Gln385 390 395 400His Gly Ser Leu Glu Gly Gly Phe Gly Ile Trp
Asn Thr Ala Val Gln 405 410 415Ala Ala Ile Arg Val Gly Glu Trp Lys
Leu Leu Thr Gly Asp Pro Gly 420 425 430Tyr Gly Asp Trp Ile Pro Pro
Gln Thr Leu Ala Thr Phe Pro Gly Ser 435 440 445Trp Trp Asn Leu Glu
Arg Met Ala Ser Val Arg Gln Ala Val Trp Leu 450 455 460Phe Asn Ile
Ser Ala Asp Pro Tyr Glu Arg Glu Asp Leu Ala Gly Gln465 470 475
480Arg Pro Asp Val Val Arg Thr Leu Leu Ala Arg Leu Ala Glu Tyr Asn
485 490 495Arg Thr Ala Ile Pro Val Arg Tyr Pro Ala Glu Asn Pro Arg
Ala His 500 505 510Pro Asp Phe Asn Gly Gly Ala Trp Gly Pro Trp Ala
Ser Asp Glu Glu 515 520 525Glu Glu Glu Glu Glu Gly Arg Ala Arg Ser
Phe Ser Arg Gly Arg Arg 530 535 540Lys Lys Lys Cys Lys Ile Cys Lys
Leu Arg Ser Phe Phe Arg Lys Leu545 550 555 560Asn Thr Arg Leu Met
Ser Gln Arg Ile 565942067DNAHomo sapiensCDS(1)..(2064) 94atg cta
att tca gga aga gaa gag aac caa ata gac ata tcc aag acc 48Met Leu
Ile Ser Gly Arg Glu Glu Asn Gln Ile Asp Ile Ser Lys Thr1 5 10 15aca
gag gta gat tgt ttt gtg gtt gaa tta gga agt cta cac aat cct 96Thr
Glu Val Asp Cys Phe Val Val Glu Leu Gly Ser Leu His Asn Pro 20 25
30aca cgg aac cca cag cga att ttc acc aag cac gtg gcc acc aag tca
144Thr Arg Asn Pro Gln Arg Ile Phe Thr Lys His Val Ala Thr Lys Ser
35 40 45tcc agc tcc aaa tgt cag ctg gac caa ggt gga aaa agc ctg gtc
cag 192Ser Ser Ser Lys Cys Gln Leu Asp Gln Gly Gly Lys Ser Leu Val
Gln 50 55 60tgc att tta ccc aga tct tca aag ctc ctc tca ccc ttg tgt
ctc ccc 240Cys Ile Leu Pro Arg Ser Ser Lys Leu Leu Ser Pro Leu Cys
Leu Pro65 70 75 80cat ccg tgt gga gct tta ctt ctg tat aga tcc tca
gga atc gcc tct 288His Pro Cys Gly Ala Leu Leu Leu Tyr Arg Ser Ser
Gly Ile Ala Ser 85 90 95gct ctt gct gcc ttt aca gac tcc ctc tct agg
agc tgc tgg ctg tca 336Ala Leu Ala Ala Phe Thr Asp Ser Leu Ser Arg
Ser Cys Trp Leu Ser 100 105 110gtg tcc ctg tgc tgt ttg ttt tgc ggt
gtt gat ggc aca ttt atg aca 384Val Ser Leu Cys Cys Leu Phe Cys Gly
Val Asp Gly Thr Phe Met Thr 115 120 125aga aac gcc aga ccc aac att
gtc ctg ctg atg gca gat gac ctt gga 432Arg Asn Ala Arg Pro Asn Ile
Val Leu Leu Met Ala Asp Asp Leu Gly 130 135 140gtg ggg gat ttg tgc
tgc tac ggt aat aac tca gtg agc aca cct aat 480Val Gly Asp Leu Cys
Cys Tyr Gly Asn Asn Ser Val Ser Thr Pro Asn145 150 155 160att gac
cgc ctg gca agt gaa gga gtg agg ctt acc cag cat ctc gca 528Ile Asp
Arg Leu Ala Ser Glu Gly Val Arg Leu Thr Gln His Leu Ala 165 170
175gct gct tcc atg tgc acc cca agt cgg gct gcc ttc ctg acc ggc cgg
576Ala Ala Ser Met Cys Thr Pro Ser Arg Ala Ala Phe Leu Thr Gly Arg
180 185 190tac ccc atc aga tca ggg atg gtg tct gcc tac aac ctg aac
