U.S. patent application number 12/038018 was filed with the patent office on 2008-07-24 for glcnac phosphotransferase of the lysosomal targeting pathway.
This patent application is currently assigned to Genzyme Glycobiology Research Institute, Inc.. Invention is credited to William M. CANFIELD.
Application Number | 20080176285 12/038018 |
Document ID | / |
Family ID | 22548927 |
Filed Date | 2008-07-24 |
United States Patent
Application |
20080176285 |
Kind Code |
A1 |
CANFIELD; William M. |
July 24, 2008 |
GLCNAC PHOSPHOTRANSFERASE OF THE LYSOSOMAL TARGETING PATHWAY
Abstract
The present invention provides nucleotide and amino sequences of
the lysosomal targeting pathway enzyme GlcNAc-phosphotransferase,
methods of producing and methods of purifying this enzyme.
Inventors: |
CANFIELD; William M.;
(Oklahoma City, OK) |
Correspondence
Address: |
OBLON, SPIVAK, MCCLELLAND MAIER & NEUSTADT, P.C.
1940 DUKE STREET
ALEXANDRIA
VA
22314
US
|
Assignee: |
Genzyme Glycobiology Research
Institute, Inc.
Oklahoma City
OK
|
Family ID: |
22548927 |
Appl. No.: |
12/038018 |
Filed: |
February 27, 2008 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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11199233 |
Aug 9, 2005 |
7371366 |
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12038018 |
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10657280 |
Sep 9, 2003 |
7067127 |
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11199233 |
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09636060 |
Aug 10, 2000 |
6642038 |
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10657280 |
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60153831 |
Sep 14, 1999 |
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Current U.S.
Class: |
435/69.1 ;
435/194; 435/320.1; 435/325; 530/387.1; 536/23.2 |
Current CPC
Class: |
C12N 9/12 20130101; A61P
43/00 20180101; C12N 9/16 20130101; C07K 16/40 20130101; C12N
9/1205 20130101; A61K 38/00 20130101; C12N 9/1223 20130101; C12Y
301/04045 20130101 |
Class at
Publication: |
435/69.1 ;
435/194; 536/23.2; 435/320.1; 435/325; 530/387.1 |
International
Class: |
C12P 21/04 20060101
C12P021/04; C12N 15/11 20060101 C12N015/11; C12N 15/00 20060101
C12N015/00; C12N 9/12 20060101 C12N009/12; C07K 16/18 20060101
C07K016/18; C12N 5/06 20060101 C12N005/06 |
Claims
1. An isolated GlcNAc-phosphotransferase.
2. The GlcNAc-phosphotransferase of claim 1 which comprises an
.alpha., .beta., and .gamma. subunit.
3. The GlcNAc-phosphotransferase wherein the .alpha.-subunit
comprises the amino acid sequence in SEQ ID NO:1, the
.beta.-subunit comprises the amino acid sequence in SEQ ID NO:2,
and the .gamma.-subunit comprises the amino acid sequence in SEQ ID
NO:3.
4. The GlcNAc-phosphotransferase of claim 3, wherein the
.alpha.-subunit comprises amino acids 1 to 928 of SEQ ID NO:1, the
.beta.-subunit comprises amino acids 1 to 328 of SEQ ID NO:2, and
the .gamma.-subunit comprises amino acids 25 to 305 of SEQ ID
NO:3.
5. An isolated nucleic acid encoding the GlcNAc-phosphotransferase
of claim 3.
6. The isolated nucleic acid of claim 5, comprising SEQ ID NO:4 and
SEQ ID NO:5.
7. The isolated nucleic acid of claim 5, wherein an .alpha.-subunit
coding region is contained in nucleotides 165 to 2948 of SEQ ID
NO:4, an .beta.-subunit coding region is contained in nucleotides
2949 to 3952 of SEQ ID NO:6, and a .gamma.-subunit coding region is
contained in nucleotides 96 to 941 of SEQ ID NO:5.
8. An isolated nucleic acid which hybridizes under stringent
conditions to at least one of the isolated nucleic acids of claim
6, wherein said stringent conditions comprise washing in
0.2.times.SSC and 0.1% SDS at 65.degree. C.
9. A vector comprising the isolated nucleic acid of claim 5.
10. A host cell comprising the isolated nucleic acid of claim
5.
11. A method of producing biologically active
GlcNAc-phosphotransferase comprising culturing the cell of claim 10
under conditions suitable for expression of the isolated nucleic
acid molecule; and recovering the biologically active
GlcNAc-phosphotransferase.
12. The isolated GlcNAc-phosphotransferase of claim 2, wherein the
.alpha.-subunit has the amino acid sequence in SEQ ID NO:15, the
.beta.-subunit has the amino acid sequence in SEQ ID NO:8, and the
7-subunit has the amino acid sequence in SEQ ID NO:9.
13. An isolated nucleic acid encoding the GlcNAc-phosphotransferase
of claim 12.
14. The isolated nucleic acid of claim 13, comprising SEQ ID NO:16
and SEQ ID NO:10.
15. An isolated nucleic acid comprising a nucleic acid which
hybridizes under stringent conditions to at least one of the
isolated nucleic acids of claim 14, wherein said stringent
conditions comprise washing in 0.2.times.SSC and 0.1% SDS at
65.degree. C.
16. A vector comprising the isolated nucleic acid of claim 13.
17. A cell comprising the isolated nucleic acid of claim 13.
18. A method of producing biologically active
GlcNAc-phosphotransferase comprising culturing the cell of claim 17
under conditions suitable for expression of the isolated nucleic
acid molecule; and recovering the biologically active
GlcNAc-phosphotransferase.
19. An antibody which binds the GlcNAc-phosphotransferase of claim
1, wherein said antibody is produced by the PT18 hybridoma
deposited at the ATCC under the accession No. ______.
20. A method of isolating GlcNAc-phosphotransferase comprising
contacting a cellular lysate containing said
GlcNAc-phosphotransferase with the antibody of claim 19, wherein an
the GlcNAc-phosphotransferase and the antibody form a complex; and
isolating the antibody-GlcNAc-phosphotransferase complex.
21. A method of producing a GlcNAc-phosphotransferase in a cell
comprising transfecting into said cell a DNA construct comprising a
targeting sequence homologous to a target site within or upstream
of a endogenous GlcNAc-phosphotransferase gene contained in the
cell, wherein said endogenous gene comprises the sequence in SEQ ID
NO:4 or SEQ ID NO:5, an exogenous regulatory sequence, an exon, and
an unpaired splice-donor site at the 3' end of the exon, wherein
said transfecting generates a homologously recombinant cell in
which the splice-donor site is operatively linked to the second
exon of the endogenous gene, and the exogenous regulatory sequence
controls transcription of the construct driven, the endogenous
gene, and any sequence lying between the construct-driven exon and
the endogenous gene, to produce a RNA transcript that encodes the
GlcNAc-phosphotransferase, so that the homologously recombinant
cell produces the GlcNAc-phosphotransferase.
22. An isolated amino acid sequence comprising SEQ ID NO:1
23. An isolated nucleic acid which encodes the amino acid sequence
of claim 22.
24. The isolated nucleic acid of claim 23 comprising nucleotides
165 to 2948 of SEQ ID NO:4.
25. An isolated nucleic acid which hybridizes under stringent
conditions to the isolated nucleic acid of claim 24, wherein said
stringent conditions comprise washing in 0.2.times.SSC and 0.1%
SDS.
26. A vector comprising the isolated nucleic acid of 23.
27. A host cell comprising the isolated nucleic acid of claim
23.
28. An isolated amino acid sequence comprising SEQ ID NO:2
29. An isolated nucleic acid which encodes the amino acid sequence
of claim 28.
30. The isolated nucleic acid of claim 29 comprising nucleotides
2949 to 3952 of SEQ ID NO:4.
31. An isolated nucleic acid which hybridizes under stringent
conditions to the isolated nucleic acid of claim 30, wherein said
stringent conditions comprise washing in 0.2.times.SSC and 0.1%
SDS.
32. A vector comprising the isolated nucleic acid of 29.
33. A host cell comprising the isolated nucleic acid of claim
29.
34. An isolated amino acid sequence comprising SEQ ID NO:3.
35. An isolated nucleic acid which encodes the amino acid sequence
of claim 34.
36. The isolated nucleic acid of claim 35 comprising nucleotides 25
to 305 of SEQ ID NO:3.
37. An isolated nucleic acid which hybridizes under stringent
conditions to the isolated nucleic acid of claim 36, wherein said
stringent conditions comprise washing in 0.2.times.SSC and 0.1%
SDS.
38. A vector comprising the isolated nucleic acid of 35.
39. A host cell comprising the isolated nucleic acid of claim 35.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] This invention relates generally to enzymes involved in the
lysosomal targeting pathway and particularly to isolated and
purified GlcNAc-phosphotransferase and phosphodiester
.alpha.-GlcNAcase, nucleic acids encoding the enzymes, processes
for production of recombinant GlcNAc-phosphotransferase and
phosphodiester .alpha.-GlcNAcase, and the use of the enzymes for
the preparation of highly phosphorylated lysosomal enzymes that are
useful for the treatment of lysosomal storage diseases.
[0003] 2. Description of the Prior Art
Lysosomes and Lysosomal Storage Diseases
[0004] Lysosomes are organelles in eukaryotic cells that function
in the degradation of macromolecules into component parts that can
be reused in biosynthetic pathways or discharged by the cell as
waste. Normally, these macromolecules are broken down by enzymes
known as lysosomal enzymes or lysosomal hydrolases. However, when a
lysosomal enzyme is not present in the lysosome or does not
function properly, the enzymes specific macromolecular substrate
accumulates in the lysosome as "storage material" causing a variety
of diseases, collectively known as lysosomal storage diseases.
[0005] Lysosomal storage diseases can cause chronic illness and
death in hundreds of individuals each year. There are approximately
50 known lysosomal storage diseases, e.g., Pompe Disease, Hurler
Syndrome, Fabry Disease, Maroteaux-Lamy Syndrome
(mucopolysaccharidosis VI), Morquio Syndrome (mucopolysaccharidosis
IV), Hunter Syndrome (mucopolysaccharidosis II), Farber Disease,
Acid Lipase Deficiency, Krabbe Disease, and Sly Syndrome
(mucopolysaccharidosis VII). In each of these diseases, lysosomes
are unable to degrade a specific compound or group of compounds
because the enzyme that catalyzes a specific degradation reaction
is missing from the lysosome, is present in low concentrations in
the lysosome, or is present at sufficient concentrations in the
lysosome but is not functioning properly.
[0006] Lysosomal storage diseases have been studied extensively and
the enzymes (or lack thereof) responsible for particular diseases
have been identified. Most of the diseases are caused by a
deficiency of the appropriate enzyme in the lysosome, often due to
mutations or deletions in the structural gene for the enzyme. For
some lysosomal storage diseases, the enzyme deficiency is caused by
the inability of the cell to target and transport the enzymes to
the lysosome, e.g., I-cell disease and pseudo-Hurler
polydystrophy.
[0007] Lysosomal Storage diseases have been studied extensively and
the enzymes (or lack thereof) responsible for particular diseases
have been identified (Scriver, Beaudet, Sly, and Vale, eds., The
Metabolic Basis of Inherited Disease, 6th Edition, 1989, Lysosomal
Enzymes, Part 11, Chapters 61-72, pp. 1565-1839). Within each
disease, the severity and the age at which the disease presents may
be a function of the amount of residual lysosomal enzyme that
exists in the patient.
Lysosomal Targeting Pathway
[0008] The lysosomal targeting pathways have been studied
extensively and the process by which lysosomal enzymes are
synthesized and transported to the lysosome has been well
described. Komfeld, S. (1986). "Trafficking of lysosomal enzymes in
normal and disease states." Journal of Clinical Investigation 77:
1-6 and Komfeld, S. (1990). "Lysosomal enzyme targeting." Biochem.
Soc. Trans. 18: 367-374. Generally, lysosomal enzymes are
synthesized by membrane-bound polysomes in the rough endoplastic
reticulum ("RER") along with secretory glycoproteins. In the RER,
lysosomal enzymes acquire N-linked oligosaccharides by the en-bloc
transfer of a preformed oligosaccharide from dolichol phosphate
containing 2 N-acetylglucosamine, 9-mannose and 3-glucose.
Glycosylated lysosomal enzymes are then transported to the Golgi
apparatus along with secretory proteins. In the cis-Golgi or
intermediate compartment lysosomal enzymes are specifically and
uniquely modified by the transfer of GlcNAc-phosphate to specific
mannoses. In a second step, the GlcNAc is removed thereby exposing
the mannose 6-phosphate ("M6P") targeting determinant. The
lysosomal enzymes with the exposed M6P binds to M6P receptors in
the trans-Golgi and is transported to the endosome and then to the
lysosome. In the lysosome, the phosphates are rapidly removed by
lysosomal phosphatases and the mannoses are removed by lysosomal
mannosidases (Einstein, R. and Gabel, C. A. (1991). "Cell- and
ligand-specific deposphorylation of acid hydrolases: evidence that
the mannose 6-phosphate is controlled by compartmentalization."
Journal of Cell Biology 112: 81-94).
[0009] The synthesis of lysosomal enzymes having exposed M6P is
catalyzed by two different enzymes, both of which are essential if
the synthesis is to occur. The first enzyme is
UDP-N-acetylglucosamine: lysosomal enzyme
N-Acetylglucosamine-1-phosphotransferase
("GlcNAc-phosphotransferase") (E.C. 2.7.8.17).
GlcNAc-phosphotransferase catalyzes the transfer of
N-acetylglucosamine-1-phosphate from UDP-GlcNAc to the 6 position
of a 1,2-linked mannoses on the lysosonial enzyme. The recognition
and addition of N-acetylgluocosamine-1-phosphate to lysosomal
hydrolases by GlcNAc-phosphotransferase is the critical and
determining step in lysosomal targeting. The second step is
catalyzed by N-acetylglucosamine-1-phosphodiester
.alpha.-N-Acetylglucosaminidase ("phosphodiester
.alpha.-GlcNAcase") (E.C. 3.1.4.45). Phosphodiester
.alpha.-GlcNAcase catalyzes the removal of N-Acetylglucosamine from
the GlcNAc-phosphate modified lysosomal enzyme to generate a
terminal M6P on the lysosomal enzyme. Preliminary studies of these
enzymes have been conducted. Bao et al., in The Journal of
Biological Chemistry, Vol. 271, Number 49, Issue of Dec. 6, 1996,
pp. 31437-31445, relates to a method for the purification of bovine
UDP-N-acetylglucosamine: Lysosomal enzyme
N-Acetylglucosamine-1-phosphotransferase and proposes a
hypothetical subunit structure for the protein. Bao et al., in The
Journal of Biological Chemistry, Vol. 271, Number 49, Issue of Dec.
6, 1996, pp. 31446-31451, relates to the enzymatic characterization
and identification of the catalytic subunit for bovine
UDP-N-acetylglucosamine: Lysosomal enzyme
N-Acetylglucosamine-1-phosphotransferase. Komfeld et al., in The
Journal of Biological Chemistry, Vol. 273, Number 36, Issue of Sep.
4, 1998, pp. 23203-23210, relates to the purification and
multimeric structure of bovine N-Acetylglucosamine-1-phosphodiester
.alpha.-N-Acetylglucosaminidase. However, the proprietary
monoclonal antibodies required to isolate these proteins have not
been made available to others and the protein sequences for the
enzymes used in these preliminary studies have not been
disclosed.
[0010] Although the lysosomal targeting pathway is known and the
naturally occurring enzymes involved in the pathway have been
partially studied, the enzymes responsible for adding M6P in the
lysosomal targeting pathway are difficult to isolate and purify and
are poorly understood. A better understanding of the lysosomal
targeting pathway enzymes and the molecular basis for their action
is needed to assist with the development of effective techniques
for the utilization of these enzymes in methods for the treatment
of lysosomal storage diseases, particularly in the area of targeted
enzyme replacement therapy.
Treatment of Lysosomal Storage Diseases
[0011] Lysosomal storage diseases caused by the lack of enzymes can
in theory be treated using enzyme replacement therapy, i.e., by
administering isolated and purified enzymes to the patient to treat
the disease. However, to be effective, the lysosomal enzyme
administered must be internalized by the cell and transported to
the lysosome. Naturally occurring enzymes and their recombinant
equivalents, however, have been of limited value in enzyme
replacement therapy because the purified or recombinant lysosomal
enzymes do not contain adequate amounts of exposed M6P, or contain
undesirable oligosaccharides which mediates their destruction.
Without sufficient M6P, the administered lysosomal enzyme cannot
efficiently bind to M6P receptors and be transported to the
lysosome. For example, human acid .alpha.-glucosidase purified from
placenta contains oligomannose oligosaccharides which are not
phosphorylated (Mutsaers, J. H. G. M., Van Halbeek, H.,
Vliegenthart, J. F. G., Tager, J. M., Reuser, A. J. J., Kroos, M.,
and Galjaard, H. (1987). "Determination of the structure of the
carbohydrate chains of acid .alpha.-glucosidase from human
placenta." Biochimica et Biophysica Acta 911: 244-251), and this
glycoform of the enzyme is not efficiently internalized by cells
(Reuser, A. J., Kroos, M. A., Ponne, N. J., Wolterman, R. A.,
Loonen, M. C., Busch, H. F., Visser, W. J., and Bolhuis, P. A.
(1984). "Uptake and stability of human and bovine acid
alpha-glucosidase in cultured fibroblasts and skeletal muscle cells
from glycogenosis type II patients." Experimental Cell Research
155:178-189). As a result of the inability to purify or synthesize
lysosomal enzymes with the desired oligosaccharide structures,
these enzyme preparations are inefficiently targeted to affected
cells and are of limited effectiveness in the treatment of these
diseases. There exists, therefore, a need for enzymes that can be
used in enzyme replacement therapy procedures, particularly highly
phosphorylated enzymes that will be efficiently internalized by the
cell and transported to the lysosome.
SUMMARY OF THE INVENTION
[0012] It is, therefore, an object of the present invention to
provide biologically active GlcNAc-phosphotransferase and
phosphodiester .alpha.-GlcNAcase as isolated and purified
polypeptides.
[0013] It is another object of the present invention to provide
nucleic acid molecules encoding GlcNAc-phosphotransferase and
phosphodiester .alpha.-GlcNAcase.
[0014] It is another object of the present invention to provide
expression vectors having DNA that encodes
GlcNAc-phosphotransferase and phosphodiester .alpha.-GlcNAcase.
[0015] It is a further object of the present invention to provide
host cells that have been transfected with expression vectors
having DNA that encodes GlcNAc-phosphotransferase or phosphodiester
.alpha.-GlcNAcase.
[0016] It is another object of the present invention to provide
methods for producing recombinant GlcNAc-phosphotransferase and
recombinant phosphodiester .alpha.-GlcNAcase by culturing host
cells that have been transfected or transformed with expression
vectors having DNA that encodes GlcNAc-phosphotransferase or
phosphodiester .alpha.-GlcNAcase.
[0017] It is another object of the present invention to provide
isolated and purified recombinant GlcNAc-phosphotransferase and
recombinant phosphodiester .alpha.-GlcNAcase.
[0018] It is another object of the present invention to provide
methods for the preparation of highly phosphorlyated lysosomal
enzymes that are useful for the treatment of lysosomal storage
diseases.
[0019] It is a further object of the present invention to provide
highly phosphorlyated lysosomal hydrolases that are useful for the
treatment of lysosomal storage diseases.
[0020] It is still another object of the present invention to
provide methods for the treatment of lysosomal storage
diseases.
[0021] It is still another object of the present invention to
provide monoclonal antibodies capable of selectively binding to
bovine GlcNAc-phosphotransferase and to bovine phosphodiester
.alpha.-GlcNAcase.
[0022] These and other objects are achieved by recovering isolated
and purified biologically active GlcNAc-phosphotransferase and
phosphodiester .alpha.-GlcNAcase and using the enzymes to obtain
nucleic acid molecules that encode for the enzymes. The nucleic
acid molecules coding for either enzyme are incorporated into
expression vectors that are used to transfect host cells that
express the enzyme. The expressed enzyme is recovered and used to
prepare highly phosphorylated lysosomal hydrolases useful for the
treatment of lysosomal storage diseases. In particular, the enzymes
are used to produce highly phosphorylated-lysosomal hydrolases that
can be effectively used in enzyme replacement therapy
procedures.
[0023] Lysosomal hydrolases having high mannose structures are
treated with GlcNAc-phosphotransferase and phosphodiester
.alpha.-GlcNAcase resulting in the production of asparagine-linked
oligosaccharides that are highly modified with mannose 6-phosphate
("M6P"). The treated hydrolase binds to M6P receptors on the cell
membrane and is transported into the cell and delivered to the
lysosome where it can perform its normal or a desired function.
[0024] Other aspects and advantages of the present invention will
become apparent from the following more detailed description of the
invention taken in conjunction with the accompanying drawings.
BRIEF OF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows a model of the subunit structure of
GlcNAc-phosphotransferase. The enzyme is a complex of six
polypeptides. The .alpha.- and .beta.-subunits are the product of a
single gene. Following translation, the .alpha.- and
.beta.-subunits are separated by proteolytic cleavage between
Lys.sup.929 and Asp.sup.930. The .alpha.-subunit is a type II
membrane glycoprotein with a single amino terminal membrane
spanning domain. The .beta.-subunit is a type I membrane spanning
glycoprotein with a single carboxyl terminal membrane spanning
domain. The .gamma.-subunit is the product of a second gene. The
.gamma.-subunit is a soluble protein with a cleaved signal peptide.
The .alpha.-, .beta.-, and .gamma.-subunits are all tightly
associated.
[0026] FIG. 2 shows a model of the subunit structure of
phosphodiester .alpha.-GlcNAcase. The enzyme is a tetramer composed
of four identical subunits arranged as two
non-covalently-associated dimers which are themselves
disulfide-linked. The single subunit is a type I membrane protein
containing a signal peptide, a pro region not present in the mature
enzyme and a single carboxyl terminal membrane spanning domain.