cgt gcc 624Tyr Pro Ile Arg Ser Gly Met Val Ser Ala Tyr Asn Leu Asn
Arg Ala 195 200 205ttc acg tgg ctt ggt ggg tca ggt ggt ctt ccc acc
aat gaa acg act 672Phe Thr Trp Leu Gly Gly Ser Gly Gly Leu Pro Thr
Asn Glu Thr Thr 210 215 220ttt gcc aag ctg ctg cag cac cgt ggc tac
cgc acg gga ctc ata ggc 720Phe Ala Lys Leu Leu Gln His Arg Gly Tyr
Arg Thr Gly Leu Ile Gly225 230 235 240aaa tgg cac ctg ggt ttg agc
tgc gcc tct cgg aat gat cac tgt tac 768Lys Trp His Leu Gly Leu Ser
Cys Ala Ser Arg Asn Asp His Cys Tyr 245 250 255cac ccg ctc aac cat
ggt ttt cac tac ttt tac ggg gtg cct ttt gga 816His Pro Leu Asn His
Gly Phe His Tyr Phe Tyr Gly Val Pro Phe Gly 260 265 270ctt tta agc
gac tgc cag gca tcc aag aca cca gaa ctg cac cgc tgg 864Leu Leu Ser
Asp Cys Gln Ala Ser Lys Thr Pro Glu Leu His Arg Trp 275 280 285ctc
agg atc aaa ctg tgg atc tcc acg gta gcc ctt gcc ctg gtt cct 912Leu
Arg Ile Lys Leu Trp Ile Ser Thr Val Ala Leu Ala Leu Val Pro 290 295
300ttt ctg ctt ctc att ccc aag ttc gcc cgc tgg ttc tca gtg cca tgg
960Phe Leu Leu Leu Ile Pro Lys Phe Ala Arg Trp Phe Ser Val Pro
Trp305 310 315 320aag gtc atc ttt gtc ttt gct ctc ctc gcc ttt ctg
ttt ttc act tcc 1008Lys Val Ile Phe Val Phe Ala Leu Leu Ala Phe Leu
Phe Phe Thr Ser 325 330 335tgg tac tct agt tat gga ttt act cga cgt
tgg aat tgc atc ctt atg 1056Trp Tyr Ser Ser Tyr Gly Phe Thr Arg Arg
Trp Asn Cys Ile Leu Met 340 345 350agg aac cat gaa att atc cag cag
cca atg aaa gag gag aaa gta gct 1104Arg Asn His Glu Ile Ile Gln Gln
Pro Met Lys Glu Glu Lys Val Ala 355 360 365tcc ctc atg ctg aag gag
gca ctt gct ttc att gaa agg tac aaa agg 1152Ser Leu Met Leu Lys Glu
Ala Leu Ala Phe Ile Glu Arg Tyr Lys Arg 370 375 380gaa cct ttt ctc
ctc ttt ttt tcc ttc ctg cac gta cat act cca ctc 1200Glu Pro Phe Leu
Leu Phe Phe Ser Phe Leu His Val His Thr Pro Leu385 390 395 400atc
tcc aaa aag aag ttt gtt ggg cgc agt aaa tat ggc agg tat ggg 1248Ile
Ser Lys Lys Lys Phe Val Gly Arg Ser Lys Tyr Gly Arg Tyr Gly 405 410
415gac aat gta gaa gaa atg gat tgg atg gtg ggt aaa atc ctg gat gcc
1296Asp Asn Val Glu Glu Met Asp Trp Met Val Gly Lys Ile Leu Asp Ala
420 425 430ctg gac cag gag cgc ctg gcc aac cac acc ttg gtg tac ttc
acc tct 1344Leu Asp Gln Glu Arg Leu Ala Asn His Thr Leu Val Tyr Phe
Thr Ser 435 440 445gac aac ggg ggc cac ctg gag ccc ctg gac ggg gct
gtt cag ctg ggt 1392Asp Asn Gly Gly His Leu Glu Pro Leu Asp Gly Ala
Val Gln Leu Gly 450 455 460ggc tgg aac ggg atc tac aaa ggt ggc aaa
gga atg gga gga tgg gaa 1440Gly Trp Asn Gly Ile Tyr Lys Gly Gly Lys
Gly Met Gly Gly Trp Glu465 470 475 480gga ggt atc cgt gtg cca ggg