[0027] FIG. 3 shows a diagram of recombinant glycoprotein
expression in CHO cells. In overexpressing CHO cells, the rh-GAA is
processed along the pathways 1 and 2, depending on whether or not
the enzyme is acted upon by GlcNAc-phosphotransferase (GnPT).
Secreted GAA contains predominantly sialylated biantenniary
complex-type glycans and is not a substrate for
GlcNAc-phosphotransferase. In the presence of the
.alpha.1,2-mannosidase inhibitors, 1-deoxymannojirimycin or
kifunensine conversion of MAN9 to MAN5 structures is blocked,
resulting in secretion of GAA-bearing MAN7-9 structures which can
be modified with GlcNAc-phosphotransferase and phosphodiester
.alpha.-GlcNAcase (UCE) generating phosphorylated species (pathway
3).
[0028] FIG. 4 shows transient expression analysis of various
plasmid constucts of the .alpha./.beta. and .gamma. subunits of
human GlcNAc-phosphotransferase. Plasmids containing the
.alpha./.beta. and/or the .gamma. subunits were transfected into
293T cells, the expressed protein was purified from the culture at
23, 44.5 and 70 hours after transfection and relative amounts of
expression were assessed by measuring phosphotransferase activity
using methyl-.alpha.-D-mannoside and [.beta.-.sup.32P] UDP-GlcNAc
as substrates.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The term "GlcNAc-phosphotransferase" as used herein refers
to enzymes that are capable of catalyzing the transfer of
N-acetylglucosamine-1-phosphate from UDP-GlcNAc to the 6' position
of .alpha.1,2-linked mannoses on lysosomal enzymes.
[0030] The term "phosphodiester .alpha.-GlcNAcase" as used herein
refers to enzymes that are capable of catalyzing the removal of
N-Acetylglucosamine from GlcNAc-phosphate-mannose diester modified
lysosomal enzymes to generate terminal M6P.
[0031] The terms "GlcNAc-phosphotransferase" and "phosphodiester
.alpha.-GlcNAcase" as used herein refer to enzymes obtained from
any eukaryotic species, particularly mammalian species such as
bovine, porcine, murine, equine, and human, and from any source
whether natural, synthetic, semi-synthetic, or recombinant. The
terms encompass membrane-bound enzymes and soluble or truncated
enzymes having less than the complete amino acid sequence and
biologically active variants and gene products.
[0032] The term "naturally occurring" as used herein means an
endogenous or exogenous protein isolated and purified from animal
tissue or cells.
[0033] The term "isolated and purified" as used herein means a
protein that is essentially free of association with other proteins
or polypeptides, e.g., as a naturally occurring protein that has
been separated from cellular and other contaminants by the use of
antibodies or other methods or as a purification product of a
recombinant host cell culture.
[0034] The term "biologically active" as used herein means an
enzyme or protein having structural, regulatory, or biochemical
functions of a naturally occurring molecule.
[0035] The term "nucleotide sequence" as used herein means a
polynucleotide molecule in the form of a separate fragment or as a
component of a larger nucleic acid construct that has been derived
from DNA or RNA isolated at least once in substantially pure form
(i.e., free of contaminating endogenous materials) and in a
quantity or concentration enabling identification, manipulation,
and recovery of its component nucleotide sequences by standard
biochemical methods. Such sequences are preferably provided in the
form of an open reading frame uninterrupted by internal
non-translated sequences, or introns that are typically present in
eukaryotic genes. Sequences of non-translated DNA may be present 5'
or 3' from an open reading frame where the same do not interfere
with manipulation or expression of the coding region.
[0036] The term "nucleic acid molecule" as used herein means RNA or
DNA, including cDNA, single or double stranded, and linear or
covalently closed molecules. A nucleic acid molecule may also be
genomic DNA corresponding to the entire gene or a substantial
portion therefor to fragments and derivatives thereof. The
nucleotide sequence may correspond to the naturally occurring
nucleotide sequence or may contain single or multiple nucleotide
substitutions, deletions and/or additions including fragments
thereof. All such variations in the nucleic acid molecule retain
the ability to encode a biologically active enzyme when expressed
in the appropriate host or an enzymatically active fragment
thereof. The nucleic acid molecule of the present invention may
comprise solely the nucleotide sequence encoding an enzyme or may
be part of a larger nucleic acid molecule that extends to the gene
for the enzyme. The non-enzyme encoding sequences in a larger
nucleic acid molecule may include vector, promoter, terminator,
enhancer, replication, signal sequences, or non-coding regions of
the gene.
[0037] The term "variant" as used herein means a polypeptide
substantially homologous to a naturally occurring protein but which
has an amino acid sequence different from the naturally occurring
protein (human, bovine, ovine, porcine, murine, equine, or other
eukaryotic species) because of one or more deletions, insertions,
derivations, or substitutions. The variant amino acid sequence
preferably is at least 50% identical to a naturally occurring amino
acid sequence but is most preferably at least 70% identical.
Variants may comprise conservatively substituted sequences wherein
a given amino acid residue is replaced by a residue having similar
physiochemical characteristics. Conservative substitutions are well
known in the art and include substitution of one aliphatic residue
for another, such as Ile, Val, Leu, or Ala for one another, or
substitutions of one polar residue for another, such as between Lys
and Arg; Glu and Asp; or Gln and Asn. Conventional procedures and
methods can be used for making and using such variants. Other such
conservative substitutions such as substitutions of entire regions
having similar hydrophobicity characteristics are well known.
Naturally occurring variants are also encompassed by the present
invention. Examples of such variants are enzymes that result from
alternate mRNA splicing events or from proteolytic cleavage of the
enzyme that leave the enzyme biologically active and capable of
performing its catalytic function. Alternate splicing of mRNA may
yield a truncated but biologically active protein such as a
naturally occurring soluble form of the protein. Variations
attributable to proteolysis include differences in the N- or
C-termini upon expression in different types of host cells due to
proteolytic removal of one or more terminal amino acids from the
protein.
[0038] The term "substantially the same" as used herein means
nucleic acid or amino acid sequences having sequence variations
that do not materially affect the nature of the protein, i.e., the
structure and/or biological activity of the protein. With
particular reference to nucleic acid sequences, the term
"substantially the same" is intended to refer to the coding region
and to conserved sequences governing expression and refers
primarily to degenerate codons encoding the same amino acid or
alternate codons encoding conservative substitute amino acids in
the encoded polypeptide. With reference to amino acid sequences,
the term "substantially the same" refers generally to conservative
substitutions and/or variations in regions of the polypeptide nor
involved in determination of structure or function.
[0039] The term "percent identity" as used herein means comparisons
among amino acid sequences as defined in the UWGCG sequence
analysis program available from the University of Wisconsin.
(Devereaux et al., Nucl. Acids Res. 12: 387-397 (1984)).
[0040] The term "highly phosphorylated lysosomal hydrolase" as used
to herein means a level of phosphorylation on a purified lysosomal
hydrolase which could not be obtained by only isolating the
hydrolase from a natural source and without subsequent treatment
with the GlcNAc-phosphotransferase and
phosphodiester-.alpha.-GlcNAcase. In particular, "highly
phosphorylated lysosomal hydrolase" means a lysosomal hydrolase
that contains from about 6% to about 100% bis-phosphorylated
oligosaccharides.
[0041] This invention is not limited to the particular methodology,
protocols, cell lines, vectors, and reagents described because
these may vary. Further, the terminology used herein is for the
purpose of describing particular embodiments only and is not
intended to limit the scope of the present invention. As used
herein and in the appended claims, the singular forms "a," "an,"
and "the" include plural reference unless the context clearly
dictates otherwise, e.g., reference to "a host cell") includes a
plurality of such host cells.
[0042] Because of the degeneracy of the genetic code, a multitude
of nucleotide sequences encoding GlcNAc-phosphotransferase,
phosphodiester .alpha.-GlcNAcase, or other sequences referred to
herein may be produced. Some of these sequences will be highly
homologous and some will be minimally homologous to the nucleotide
sequences of any known and naturally occurring gene. The present
invention contemplates each and every possible variation of
nucleotide sequence that could be made by selecting combinations
based on possible codon choices. These combinations are made in
accordance with the standard triplet genetic code as applied to the
nucleotide sequence of naturally occurring
GlcNAc-phosphotransferase or phosphodiester .alpha.-GlcNAcase, and
all such variations are to be considered as being specifically
disclosed.
[0043] Unless defined otherwise, all technical and scientific terms
and any acronyms used herein have the same meanings as commonly
understood by one of ordinary skill in the art in the field of the
invention. Although any methods and materials similar or equivalent
to those described herein can be used in the practice of the
present invention, the preferred methods, devices, and materials
are described herein.
[0044] All patents and publications mentioned herein are
incorporated herein by reference to the extent allowed by law for
the purpose of describing and disclosing the proteins, enzymes,
vectors, host cells, and methodologies reported therein that might
be used with the present invention. However, nothing herein is to
be construed as an admission that the invention is not entitled to
antedate such disclosure by virtue of prior invention.
The Invention
GlcNAc-phosphotransferase
[0045] In one aspect, the present invention provides isolated and
purified biologically active GlcNAc-phosphotransferase, nucleic
acid molecules encoding GlcNAc-phosphotransferase and its subunits,
expression vectors having a DNA that encodes
GlcNAc-phosphotransferase, host cells that have been transfected or
transformed with expression vectors having DNA that encodes
GlcNAc-phosphotransferase, methods for producing recombinant
GlcNAc-phosphotransferase by culturing host cells that have been
transfected or transformed with expression vectors having DNA that
encodes GlcNAc-phosphotransferase, isolated and purified
recombinant GlcNAc-phosphotransferase, and methods for using
GlcNAc-phosphotransferase for the preparation of highly
phosphorylated lysosomal enzymes that are useful for the treatment
of lysosomal storage diseases.
[0046] To obtain isolated and purified GlcNAc-phosphotransferase
and its subunits and the nucleic acid molecules encoding the enzyme
according to the present invention, bovine GlcNAc
phosphotransferase was obtained and analyzed as follows.
Splenocytes from mice immunized with a partially purified
preparation of bovine GlcNAc-phosphotransferase were fused with
myeloma cells to generate a panel of hybridomas. Hybridomas
secreting monoclonal antibodies specific for
GlcNAc-phosphotransferase were identified by immunocapture assay.
In this assay, antibodies which could capture
GlcNAc-phosphotransferase from a crude source were identified by
assay of immunoprecipitates with a specific
GlcNAc-phosphotransferase enzymatic assay. Hybridomas were
subcloned twice, antibody produced in ascites culture, coupled to a
solid support and evaluated for immunoaffinity chromatography.
Monoclonal PT18-Emphaze was found to allow a single step
purification of GlcNAc-phosphotransferase to homogeneity. Bao,
et.al., The Journal of Biological Chemistry, Vol. 271, Number 49,
Issue of Dec. 6, 1996, pp. 31437-31445 relates to a method for the
purification of bovine UDP-N-acetylglucosamine:Lysosomal-enzyme
N-Acetylglucosamine-1-phosphotransferase and proposes a
hypothetical subunit structure for the protein. Bao, et. al., The
Journal of Biological Chemistry, Vol. 271, Number 49, Issue of Dec.
6, 1996, pp. 31446-31451. Using this technique, the enzyme was
purified 488.000-fold in 29% yield. The eluted
GlcNAc-phosphotransferase has a specific activity of >10.sup.6,
preferably >5.times.10.sup.6, more preferably
>12.times.10.sup.6 pmol/h/mg and is apparently a homogenous,
multi-subunit enzyme based on silver-stained SDS-PAGE. The
monoclonal antibody labeled PT18 was selected for use in further
experiments. A hybridoma secreting monoclonal antibody PT 18 was
deposited with the American Type Culture Collection, 10801
University Blvd., Manassas, Va. 20110 and assigned ATCC Accession
No. ______.
[0047] GlcNAc-phosphotransferase was determined to be a complex of
six polypeptides with a subunit structure
.alpha..sub.2.beta..sub.2.gamma..sub.2. FIG. 1 shows a model of the
subunit structure obtained from quantitative amino acid sequencing,
immunoprecipitation with subunit-specific monoclonal antibodies,
SDS-PAGE, and cDNA sequences. The evidence for the model is
summarized below. The molecular mass of the complex estimated by
gel filtration is 570,000 Daltons. The 166,000 Dalton
.alpha.-subunit is found as a disulfide-linked homodimer. Likewise,
the 51,000 Dalton 1-subunit is found as a disulfide-linked
homodimer. Because both the .alpha.- and .gamma.-subunits are found
in disulfide-linked homodimers, each molecule must contain at least
one .alpha.- and one .gamma. homodimer. Although the 56,000 Dalton
.beta.-subunit is not found in a disulfide-linked homodimer, two
independent lines of evidence strongly suggest each complex
contains two .beta.-subunits as well. First, quantitative
aminoterminal sequencing demonstrates a 1:1 molar ratio between the
.beta.- and .gamma.-subunits. Secondly, since the .alpha.- and
.beta.-subunits are encoded by a single cDNA and divided by
proteolytic processing, two .beta.-subunits are produced for each
.alpha.-subunit dimer. The predicted mass of the complex based on
the composition .alpha..sub.2.beta..sub.2.gamma..sub.2 is 546,000
Daltons (2.times.166,000+2.times.56,000+2.times.51,000) in
excellent agreement with the mass estimated by gel filtration.
[0048] GlcNAc-phosphotransferase was purified using an assay for
the transfer of GlcNAc-1-Phosphate to the synthetic acceptor
.alpha.-methylmannoside. However, the natural acceptors for
GlcNAc-phosphotransferase are the high mannose oligosaccharides of
lysosomal hydrolases. To evaluate the ability of the purified
GlcNAc-phosphotransferase to utilize glycoproteins as acceptors,
the transfer of GlcNAc-1-P to the lysosomal enzymes uteroferrin and
cathepsin D, the nonlysosomal glycoprotein RNAse B, and the
lysosomal hydrolase .beta.-glucocerebrosidase (which is trafficked
by a M6P independent pathway), were investigated. Both uteroferrin
and cathepsin D are effectively utilized as acceptors by purified
GlcNAc-phosphotransferase with K.sub.ms below 20 .mu.m. In
contrast, neither RNAse B nor .beta.-glucocerebrosidase is an
effective acceptor.
[0049] The ineffectiveness of RNAse B, which contains a single high
mannose oligosaccharide, as an acceptor is especially notable since
the K.sub.m was not reached at the solubility limit of the protein
(at 600 .mu.m). This data clearly demonstrates the specific
phosphorylation of Lysosomal hydrolases previously observed with
crude preparations (Waheed, Pohlmann A., R., et al. (1982).
"Deficiency of UDP-N-acetylglucosamine:lysosomal enzyme
N-Acetylglucosamine-1phosphotransferase in organs of I-Cell
patients." Biochemical and Biophysical Research Communications
105(3): 1052-10580 is a property of the GlcNAc-phosphotransferase
itself.
[0050] The .alpha.-subunit was identified as containing the
UDP-GlcNAc binding site since this subunit was specifically
photoaffinity-labeled with [.beta.-.sup.32P]-5-azido-UDP-Glc.
[0051] The amino-terminal and internal (tryptic) protein sequence
data was obtained for each subunit. N-terminal sequence was
obtained from each subunit as follows. Individual subunits of
GlcNAc-phosphotransferase were resolved by polyacrylamide gel
electrophoresis in the presence of sodium dodecyl sulfate before
and after disulfide bond reduction. Subunits were then transferred
to a PVDF membrane by electroblotting, identified by Coomassie blue
staining, excised, and subjected to N-terminal sequencing. To
obtain internal sequence, GlcNAc-phosphotransferase was denatured,
reduced, and alkylated, and individual subunits were resolved by
gel filtration chromatography. Isolated subunits were then digested
with trypsin and the tryptic peptides fractionated by reverse phase
HPLC. Peaks which appeared to contain only a single peptide were
analyzed for purity by MALDI and subjected to N-terminal amino acid
sequencing.
[0052] The amino acid sequence for the human .alpha.-subunit is
shown in amino acids 1-928 of SEQ ID NO: 1; the human
.beta.-subunit in amino acids 1-328 of SEQ ID NO:2; and the human
.gamma.-subunit in amino acids 25-305 of SEQ ID NO:3. The
.gamma.-subunit has a signal sequence shown in amino acids 1-24 of
SEQ ID NO:3.
[0053] Comparison with the databases using the blast algorithms
demonstrate these proteins have not been previously described
although several EST sequences of the corresponding cDNAs are
present.
[0054] Using these peptide sequences and a combination of library
screening, RACE, PCR and Blast searching of expressed sequence tag
("EST") files, full-length human cDNAs encoding each subunit were
cloned and sequenced.
[0055] The nucleotide sequence for the human .alpha./.beta.-subunit
precursor cDNA is shown in nucleotides 165-3932 of SEQ ID NO:4; the
nucleotide sequence for the .alpha.-subunit is shown in nucleotides
165-2948 of SEQ ID NO:4; the nucleotide sequence for the
.beta.-subunit is shown in nucleotides 2949-3932 of SEQ ID NO:4;
and the nucleotide sequence for the .gamma.-subunit is shown in
nucleotides 96-941 of SEQ ID NO:5. The nucleotide sequence for the
.gamma.-Subunit signal peptide is shown in nucleotides 24-95 of SEQ
ID NO:5.
[0056] For each subunit a N-terminal peptide and two internal
peptide sequences have been identified in the respective cDNA
sequence. Although the protein sequence data is from the bovine
protein and the cDNA sequences are human, the sequences are highly
homologous (identities: .alpha.-subunit 43/50; .beta.-subunit
64/64; .gamma.-subunit 30/32), confirming the cloned cDNAs
represent the human homologs of the bovine
GlcNAc-phosphotransferase subunits. The .alpha.- and
.beta.-subunits were found to be encoded by a single cDNA whose
gene is on chromosome 12. The .gamma.-subunit is the product of a
second gene located on chromosome 16. The .alpha./.beta.-subunits
precursor gene has been cloned and sequenced. The gene spans
.about.80 kb and contains 21 exons. The .gamma.-subunit gene has
also been identified in data reported from a genome sequencing
effort. The .gamma.-subunit gene is arranged as 11 exons spanning
12 kb of genomic DNA.
[0057] Using the human cDNAs, the homologous murine cDNAs for the
.alpha.-, .beta.- and .gamma.-subunits were isolated and sequenced
using standard techniques. The murine .alpha.- .beta.-subunit
precursor cDNA is shown in SEQ ID NO:16. The deduced amino acid
sequence for the murine .alpha.-subunit is shown in SEQ ID NO:15
and the .beta.-subunit in SEQ ID NO:8.
[0058] The mouse .gamma.-subunit cDNA was isolated from a mouse
liver library in .lamda.Zap II using the .gamma.-human
.gamma.-subunit cDNA as a probe. The human .gamma.-subunit cDNA was
random hexamer-labeled with .sup.32P-dCTP and used to screen a
mouse liver cDNA library in .lamda.Zap II. The probe hybridized to
three of 500,000 plaques screened. Each was subcloned to
homogeneity, the insert excised, cloned into pUC19, and sequenced
using standard methods Sambrook, J., Fritsch E. F., et al. (1989).
Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, Cold
Spring Harbor Laboratory Press. The mouse .gamma.-subunit cDNA
sequence is shown in SEQ ID NO:10 and the deduced amino acid
sequence for the mouse .gamma.-subunit is shown in SEQ ID NO:9.
[0059] Comparison of the deduced amino acid sequences of the human
and mouse .alpha.-, .beta.-, and .gamma.-subunits demonstrates that
the proteins are highly homologous with about an 80 percent
identity.
[0060] To confirm that these enzymes were substantially the same
between species, a partial homologous rat cDNA for the .alpha.- and
.beta.-subunits was isolated and sequenced using standard
techniques. The partial rat .alpha.- and .beta.-subunit cDNA is
shown in SEQ ID NO:12. The deduced amino acid sequence
corresponding to the cDNA is shown in SEQ ID NO:11. Further, a
partial homologous Drosophila cDNA for the .alpha.- and
.beta.-subunits was isolated and sequenced using standard
techniques. The partial Drosophila .alpha.- and .beta.-subunit cDNA
is shown in SEQ ID NO:17. The deduced amino acid sequence
corresponding to the cDNA is shown in SEQ ID NO:13. Comparisons of
the deduced amino acid sequences of the partial human, rat, and
Drosophila .alpha.- and .beta.-subunits show that the proteins are
highly homologous.
Phosphodiester .alpha.-GlcNAcase
[0061] In another aspect, the present invention provides isolated
and purified biologically active phosphodiester .alpha.-GlcNAcase,
nucleic acid molecules encoding phosphodiester .alpha.-GlcNAcase,
expression vectors having a DNA that encodes phosphodiester
.alpha.-GlcNAcase, host cells that have been transfected or
transformed with expression vectors having DNA that encodes
phosphodiester .alpha.-GlcNAcase, methods for producing recombinant
phosphodiester .alpha.-GlcNAcase by culturing host cells that have
been transfected or transformed with expression vectors having DNA
that encodes phosphodiester .alpha.-GlcNAcase, isolated and
purified recombinant phosphodiester .alpha.-GlcNAcase, and methods
for using phosphodiester .alpha.-GlcNAcase for the preparation of
highly phosphorylated lysosomal enzymes that are useful for the
treatment of lysosomal storage diseases.
[0062] To obtain isolated and purified phosphodiester
.alpha.-GlcNAcase and the nucleic acid molecules encoding the
enzyme according to the present invention, bovine phosphodiester
.alpha. GlcNAcase was obtained and analyzed as follows. Mice were
immunized with a partially purified preparation of phosphodiester
.alpha.-GlcNAcase and a functional screening strategy was utilized
to identify and isolate a monoclonal antibody specific for
phosphodiester .alpha.-GlcNAcase. Immunogen was prepared by
partially purifying phosphodiester .alpha.-GlcNAcase 6000-fold from
a bovine pancreas membrane pellet using chromatography on
DEAE-Sepharose, iminodiacetic acid Sepharose, and Superose 6. Two
BALB/c mice were each injected intraperitoneally with 5 .mu.g
partially purified phosphodiester .alpha.-GlcNAcase emulsified in
Freunds complete adjuvant. On day 28, the mice were boosted
intraperitoneally with 5 .mu.g phosphodiester .alpha.-GlcNAcase
emulsified in Freunds incomplete adjuvant. On day 42 the mice were
bled and an phosphodiester .alpha.-GlcNAcase specific immune
response was documented by "capture assay." To perform the capture
assay, serum (5 .mu.l) was incubated overnight with 1.2 units
partially purified phosphodiester .alpha.-GlcNAcase. Mouse antibody
was then captured on rabbit antimouse IgG bound to protein
A-Ultralink.TM. resin. Following extensive washing, bound
phosphodiester .alpha.-GlcNAcase was determined in the Ultralink
pellet by assay of cleavage of [.sup.3H]-GlcNAc-1-phosphomannose
.alpha.-methyl.
[0063] Following a second intravenous boost with phosphodiester
.alpha.-GlcNAcase, the spleen was removed and splenocytes fused
with SP2/0 myeloma cells according to our modifications (Bag, M.,
Booth J. L., et al. (1996). "Bovine UDP-N-acetylglucosamine:
lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. I.
Purification and subunit structure." Journal of Biological
Chemistry 271: 31437-31445) of standard techniques; Harlow, E. and
Lane, D. (1988). Antibodies: a laboratory manual, Cold Spring
Harbor Laboratory). The fusion was plated in eight 96-well plates
in media supplemented with recombinant human IL-6 (Bazin, R. and
Lemieux, R. (1989). "Increased proportion of B cell hybridomas
secreting monoclonal antibodies of desired specificity in cultures
containing macrophage-derived hybridoma growth factor (IL-6)."
Journal of Immunological Methods 116: 245-249) and grown until
hybridomas were just visible. Forty-eight pools of 16-wells were
constructed and assayed for antiphosphodiester .alpha.-GlcNAcase
activity using the capture assay. Four pools were positive.
Subpools of 4-wells were then constructed from the wells present in
the positive 16-well pools. Three of the four 16-well pools
contained a single 4-well pool with anti-phosphodiester
.alpha.-GlcNAcase activity. The 4 single wells making up the 4-well
pools were then assayed individually identifying the well
containing the anti-phosphodiester .alpha.-GlcNAcase secreting
hybridomas. Using the capture assay, each hybridoma was subcloned
twice and antibody prepared by ascites culture. Monoclonals UC2 and
UC3 were found to be low affinity antibodies. UC1, a high affinity
IgG monoclonal antibody, was prepared by ascites culture and
immobilized on Emphaze for purification of phosphodiester
.alpha.-GlcNAcase. The monoclonal antibody labeled UC1 was selected
for use in further experiments. A hybridoma secreting monoclonal
antibody UC1 was deposited with the American Type Culture
Collection, 10801 University Blvd., Manassas, Va. 20110 and
assigned ATCC Accession No. ______.
[0064] To purify phosphodiester .alpha.-GlcNAcase, a solubilized
membrane fraction was prepared from bovine liver. Phosphodiester
.alpha.-GlcNAcase was absorbed to monoclonal antibody UC1 coupled
to Emphaze resin by incubation overnight with gentle rotation. The
UC1-Emphaze was then packed in a column, washed sequentially with
EDTA and NaHCO.sub.3 at pH 7.0, then phosphodiester
.alpha.-GlcNAcase was eluted with NaHCO.sub.3 at pH 10. Fractions
containing phosphodiester .alpha.-GlcNAcase at specific activities
>50,000 .mu./mg were pooled and adjusted to pH 8.0 with 1/5th
volume of 1 M Tris HCl, pH 7.4. Following chromatography on
UC1-Emphaze the phosphodiester .alpha.-GlcNAcase was purified
92,500-fold in 32% yield.
[0065] The phosphodiester .alpha.-GlcNAcase from UC1-Emphaze was
concentrated and chromatographed on Superose 6. Phosphodiester
.alpha.-GlcNAcase eluted early in the chromatogram as a symmetric
activity peak with a coincident protein peak. Following
chromatography on Superose 6, the enzyme was purified
.about.715,000-fold in 24% yield. The purified enzyme catalyzed the
cleavage of 472 .mu.mols/hr/mg
[.sup.3H]-GlcNAc-1-phosphomannose-.alpha.-methyl, corresponding to
a specific activity of 472,000 units/mg.
[0066] The purified phosphodiester .alpha.-GlcNAcase was subjected
to SDS-PAGE and protein was detected by silver staining (Blum, H.,
Beier H., et al. (1987). "Improved silver staining of plant
proteins, RNA and DNA in polyacrylamide gels." Electrophoresis:
93-99). A diffuse band was observed with a molecular mass of
approximately 70 kDa whose intensity varies with the measured
phosphodiester .alpha.-GlcNAcase activity. The diffuse appearance
of the band suggests the protein may be heavily glycosylated. A
faint band with a molecular mass of 150,000, which does not
correlate with activity, was also present.
[0067] A model for the subunit structure of phosphodiester
.alpha.-GlcNAcase was determined by gel filtration chromatography
and SDS-PAGE with and without disulfide bond reduction. The mass by
gel filtration is about 300,000. SDS-PAGE without disulfide bond
reduction is .about.140,000. Following disulfide bond reduction,
the apparent mass is 70,000. Together these data show
phosphodiester .alpha.-GlcNAcase is a tetramer composed of
disulfide linked homodimers. FIG. 2 shows a model of the subunit
structure of phosphodiester .alpha.-GlcNAcase.
[0068] The amino terminal amino acid sequence of affinity purified,
homogeneous bovine phosphodiester .alpha.-GlcNAcase was determined
using standard methods (Matsudaira, P., Ed. (1993). A Practical
Guide to Protein and Peptide Purification for Microsequencing. San
Diego, Academic Press, Inc.). The pure enzyme was also subjected to
trypsin digestion and HPLC to generate two internal tryptic
peptides which were sequenced. The amino acid sequences of these
three peptides are:
TABLE-US-00001 Peptide 1 - Amino Terminal
DXTRVHAGRLEHESWPPAAQTAGAHRPSVRTFV; (SEQ ID NO:23) Peptide 2 -
Tryptic RDGTLVTGYLSEEEVLDTEN: (SEQ ID NO:24) and Peptide 3 -
Tryptic GINLWEMAEFLLK. (SEQ ID NO:25)
[0069] The protein, nucleotide, and EST data bases were searched
for sequences that matched these peptide sequences and several
human and mouse ESTs were found that had the sequence of the third
peptide at their amino termini. Three human infant brain EST clones
and one mouse embryo clone were obtained from ATCC and sequenced.
The three human clones were all identical except for total length
at their 3' ends and virtually identical to the mouse clone, except
that the mouse EST contained a 102 bp region that was absent from
all three human brain ESTs. An EcoR I-Hind III fragment of about
700 bp was excised from the human cDNA clone (ATCC #367524) and
used to probe a human liver cDNA library directionally cloned in
TriplEx vector (Clontech). Of the positive clones isolated from the
library and converted to plasmids (pTriplEx), the largest (2200 bp)
was represented by clone 6.5 which was used for the rest of the
analysis.
[0070] The cDNA clone has been completely sequenced on both strands
and is a novel sequence that predicts a mature protein of about 50
kDa which is in agreement with the size of the deglycosylated
mature bovine liver phosphodiester .alpha.-GlcNAcase.
[0071] There is a unique BamH I site at base #512 and a unique Hind
ID site at base #1581. All three bovine peptide sequences (peptides
1, 2, and 3) were found. Although the sequences of peptides 2 and 3
in the human are 100% identical to the bovine sequences, the
amino-terminal peptide in humans is only 67% identical to the
bovine sequence. The human liver clone contains the 102 base pair
insert that has the characteristics of an alternatively spliced
segment that was missing in the human brain EST. The hydrophilicity
plot indicates the presence of a hydrophobic membrane spanning
region from amino acids 448 to 474 and another hydrophobic region
from amino acid 8 to 24 which fits the motif for a signal sequence
and there is a likely signal sequence cleavage site between G24 and
G25. There are six Asn-X-Ser/Thr potential N-linked glycosylation
sites, one of which is within the 102 bp insert. All of these sites
are amino terminal of the putative trans-membrane region. These
features indicate that the phosphodiester .alpha.-GlcNAcase is a
type I membrane spanning glycoprotein with the amino terminus in
the lumen of the Golgi and the carboxyl terminus in the cytosol.
This orientation is different from that of other
glycosyltransferases and glycosidases involved in glycoprotein
processing, which to date have been shown to be type II membrane
spanning proteins.
[0072] The amino acid sequence for the phosphodiester
.alpha.-GlcNAcase monomer is shown in amino acids 50-515 of SEQ ID
NO:6. The signal peptide is shown in amino acids 1-24 of SEQ ID
NO:6 and the pro segment is shown in amino acids 25-49 of SEQ ID
NO:6. The human cDNA was cloned using the techniques described
above. The nucleotide sequence for the monomer that associates to
form the phosphodiester .alpha.-GlcNAcase tetramer is shown in
nucleotides 151-1548 of SEQ ID NO:7. The nucleotide sequence for
the signal sequence is shown in nucleotides 1-72 of SEQ ID NO:7.
The nucleotide sequence for the propeptide is shown in nucleotides
73-150 of SEQ ID NO:7.
[0073] The murine cDNA for phosphodiester .alpha.-GlcNAcase is
shown in SEQ ID NO:18. The deduced amino acid sequence for the
murine phosphodiester .alpha.-GlcNAcase is shown in SEQ ID NO:19.
Comparison of the deduced amino acid sequences of the human and
mouse enzymes demonstrates that the proteins are highly homologous
with about an 80 percent identity. This is especially true in the
region of the active site where identity exceeds 90%. The murine
gene for phosphodiester .alpha.-GlcNAcase is shown in SEQ ID
NO:14.
[0074] The human phosphodiester .alpha.-GlcNAcase gene has been
identified by database searching. The sequence was determined
during the sequencing of clone 165E7 from chromosome 16.13.3,
GenBank AC007011.1, gi4371266. Interestingly, the phosphodiester
.alpha.-GlcNAcase gene was not identified by the SCAN program used
to annotate the sequence.
[0075] Because of the degeneracy of the genetic code, a DNA
sequence may vary from that shown in SEQ ID NO:4, SEQ ID NO:5, and
SEQ ID NO:7 and still encode a GlcNAc phosphotransferase and a
phosphodiester .alpha.-GlcNAcase enzyme having the amino acid
sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID
NO:6. Such variant DNA sequences may result from silent mutations,
e.g., occurring during PCR amplification, or may be the product of
deliberate mutagenesis of a native sequence. The invention,
therefore, provides equivalent isolated DNA sequences encoding
biologically active GlcNAc-phosphotransferase and phosphodiester
.alpha.-GlcNAcase selected from: (a) the coding region of a native
mammalian GlcNAc-phosphotransferase gene and phosphodiester
.alpha.-GlcNAcase gene; (b) cDNA comprising the nucleotide sequence
presented in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:7; (c) DNA
capable of hybridization to the native mammalian
GlcNAc-phosphotransferase gene and phosphodiester .alpha.-GlcNAcase
gene under moderately stringent conditions and which encodes
biologically active GlcNAc-phosphotransferase and phosphodiester
.alpha.-GlcNAcase; and (d) DNA which is degenerate as a result of
the genetic code to a DNA defined in (a), (b), or (c) and which
encodes biologically active GlcNAc-phosphotransferase and
phosphodiester .alpha.-GlcNAcase. GlcNAc-phosphotransferase and
phosphodiester .alpha.-GlcNAcase proteins encoded by such DNA
equivalent sequences are encompassed by the invention.
[0076] Those sequences which hybridize under stringent conditions
and encode biologically functional GlcNAc-phosphotransferase and
phosphodiester .alpha.-GlcNAcase are preferably at least 50-100%
homologous, which includes 55, 60, 65, 70, 75, 75, 80, 85, 90, 95,
99% and all values and subranges therebetween. Homology may be
determined with the software UWCG as described above. Stringent
hybridization conditions are known in the art and are meant to
include those conditions which allow hybridization to those
sequences with a specific homology to the target sequence. An
example of such stringent conditions are hybridization at
65.degree. C. in a standard hybridization buffer and subsequent
washing in 0.2.times. concentrate SSC and 0.1% SDS at 42-65.degree.
C., preferably 60.degree. C. This and other hybridization
conditions are disclosed in Sambrook, J., Fritsch E. F., et al.
(1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor,
Cold Spring Harbor Laboratory Press. Alternatively, the temperature
for hybridization conditions may vary dependent on the percent GC
content and the length of the nucleotide sequence, concentration of
salt in the hybridization buffer and thus the hybridization
conditions may be calculated by means known in the art.
[0077] Recombinant Expression for GlcNAc-phosphotransferase and
Phosphodiester .alpha.-GlcNAcase Isolated and purified recombinant
GlcNAc-phosphotransferase and phosphodiester .alpha.-GlcNAcase
enzymes are provided according to the present invention by
incorporating the DNA corresponding to the desired protein into
expression vectors and expressing the DNA in a suitable host cell
to produce the desired protein.
Expression Vectors
[0078] Recombinant expression vectors containing a nucleic acid
sequence encoding the enzymes can be prepared using well known
techniques. The expression vectors include a DNA sequence operably
linked to suitable transcriptional or translational regulatory
nucleotide sequences such as those derived from mammalian,
microbial, viral, or insect genes. Examples of regulatory sequences
include transcriptional promoters, operators, enhancers, mRNA
ribosomal binding sites, and appropriate sequences which control
transcription and translation initiation and termination.
Nucleotide sequences are "operably linked" when the regulatory
sequence functionally relates to the DNA sequence for the
appropriate enzyme. Thus, a promoter nucleotide sequence is
operably linked to a GlcNAc-phosphotransferase or phosphodiester a
GlcNAcase DNA sequence if the promoter nucleotide sequence controls
the transcription of the appropriate DNA sequence.
[0079] The ability to replicate in the desired host cells, usually
conferred by an origin of replication and a selection gene by which
transformants are identified, may additionally be incorporated into
the expression vector.
[0080] In addition, sequences encoding appropriate signal peptides
that are not naturally associated with GlcNAc-phosphotransferase or
phosphodiester .alpha.-GlcNAcase can be incorporated into
expression vectors. For example, a DNA sequence for a signal
peptide (secretory leader) may be fused in-frame to the enzyme
sequence so that the enzyme is initially translated as a fusion
protein comprising the signal peptide. A signal peptide that is
functional in the intended host cells enhances extracellular
secretion of the appropriate polypeptide. The signal peptide may be
cleaved from the polypeptide upon secretion of enzyme from the
cell.
Host Cells
[0081] Suitable host cells for expression of
GlcNAc-phosphotransferase and phosphodiester at .alpha.-GlcNAcase
include prokaryotes, yeast, archae, and other eukaryotic cells.
Appropriate cloning and expression vectors for use with bacterial,
fungal, yeast, and mammalian cellular hosts are well known in the
art, e.g., Pouwels et al. Cloning Vectors: A Laboratory Manual,
Elsevier, N.Y. (1985). The vector may be a plasmid vector, a single
or double-stranded phage vector, or a single or double-stranded RNA
or DNA viral vector. Such vectors may be introduced into cells as
polynucleotides, preferably DNA, by well known techniques for
introducing DNA and RNA into cells. The vectors, in the case of
phage and viral vectors also may be and preferably are introduced
into cells as packaged or encapsulated virus by well known
techniques for infection and transduction. Viral vectors may be
replication competent or replication defective. In the latter case
viral propagation generally will occur only in complementing host
cells. Cell-free translation systems could also be employed to
produce the enzymes using RNAs derived from the present DNA
constructs.
[0082] Prokaryotes useful as host cells in the present invention
include gram negative or gram positive organisms such as E. coli or
Bacilli. In a prokaryotic host cell, a polypeptide may include a
N-terminal methionine residue to facilitate expression of the
recombinant polypeptide in the prokaryotic host cell. The
N-terminal Met may be cleaved from the expressed recombinant
GlcNAc-phosphotransferase or phosphodiester .alpha.-GlcNAcase
polypeptide. Promoter sequences commonly used for recombinant
prokaryotic host cell expression vectors include .beta.-lactamase
and the lactose promoter system.
[0083] Expression vectors for use in prokaryotic host cells
generally comprise one or more phenotypic selectable marker genes.
A phenotypic selectable marker gene is, for example, a gene
encoding a protein that confers antibiotic resistance or that
supplies an autotrophic requirement. Examples of useful expression
vectors for prokaryotic host cells include those derived from
commercially available plasmids such as the cloning vector pBR322
(ATCC 37017). pBR322 contains genes for ampicillin and tetracycline
resistance and thus provides simple means for identifying
transformed cells. To construct an expression vector using pBR322,
an appropriate promoter and a DNA sequence are inserted into the
pBR322 vector.
[0084] Other commercially available vectors include, for example,
pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1
(Promega Biotec, Madison, Wis., USA).
[0085] Promoter sequences commonly used for recombinant prokaryotic
host cell expression vectors include .beta.-lactamase
(penicillinase), lactose promoter system (Chang et al., Nature
275:615, (1978); and Goeddel et al., Nature 281:544, (1979)),
tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res.
8:4057, (1980)), and tac promoter (Maniatis, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, p. 412
(1982)).
[0086] Yeasts useful as host cells in the present invention include
those from the genus Saccharomyces, Pichia, K. Actinomycetes and
Kluyveromyces. Yeast vectors will often contain an origin of
replication sequence from a 2.mu. yeast plasmid, an autonomously
replicating sequence (ARS), a promoter region, sequences for
polyadenylation, sequences for transcription termination, and a
selectable marker gene. Suitable promoter sequences for yeast
vectors include, among others, promoters for metallothionein,
3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem.
255:2073, (1980)) or other glycolytic enzymes (Holland et al.,
Biochem. 17:4900, (1978)) such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatee
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase. Other
suitable vectors and promoters for use in yeast expression are
further described in Fleer et al., Gene, 107:285-195 (1991). Other
suitable promoters and vectors for yeast and yeast transformation
protocols are well known in the art.
[0087] Yeast transformation protocols are known to those of skill
in the art. One such protocol is described by Hinnen et al.,
Proceedings of the National Academy of Sciences USA, 75:1929
(1978). The Hinnen protocol selects for Trp.sup.+ transformants in
a selective medium, wherein the selective medium consists of 0.67%
yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 .mu.g/ml
adenine, and 20 .mu.g/ml uracil.
[0088] Mammalian or insect host cell culture systems well known in
the art could also be employed to express recombinant
GlcNAc-phosphotransferase or phosphodiester .alpha.-GlcNAcase
polypeptides, e.g., Baculovirus systems for production of
heterologous proteins in insect cells (Luckow and Summers,
Bio/Technology 6:47 (1988)) or Chinese hamster ovary (CHO) cells
for mammalian expression may be used. Transcriptional and
translational control sequences for mammalian host cell expression
vectors may be excised from viral genomes. Commonly used promoter
sequences and enhancer sequences are derived from Polyoma virus,
Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.
DNA sequences derived from the SV40 viral genome may be used to
provide other genetic elements for expression of a structural gene
sequence in a mammalian host cell, e.g., SV40 origin, early and
late promoter, enhancer, splice, and polyadenylation sites. Viral
early and late promoters are particularly useful because both are
easily obtained from a viral genome as a fragment which may also
contain a viral origin of replication. Exemplary expression vectors
for use in mammalian host cells are well known in the art.
[0089] The enzymes of the present invention may, when beneficial,
be expressed as a fusion protein that has the enzyme attached to a
fusion segment. The fusion segment often aids in protein
purification, e.g., by permitting the fusion protein to be isolated
and purified by affinity chromatography. Fusion proteins can be
produced by culturing a recombinant cell transformed with a fusion
nucleic acid sequence that encodes a protein including the fusion
segment attached to either the carboxyl and/or amino terminal end
of the enzyme. Preferred fusion segments include, but are not
limited to, glutathione-S-transferase, .beta.-galactosidase, a
poly-histidine segment capable of binding to a divalent metal ion,
and maltose binding protein. In addition, the HPC-4 epitope
purification system may be employed to facilitate purification of
the enzymes of the present invention. The HPC-4 system is described
in U.S. Pat. No. 5,202,253, the relevant disclosure of which is
herein incorporated by reference.
Expression by Gene Activation Technology
[0090] In addition to expression strategies involving transfection
of a cloned cDNA sequence, the endogenous GlcNAc-phophotransfease
and phosphodiester .alpha.-GlcNAcase genes can be expressed by
altering the promoter.
[0091] Methods of producing the enzymes of the present invention
can also be accomplished according to the methods of protein
production as described in U.S. Pat. No. 5,968,502, the relevant
disclosure of which is herein incorporated by reference, using the
sequences for GlcNAc-phosphotransferase and phosphodiester
.alpha.-GlcNAcase as described herein.
Expression and Recovery
[0092] According to the present invention, isolated and purified
GlcNAc-phosphotransferase or phosphodiester .alpha.-GlcNAcase
enzymes may be produced by the recombinant expression systems
described above. The method comprises culturing a host cell
transformed with an expression vector comprising a DNA sequence
that encodes the enzyme under conditions sufficient to promote
expression of the enzyme. The enzyme is then recovered from culture
medium or cell extracts, depending upon the expression system
employed. As is known to the skilled artisan, procedures for
purifying a recombinant protein will vary according to such factors
as the type of host cells employed and whether or not the
recombinant protein is secreted into the culture medium. When
expression systems that secrete the recombinant protein are
employed, the culture medium first may be concentrated. Following
the concentration step, the concentrate can be applied to a
purification matrix such as a gel filtration medium. Alternatively,
an anion exchange resin can be employed, e.g., a matrix or
substrate having pendant diethylaminoethyl (DEAE) groups. The
matrices can be acrylamide, agarose, dextran, cellulose, or other
types commonly employed in protein purification. Also, a cation
exchange step can be employed. Suitable cation exchangers include
various insoluble matrices comprising sulfopropyl or carboxymethyl
groups. Further, one or more reversed-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media
(e.g., silica gel having pendant methyl or other aliphatic groups)
can be employed to further purify the enzyme. Some or all of the
foregoing purification steps, in various combinations, are well
known in the art and can be employed to provide an isolated and
purified recombinant protein.