ata ttc cgg tgg ccg tca gtc ttg gag 1488Gly Gly Ile Arg
Val Pro Gly Ile Phe Arg Trp Pro Ser Val Leu Glu 485 490 495gct ggg
aga gtg atc aat gag ccc acc agc tta atg gac atc tat ccg 1536Ala Gly
Arg Val Ile Asn Glu Pro Thr Ser Leu Met Asp Ile Tyr Pro 500 505
510acg ctg tct tat ata ggc gga ggg atc ttg tcc cag gac aga gtg att
1584Thr Leu Ser Tyr Ile Gly Gly Gly Ile Leu Ser Gln Asp Arg Val Ile
515 520 525gac ggc cag aac cta atg ccc ctg ctg gaa gga agg gcg tcc
cac tcc 1632Asp Gly Gln Asn Leu Met Pro Leu Leu Glu Gly Arg Ala Ser
His Ser 530 535 540gac cac gag ttc ctc ttc cac tac tgt ggg gtc tat
ctg cac acg gtc 1680Asp His Glu Phe Leu Phe His Tyr Cys Gly Val Tyr
Leu His Thr Val545 550 555 560agg tgg cat cag aag gac tgt gca act
gtg tgg aaa gct cat tat gtg 1728Arg Trp His Gln Lys Asp Cys Ala Thr
Val Trp Lys Ala His Tyr Val 565 570 575act cct aaa ttc tac cct gaa
gga aca ggt gcc tgc tat ggg agt gga 1776Thr Pro Lys Phe Tyr Pro Glu
Gly Thr Gly Ala Cys Tyr Gly Ser Gly 580 585 590ata tgt tca tgt tcg
ggg gat gta acc tac cac gac cca cca ctc ctc 1824Ile Cys Ser Cys Ser
Gly Asp Val Thr Tyr His Asp Pro Pro Leu Leu 595 600 605ttt gac atc
tca aga gac cct tca gaa gcc ctt cca ctg aac cct gac 1872Phe Asp Ile
Ser Arg Asp Pro Ser Glu Ala Leu Pro Leu Asn Pro Asp 610 615 620aat
gag cca tta ttt gac tcc gtg atc aaa aag atg gag gca gcc ata 1920Asn
Glu Pro Leu Phe Asp Ser Val Ile Lys Lys Met Glu Ala Ala Ile625 630
635 640aga gag cat cgt agg aca cta aca cct gtc cca cag cag ttc tct
gtg 1968Arg Glu His Arg Arg Thr Leu Thr Pro Val Pro Gln Gln Phe Ser
Val 645 650 655ttc aac aca att tgg aaa cca tgg ctg cag cct tgc tgt
ggg acc ttc 2016Phe Asn Thr Ile Trp Lys Pro Trp Leu Gln Pro Cys Cys
Gly Thr Phe 660 665 670ccc ttc tgt ggg tgt gac aag gaa gat gac atc
ctt ccc atg gct ccc 2064Pro Phe Cys Gly Cys Asp Lys Glu Asp Asp Ile
Leu Pro Met Ala Pro 675 680 685tga 206795688PRTHomo sapiens 95Met
Leu Ile Ser Gly Arg Glu Glu Asn Gln Ile Asp Ile Ser Lys Thr1 5 10
15Thr Glu Val Asp Cys Phe Val Val Glu Leu Gly Ser Leu His Asn Pro
20 25 30Thr Arg Asn Pro Gln Arg Ile Phe Thr Lys His Val Ala Thr Lys
Ser 35 40 45Ser Ser Ser Lys Cys Gln Leu Asp Gln Gly Gly Lys Ser Leu
Val Gln 50 55 60Cys Ile Leu Pro Arg Ser Ser Lys Leu Leu Ser Pro Leu
Cys Leu Pro65 70 75 80His Pro Cys Gly Ala Leu Leu Leu Tyr Arg Ser
Ser Gly Ile Ala Ser 85 90 95Ala Leu Ala Ala Phe Thr Asp Ser Leu Ser
Arg Ser Cys Trp Leu Ser 100 105 110Val Ser Leu Cys Cys Leu Phe Cys
Gly Val Asp Gly Thr Phe Met Thr 115 120 125Arg Asn Ala Arg Pro Asn
Ile Val Leu Leu Met Ala Asp Asp Leu Gly 130 135 140Val Gly Asp Leu
Cys Cys Tyr Gly Asn Asn Ser Val Ser Thr Pro Asn145 150 155 160Ile