[0093] Recombinant protein produced in bacterial culture is usually
isolated by initial disruption of the host cells, centrifugation,
extraction from cell pellets if an insoluble polypeptide, or from
the supernatant fluid if a soluble polypeptide, followed by one or
more concentration, salting-out, ion exchange, affinity
purification, or size exclusion chromatography steps. Finally,
RP-HPLC can be employed for final purification steps. Microbial
cells can be disrupted by any convenient method, including
freeze-thaw cycling, sonication, mechanical disruption, or use of
cell lysing agents.
Preparation of Highly Phosphorylated Lysosomal Enzymes
[0094] In another aspect, the present invention provides highly
phosphorylated lysosomal hydrolases and methods for the preparation
of such hydrolases. The highly phosphorylated lysosomal hydrolases
can be used in clinical applications for the treatment of lysosomal
storage diseases.
[0095] The method comprises obtaining lysosomal hydrolases having
asparagine-linked oligosaccharides with high mannose structures and
modifying the .alpha.1,2-linked or other outer mannoses by the
addition of M6P in vitro to produce a hydrolase that can be used
for the treatment of lysosomal storage diseases because it binds to
cell membrane M6P receptors and is readily taken into the cell and
into the lysosome. Typically, the high mannose structures consist
of from six to nine molecules of mannose and two molecules of
N-acetylglucosamine (GlcNAc). In the preferred embodiment, the high
mannose structure is a characteristic MAN7(D.sub.2D.sub.3) isomer
structure consisting of seven molecules of mannose and two
molecules of N-acetylglucosamine (GlcNAc).
[0096] Highly phosphorylated Lysosomal hydrolases are produced by
treating the high mannose hydrolases with GlcNAc-phosphotransferase
which catalyzes the transfer of N-acetylglucosamine-1-phosphate
from UDP-GlcNAc to the 6' position of .alpha.1,2-linked or other
outer mannoses on the hydrolase. This GlcNAc-phosphotransferase
modified hydrolase is then treated with phosphodiester
.alpha.-GlcNAcase which catalyzes the removal of
N-Acetylglucosamine to generate terminal M6P on the hydrolase.
[0097] In one embodiment of the invention, the
GlcNAc-phosphotransferase treated hydrolase may be isolated and
stored without any subsequent treatment. Subsequently, the
GlcNAc-phosphotransferase treated hydrolase may be modified further
by treating the hydrolase with a phosphodiester
.alpha.-GlcNAcase.
[0098] Surprisingly, it has been found that the hydrolases
containing M6P generated by this method are highly phosphorylated
when compared to naturally occurring or known recombinant
hydrolases. The highly phosphorylated lysosomal hydrolases of the
present invention contain from about 6% to about 100%
bis-phosphorylated oligosaccharides compared to less that about 5%
bis-phosphorylated oligosaccharides on known naturally occurring or
recombinant hydrolases.
[0099] These highly phosphorylated hydrolases have a higher
affinity for the M6P receptor and are therefore more efficiently
taken into the cell by plasma membrane receptors. (Reuser, A. J.,
Kroos, M. A., Ponne, N. J., Wolterman, R. A., Loonen, M. C., Busch,
H. F., Visser, W. J., and Bolhuis, P. A. (1984). "Uptake and
stability of human and bovine acid alpha-glucosidase in cultured
fibroblasts and skeletal muscle cells from glycogenosis type II
patients." Experimental Cell Research 155: 178-189).
[0100] The high-affinity ligand for the cation-independent M6P
receptor is an oligosaccharide containing two M6P groups (i.e., a
bis-phosphorylated oligosaccharide). Since a bisphosphorylated
oligosaccharides binds with an affinity 3500-fold higher than a
monophosphorylated oligosaccharides, virtually all the
high-affinity binding of a lysosomal enzyme to the M6P receptor
will result from the content of bis-phosphorylated oligosaccharides
(Tong, P. Y., Gregory, W., and Komfeld, S. (1989)). "Ligand
interactions of the cation-independent mannose 6-phosphate
receptor. The stoichiometry of mannose 6-phosphate binding."
Journal of Biological Chemistry 264: 7962-7969). It is therefore
appropriate to use the content of bis-phosphorylated
oligosaccharides to compare the binding potential of different
preparations of lysosomal enzymes.
[0101] The extent of mannose 6-phosphate modification of two
different lysosomal enzymes has been published. The oligosaccharide
composition of human .alpha.-galactosidase A secreted from Chinese
hamster ovary cells has been published (Matsuura, F., Ohta, M.,
Ioannou, Y. A., and Desnick, R. I. (1998). "Human
alpha-galactosidase A: characterization of the N-linked
oligosaccharides on the intracellular and secreted glycoforms
overexpressed by Chinese hamster ovary cells." Glycobiology 8(4):
329-39). Of all oligosaccharides on .alpha.-gal A released by
hydrazinolysis, only 5.2% were bis-phosphorylated. Zhao et al.
partially characterized the oligosaccharide structures on
recombinant human .alpha.-iduronidase secreted by CHO cells (Zhao,
K. W., Faull, K. F., Kakkis, E. D., and Neufeld, E. F. (1997).
"Carbohydrate structures of recombinant human alpha-L-iduronidase
secreted by Chinese hamster ovary cells." J Biol Chem 272(36):
22758-65) and demonstrated a minority of the oligosaccharides were
bisphosphorylated. The qualitative techniques utilized precluded
the determination of the fraction of oligosaccharides
phosphorylated.
[0102] The production and secretion of human acid
.alpha.-glucosidase by CHO cells has been reported (Van Hove, J.
L., Yang, H. W., Wu, J. Y., Brady, R. O., and Chen, Y. T. (1996).
"High level production of recombinant human lysosomal acid
alpha-glucosidase in Chinese hamster ovary cells which targets to
heart muscle and corrects glycogen accumulation in fibroblasts from
patients with Pompe disease." Proceedings of the National Academy
of Sciences USA, 93(1): 6570). The carbohydrate structures of this
preparation were not characterized in this publication. However,
this preparation was obtained and analyzed. The results, given in
the examples below, showed that less than 1% of the
oligosaccharides contained any M6P and bis-phosphorylated
oligosaccharides were not detectable. Together, these data show
that known preparations of recombinant lysosomal enzymes contain no
more than 5.2% phosphorylated oligosaccharides. It appears that the
preparation of more highly phosphorylated lysosomal enzymes is
unlikely to be achieved with known techniques. Naturally occurring
human acid .alpha.-glucosidase purified from human placenta
contains very low levels of M6P (Mutsaers, I. H. G. M., Van
Halbeek, H., Vliegenthart, J. F. G., Tager, J. M., Reuser, A. J.
J., Kroos, M., and Galjaard, H. (1987). "Determination of the
structure of the carbohydrate chains of acid .alpha.-glucosidase
from human placenta." Biochimica et Biophysica Acta 911: 244-251).
The arrangement of the phosphates as either bis- or
monophosphorylated oligosaccharides has not been determined, but
less than 1% of the oligosaccharides contain any M6P.
[0103] The highly phosphorylated hydrolases of the present
invention are useful in enzyme replacement therapy procedures
because they are more readily taken into the cell and the lysosome.
(Reuser, A. J., Kroos, M. A., Ponne, N. J., Wolterman, R. A.,
Loonen, M. C., Busch, H. F., Visser, W. J. and Bolhuis, P. A.
(1984). "Uptake and stability of human and bovine acid
alpha-glucosidase in cultured fibroblasts and skeletal muscle cells
from glycogenosis type II patients." Experimental Cell Research
155: 178-189).
[0104] Any lysosomal enzyme that uses the M6P transport system can
be treated according to the method of the present invention.
Examples include .alpha.-glucosidase (Pompe Disease),
.alpha.-L-iduronidase (Hurler Syndrome), .alpha.-galactosidase A
(Fabry Disease), arylsulfatase (Maroteaux-Lamy Syndrome),
N-acetylgalactosamine-6-sulfatase or .beta.-galactosidase (Morquio
Syndrome), iduronate 2-sulfatase (Hunter Syndrome), ceramidase
(Farber Disease), galactocerebrosidase (Krabbe Disease),
.beta.-glucuronidase (Sly Syndrome), Heparan N-sulfatase
(Sanfilippo A), N-Acetyl-.alpha.-glucosaminidase (Sanfilippo B),
Acetyl CoA-.alpha.-glucosaminide N-acetyl transferase,
N-acetyl-glucosamine-6 sulfatase (Sanfilippo D), Galactose
6-sulfatase (Morquio A), Arylsulfatase A, B, and C (Multiple
Sulfatase Deficiency), Arylsulfatase A Cerebroside (Metachromatic
Leukodystrophy), Ganglioside (Mucolipidosis IV), Acid
.beta.-galactosidase G.sub.M1 Galglioside (G.sub.M1
Gangliosidosis), Acid .beta.-galactosidase (Galactosialidosis),
Hexosaminidase A (Tay-Sachs and Variants), Hexosaminidase B
(Sandhoff), .alpha.-fucosidase (Fucsidosis), .alpha.-N-Acetyl
galactosaminidase (Schindler Disease), Glycoprotein Neuraminidase
(Sialidosis), Aspartylglucosamine amidase (Aspartylglucosaminuria),
Acid Lipase (Wolman Disease), Acid Ceramidase (Farber
Lipogranulomatosis), Lysosomal Sphingomyelinase and other
Sphingomyelinase (Nieman-Pick).
[0105] Methods for treating any particular lysosomal hydrolase with
the enzymes of the present invention are within the skill of the
artisan. Generally, the lysosomal hydrolase at a concentration of
about 10 mg/ml and GlcNAc-phosphotransferase at a concentration of
about 100,000 units/mL are incubated at about 37.degree. C. for 2
hours in the presence of a buffer that maintains the pH at about
6-7 and any stabilizers or coenzymes required to facilitate the
reaction. Then, phosphodiester .alpha.-GlcNAcase is added to the
system to a concentration of about 1000 units/mL and the system is
allowed to incubate for about 2 more hours. The modified lysosomal
enzyme having highly phosphorylated oligosaccharides is then
recovered by conventional means.
[0106] In a preferred embodiment, the lysosomal hydrolase at 10
mg/ml is incubated in 50 mm Tris-HCl, pH 6.7, 5 mM MgCl.sub.2, 5 mM
MnCl.sub.2, 2 mM UDP-GlcNAc with GlcNAc phosphotransferase at
100,000 units/mL at 37.degree. C. for 2 hours. Phosphodiester
.alpha.-GlcNAcase, 1000 units/mL, is then added and the incubation
continued for another 2 hours. The modified enzyme is then
repurified by chromatography on Q-Sepharose and step elution with
NaCl.
Methods for Obtaining High Mannose Lysosomal Hydrolases
[0107] High mannose lysosomal hydrolases for treatment according to
the present invention can be obtained from any convenient source,
e.g., by isolating and purifying naturally occurring enzymes or by
recombinant techniques for the production of proteins. High mannose
lysosomal hydrolases can be prepared by expressing the DNA encoding
a particular hydrolase in any host cell system that generates a
oligosaccharide modified protein having high mannose structures,
e.g., yeast cells, insect cells, other eukaryotic cells,
transformed Chinese Hamster Ovary (CHO) host cells, or other
mammalian cells.
[0108] In one embodiment, high mannose lysosomal hydrolases are
produced using mutant yeast that are capable of expressing peptides
having high mannose structures. These yeast include the mutant S.
cervesiae .DELTA. ochl, .DELTA. mnnl (Nakanishi-Shindo, Y.,
Nakayama, K. I., Tanaka, A., Toda, Y. and Jigami, Y. (1993).
"Structure of the N-linked oligosaccharides that show the complete
loss of .alpha.-1,6-polymannose outer chain from ochl, ochl mnnl,
and ochl mnnl alg3 mutants of Saccharomyces cerevisiae." Journal of
Biological Chemistry 268: 26338-26345).
[0109] Preferably, high mannose lysosomal hydrolases are produced
using over-expressing transformed insect, CHO, or other mammalian
cells that are cultured in the presence of certain inhibitors.
Normally, cells expressing lysosomal hydrolases secrete acid
.alpha.-glucosidase that contains predominantly sialylated
biantenniary complex type glycans that do not serve as a substrate
for GlcNAc-phosphotransferase and therefore cannot be modified to
use the M6P receptor.
[0110] According to the present invention, a new method has been
discovered for manipulating transformed cells containing DNA that
expresses a recombinant hydrolase so that the cells secrete high
mannose hydrolases that can be modified according to the above
method. In this method, transformed cells are cultured in the
presence of .alpha.1,2-mannosidase inhibitors and the high mannose
recombinant hydrolases are recovered from the culture medium.
Inhibiting alpha 1,2-mannosidase prevents the enzyme from trimming
mannoses and forces the cells to secrete glycoproteins having the
high mannose structure. High mannose hydrolases are recovered from
the culture medium using known techniques and treated with
GlcNAc-phosphotransferase and phosphodiester .alpha.-GlcNAcase
according to the method herein to produce hydrolases that have M6P
and can therefore bind to membrane M6P receptors and be taken into
the cell. Preferably, the cells are CHO cells and the hydrolases
are secreted with the MAN7(D.sub.2D.sub.3) structure. FIG. 3 shows
the reaction scheme for this method.
[0111] In a preferred embodiment, recombinant human acid alpha
glucosidase ("rh-GAA") is prepared by culturing CHO cells secreting
rh-GAA in Iscove's Media modified by the addition of an alpha
1,2-mannosidase inhibitor. Immunoprecipitation of rh-GAA from the
media followed by digestion with either N-glycanase or
endoglycosidase-H demonstrates that in the presence of the alpha
1,2-mannosidase inhibitor the rh-GAA retains high mannose
structures rather than the complex structures found on a
preparation secreted in the absence of the inhibitor. The secreted
rh-GAA bearing high mannose structures is then purified to
homogeneity, preferably by chromatography beginning with ion
exchange chromatography on ConA-Sepharose, Phenyl-Sepharose and
affinity chromatography on Sephadex G-100. The purified rh-GAA is
then treated in vitro with GlcNAc-phosphotransferase to convert
specific mannoses to GlcNAc-phospho-mannose diesters. The
GlcNAcphosphomannose diesters are then converted to M6P groups by
treatment with phosphodiester .alpha. GlcNAcase. Experiments show
that 74% of the rh-GAA oligosaccharides were phosphorylated, 62%
being bis-phosphorylated, and 12% monophosphorylated. Since each
molecule of rh-GAA contains 7 N-linked oligosaccharides, 100% of
the rh-GAA molecules are likely to contain the mannose-phosphate
modification.
[0112] Any alpha 1,2-mannosidase inhibitor can function in the
present invention. Preferably, the inhibitor is selected from the
group consisting of deoxymannojirimycin (dMM), kifunensine,
D-Mannonolactam amidrazone, and N-butyl-deoxymannojirimycin. Most
preferably the inhibitor is deoxymannojimycin.
Treatment of Lysosomal Storage Diseases
[0113] In a further aspect, the present invention provides a method
for the treatment of lysosomal storage diseases by administering a
disease treating amount of the highly phosphorylated lysosomal
hydrolases of the present invention to a patient suffering from the
corresponding lysosomal storage disease. While dosages may vary
depending on the disease and the patient, the enzyme is generally
administered to the patient in amounts of from about 0.1 to about
1000 milligrams per 50 kg of patient per month, preferably from
about 1 to about 500 milligrams per 50 kg of patient per month. The
highly phosphorylated enzymes of the present invention are more
efficiently taken into the cell and the lysosome than the naturally
occurring or less phosphorylated enzymes and are therefore
effective for the treatment of the disease. Within each disease,
the severity and the age at which the disease presents may be a
function of the amount of residual lysosomal enzyme that exists in
the patient. As such, the present method of treating lysosomal
storage diseases includes providing the highly phosphorylated
lysosomal hydrolases at any or all stages of disease
progression.
[0114] The lysosomal enzyme is administered by any convenient
means. For example, the enzyme can be administered in the form of a
pharmaceutical composition containing the enzyme and any
pharmaceutically acceptable carriers or by means of a delivery
system such as a liposome or a controlled release pharmaceutical
composition. The term "pharmaceutically acceptable" refers to
molecules and compositions that are physiologically tolerable and
do not typically produce an allergic or similar unwanted reaction
such as gastric upset or dizziness when administered. Preferably,
"pharmaceutically acceptable" means approved by a regulatory agency
of the Federal or a state government or listed in the U.S.
Pharmacopoeia or other generally recognized pharmacopoeia for use
in animals, preferably humans. The term "carrier" refers to a
diluent, adjuvant, excipient, or vehicle with which the compound is
administered. Such pharmaceutical carriers can be sterile liquids,
such as saline solutions, dextrose solutions, glycerol solutions,
water and oils emulsions such as those made with oils of petroleum,
animal, vegetable, or synthetic origin (peanut oil, soybean oil,
mineral oil, or sesame oil). Water, saline solutions, dextrose
solutions, and glycerol solutions are preferably employed as
carriers, particularly for injectable solutions.
[0115] The enzyme or the composition can be administered by any
standard technique compatible with enzymes or their compositions.
For example, the enzyme or composition can be administered
parenterally, transdermally, or transmucosally, e.g., orally or
nasally. Preferably, the enzyme or composition is administered by
intravenous injection.
[0116] The following Examples provide an illustration of
embodiments of the invention and should not be construed to limit
the scope of the invention which is set forth in the appended
claims. In the following Examples, all methods described are
conventional unless otherwise specified.
EXAMPLES
Materials and Methods
[0117] Lactating bovine udders were obtained from Mikkelson Beef,
Inc. (Oklahoma City, Okla.). Ultrasphere ODS columns were obtained
from Beckman Instruments. Microsorb MV-NH.sub.2 columns were
obtained from Rainin Instrument Co., Inc. (Woburn, Mass.).
[.gamma..sup.32P]ATP (7000 Ci/mmol; end labeling grade),
Na.sup.125I, and Lubrol
(C.sub.16H.sub.33(CH.sub.2CH.sub.2O).sub.23H) were obtained from
ICN (Costa Mesa, Calif.). Superose 6 (prep grade), DEAE-Sepharose
FF, QAE-Sephadex A-25, molecular mass standards for SDS-PAGE,
HiTrap-protein G columns, and Mono Q columns were obtained from
Pharmacia Biotech Inc. 3M-Emphaze Biosupport Medium AB1, IODO GEN
iodination reagent, and the BCA protein assay reagent were obtained
from Pierce. Glycerol, sucrose, .alpha.-methylmannoside,
.alpha.-methylglucoside, reactive green 19-agarose, sodium
deoxycholate, benzamidine, UDP-GlcNAc, phenylmethylsulfonyl
fluoride, Tris, rabbit anti-mouse IgG, and mouse monoclonal
antibody isotyping reagents were obtained from Sigma.
[0118] POROS 50 HQ was obtained from PerSeptive Biosystems
(Cambridge, Mass.). ProBlott polyvinylidene difluoride membranes
were obtained from Applied Biosystems Inc. (Foster City, Calif.). A
Model QT12 rotary tumbler was obtained from LORTONE, Inc. (Seattle,
Wash.). A mouse immunoglobulin standard panel was obtained from
Southern Biotechnology Associates, Inc. (Birmingham, Ala.).
Recombinant interleukin-6, porcine uteroferrin, and monoclonal
antibody BP95 were gifts from colleagues. Other chemicals were
reagent grade or better and were from standard suppliers.
Example 1
Preparation of Monoclonal Antibodies Specific for Bovine
GlcNAc-Phosphotransferase
[0119] Bovine GlcNAc-phosphotransferase was partially purified
30,000 fold as described (Bao, M., Booth J. L., et al. (1996).
"Bovine UDP-N-acetylglucosamine: Lysosomal enzyme
N-acetylglucosamine-1-phosphotransferase. 1. Purification and
subunit structure." Journal of Biological Chemistry 271:
31437-31445) and used to immunize mice. Spleens of immune mice were
removed and spenocytes fused with SP2/0 myeloma cells according to
Harlow (Harrow, E. and Lane, D. (1988). Antibodies: a laboratory
manual, Cold Spring Harbor Laboratory). The fusion was plated into
96 well plates and cultured in HAT media until hybridomas were
visible.
[0120] Hybridomas secreting monoclonal antibodies capable of
capturing GlcNAc-phosphotransferase from a crude sample were
identified by incubation of hybridoma media (200 .mu.l) with 200
units. Partially purified GlcNAc-phosphotransferase and capturing
the resulting immune complex on rabbit anti-mouse IgG bound to
protein A coupled to Ultralink.TM. matrix. Immune complexes which
contained monoclonal antibodies directed against
GlcNAc-phosphotransferase were then identified by assay of the
immune complex for GlcNAc-phosphotransferase activity. By this
strategy, four monoclonals directed against
GlcNAc-phosphotransferase were identified in the fifth fusion
screened. The hybridomas identified were subcloned twice using the
same assay and ascites was produced in BALBc mice according to
standard techniques (Harlow, E. and Lane, D. (1988). Antibodies: a
laboratory manual, Cold Spring Harbor Laboratory). The monoclonal
antibody labeled PT18 was selected for use in further
experiments.
Example 2
Purification of Bovine GlcNAc-Phosphotransferase
[0121] Lactating bovine mammary gland (6 kg) was collected at
slaughter and immediately sliced into 10 cm thick slices and
chilled in ice. Following homogenization in a Waring commercial
blender, the post-nuclear supernatant fraction was prepared by
centrifugation. Membrane fragments were collected by high speed
centrifugation (39,000.times.g, 45 minutes) and membrane proteins
were solubilized in 4% Lubrol, 0.5% deoxycholate.
GlcNAc-phosphotransferase was specifically adsorbed from the
solubilized membrane fraction by incubation overnight with 10 ml of
monoclonal antibody PT18 coupled to Ultralink.TM. matrix
(substitution 5 mg/ml). The matrix was then collected by low speed
centrifugation, washed with 0.025 M Tris-HCl, pH 7.4, 0.005 M
MgCl.sub.2, 0.3% Lubrol buffer containing 1 M NaCl. The column was
then washed with 2 column volumes of 0.01 M Tris-HCl, pH 7.4, 0.005
M MgCl2, 0.3% Lubrol buffer. GlcNAc-phosphotransferase was then
eluted from the column with 0.10 M Tris-HCl, pH 10.0, 0.005 M
MgCl2, 0.3% Lubrol and neutralized with 1/10th volume of 1 M
Tris-HCl, pH 6.0. Recovery is typically 20-50% of the
GlcNAc-phosphotransferase activity present in the homogenized
tissue, and approximately 0.5 mg of enzyme is recovered per 10 kg
of tissue processed.
Example 3
Amino Acid Sequencing of Bovine GlcNAc-Phosphotransferase
Example 3A
Reduction, Alkylation and Separation of Individual Subunits
[0122] Bovine GlcNAc-phosphotransferase, 1.9 mg was desalted on a
column of Sephadex G-25 superfine equilibrated in 9% formic acid
and lyophilized. The lyophilized protein was dissolved in 1 ml of
500 mM Tris-HCl, pH 8.6, 6 M guanidine-HCl, 10 mM EDTA, 2 mM DTT
degassed by bubbling N.sub.2 gas through the solution and incubated
at 37.degree. C. for 1 hour. The solution was made 5 mM in
iodoacetic acid and incubated at 37.degree. C. in the dark for a
further 21/2 hours. The solution was then made 15 mM in
.beta.-mercaptoethanol and chromatographed on a column of Sephadex
G-25 superfine equilibrated in 9% formic acid. The void fraction
was pooled and lyophilized. The individual subunits were resolved
by chromatography on a 1.0.times.30 cm column of Superose 12
equilibrated with 9% formic acid.
Example 3B
Amino Terminal Sequencing of Individual Subunits
[0123] Bovine GlcNAc-phosphotransferase, 0.5 mg was equilibrated
with sodium dodecyl sulfate, electrophoresed on a 6% polyacrylamide
gel in the presence of sodium dodecyl sulfate. The resolved
subunits were then electro-transferred to a PVDF membrane and the
protein bands detected by staining with Coomassie Blue. The bands
corresponding to the individual subunits were then excised with a
razor blade and subjected to amino-terminal sequencing in an
Applied Biosystems Model 492 protein sequencer. The amino terminal
sequence of the .alpha.-subunit was Met Leu Leu Lys Leu Leu Gln Arg
Gln Arg Gln Thr Tyr (SEQ ID NO:26). The amino terminal sequence of
the .beta. Subunit is Asp Thr Phe Ala Asp Ser Leu Arg Tyr Val Asn
Lys Ile Leu Asn Ser Lys Phe Gly Phe Thr Ser Arg Lys Val Pro Ala His
(SEQ ID NO:27). The amino terminal sequence of the .gamma.-subunit
is Ala Lys Met Lys Val Val Glu Glu Pro Asn Thr Phe Gly Leu Asn Asn
Pro Phe Leu Pro Gln (SEQ ID NO:28).
Example 3C
Internal Amino Acid Sequence of the .beta.- and
.gamma.-Subunits
[0124] The resolved .beta.- and .gamma.-subunits from example 3B
were treated with trypsin at a 1/40 mass ratio overnight at
37.degree. C. in 0.1 M Tris-HCl, pH 8.0. The tryptic fragments were
then resolved by reverse phase chromatography on a C18 column
equilibrated with 0.1% trifluoroacetic acid and developed with a
linear gradient in acetonitrile. Well resolved peaks were then
subjected to amino terminal sequencing as described in example 3B.
The peptides sequenced from the .beta.-subunit had the sequences
Ile Leu Asn Ser Lys (SEQ ID NO:29), Thr Ser Phe His Lys (SEQ ID
NO:30), Phe Gly Phe The Ser Arg (SEQ ID NO:31), and Ser Leu Val Thr
Asn Cys Lys Pro Val Thr Asp Lys (SEQ ID NO:32). The peptide
sequenced from the .gamma.-subunit had the sequence Leu Ala His Val
Ser Glu Pro Ser Thr Cys Val Tyr (SEQ ID NO:33). A second peptide
sequence from the .gamma.-subunit was obtained by chymotryptic
digestion with the sequence Asn Asn Pro Phe Leu Pro Gln Thr Ser Arg
Leu Gln Pro (SEQ ID NO:34).
Example 3D
Internal Amino Acid Sequence of the .alpha.-Subunit
[0125] Internal peptide sequences of the .alpha.-subunit were
obtained as follows. Bovine GlcNAc phosphotransferase was reduced,
alkylated, electrophoresed and transferred to PVDF as previously
described. The .alpha.-subunit band was excised and tryptic
peptides generated by in situ digestion with trypsin, eluted with
acetonitrile/trifluoroacetic acid and fractionated by reverse phase
HPLC. Individual peaks were then examined by Matrix Associated
Laser Desorption-Ionization-Mass Spectroscopy (MALDI-MS) and peaks
containing a single mass were subjected to amino terminal
sequencing as above. The peptide sequences determined from the
.alpha.-subunit are Val Pro Met Leu Val Leu Asp Xaa Ala Xaa Pro Thr
Xaa Val Xaa Leu Lys (SEQ ID NO:35) and Glu Leu Pro Ser Leu Tyr Pro
Ser Phe Leu Ser Ala Ser Asp Val Phe Asn Val Ala Lys Pro Lys (SEQ ID
NO:36).
Example 4
Cloning the Human GlcNAc-Phosphotransferase .alpha./.beta.-Subunit
cDNA
[0126] The amino-terminal protein sequence determined from the
isolated bovine .beta.-subunit was used to search the Expressed
Sequence Tag (EST) data base using the program tblastn. Altschul,
S. F., Gish W., et al. (1990). "Basic Local Alignment Search Tool."
Journal of Molecular Biology 215: 403-410. This search identified a
partial mouse cDNA previously identified during a positional
cloning strategy. Cordes, S. P. and Barsh, G. S. (1994). "The mouse
segmentation gene kr encodes a novel basic domain-leucine zipper
transcription factor." Cell 79: 1025-11034.
[0127] A forward PCR primer was designed based on the mouse
sequence and used with an oligo dT reverse primer for RT-PCR
amplification of a 1,848 bp product using mouse liver poly A RNA as
template. The PCR product was cloned and sequenced and proved to
contain all the determined .beta.-subunit sequences, demonstrating
it encoded the murine .beta.-subunit.
[0128] The human .beta.-subunit cDNA was cloned by screening a size
selected human placental cDNA library (Fischman, K., Edman J. C.,
et al. (1990). "A murine fer testis-specific transcript (ferT
encodes a truncated fer protein." Molecular and Cellular Biology
10: 146-153) obtained from ATCC with the random hexamer labeled
murine .beta.-subunit cDNA under conditions of reduced stringency
(55.degree. C., 2.times.SSC). The remaining portion of the
.alpha./.beta.-subunit precursor cDNA was cloned by a combination
of a walking strategy beginning with the portion of the cDNA
encoding the human .beta.-subunit and standard library screening
strategies. Additionally, EST data base searches were used to
identify clones containing portions of the human .alpha./.beta.
cDNA, which were obtained from the corresponding repositories and
sequenced. Together these strategies allowed the determination of
the full length human .alpha./.beta.-subunits precursor cDNA
sequence. A clone containing this sequence was assembled using the
appropriate fragments and cloned into pUC19. The 5597 bp sequence
is given in Sequence NO:4 and contains DNA sequences predicted to
encode protein sequences homologous to all of the amino terminal
and internal peptide sequences determined from the bovine .alpha.-
and .beta.-subunits.
Example 5
Cloning the Human GlcNAc-Phosphotransferase .gamma.-Subunit
cDNA
[0129] The .gamma.-subunit amino terminal and tryptic peptide
sequences were used to search the Expressed Sequence Tag (EST) data
base using the program tblastn. Altschul, S. F., Gish W., et al.
(1990). "Basic Local Alignment Search Tool." Journal of Molecular
Biology 215: 403-10. Three human EST sequences were identified
which were highly homologous to the determined bovine protein
sequences. cDNA clone 48250 from which EST sequence 280314 was
determined was obtained from Genome Systems and sequenced using
standard techniques. This clone contained a 1191 bp insert which
contained all the determined protein sequences and appeared to
contain a signal sequence 5' of the determined amino terminal
sequence. The clone however lacked an initiator methionine or any
5' non-coding sequence. The 5' portion of the cDNA was obtained by
PCR. the reverse primer 5'-GCGAAGATGAAGGTGGTGGAGGACC-3' (SEQ ID
NO:37) and a T7 promoter primer were used in a reaction along with
template DNA from a human brain cDNA library in pCMV-SPORT(GIBCO).
A 654 bp product was obtained, cloned in pCR2.1 and sequenced. The
sequence demonstrated the amplified product contained 23 bp of 5'
non-coding sequence, the initiator methionine and the signal
peptide identified in EST 280314. A full length cDNA for the
.gamma.-subunit (pBC36) was assembled by ligating a 75 bp
EcoRI-ApaI fragment from the cloned PCR product, an ApaI-NotI
fragment from clone 48250 and EcoRI-NotI cut pcDNA3
(Invitrogen).
Example 6
Cloning the Human GlcNAc-Phosphotransferase .alpha./.beta.-Subunit
Gene
[0130] Plasmid DNA was prepared from a human brain cDNA library
(Life Technologies) according to the manufacturers protocol. This
DNA was used as template for PCR using primers with the sequences
5'-TGCAGAGACAGACCTATACCTGCC-3' (SEQ ID NO:38) and 5'
ACTCACCTCTCCGAACTG-GAAAG-3' (SEQ ID NO:39) using Taq DNA polymerase
and buffer A from Fischer Scientific using 35 cycles of 94.degree.
C. 1 minute, 55.degree. C. 1 minute, and 79.degree. C. 1 minute. A
106 bp product was obtained, purified by agarose gel
electrophoresis, isolated by GeneClean (Bio101) and cloned into
pCR2. DNA sequencing determined the resulting plasmid pAD39
contained a 106 bp insert which was excised by digestion with EcoRI
and submitted to Genome Systems for screening of a human genomic
BAC library. Four human BACs were identified and BAC #14951 was
sequenced. For sequencing BAC #14951 was submitted to a colleague's
laboratory at the University of Oklahoma. The BAC was then
fragmented by nebulization, and fragments cloned into pUC18 and
shotgun sequenced. Contigs were generated by computer analysis and
gaps closed by primer walking strategies. The sequence of the BAC
spans 177,364 bp. The GlcNAc-phosphotransferase
.alpha./.beta.-subunits precursor gene spans .sup..about.80 kb and
is arranged as 21 exons.
Example 7
Cloning the Human GlcNAc-Phosphotransferase .gamma.-Subunit
Gene
[0131] The human .gamma.-subunit gene was identified by blastn
searching of the NCBI High Throughput Genomic Sequence (HGTS)
database with the full length human Subunit cDNA sequence. The
search identified a clone HS316G12(gi 4495019) derived from human
chromosome 16 which contained the human .gamma.-subunit gene. The
human GlcNAc-phosphotransferase .gamma.-subunit gene spans about 12
kb and is arranged as 11 exons. Exons 1-3 and 4-11 are separated by
a large intron of about 9 kb.
Example 8
Preparation of Modified Expression Plasmid for the Human
GlcNAc-Phosphotransferase .alpha./.beta.-Subunits Precursor
cDNA
[0132] An expression vector for the GlcNAc-phosphotransferase
.alpha./.beta. cDNA was constructed in pcDNA3.1(+) as follows. Two
upstream ATG's in the 5'-noncoding sequence of the human
GlcNAc-phosphotransferase cDNA were removed and the Kozak sequence
were modified as follows. Two fragments from pAD98, which was the
human GlcNAc-phosphotransferase ct/p cDNA cloned into pcDNA3.1(+),
were excised. A 1068 bp XhoI-PstI fragment and a 9746 bp NheI-XhoI
fragment were ligated with oligonucleotides with sequences
5'-CTAGCCACCATGGGGTTCAAGCTCTTGCA-3' (SEQ ID NO:40) and
5'-AGAGCTTGAACCCCATGGTGG-3' (SEQ ID NO:41) generating pAD105. The
poly A sequence near the 3' end of the cDNA clone was removed by
ligating a NheI-BglII fragment from the cDNA with NheI-BamHI cut
vector pcDNA3.1(+) generating pAD128.
Example 9
Preparation of an Expression Plasmids for the Human
GlcNAc-Phosphotransferase .alpha./.beta. Subunits Precursor
cDNA
[0133] DNA sequencing of pAD128 identified deletion of an A in an
AAAAA sequence (positions 2761-2765 shown in SEQ ID NO:4) that
disrupted the coding sequence. Plasmid pAD130 was constructed in an
attempt to correct this by ligating a 5929 bp NheI-MfeI fragment
and a 2736 bp NheI-AgeI fragment (both from pAD128 with a 515 bp
MfeI-AgeI fragment derived from pAD124). Plasmid pAD130 was then
grown and subsequent sequencing of plasmid pAD130 demonstrated that
the AAAAA sequence had reverted to AAAA again indicating
instability in the sequence at this point.
[0134] In order to eliminate this instability the first AAA
(position 2761-2763 shown in SEQ ID NO:4) that codes for lysine was
changed to AAG (also coding for lysine) so that the unstable AAAAA
sequence was changed to a stable AAGAA without altering the encoded
amino acid. Plasmid pAD130 was corrected by removing a 214 bp
MfeI-DraIII fragment and replacing it with a fragment with the
correct sequence. The correct MfeI-DraIII fragment was prepared by
PCR using pAD130 as a template with forward primer
5'-GAAGACACAATTGGCATACTTCACTGATAGCAAGAATACTGGGAGGCAACTAAAAGATAC-3'
(SEQ ID NO:42) (oligo TTI 25 with desired AAGAA sequence as
underlined) and reverse primer 5'-ACTGCATATCCTCAGAATGG-3' (SEQ ID
NO:43) (oligo TTI 24). The PCR fragment was subcloned into the
EcoRV site of pBluescript KS II(+) (Stratagene) generating pMK16.
The insert was sequenced for confirmation and the 215 bp
MfeI-DraIII fragment was prepared. To avoid MfeI-DraIII sites on
the vector pcDNA 3.1(+) (Invitrogen), the NheI-XbaI fragment was
prepared from pAD130 and subcloned into the XbaI site of pUC19
(Life Technologies) to construct pMK15. pMK15 was cleaved with MfeI
and DraIII and the 6317 bp fragment was purified and ligated with
the MfeI-DraIII fragment from pMK16 to form pMK19 containing the
desired stable sequence in pUC19.
[0135] The corrected cDNA for the .alpha./.beta. subunit was
excised from pMK19 as a KpnI-XbaI fragment and subcloned between
the KpnI and XbaI sites of pcDNA6N5/His-A and designated pMK25.
Plasmid pMK25 containing the cDNA as shown in SEQ ID NO:20 where
the nucleotide sequence for the modified human
.alpha./.beta.-subunit precursor cDNA is shown in nucleotides
1-3768. This sequence corresponds to and is a modification of the
nucleotide sequence 165-3932 shown in SEQ ID NO:4.
Example 10
Construction of Expression Vectors for Soluble, Human
GlcNAc-Phosphotransferase .alpha./.beta. Subunits Precursor
cDNA
[0136] Plasmid pMK19 was digested with BglII (cutting at positions
255 and 2703 shown in SEQ ID NO:20) and self-ligated to reduce the
length of the cDNA to be amplified from approx. 3.5 kb to 1 kb so
that the 5' and 3' ends of the cDNA can be modified by PCR to
remove the transmembrane domains of the .alpha. and .beta. subunits
of human GlcNAc-phosphotransferase and used to construct expression
vectors to produce soluble GlcNAc-phosphotransferase. This plasmid
was designated pMK21. The strategy is that the nucleotides encoding
the first 44 amino acids containing the transmembrane domain of the
.alpha. subunit (nucleotides 1-132 of SEQ ID NO:20) are replaced
with a HindIII site, and nucleotides encoding the last 47 amino
acids containing the transmembrane domain of the .beta. subunit
(nucleotides 3628-3768 of SEQ ID NO:21) are replaced with a stop
codon and a XbaI site.
[0137] Plasmid pMK21 was used as a template for PCR with the
following primers: A forward primer
(5'-TGGTTCTGAAGCTTAGCCGAGATCAATACCA TG-3' (SEQ ID NO:44), oligo TTI
76) containing a HindIII site (underlined) and a sequence
complementary to nucleotides 133 to 151 of SEQ ID NO:20 (italics),
which will produce the 5'-end of a PCR fragment that removes the
coding sequence of the first 44 amino acids comprising the putative
transmembrane domain of the .alpha. subunit. A reverse primer
(5'-TAGTACACTCTAGActactaCTTCAATTTGTCTCGATAAG-3' (SEQ ID NO:45),
oligo TTI 78) containing a XbaI site (underlined), two stop codons
(lower case) and a sequence complementary to nucleotides 3608 to
3627 of SEQ ID NO:21 (italics), which will produce the 3'-end of a
PCR fragment that removes the coding sequence of the last 47 amino
acids comprising the putative transmembrane domain of the .beta.
subunit and replaces it with two stop codons. The resulting PCR
fragment was subcloned into the EcoRV site of pBluescript KS II+
(Stratagene). This plasmid, designated pMK42, was sequenced to
ensure no errors were introduced by PCR. The BglII-BglII fragment
(positions 255-2703 shown in SEG ID NO:20) which was previously
removed was subcloned back into the BglII site of pMK42. The
orientation of this fragment was determined to be correct and this
plasmid was designated pMK49. Thus, plasmid pMK49 contained a cDNA
comprising a 5' HindIII site and a 3' XbaI site flanking a coding
region for the human GlcNAc-phosphotransferase .alpha./.beta.
subunits precursor cDNA with the .alpha. subunit putative
transmembrane domain deleted and the putative transmembrane domain
of the .beta. subunit replaced with two stop codons (soluble
.alpha./.beta.-cDNA).
[0138] This "soluble .alpha./.beta.-cDNA" can now be conveniently
sub-cloned into vectors constructed to contain the HPC4 epitope
(used for rapid purification of the soluble enzyme) and different
secretion signal peptides. These pcDNA6/V5/His-A+tag) vectors were
constructed as follows:
[0139] Synthetic oligonucleotide cassettes containing a 5'-NheI
site and a 3'-HindIII site flanking nucleotide sequences coding for
different secretion signal peptides and the nucleotide sequence
coding for the HPC4 epitope were inserted into plasmid
pcDNA6/V5/His-A cut with NheI and HindIII. The following plasmids
were prepared with the indicated cassette:
1. pMK45--mouse immunoglobulin Kappa chain signal peptide (sequence
in italics) and HPC4 epitope (sequence underlined)
TABLE-US-00002 (SEQ ID NO:46) CTAGCCGCCACC ATGGAGACAGACACACTC
CTGCTATGGGTACTGCTG CTCGGCGGTGGTACC TC TGTCT
GTGTGAGGACGATACCCATGACGAC GAGTGGGTTCC AGGT TC CACTGGTGA
CGAAGATCAGGTAGATCCGC GGTT AATCACCCAAGGTCCAAGGTGACCACTGCTTC TAGTCCAT
CTA GGCGCCAATTAGGACGGTACT GCCATTCGA
1. pMK44--a transferrin signal peptide sequence (in italics) and
HPC4 epitope (sequence underlined)
TABLE-US-00003 (SEQ ID NO:47) CTAGCGGTACCATGAGATT AGCAGTAGGCGCC TT
ATTAG TATGCGC AGTACT CCGCCATGGTACTCTAATCGTCATCCGCGGAATAATCATACGC
GTCATGAGGGATTAT GTC TCGCAG AAGATCAGGTAGATCCGC GGTT
AATCGACGGTACCTTATACAGAGCGTCTTCTAG TCCAT CTAGGCGCCA AT
TAGCTGCCATTCGA
1. pMK43--a transferrin secretion peptide sequence modified to
satisfy a Kozak's sequence (sequence in italics) and HPC4 epitope
(sequence underlined),
TABLE-US-00004 (SEQ ID NO:48) CTAGCCGCCACCATGGGATT AGCAGTAGGCGCCTT
ATT AGT ATGCG C AGTCGCCGGTGGTACCCTAATCGTCATCCGCGGAATAATCATACGCGT
CAACT CGGATTAT GT C TCGCA GAAGATCAGGTAGATCCGC GGTT
AATCGACGTGAGCCTAATACAGAGCGTCTT CTAGT CCATCTAGGCGCC AAT
TAGCTGCGTACATTCGA
[0140] The cDNA encoding "soluble .alpha./.beta. subunits" can be
obtained as a HindIII-XbaI fragment from pMK49 and inserted into
the plasmid pMK43 to form pMK50; pMK44 to form pMK51, and into
pMK45 to form pMK52, plasmids capable of encoding the
.alpha./.beta. subunits of human GlcNAc-phosphotransferase with
putative transmembrane domains deleted, with different signal
peptides and all having the HPC4 epitope tag to facilitate
purification of the soluble, secreted enzyme.