Asp Arg Leu Ala Ser Glu Gly Val Arg Leu Thr Gln His Leu Ala 165 170
175Ala Ala Ser Met Cys Thr Pro Ser Arg Ala Ala Phe Leu Thr Gly Arg
180 185 190Tyr Pro Ile Arg Ser Gly Met Val Ser Ala Tyr Asn Leu Asn
Arg Ala 195 200 205Phe Thr Trp Leu Gly Gly Ser Gly Gly Leu Pro Thr
Asn Glu Thr Thr 210 215 220Phe Ala Lys Leu Leu Gln His Arg Gly Tyr
Arg Thr Gly Leu Ile Gly225 230 235 240Lys Trp His Leu Gly Leu Ser
Cys Ala Ser Arg Asn Asp His Cys Tyr 245 250 255His Pro Leu Asn His
Gly Phe His Tyr Phe Tyr Gly Val Pro Phe Gly 260 265 270Leu Leu Ser
Asp Cys Gln Ala Ser Lys Thr Pro Glu Leu His Arg Trp 275 280 285Leu
Arg Ile Lys Leu Trp Ile Ser Thr Val Ala Leu Ala Leu Val Pro 290 295
300Phe Leu Leu Leu Ile Pro Lys Phe Ala Arg Trp Phe Ser Val Pro
Trp305 310 315 320Lys Val Ile Phe Val Phe Ala Leu Leu Ala Phe Leu
Phe Phe Thr Ser 325 330 335Trp Tyr Ser Ser Tyr Gly Phe Thr Arg Arg
Trp Asn Cys Ile Leu Met 340 345 350Arg Asn His Glu Ile Ile Gln Gln
Pro Met Lys Glu Glu Lys Val Ala 355 360 365Ser Leu Met Leu Lys Glu
Ala Leu Ala Phe Ile Glu Arg Tyr Lys Arg 370 375 380Glu Pro Phe Leu
Leu Phe Phe Ser Phe Leu His Val His Thr Pro Leu385 390 395 400Ile
Ser Lys Lys Lys Phe Val Gly Arg Ser Lys Tyr Gly Arg Tyr Gly 405 410
415Asp Asn Val Glu Glu Met Asp Trp Met Val Gly Lys Ile Leu Asp Ala
420 425 430Leu Asp Gln Glu Arg Leu Ala Asn His Thr Leu Val Tyr Phe
Thr Ser 435 440 445Asp Asn Gly Gly His Leu Glu Pro Leu Asp Gly Ala
Val Gln Leu Gly 450 455 460Gly Trp Asn Gly Ile Tyr Lys Gly Gly Lys
Gly Met Gly Gly Trp Glu465 470 475 480Gly Gly Ile Arg Val Pro Gly
Ile Phe Arg Trp Pro Ser Val Leu Glu 485 490 495Ala Gly Arg Val Ile
Asn Glu Pro Thr Ser Leu Met Asp Ile Tyr Pro 500 505 510Thr Leu Ser
Tyr Ile Gly Gly Gly Ile Leu Ser Gln Asp Arg Val Ile 515 520 525Asp
Gly Gln Asn Leu Met Pro Leu Leu Glu Gly Arg Ala Ser His Ser 530 535
540Asp His Glu Phe Leu Phe His Tyr Cys Gly Val Tyr Leu His Thr
Val545 550 555 560Arg Trp His Gln Lys Asp Cys Ala Thr Val Trp Lys
Ala His Tyr Val 565 570 575Thr Pro Lys Phe Tyr Pro Glu Gly Thr Gly
Ala Cys Tyr Gly Ser Gly 580 585 590Ile Cys Ser Cys Ser Gly Asp Val
Thr Tyr His Asp Pro Pro Leu Leu 595 600 605Phe Asp Ile Ser Arg Asp
Pro Ser Glu Ala Leu Pro Leu Asn Pro Asp 610 615 620Asn Glu Pro Leu
Phe Asp Ser Val Ile Lys Lys Met Glu Ala Ala Ile625 630 635 640Arg
Glu His Arg Arg Thr Leu Thr Pro Val Pro Gln Gln Phe Ser Val 645 650
655Phe Asn Thr Ile Trp Lys Pro Trp Leu Gln Pro Cys Cys Gly Thr Phe
660 665 670Pro Phe Cys Gly Cys Asp Lys Glu Asp Asp Ile Leu Pro Met
Ala Pro 675 680 685964PRTHomo sapiens 96Lys Asp Glu Leu1
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