Example 111
Construction of Expression Vectors for the Human
GlcNAc-Phosphotransferase .gamma. Subunit Precursor cDNA
[0141] The human GlcNAc-phosphotransferase .gamma.-subunit
precursor cDNA was obtained from plasmid pAD133 in pAC5.1/V5-His by
cutting with EcoRI. This cDNA was inserted into EcoRI digested
pcDNA6/V5/His-A to form plasmid pMK17 containing cDNA as shown in
SEQ ID NO:5. Plasmid pMK17 was digested with MluI (position 124-129
as shown in SEQ ID NO:5) and EcoRI (position 1103-1108 as shown in
SEQ ID NO:5) and the 980 bp MluI-EcoRI fragment was then subcloned
in pBluescriptKSII(+) with a synthetic double stranded cassette
having an HindIII site and a MluI site flanking a nucleotide
sequence including positions corresponding to 95-123 as shown in
SEQ ID NO:5 thereby removing the nucleotide sequence encoding the
amino terminal, 24-amino acid signal peptide in plasmid pMK26.
Plasmid pMK26 was sequenced to ensure its sequence. The correct
cDNA from pMK26, which encodes amino acids for the human
GlcNAc-phosphotransferase .gamma. subunit with the signal peptide
removed, is then excised from pMK26 by HindIII and EcoRI digestion
and placed into plasmids pMK43 to form pMK58; pMK44 to form pMK59,
and into pMK45 to form pMK64, plasmids capable of encoding the
.gamma. subunit of human GlcNAc-phosphotransferase with its signal
peptide deleted, with different signal peptides and all having the
HPC4 epitope tag to facilitate purification of the soluble, .gamma.
subunit.
[0142] To evaluate the behavior of .alpha./.beta./.gamma. secreted
products, the .alpha./.beta. subunit precursor and the .gamma.
subunit were co-expressed in the bi-cistronic vector pIRES
(Clontech). This was accomplished by subcloning .alpha./.beta. and
.gamma. cDNAs expressing the desired subunit with a selected signal
peptide and the HPC4 Tag into NheI site (MCS-A) and XbaI site
(MCS-B) of pIRES, respectively.
Example 12
Transient Expression of the .alpha./.beta. and .gamma. Subunits of
Human GlcNAc-Phosphotransferase in 293T Cells
[0143] Plasmids were transfected into 293T cells using Fugene6
(Roche) according to manufacturer's instructions. Culture media was
collected 23 h, 44.5 h and 70 h after transfection. Aliquots of
media containing expressed protein was captured on anti-HPC4
monoclonal antibody (U.S. Pat. No. 5,202,253) conjugated with
Ultralink beads (Pierce) by overnight incubation at 4.degree. C.
The beads were washed to remove unbound protein and assayed
directly for phosphotransferase activity as described previously
(REF).
[0144] Plasmids used for expression all containing a sequence
encoding for the HPC4 tag were as follows: [0145] 1.
pMK50--modified transferrin secretion peptide and .alpha./.beta.
subunit in pcDNA6/V5/His-4 [0146] 2. pMK51--transferrin secretion
peptide and .alpha./.beta. subunit in pcDNA6N5/His-4 [0147] 3.
pMK52--mouse immunoglobulin secretion peptide and .alpha./.beta.
subunit in pcDNA6/V5/His-4 [0148] 4. pMK75--modified transferrin
secretion peptide and .alpha./.beta. subunit and modified
transferrin secretion peptide and .gamma. subunit in pIRES [0149]
5. pMK81--transferrin secretion peptide and .alpha./.beta. subunit
and transferrin secretion peptide and .gamma. subunit in pIRES
[0150] 6. pMK76--mouse immunoglobulin secretion peptide and
.alpha./.beta. subunit and mouse immunoglobulin secretion peptide
and .gamma. in pIRES
[0151] The relative amounts of expression detected by assay for
phosphotransferase using methyl-.alpha.-D-mannoside and
UDP-[.beta.-.sup.32P]-GlcNAc as substrates with cell transfected
with pcDNA6/V5/His-4 as controls is shown in FIG. 4.
Example 13
Expression and Purification GlcNAc-Phosphotransferase
.alpha./.beta./.gamma.
[0152] For expression and purification of the enzyme, a modified
expression plasmid is constructed in a modified expression vector
derived from pEE14. The plasmid directs the synthesis of a soluble
epitope tagged GlcNAc-phosphotransferase molecule. The
.alpha./.beta.-subunit precursor is modified as follows: The 5'
portion of the cDNA which encodes the .alpha.-subunit cytoplasmic
and transmembrane domain is deleted and replaced with nucleotides
which encode the transferrin signal peptide followed by amino acids
which encode the epitope for monoclonal antibody HPC4. The 3'
portion of the cDNA is modified by the insertion of a stop codon
before the .beta.-subunit transmembrane segment. The vector pEE14.1
(Lonza Biologics) is modified by the insertion of a 850 bp
MluI-NcoI fragment containing a modified vascular endothelial
growth factor (VEGF) promoter at the unique MluI site in pEE14.1.
This vector encoding the modified GlcNAc-phosphotransferase
.alpha./.beta.-subunit precursor is co-transfected with a wild type
.gamma.-subunit construct containing the VEGF promoter in pEE14.1
into CHO-K1 cells using Fugene6 and plated into 96 well plates.
Transfectants are selected in 25 .mu.m methionine sulfoximine and
the plasmid amplified by selection in 96 well plates with 50 .mu.M,
100 .mu.M, 250 .mu.M, and 500 .mu.M methionine sulfoximine. Clones
are picked into duplicate 96 well plate and the highest expressing
clones selected by dot blotting media and immuno-detection with
monoclonal antibody HPC4. The highest expressing clone is expanded
into cell factories. The recombinant soluble epitope tagged
GlcNAc-phosphotransferase is purified from the media by
chromatography on monoclonal antibody HPC4 coupled to Ultralink in
the presence of 5 mM MgCl.sub.2 and 1 mM CaCl.sub.2. The soluble
epitope tagged GlcNAc-phosphotransferase is eluted with 5 mM EGTA
and 5 mM MgCl.sub.2.
Example 14
Preparation of Monoclonal Antibodies Specific for Bovine
Phosphodiester .alpha.-GlcNAcase
[0153] Murine monoclonal antibodies specific for bovine
phosphodiester .alpha.-GlcNAcase were generated by immunization of
mice with a partially purified preparation of phosphodiestcr
.alpha.-GlcNAcase. Spleens were then removed from immune mice and
fused with SP2/O myeloma cells according to standard techniques
(Harrow, E. and Lane, D. (1988). Antibodies: a laboratory manual,
Cold Spring Harbor Laboratory). Hybridomas were plated in eight
96-well plates and grown until hybridomas were visible. Hybridomas
secreting antibodies to phosphodiester .alpha.-GlcNAcase were
identified measuring phosphodiester .alpha.-GlcNAcase activity in
immunoprecipitates prepared by incubation of a partially purified
preparation of phosphodiester .alpha.-GlcNAcase with pooled
hybridoma supernatants. Pools from 16 and 4 wells were assayed
followed by individual wells. Monoclonal UC1 was identified by this
protocol and coupled to Ultralink.TM. for use in purification of
phosphodiester .alpha.-GlcNAcase.
Example 15
Purification of Bovine Phosphodiester .alpha.-GlcNAcase
[0154] Bovine calf liver (1 kg) was homogenized in 0.05 M
Imidazole-HCl, pH 7.0, 0.15 M NaCl, 0.01 M EDTA and a washed
post-nuclear supernatant was prepared. Membranes were collected by
centrifugation at 30,000.times.g for 30 minutes and washed three
times with the above buffer. Membrane proteins were then
solubilized in buffer containing 2% Triton X-100, 0.05%
deoxycholate and insoluble material removed by centrifugation, as
before. The solubilized membrane fraction was incubated with 20 ml
of monoclonal antibody UC1 coupled to Ultralink.TM. (substitution 5
mg/ml) with constant rotation for 16 hours at 4.degree. C. The
UC1-Ultralink.TM. was collected by low speed centrifugation. packed
into a column and washed with 0.025 M Tris-HCl, pH 7.4, 0.3%
Lubrol, followed by two column volumes of 0.5 M NaHCO3, pH 8.0,
0.3% Lubrol. Phosphodiester .alpha.-GlcNAcase was then eluted with
0.5 M NaHCO3, pH 10.0, 0.3% Lubrol and collected in 1/10 volume of
1.0 M Tris-HCl, pH 5.5.
Example 16
Amino Acid Sequencing of Bovine Phosphodiester
.alpha.-GlcNAcase
Example 16A
Amino-Terminal Sequence of Bovine Phosphodiester
.alpha.-GlcNAcase
[0155] Bovine phosphodiester .alpha.-GlcNAcase was bound to a 0.25
ml column of POROS HQ and step-eluted with buffer containing 0.5 M
NaCl. Fractions containing phosphodiester .alpha.-GlcNAcase
activity were identified by phosphodiester .alpha.-GlcNAcase assay,
pooled and absorbed to a ProSorb Sample Preparation Cartridge
(Perkin Elmer) and subjected to amino acid sequencing in an Applied
Biosystems Model 492 Protein Sequencer operated according to the
manufacturer's instructions. The sequence
Asp-Xaa-Thr-Arg-Val-His-Ala-Gly-Arg-Leu-Glu-His-Glu-Ser-Trp-Pro--
Pro-Ala-Ala-Gln-Thr-Ala-Gly-Ala-His-Arg-Pro-Ser-Val-Arg-Thr-Phe-Val
was obtained.
Example 16B
Internal Sequence of Bovine Phosphodiester .alpha.-GlcNAcase
[0156] Bovine liver phosphodiester .alpha.-GlcNAcase was
concentrated to 10 .mu.l in a Speed Vac, combined with 30 .mu.l 0.1
M Tris-HCl, pH 7.4, 8 M guanidine-HCl, and 2-4 .mu.l 25 mM DTT and
incubated at 50.degree. C. for 1 hour. Iodoacetamide 2.4 .mu.l 50
.mu.M was then added and the incubation was continued for 1 hour.
The reaction mixture was then desalted on a column of Sephadex G25
superfine as described for GlcNAc-phosphotransferase and digested
with trypsin. The peptides were fractionated by HPLC and sequenced
as described for GlcNAc-phosphotransferase. The sequences
determined are Arg Asp Gly Thr Leu Val Thr Gly Tyr Leu Ser Glu Glu
Glu Val Leu Asp Thr Glu Asn and Gly Ile Asn Leu Trp Glu Met Ala Glu
Phe Leu Leu Lys.
Example 17
Cloning the Human Phosphodiester .alpha.-GlcNAcase cDNA
[0157] The phosphodiester .alpha.-GlcNAcase tryptic peptide
sequences were used to search the EST data bases as described for
GlcNAc-phosphotransferase above. Three EST sequences were
identified which contained the human phosphodiester
.alpha.-GlcNAcase cDNA and clone ATCC #367524 was obtained and a
.about.700 bp EcoRI-NotI fragment was excised from this clone and
used to probe a human liver cDNA library in the vector TriplEx.
Several clones were identified and sequenced, one of which (clone
6.5) proved to contain a nearly full length cDNA for the human
phosphodiester .alpha.-GlcNAcase. The genomic clone described in
Example 18 demonstrated that clone 6.5 was missing only the
initiator methionine.
Example 18
Cloning the Human Phosphodiester .alpha.-GlcNAcase Gene
[0158] The human phosphodiester .alpha.-GlcNAcase gene was
identified by searching the NCBI database nr with the human
phosphodiester .alpha.-GlcNAcase cDNA using the program blastn. The
genomic sequence was determined during the sequencing of a clone
from chromosome 16p13.3 and deposited 6 Mar. 1999 in GenBank as an
unidentified sequence of 161264 bp with the accession number
AC007011. The gene spans about 12 kb of genomic DNA on chromosome
16.13 and is arranged in 11 exons.
Example 19
Construction of an Expression Vector for Human Phosphodiester or
.alpha.-GlcNAcase
[0159] An expression vector for human phosphodiester
.alpha.-GlcNAcase was prepared as follows: The 5' end of the
sequence of clone 6.5 was modified by PCR amplification of the 5'
end of the cDNA with a forward primer with the sequence
5'-GGAATTCCACCATGGCGACCTCCACGGGTCG-3' (SEQ ID NO:49) and a reverse
primer 5'-TGACCAGGGTCCCGTCGCG-3' (SEQ ID NO:49). This served to add
a consensus Kozak sequence and initiator methionine to the sequence
of clone 6.5. The .about.500 bp PCR product was purified, digested
with EcoRI and BamHI and ligated into pcDNA3.1(-) which was
sequenced. This construct was then digested with BamHI and HindIII
and ligated with a .about.1600 bp BamHI-HindIII fragment containing
the 3' portion of the cDNA from clone 6.5 generating the full
length expression plasmid.
Example 20
Host Cell Preparation for Human Phosphodiester
.alpha.-GlcNAcase
[0160] Cos cells were grown in 60 mm plates in Dulbeccos minimal
essential media (DMEM) at 37.degree. C. in 5% CO.sub.2 until they
reached 50-80% confluence. The plates were then washed with OptiMEM
I and the cells transfected with the expression vector described in
Example 19 using Lipofectamine Plus (GIBCO BRL Life Technologies)
according to the manufacturers instructions. Cells were harvested
at 48 hours, a solubilized membrane fraction prepared and assayed
for phosphodiester .alpha.-GlcNAcase activity.
Example 21
Expression and Purification of Soluble Recombinant Human
Phosphodiester .alpha.-GlcNAcase
[0161] For expression and purification of the enzyme, a modified
expression plasmid is constructed in a modified expression vector
derived from pEE14.1. The plasmid directs the synthesis of a
soluble epitope tagged phosphodiester .alpha.-GlcNAcase molecule.
The phosphodiester .alpha.-GlcNAcase precursor is modified as
follows: The 3' portion of the cDNA which encodes the
phosphodiester .alpha.-GlcNAcase transmembrane and cytoplasmic
domains is deleted and replaced with nucleotides which encode the
epitope for monoclonal antibody HPC4 followed by a stop codon. The
vector pEE14.1 (Lonza Biologics) is modified by the insertion of a
850 bp MluI-NcoI fragment containing a modified vascular
endothelial growth factor (VEGF) promoter at the unique MluI site
in pEE14.1. This vector encoding the epitope tagged soluble
phosphodiester .alpha.-GlcNAcase precursor is transfected into
CHO-K1 cells using Fugene6 and plated into 96 well plates.
Transfectants are selected in 25 .mu.m methionine sulfoximine, and
the plasmid amplified by selection in 96 well plates with 50 M, 100
.mu.M, 250 .mu.M, and 500 .mu.M methionine sulfoximine. Clones are
picked into duplicate 96 well plate and the highest expressing
clones selected by dot blotting media and immuno-detection with
monoclonal antibody HPC4. Media from clones demonstrating the
highest level of epitope tag expression is assayed for
phosphodiester .alpha.-GlcNAcase activity. The highest expressing
clone is expanded into cell factories. The recombinant soluble
epitope tagged phosphodiester .alpha.-GlcNAcase is purified from
the media by chromatography on monoclonal antibody HPC4 coupled to
Ultralink.TM. in the presence of 5 mM MgCl.sub.2 and 1 mM
CaCl.sub.2. The soluble epitope tagged phosphodiester
.alpha.-GlcNAcase is eluted with 5 mM EGTA and 5 mM MgCl.sub.2.
Example 22
Construction of an Expression Vector for Soluble, Human
Phosphodiester .alpha.-GlcNAcase
[0162] For expression and purification of the enzyme, a modified
expression plasmid is constructed in a modified expression vector
derived from the pEE14.1 vector (Lonza Biologics). The plasmid
directs the synthesis of a soluble epitope tagged phosphodiester
.alpha.-GlcNAcase molecule. The phosphodiester .alpha.-GlcNAcase
precursor is modified as follows: The 3' portion of the cDNA
(1342-1548 of SEQ ID NO: 7) which encodes the phosphodiester
.alpha.-GlcNAcase transmembrane and cytoplasmic domains was deleted
and replaced with nucleotide sequence
GAGGACCAGGTGGACCCCAGGCTGATCCACGGCAAGGAT (SEQ ID NO:51) that encodes
the epitope for monoclonal antibody HPC4 (EDQVDPRLIDGKD (SEQ ID
NO:52)) followed by a stop codon.
[0163] This expression vector was constructed by generating two
intermediate plasmids and ligating a fragment from each into
pEE14.1 vector (Lonza Biologics) to yield the final expression
vector. The first intermediate plasmid designated pKB4 was
constructed by ligating the 1034 bp FseI<<Bsu36I fragment of
phosphodiester .alpha.-GlcNAcase (lacking the C-terminal
transmembrane and cytoplasmic domains) from clone 6.5, and a
Bsu36I-XbaI oligonucleotide fragment that contains the HPC4 epitope
into a modified pUC19 vector. The second intermediate plasmid
designated pKB5, was constructed by ligating a 850 bp MluI-NcoI
fragment containing a modified vascular endothelial growth factor
(VEGF) promoter from pcDNA4/HisMax (Invitrogen), a 256 bp Bsel-FseI
fragment encoding the N-terminus of human phosphodiester
.alpha.-GlcNAcase from clone 6.5, and an oligonucleotide linker
into a modified pUC19 vector. The final expression vector
designated pKB6 was constructed by ligating the MluI-FseI fragment
from pKB5, and the FseI-HindIII fragment from pKB4 into a
MluI/HindIII digested pEE14.1 vector. The plasmid pKB6 contains the
nucleotide sequence shown in SEQ ID NO:22.
Expression and Purification of Soluble Recombinant Human
Phosphodiester .alpha.-GlcNAcase
[0164] Approximately 10.sup.8 293T cells were plated in a cell
factory using Dulbecco's modified eagle's medium (DMEM) containing
10% fetal bovine serum in a humidified atmosphere at 37.degree. C.
with 5% CO2. These cells were transfected with approximately 700 g
of pKB6 using 2 ml of transfection reagent Fugene-6 (Roche) for the
transient expression of soluble human phosphodiester
.alpha.-GlcNAcase. After three days of culturing the transfected
cells, the medium containing soluble, epitope-tagged, human
phosphodiester .alpha.-GlcNAcase was collected and applied in the
presence of 1 mM CaCl2 to a column of monoclonal antibody HPC4
coupled to Ultralink (Pierce). Affinity purified, epitope-tagged,
human phosphodiester .alpha.-GlcNAcase (approximately 11 mg) was
eluted with buffer containing 5 mM EDTA and stored at -20.degree.
C. in 50 mM Tris, 150 mM NaCl, 2 mM CaCl2, 50% glycerol, pH 7.2.
The enzyme had a specific activity of 500,000 units/mg with
[.sup.3H]GlcNAc-phosphomannose-.alpha.-methyl as a substrate
(Komfeld R, et al., JBC 273:23203-23210).
Example 23
CHO Cells Expressing Recombinant Human Acid .alpha.-Glucosidase
[0165] The human acid .alpha.-glucosidase cDNA was obtained from
Dr. Frank Martinuk (Martiniuk, F., Mehler, M., Tzall, S., Meredith,
G. and Hirschhorn, R. (1990). "Sequence of the cDNA and 5'-flanking
region for human acid alpha-glucosidase, detection of an intron in
the 5' untranslated leader sequence, definition of 18-bp
polymorphisms, and differences with previous cDNA and amino acid
sequences." DNA Cell Biol 9(2): 85-94) and cloned into the
expression vector pEE14.1. This vector was used to transfect CHO-K1
cells using Fugene6 and plated into 96 well plates. Transfectants
were selected in 25 .mu.m methionine sulfoximine, and clones picked
and plated into 96 well plates. The plasmid was amplified by
selection with 50 .mu.M, 100 .mu.M, 250 .mu.M, and 500 .mu.M
methionine sulfoximine. Clones were picked into duplicate 96 well
plates and the highest expressing clones selected by assay of the
media for acid .alpha.-glucosidase activity and the cells for DNA
content. The highest expressing clone (Clone 3.49.13) based on acid
.alpha.-glucosidase activity to DNA content ratio was then expanded
into a cell factory. This clone was incubated at 37.degree. C. in
5% CO.sub.2 and maintained in Glasgow Minimal Essential Media
containing 20 mM TES, pH 7.2, 5% fetal bovine serum.
Example 24
Growth of CHO Cells Expressing Recombinant Human Acid
.alpha.-Glucosidase in the Presence of .alpha.-1,2 Mannosidase
Inhibitors
[0166] CHO cells expressing human acid .alpha.-glucosidase were
cultured in Glasgow Modified Minimal Essential Media containing 5%
Fetal Bovine Serum, 25 .mu.M methionine sulfoximine, 20 mM TES, pH
7.2, and 7.5 mM 1-deoxymannojirimycin-HCl. Alternatively, the cells
can be cultured in the above media containing 100 .mu.g/mL
1-deoxymannojirimycin-HCl and 25 .mu.g/mL kifunensine.
Example 25
Isolation of Recombinant Human Acid .alpha.-Glucosidase
[0167] Recombinant human acid .alpha.-glucosidase was purified from
spent tissue culture media as follows: Media was concentrated 10
fold by tangential ultrafiltration with a 30,000 dalton cutoff
membrane and dialyzed into 50 mM sodium phosphate, pH 6.5, and
applied to a column of ConA Sepharose (Pharmacia). Following a wash
with the same buffer to remove the unbound proteins, acid
.alpha.-glucosidase was eluted with 1.0 M .alpha.-methyl glucoside,
pooled, concentrated and dialyzed as before. The acid
.alpha.-glucosidase was then applied to a column of Sephadex G-200
equilibrated with 50 mM sodium phosphate, pH 6.5 and eluted
isocratically with the same buffer.
Example 26
Treatment of Recombinant Human Acid .alpha.-Glucosidase with
GlcNAc-Phosphotransferase and Phosphodiester .alpha.-GlcNAcase
[0168] Human acid .alpha.-glucosidase at 10 mg/ml was incubated in
50 mm Tris-HCl, pH 6.7, 5 mM MgCl.sub.2, 5 mM MnCl.sub.2, 2 mM
UDP-GlcNAc with GlcNAc-phosphotransferase at 100,000 u/mL at
37.degree. C. for 2 hours. Phosphodiester .alpha.-GlcNAcase, 1000
u/mL was then added and the incubation continued for another 2
hours. The acid .alpha.-glucosidase was then repurified by
chromatography on Q-Sepharose, and step elution with NaCl.
Example 27
Characterization of the Oligosaccharide Structures on Modified
Recombinant Human Acid .alpha.-Glucosidase
[0169] Recombinant acid .alpha.-glucosidase treated or untreated
with GlcNAc-phosphotransferase and phosphodiester .alpha.-GlcNAcase
was digested with N-glycanase (New England Biolabs) or
endomannosidase H (New England Biolabs) according to the
manufacturer's conditions. The released oligosaccharides were then
labeled on the reducing terminus with 2-aminobenzamide and
fractionated by HPLC with fluorescent detection according to the
manufacturer's instructions (Oxford Glycosystems). Peaks were
identified by comparison with standards chromatographed on the same
system, and confirmed by digestion with linkage specific
glycosidases and/or mass determination by MALDI. The results are
shown in Table 1.
TABLE-US-00005 TABLE 1 Enzyme Preparation Com- M6 M7 M8 M9 1P-Gn
2P-Gn 1M6P plex Rh-GAA 0 0 0 0 0 0 1 99 (Secreted) Rh-GAA (dMM/ 23
31 23 6 0 0 17 0 intracellular) Rh-GAA (dMM/ 6 11 7 2 12 62 0 0
intracellular) Ptase-treated
[0170] Referring to Table 1, the data (given in mole percent) show
that the Lysosomal enzymes prepared using the
GlcNAc-phosphotransferase and phosphodiester .alpha.-GlcNAcase of
the present invention are highly phosphorylated The data shows that
the present invention produces lysosomal enzymes having about 5-10
M6P groups per enzyme compared to about 0-2 for untreated enzymes
and enzymes known in the art. When compared to naturally occurring
or recombinant lysosomal enzymes, the in vitro-modified preparation
is very highly phosphorylated. In the most highly phosphorylated
lysosomal enzyme known in the art, the .alpha.-galactosidase A
described by Matsuura, F., Ohta, M., Ioannou, Y. A. and Desnick. R.
J. (1998). "Human alpha-galactosidase A: characterization of the
N-linked oligosaccharides on the intracellular and secreted
glycoforms overexpressed by Chinese hamster ovary cells."
Glycobiology 8(4): 329-39, 5.2% of the oligosaccharides are
bis-phosphorylated. In marked contrast, 62% of the oligosaccharides
on the in vitro-phosphorylated acid .alpha.-glucosidase,
preparation described here contains bis-phosphorylated
oligosaccharides. This represents about a 12 fold increase. When
the in vitro phosphorylated preparation of rh-GAA shown in Table 1
is compared with GAA secreted from CHO cells by methods known in
the art, an even greater increase in phosphorylation is evident,
about a 62 fold increase.
[0171] Thus, the in vitro-phosphorylated GAA is 12-62 fold more
phosphorylated than any other described preparation of natural or
recombinant lysosomal enzyme. This difference has a major influence
on the rate and extent of internalization (Reuser, A. J., Kroos, M.
A., Ponne, N. J., Wolterman, R. A., Loonen, M. C., Busch, H. F.,
Visser, W. J. and Bolhuis, P. A. (1984). "Uptake and stability of
human and bovine acid alpha-glucosidase in cultured fibroblasts and
skeletal muscle cells from glycogenosis type II patients."
Experimental Cell Research 155: 178-189).
Example 28
Comparison of Cell Uptake of Recombinant Human Acid
.alpha.-Glucosidase with or without Modification by
GlcNAc-Phosphotransferase and Phosphodiester .alpha.-GlcNAcase
[0172] Human Pompe disease fibroblasts are obtained from ATCC and
cultured in DMEM with 10% fetal bovine serum in 6 well plates and
incubated at 37.degree. C. in 5% CO.sub.2. Recombinant human acid
.alpha.-glucosidase with different carbohydrate structures are
compared for the rate and extent of internalization. Controls
include each preparation incubated with 5 mM mannose 6-phosphate
and incubations without added recombinant human acid
.alpha.-glucosidase. The different preparations to be examined
include acid .alpha.-glucosidase secreted from CHO cells, acid
.alpha.-glucosidase secreted from CHO cells in the presence of a
1,2-mannosidase inhibitors, acid .alpha.-glucosidase secreted from
CHO cells in the presence of .alpha.1,2-mannosidase inhibitors
treated with GlcNAc-phosphotransferase, and acid
.alpha.-glucosidase secreted from CHO cells in the presence of or
1,2-mannosidase inhibitors treated with GlcNAc-phosphotransferase
and phosphodiester .alpha.-GlcNAcase. Equal amounts of the four
different preparations are added to each well and incubated at
37.degree. C. for periods varying from 5 minutes to 4 hours. At the
end of each incubation period the cell monolayers are washed with
phosphate buffered saline containing 5 mM mannose 6-phosphate and
the monolayer solubilized in 1% Triton X-100 and assayed for
internalized acid .alpha.-glucosidase by enzymatic assay.
[0173] Applicant and the assignee acknowledge their responsibility
to replace these cultures should they die before the end of the
term of a patent issued hereon, 5 years after the last request for
a culture, or 30 years, whichever is the longer, and their
responsibility to notify the depository of the issuance of such a
patent, at which time the deposit will be made irrevocably
available to the public. Until that time the deposit will be made
available to the Commissioner of Patents under the terms of 37
C.F.R. 1.14 and 35 U.S.C. 112.
[0174] While the preferred embodiments are shown to illustrate the
invention, numerous changes to the materials and methods can be
made by those skilled in the art. All such changes are encompassed
within the spirit of the invention as defined by the appended
claims.
[0175] This application claims priority to U.S. Provisional
application No. 60/153,831 filed Sep. 14, 1999, and is incorporated
herein by reference.
Sequence CWU 1
1
521928PRTHomo sapiens 1Met Leu Phe Lys Leu Leu Gln Arg Gln Thr Tyr
Thr Cys Leu Ser His1 5 10 15Arg Tyr Gly Leu Tyr Val Cys Phe Leu Gly
Val Val Val Thr Ile Val 20 25 30Ser Ala Phe Gln Phe Gly Glu Val Val
Leu Glu Trp Ser Arg Asp Gln 35 40 45Tyr His Val Leu Phe Asp Ser Tyr
Arg Asp Asn Ile Ala Gly Lys Ser 50 55 60Phe Gln Asn Arg Leu Cys Leu
Pro Met Pro Ile Asp Val Val Tyr Thr65 70 75 80Trp Val Asn Gly Thr
Asp Leu Glu Leu Leu Lys Glu Leu Gln Gln Val 85 90 95Arg Glu Gln Met
Glu Glu Glu Gln Lys Ala Met Arg Glu Ile Leu Gly 100 105 110Lys Asn
Thr Thr Glu Pro Thr Lys Lys Ser Glu Lys Gln Leu Glu Cys 115 120
125Leu Leu Thr His Cys Ile Lys Val Pro Met Leu Val Leu Asp Pro Ala
130 135 140Leu Pro Ala Asn Ile Thr Leu Lys Asp Val Pro Ser Leu Tyr
Pro Ser145 150 155 160Phe His Ser Ala Ser Asp Ile Phe Asn Val Ala
Lys Pro Lys Asn Pro 165 170 175Ser Thr Asn Val Ser Val Val Val Phe
Asp Ser Thr Lys Asp Val Glu 180 185 190Asp Ala His Ser Gly Leu Leu
Lys Gly Asn Ser Arg Gln Thr Val Trp 195 200 205Arg Gly Tyr Leu Thr
Thr Asp Lys Glu Val Pro Gly Leu Val Leu Met 210 215 220Gln Asp Leu
Ala Phe Leu Ser Gly Phe Pro Pro Thr Phe Lys Glu Thr225 230 235
240Asn Gln Leu Lys Thr Lys Leu Pro Glu Asn Leu Ser Ser Lys Val Lys
245 250 255Leu Leu Gln Leu Tyr Ser Glu Ala Ser Val Ala Leu Leu Lys
Leu Asn 260 265 270Asn Pro Lys Asp Phe Gln Glu Leu Asn Lys Gln Thr
Lys Lys Asn Met 275 280 285Thr Ile Asp Gly Lys Glu Leu Thr Ile Ser
Pro Ala Tyr Leu Leu Trp 290 295 300Asp Leu Ser Ala Ile Ser Gln Ser
Lys Gln Asp Glu Asp Ile Ser Ala305 310 315 320Ser Arg Phe Glu Asp
Asn Glu Glu Leu Arg Tyr Ser Leu Arg Ser Ile 325 330 335Glu Arg His
Ala Pro Trp Val Arg Asn Ile Phe Ile Val Thr Asn Gly 340 345 350Gln
Ile Pro Ser Trp Leu Asn Leu Asp Asn Pro Arg Val Thr Ile Val 355 360
365Thr His Gln Asp Val Phe Arg Asn Leu Ser His Leu Pro Thr Phe Ser
370 375 380Ser Pro Ala Ile Glu Ser His Ile His Arg Ile Glu Gly Leu
Ser Gln385 390 395 400Lys Phe Ile Tyr Leu Asn Asp Asp Val Met Phe
Gly Lys Asp Val Trp 405 410 415Pro Asp Asp Phe Tyr Ser His Ser Lys
Gly Gln Lys Val Tyr Leu Thr 420 425 430Trp Pro Val Pro Asn Cys Ala
Glu Gly Cys Pro Gly Ser Trp Ile Lys 435 440 445Asp Gly Tyr Cys Asp
Lys Ala Cys Asn Asn Ser Ala Cys Asp Trp Asp 450 455 460Gly Gly Asp
Cys Ser Gly Asn Ser Gly Gly Ser Arg Tyr Ile Ala Gly465 470 475
480Gly Gly Gly Thr Gly Ser Ile Gly Val Gly His Pro Trp Gln Phe Gly
485 490 495Gly Gly Ile Asn Ser Val Ser Tyr Cys Asn Gln Gly Cys Ala
Asn Ser 500 505 510Trp Leu Ala Asp Lys Phe Cys Asp Gln Ala Cys Asn
Val Leu Ser Cys 515 520 525Gly Phe Asp Ala Gly Asp Cys Gly Gln Asp
His Phe His Glu Leu Tyr 530 535 540Lys Val Ile Leu Leu Pro Asn Gln
Thr His Tyr Ile Ile Pro Lys Gly545 550 555 560Glu Cys Leu Pro Tyr
Phe Ser Phe Ala Glu Val Ala Lys Arg Gly Val 565 570 575Glu Gly Ala
Tyr Ser Asp Asn Pro Ile Ile Arg His Ala Ser Ile Ala 580 585 590Asn
Lys Trp Lys Thr Ile His Leu Ile Met His Ser Gly Met Asn Ala 595 600
605Thr Thr Ile His Phe Asn Leu Thr Phe Gln Asn Thr Asn Asp Glu Glu
610 615 620Phe Lys Met Gln Ile Thr Val Glu Val Asp Thr Arg Glu Gly
Pro Lys625 630 635 640Leu Asn Ser Thr Ala Gln Lys Gly Tyr Glu Asn
Leu Val Ser Pro Ile 645 650 655Thr Leu Leu Pro Glu Ala Glu Ile Leu
Phe Glu Asp Ile Pro Lys Glu 660 665 670Lys Arg Phe Pro Lys Phe Lys
Arg His Asp Val Asn Ser Thr Arg Arg 675 680 685Ala Gln Glu Glu Val
Lys Ile Pro Leu Val Asn Ile Ser Leu Leu Pro 690 695 700Lys Asp Ala
Gln Leu Ser Leu Asn Thr Leu Asp Leu Gln Leu Glu His705 710 715
720Gly Asp Ile Thr Leu Lys Gly Tyr Asn Leu Ser Lys Ser Ala Leu Leu
725 730 735Arg Ser Phe Leu Met Asn Ser Gln His Ala Lys Ile Lys Asn
Gln Ala 740 745 750Ile Ile Thr Asp Glu Thr Asn Asp Ser Leu Val Ala
Pro Gln Glu Lys 755 760 765Gln Val His Lys Ser Ile Leu Pro Asn Ser
Leu Gly Val Ser Glu Arg 770 775 780Leu Gln Arg Leu Thr Phe Pro Ala
Val Ser Val Lys Val Asn Gly His785 790 795 800Asp Gln Gly Gln Asn
Pro Pro Leu Asp Leu Glu Thr Thr Ala Arg Phe 805 810 815Arg Val Glu
Thr His Thr Gln Lys Thr Ile Gly Gly Asn Val Thr Lys 820 825 830Glu
Lys Pro Pro Ser Leu Ile Val Pro Leu Glu Ser Gln Met Thr Lys 835 840
845Glu Lys Lys Ile Thr Gly Lys Glu Lys Glu Asn Ser Arg Met Glu Glu
850 855 860Asn Ala Glu Asn His Ile Gly Val Thr Glu Val Leu Leu Gly
Arg Lys865 870 875 880Leu Gln His Tyr Thr Asp Ser Tyr Leu Gly Phe
Leu Pro Trp Glu Lys 885 890 895Lys Lys Tyr Phe Gln Asp Leu Leu Asp
Glu Glu Glu Ser Leu Lys Thr 900 905 910Gln Leu Ala Tyr Phe Thr Asp
Ser Lys Asn Thr Gly Arg Gln Leu Lys 915 920 9252328PRTHomo sapiens
2Asp Thr Phe Ala Asp Ser Leu Arg Tyr Val Asn Lys Ile Leu Asn Ser1 5
10 15Lys Phe Gly Phe Thr Ser Arg Lys Val Pro Ala His Met Pro His
Met 20 25 30Ile Asp Arg Ile Val Met Gln Glu Leu Gln Asp Met Phe Pro
Glu Glu 35 40 45Phe Asp Lys Thr Ser Phe His Lys Val Arg His Ser Glu
Asp Met Gln 50 55 60Phe Ala Phe Ser Tyr Phe Tyr Tyr Leu Met Ser Ala
Val Gln Pro Leu65 70 75 80Asn Ile Ser Gln Val Phe Asp Glu Val Asp
Thr Asp Gln Ser Gly Val 85 90 95Leu Ser Asp Arg Glu Ile Arg Thr Leu
Ala Thr Arg Ile His Glu Leu 100 105 110Pro Leu Ser Leu Gln Asp Leu
Thr Gly Leu Glu His Met Leu Ile Asn 115 120 125Cys Ser Lys Met Leu
Pro Ala Asp Ile Thr Gln Leu Asn Asn Ile Pro 130 135 140Pro Thr Gln
Glu Ser Tyr Tyr Asp Pro Asn Leu Pro Pro Val Thr Lys145 150 155
160Ser Leu Val Thr Asn Cys Lys Pro Val Thr Asp Lys Ile His Lys Ala
165 170 175Tyr Lys Asp Lys Asn Lys Tyr Arg Phe Glu Ile Met Gly Glu
Glu Glu 180 185 190Ile Ala Phe Lys Met Ile Arg Thr Asn Val Ser His
Val Val Gly Gln 195 200 205Leu Asp Asp Ile Arg Lys Asn Pro Arg Lys
Phe Val Cys Leu Asn Asp 210 215 220Asn Ile Asp His Asn His Lys Asp
Ala Gln Thr Val Lys Ala Val Leu225 230 235 240Arg Asp Phe Tyr Glu
Ser Met Phe Pro Ile Pro Ser Gln Phe Glu Leu 245 250 255Pro Arg Glu
Tyr Arg Asn Arg Phe Leu His Met His Glu Leu Gln Glu 260 265 270Trp
Arg Ala Tyr Arg Asp Lys Leu Lys Phe Trp Thr His Cys Val Leu 275 280
285Ala Thr Leu Ile Met Phe Thr Ile Phe Ser Phe Phe Ala Glu Gln Leu
290 295 300Ile Ala Leu Lys Arg Lys Ile Phe Pro Arg Arg Arg Ile His
Lys Glu305 310 315 320Ala Ser Pro Asn Arg Ile Arg Val
3253305PRTHomo sapiensSIGNAL(1)..(24) 3Met Ala Ala Gly Leu Ala Arg
Leu Leu Leu Leu Leu Gly Leu Ser Ala1 5 10 15Gly Gly Pro Ala Pro Ala
Gly Ala Ala Lys Met Lys Val Val Glu Glu 20 25 30Pro Asn Ala Phe Gly
Val Asn Asn Pro Phe Leu Pro Gln Ala Ser Arg 35 40 45Leu Gln Ala Lys
Arg Asp Pro Ser Pro Val Ser Gly Pro Val His Leu 50 55 60Phe Arg Leu
Ser Gly Lys Cys Phe Ser Leu Val Glu Ser Thr Tyr Lys65 70 75 80Tyr
Glu Phe Cys Pro Phe His Asn Val Thr Gln His Glu Gln Thr Phe 85 90
95Arg Trp Asn Ala Tyr Ser Gly Ile Leu Gly Ile Trp His Glu Trp Glu
100 105 110Ile Ala Asn Asn Thr Phe Thr Gly Met Trp Met Arg Asp Gly
Asp Ala 115 120 125Cys Arg Ser Arg Ser Arg Gln Ser Lys Val Glu Leu
Ala Cys Gly Lys 130 135 140Ser Asn Arg Leu Ala His Val Ser Glu Pro
Ser Thr Cys Val Tyr Ala145 150 155 160Leu Thr Phe Glu Thr Pro Leu
Val Cys His Pro His Ala Leu Leu Val 165 170 175Tyr Pro Thr Leu Pro
Glu Ala Leu Gln Arg Gln Trp Asp Gln Val Glu 180 185 190Gln Asp Leu
Ala Asp Glu Leu Ile Thr Pro Gln Gly His Glu Lys Leu 195 200 205Leu
Arg Thr Leu Phe Glu Asp Ala Gly Tyr Leu Lys Thr Pro Glu Glu 210 215
220Asn Glu Pro Thr Gln Leu Glu Gly Gly Pro Asp Ser Leu Gly Phe
Glu225 230 235 240Thr Leu Glu Asn Cys Arg Lys Ala His Lys Glu Leu
Ser Lys Glu Ile 245 250 255Lys Arg Leu Lys Gly Leu Leu Thr Gln His
Gly Ile Pro Tyr Thr Arg 260 265 270Pro Thr Glu Thr Ser Asn Leu Glu
His Leu Gly His Glu Thr Pro Arg 275 280 285Ala Lys Ser Pro Glu Gln
Leu Arg Gly Asp Pro Gly Leu Arg Gly Ser 290 295
300Leu30545597DNAHomo sapiens 4cggagccgag cgggcgtccg tcgccggagc
tgcaatgagc ggcgcccgga ggctgtgacc 60tgcgcgcggc ggcccgaccg gggcccctga
atggcggctc gctgaggcgg cggcggcggc 120ggcggctcag gctcctcggg
gcgtggcgtg gcggtgaagg ggtgatgctg ttcaagctcc 180tgcagagaca
aacctatacc tgcctgtccc acaggtatgg gctctacgtg tgcttcttgg
240gcgtcgttgt caccatcgtc tccgccttcc agttcggaga ggtggttctg
gaatggagcc 300gagatcaata ccatgttttg tttgattcct atagagacaa
tattgctgga aagtcctttc 360agaatcggct ttgtctgccc atgccgattg
acgttgttta cacctgggtg aatggcacag 420atcttgaact actgaaggaa
ctacagcagg tcagagaaca gatggaggag gagcagaaag 480caatgagaga
aatccttggg aaaaacacaa cggaacctac taagaagagt gagaagcagt
540tagagtgttt gctaacacac tgcattaagg tgccaatgct tgtactggac
ccagccctgc 600cagccaacat caccctgaag gacgtgccat ctctttatcc
ttcttttcat tctgccagtg 660acattttcaa tgttgcaaaa ccaaaaaacc
cttctaccaa tgtctcagtt gttgtttttg 720acagtactaa ggatgttgaa
gatgcccact ctggactgct taaaggaaat agcagacaga 780cagtatggag
ggggtacttg acaacagata aagaagtccc tggattagtg ctaatgcaag
840atttggcttt cctgagtgga tttccaccaa cattcaagga aacaaatcaa
ctaaaaacaa 900aattgccaga aaatctttcc tctaaagtca aactgttgca
gttgtattca gaggccagtg 960tagcgcttct aaaactgaat aaccccaagg
attttcaaga attgaataag caaactaaga 1020agaacatgac cattgatgga
aaagaactga ccataagtcc tgcatattta ttatgggatc 1080tgagcgccat
cagccagtct aagcaggatg aagacatctc tgccagtcgt tttgaagata
1140acgaagaact gaggtactca ttgcgatcta tcgagaggca tgcaccatgg
gttcggaata 1200ttttcattgt caccaacggg cagattccat cctggctgaa
ccttgacaat cctcgagtga 1260caatagtaac acaccaggat gtttttcgaa
atttgagcca cttgcctacc tttagttcac 1320ctgctattga aagtcacatt
catcgcatcg aagggctgtc ccagaagttt atttacctaa 1380atgatgatgt
catgtttggg aaggatgtct ggccagatga tttttacagt cactccaaag
1440gccagaaggt ttatttgaca tggcctgtgc caaactgtgc cgagggctgc
ccaggttcct 1500ggattaagga tggctattgt gacaaggctt gtaataattc
agcctgcgat tgggatggtg 1560gggattgctc tggaaacagt ggagggagtc
gctatattgc aggaggtgga ggtactggga 1620gtattggagt tggacacccc
tggcagtttg gtggaggaat aaacagtgtc tcttactgta 1680atcagggatg
tgcgaattcc tggctcgctg ataagttctg tgaccaagca tgcaatgtct
1740tgtcctgtgg gtttgatgct ggcgactgtg ggcaagatca ttttcatgaa
ttgtataaag 1800tgatccttct cccaaaccag actcactata ttattccaaa
aggtgaatgc ctgccttatt 1860tcagctttgc agaagtagcc aaaagaggag
ttgaaggtgc ctatagtgac aatccaataa 1920ttcgacatgc ttctattgcc
aacaagtgga aaaccatcca cctcataatg cacagtggaa 1980tgaatgccac
cacaatacat tttaatctca cgtttcaaaa tacaaacgat gaagagttca
2040aaatgcagat aacagtggag gtggacacaa gggagggacc aaaactgaat
tctacggccc 2100agaagggtta cgaaaattta gttagtccca taacacttct
tccagaggcg gaaatccttt 2160ttgaggatat tcccaaagaa aaacgcttcc
cgaagtttaa gagacatgat gttaactcaa 2220caaggagagc ccaggaagag
gtgaaaattc ccctggtaaa tatttcactc cttccaaaag 2280acgcccagtt
gagtctcaat accttggatt tgcaactgga acatggagac atcactttga
2340aaggatacaa tttgtccaag tcagccttgc tgagatcatt tctgatgaac
tcacagcatg 2400ctaaaataaa aaatcaagct ataataacag atgaaacaaa
tgacagtttg gtggctccac 2460aggaaaaaca ggttcataaa agcatcttgc
caaacagctt aggagtgtct gaaagattgc 2520agaggttgac ttttcctgca
gtgagtgtaa aagtgaatgg tcatgaccag ggtcagaatc 2580cacccctgga
cttggagacc acagcaagat ttagagtgga aactcacacc caaaaaacca
2640taggcggaaa tgtgacaaaa gaaaagcccc catctctgat tgttccactg
gaaagccaga 2700tgacaaaaga aaagaaaatc acagggaaag aaaaagagaa
cagtagaatg gaggaaaatg 2760ctgaaaatca cataggcgtt actgaagtgt
tacttggaag aaagctgcag cattacacag 2820atagttactt gggctttttg
ccatgggaga aaaaaaagta tttccaagat cttctcgacg 2880aagaagagtc
attgaagaca caattggcat acttcactga tagcaaaaat actgggaggc
2940aactaaaaga tacatttgca gattccctca gatatgtaaa taaaattcta
aatagcaagt 3000ttggattcac atcgcggaaa gtccctgctc acatgcctca
catgattgac cggattgtta 3060tgcaagaact gcaagatatg ttccctgaag
aatttgacaa gacgtcattt cacaaagtgc 3120gccattctga ggatatgcag
tttgccttct cttattttta ttatctcatg agtgcagtgc 3180agccactgaa
tatatctcaa gtctttgatg aagttgatac agatcaatct ggtgtcttgt
3240ctgacagaga aatccgaaca ctggctacca gaattcacga actgccgtta
agtttgcagg 3300atttgacagg tctggaacac atgctaataa attgctcaaa
aatgcttcct gctgatatca 3360cgcagctaaa taatattcca ccaactcagg
aatcctacta tgatcccaac ctgccaccgg 3420tcactaaaag tctagtaaca
aactgtaaac cagtaactga caaaatccac aaagcatata 3480aggacaaaaa
caaatatagg tttgaaatca tgggagaaga agaaatcgct tttaaaatga
3540ttcgtaccaa cgtttctcat gtggttggcc agttggatga cataagaaaa
aaccctagga 3600agtttgtttg cctgaatgac aacattgacc acaatcataa
agatgctcag acagtgaagg 3660ctgttctcag ggacttctat gaatccatgt
tccccatacc ttcccaattt gaactgccaa 3720gagagtatcg aaaccgtttc
cttcatatgc atgagctgca ggaatggagg gcttatcgag 3780acaaattgaa
gttttggacc cattgtgtac tagcaacatt gattatgttt actatattct
3840cattttttgc tgagcagtta attgcactta agcggaagat atttcccaga
aggaggatac 3900acaaagaagc tagtcccaat cgaatcagag tatagaagat
cttcatttga aaaccatcta 3960cctcagcatt tactgagcat tttaaaactc
agcttcacag agatgtcttt gtgatgtgat 4020gcttagcagt ttggcccgaa
gaaggaaaat atccagtacc atgctgtttt gtggcatgaa 4080tatagcccac
tgactaggaa ttatttaacc aacccactga aaacttgtgt gtcgagcagc
4140tctgaactga ttttactttt aaagaatttg ctcatggacc tgtcatcctt
tttataaaaa 4200ggctcactga caagagacag ctgttaattt cccacagcaa
tcattgcaga ctaactttat 4260taggagaagc ctatgccagc tgggagtgat
tgctaagagg ctccagtctt tgcattccaa 4320agccttttgc taaagttttg
cacttttttt ttttcatttc ccatttttaa gtagttacta 4380agttaactag
ttattcttgc ttctgagtat aacgaattgg gatgtctaaa cctattttta
4440tagatgttat ttaaataatg cagcaatatc acctcttatt gacaatacct
aaattatgag 4500ttttattaat atttaagact gtaaatggtc ttaaaccact
aactactgaa gagctcaatg 4560attgacatct gaaatgcttt gtaattattg
acttcagccc ctaagaatgc tatgatttca 4620cgtgcaggtc taatttcaac
aggctagagt tagtactact taccagatgt aattatgttt 4680tggaaatgta
catattcaaa cagaagtgcc tcattttaga aatgagtagt gctgatggca
4740ctggcacatt acagtggtgt cttgtttaat actcattggt atattccagt
agctatctct 4800ctcagttggt ttttgataga acagaggcca gcaaactttc
tttgtaaaag gctggttagt 4860aaattattgc aggccacctg tgtctttgtc
atacattctt cttgctgttg tttagtttgt 4920tttttttcaa acaaccctct
aaaaatgtaa aaaccatgtt tagcttgcag ctgtacaaaa 4980actgcccacc
agccagatgt gaccctcagg ccatcatttg ccaatcactg agaattattt
5040ttgttgttgt tgttgttgtt gtttttgaga cagagtctct ctctgttgcc
caggctggag 5100tgcagtggcg caatctcagc tcactgcaac ctccgcctcc
cgggttcaag cagttctgtc 5160tcagccttct gagtagctgg gactacaggt
gcatgccacc acaccctgct aatttttgta 5220tttttagtag agacgggggt
tccaccatat tggtcaggct tatcttgaac tcctgacctc 5280aggtgatcca
cctgcctctg cctcccaaag tgctgagatt acaggcataa gccagtgcac
5340ccagccgaga attagtattt ttatgtatgg ttaaaccttg gcgtctagcc
atattttatg 5400tcataataca atggatttgt gaagagcaga ttccatgagt
aactctgaca ggtattttag 5460atcatgatct caacaatatt cctcccaaat
ggcatacatc ttttgtacaa agaacttgaa 5520atgtaaatac tgtgtttgtg
ctgtaagagt tgtgtatttc aaaaactgaa atctcataaa 5580aagttaaatt ttgaaaa
559751219DNAHomo sapienssig_peptide(24)..(95) 5gtagagcgca
ggtgcgcggc tcgatggcgg cggggctggc gcggctcctg ttgctcctcg 60ggctctcggc
cggcgggccc gcgccggcag gtgcagcgaa gatgaaggtg gtggaggagc
120ccaacgcgtt tggggtgaac aacccgttct tgcctcaggc cagtcgcctc
caggccaaga 180gggatccttc acccgtgtct ggacccgtgc atctcttccg
actctcgggc aagtgcttca 240gcctggtgga gtccacgtac aagtatgagt
tctgcccgtt ccacaacgtg acccagcacg 300agcagacctt ccgctggaac
gcctacagtg ggatcctcgg catctggcac gagtgggaga 360tcgccaacaa
caccttcacg ggcatgtgga tgagggacgg tgacgcctgc cgttcccgga
420gccggcagag caaggtggag ctggcgtgtg gaaaaagcaa ccggctggcc
catgtgtccg 480agccgagcac ctgcgtctat gcgctgacgt tcgagacccc
cctcgtctgc cacccccacg 540ccttgctagt gtacccaacc ctgccagagg
ccctgcagcg gcagtgggac caggtagagc 600aggacctggc cgatgagctg
atcacccccc agggccatga gaagttgctg aggacacttt 660ttgaggatgc
tggctactta aagaccccag aagaaaatga acccacccag ctggagggag
720gtcctgacag cttggggttt gagaccctgg aaaactgcag gaaggctcat
aaagaactct 780caaaggagat caaaaggctg aaaggtttgc tcacccagca
cggcatcccc tacacgaggc 840ccacagaaac ttccaacttg gagcacttgg
gccacgagac gcccagagcc aagtctccag 900agcagctgcg gggtgaccca
ggactgcgtg ggagtttgtg accttgtggt gggagagcag 960aggtggacgc
ggccgagagc cctacagaga agctggctgg taggacccgc aggaccagct
1020gaccaggctt gtgctcagag aagcagacaa aacaaagatt caaggtttta
attaattccc 1080atactgataa aaataactcc atgaattctg taaaccattg
cataaatgct atagtgtaaa 1140aaaatttaaa caagtgttaa ctttaaacag
ttcgctacaa gtaaatgatt ataaatacta 1200aaaaaaaaaa aaaaaaaaa
12196515PRTHomo sapiensSIGNAL(1)..(24) 6Met Ala Thr Ser Thr Gly Arg
Trp Leu Leu Leu Arg Leu Ala Leu Phe1 5 10 15Gly Phe Leu Trp Glu Ala
Ser Gly Gly Leu Asp Ser Gly Ala Ser Arg 20 25 30Asp Asp Asp Leu Leu
Leu Pro Tyr Pro Arg Ala Arg Ala Arg Leu Pro 35 40 45Arg Asp Cys Thr
Arg Val Arg Ala Gly Asn Arg Glu His Glu Ser Trp 50 55 60Pro Pro Pro
Pro Ala Thr Pro Gly Ala Gly Gly Leu Ala Val Arg Thr65 70 75 80Phe
Val Ser His Phe Arg Asp Arg Ala Val Ala Gly His Leu Thr Arg 85 90
95Ala Val Glu Pro Leu Arg Thr Phe Ser Val Leu Glu Pro Gly Gly Pro
100 105 110Gly Gly Cys Ala Ala Arg Arg Arg Ala Thr Val Glu Glu Thr
Ala Arg 115 120 125Ala Ala Asp Cys Arg Val Ala Gln Asn Gly Gly Phe
Phe Arg Met Asn 130 135 140Ser Gly Glu Cys Leu Gly Asn Val Val Ser
Asp Glu Arg Arg Val Ser145 150 155 160Ser Ser Gly Gly Leu Gln Asn
Ala Gln Phe Gly Ile Arg Arg Asp Gly 165 170 175Thr Leu Val Thr Gly
Tyr Leu Ser Glu Glu Glu Val Leu Asp Thr Glu 180 185 190Asn Pro Phe
Val Gln Leu Leu Ser Gly Val Val Trp Leu Ile Arg Asn 195 200 205Gly
Ser Ile Tyr Ile Asn Glu Ser Gln Ala Thr Glu Cys Asp Glu Thr 210 215
220Gln Glu Thr Gly Ser Phe Ser Lys Phe Val Asn Val Ile Ser Ala
Arg225 230 235 240Thr Ala Ile Gly His Asp Arg Lys Gly Gln Leu Val
Leu Phe His Ala 245 250 255Asp Gly His Thr Glu Gln Arg Gly Ile Asn
Leu Trp Glu Met Ala Glu 260 265 270Phe Leu Leu Lys Gln Asp Val Val
Asn Ala Ile Asn Leu Asp Gly Gly 275 280 285Gly Ser Ala Thr Phe Val
Leu Asn Gly Thr Leu Ala Ser Tyr Pro Ser 290 295 300Asp His Cys Gln
Asp Asn Met Trp Arg Cys Pro Arg Gln Val Ser Thr305 310 315 320Val
Val Cys Val His Glu Pro Arg Cys Gln Pro Pro Asp Cys His Gly 325 330
335His Gly Thr Cys Val Asp Gly His Cys Gln Cys Thr Gly His Phe Trp
340 345 350Arg Gly Pro Gly Cys Asp Glu Leu Asp Cys Gly Pro Ser Asn
Cys Ser 355 360 365Gln His Gly Leu Cys Thr Glu Thr Gly Cys Arg Cys
Asp Ala Gly Trp 370 375 380Thr Gly Ser Asn Cys Ser Glu Glu Cys Pro
Leu Gly Trp His Gly Pro385 390 395 400Gly Cys Gln Arg Arg Cys Lys
Cys Glu His His Cys Pro Cys Asp Pro 405 410 415Lys Thr Gly Asn Cys
Ser Val Ser Arg Val Lys Gln Cys Leu Gln Pro 420 425 430Pro Glu Ala
Thr Leu Arg Ala Gly Glu Leu Ser Phe Phe Thr Arg Thr 435 440 445Ala
Trp Leu Ala Leu Thr Leu Ala Leu Ala Phe Leu Leu Leu Ile Ser 450 455
460Ile Ala Ala Asn Leu Ser Leu Leu Leu Ser Arg Ala Glu Arg Asn
Arg465 470 475 480Arg Leu His Gly Asp Tyr Ala Tyr His Pro Leu Gln
Glu Met Asn Gly 485 490 495Glu Pro Leu Ala Ala Glu Lys Glu Gln Pro
Gly Gly Ala His Asn Pro 500 505 510Phe Lys Asp 51572183DNAHomo
sapiens 7atggcgacct ccacgggtcg ctggcttctc ctccggcttg cactattcgg
cttcctctgg 60gaagcgtccg gcggcctcga ctcgggggcc tcccgcgacg acgacttgct
actgccctat 120ccacgcgcgc gcgcgcgcct cccccgggac tgcacacggg
tgcgcgccgg caaccgcgag 180cacgagagtt ggcctccgcc tcccgcgact
cccggcgccg gcggtctggc cgtgcgcacc 240ttcgtgtcgc acttcaggga
ccgcgcggtg gccggccacc tgacgcgggc cgttgagccc 300ctgcgcacct
tctcggtgct ggagcccggt ggacccggcg gctgcgcggc gagacgacgc
360gccaccgtgg aggagacggc gcgggcggcc gactgccgtg tcgcccagaa
cggcggcttc 420ttccgcatga actcgggcga gtgcctgggg aacgtggtga
gcgacgagcg gcgggtgagc 480agctccgggg ggctgcagaa cgcgcagttc
gggatccgcc gcgacgggac cctggtcacc 540gggtacctgt ctgaggagga
ggtgctggac actgagaacc catttgtgca gctgctgagt 600ggggtcgtgt
ggctgattcg taatggaagc atctacatca acgagagcca agccacagag
660tgtgacgaga cacaggagac aggttccttt agcaaatttg tgaatgtgat
atcagccagg 720acggccattg gccacgaccg gaaagggcag ctggtgctct
ttcatgcaga cggccatacg 780gagcagcgtg gcatcaacct gtgggaaatg
gcggagttcc tgctgaaaca ggacgtggtc 840aacgccatca acctggatgg
gggtggctct gccacctttg tgctcaacgg gaccttggcc 900agttacccgt
cagatcactg ccaggacaac atgtggcgct gtccccgcca agtgtccacc
960gtggtgtgtg tgcacgaacc ccgctgccag ccgcctgact gccacggcca
cgggacctgc 1020gtggacgggc actgccaatg caccgggcac ttctggcggg
gtcccggctg tgatgagctg 1080gactgtggcc cctctaactg cagccagcac
ggactgtgca cggagaccgg ctgccgctgt 1140gatgccggat ggaccgggtc
caactgcagt gaagagtgtc cccttggctg gcatgggccg 1200ggctgccaga
ggcgttgtaa gtgtgagcac cattgtccct gtgaccccaa gactggcaac
1260tgcagcgtct ccagagtaaa gcagtgtctc cagccacctg aagccaccct
gagggcggga 1320gaactctcct ttttcaccag gaccgcctgg ctagccctca
ccctggcgct ggccttcctc 1380ctgctgatca gcattgcagc aaacctgtcc
ttgctcctgt ccagagcaga gaggaaccgg 1440cgcctgcatg gggactatgc
ataccacccg ctgcaggaga tgaacgggga gcctctggcc 1500gcagagaagg
agcagccagg gggcgcccac aaccccttca aggactgaag cctcaagctg
1560cccggggtgg cacgtcgcga aagcttgttt ccccacggtc tggcttctgc
aggggaaatt 1620tcaaggccac tggcgtggac catctgggtg tcctcaatgg
cccctgtggg gcagccaagt 1680tcctgatagc acttgtgcct cagcccctca
cctggccacc tgccagggca cctgcaaccc 1740tagcaatacc atgctcgctg
gagaggctca gctgcctgct tctcgcctgc ctgtgtctgc 1800tgccgagaag
cccgtgcccc cgggagggct gccgcactgc caaagagtct ccctcctcct
1860ggggaagggg ctgccaacga accagactca gtgaccacgt catgacagaa
cagcacatcc 1920tggccagcac ccctggctgg agtgggttaa agggacgagt
ctgccttcct ggctgtgaca 1980cgggacccct tttctacaga cctcatcact
ggatttgcca actagaattc gatttcctgt 2040cataggaagc tccttggaag
aagggatggg gggatgaaat catgtttaca gacctgtttt 2100gtcatcctgc
tgccaagaag ttttttaatc acttgaataa attgatataa taaaaggagc
2160caccaggtgg tgtgtggatt ctg 21838328PRTMus musculus 8Asp Thr Phe
Ala Asp Ser Leu Arg Tyr Val Asn Lys Ile Leu Asn Ser1 5 10 15Lys Phe
Gly Phe Thr Ser Arg Lys Val Pro Ala His Met Pro His Met 20 25 30Ile
Asp Arg Ile Val Met Gln Glu Leu Gln Asp Met Phe Pro Glu Glu 35 40
45Phe Asp Lys Thr Ser Phe His Lys Val Arg His Ser Glu Asp Met Gln
50 55 60Phe Ala Phe Ser Tyr Phe Tyr Tyr Leu Met Ser Ala Val Gln Pro
Leu65 70 75 80Asn Ile Ser Gln Val Phe His Glu Val Asp Thr Asp Gln
Ser Gly Val 85 90 95Leu Ser Asp Arg Glu Ile Arg Thr Leu Ala Thr Arg
Ile His Asp Leu 100 105 110Pro Leu Ser Leu Gln Asp Leu Thr Gly Leu
Glu His Met Leu Ile Asn 115 120 125Cys Ser Lys Met Leu Pro Ala Asn
Ile Thr Gln Leu Asn Asn Ile Pro 130 135 140Pro Thr Gln Glu Ala Tyr
Tyr Asp Pro Asn Leu Pro Pro Val Thr Lys145 150 155 160Ser Leu Val
Thr Asn Cys Lys Pro Val Thr Asp Lys Ile His Lys Ala 165 170 175Tyr
Lys Asp Lys Asn Lys Tyr Arg Phe Glu Ile Met Gly Glu Glu Glu 180 185
190Ile Ala Phe Lys Met Ile Arg Thr Asn Val Ser His Val Val Gly Gln
195 200 205Leu Asp Asp Ile Arg Lys Asn Pro Arg Lys Phe Val Cys Leu
Asn Asp 210 215 220Asn Ile Asp His Asn His Lys Asp Ala Arg Thr Val
Lys Ala Val Leu225 230 235 240Arg Asp Phe Tyr Glu Ser Met Phe Pro
Ile Pro Ser Gln Phe Glu Leu 245 250 255Pro Arg Glu Tyr Arg Asn Arg
Phe Leu His Met His Glu Leu Gln Glu 260 265 270Trp Arg Ala Tyr Arg
Asp Lys Leu Lys Phe Trp Thr His Cys Val Leu 275 280 285Ala Thr Leu
Ile Ile Phe Thr Ile Phe Ser Phe Phe Ala Glu Gln Ile 290 295 300Ile
Ala Leu Lys Arg Lys Ile Phe Pro Arg Arg Arg Ile His Lys Glu305 310
315 320Ala Ser Pro Asp Arg Ile Arg Val 3259307PRTMus musculus 9Met
Ala Gly Arg Leu Ala Gly Phe Leu Met Leu Leu Gly Leu Ala Ser1 5 10
15Gln Gly Pro Ala Pro Ala Cys Ala Gly Lys Met Lys Val Val Glu Glu
20 25 30Pro Asn Thr Phe Gly Leu Asn Asn Pro Phe Leu Pro Gln Ala Ser
Arg 35 40 45Leu Gln Pro Lys Arg Glu Pro Ser Ala Val Ser Gly Pro Leu
His Leu 50 55 60Phe Arg Leu Ala Gly Lys Cys Phe Ser Leu Val Glu Ser
Thr Tyr Lys65 70 75 80Tyr Glu Phe Cys Pro Phe His Asn Val Thr Gln
His Glu Gln Thr Phe 85 90 95Arg Trp Asn Ala Tyr Ser Gly Ile Leu Gly
Ile Trp His Glu Trp Glu 100 105 110Ile Ile Asn Asn Thr Phe Lys Gly
Met Trp Met Thr Asp Gly Asp Ser 115 120 125Cys His Ser Arg Ser Arg
Gln Ser Lys Val Glu Leu Thr Cys Gly Lys 130 135 140Ile Asn Arg Leu
Ala His Val Ser Glu Pro Ser Thr Cys Val Tyr Ala145 150 155 160Leu
Thr Phe Glu Thr Pro Leu Val Cys His Pro His Ser Leu Leu Val 165 170
175Tyr Pro Thr Leu Ser Glu Ala Leu Gln Gln Arg Leu Asp Gln Val Glu
180 185 190Gln Asp Leu Ala Asp Glu Leu Ile Thr Pro Gln Gly Tyr Glu
Lys Leu 195 200 205Leu Arg Val Leu Phe Glu Asp Ala Gly Tyr Leu Lys
Val Pro Gly Glu 210 215 220Thr His Pro Thr Gln Leu Ala Gly Gly Ser
Lys Gly Leu Gly Leu Glu225 230 235 240Thr Leu Asp Asn Cys Arg Lys
Ala His Ala Glu Leu Ser Gln Glu Val 245 250 255Gln Arg Leu Thr Ser
Leu Leu Gln Gln His Gly Ile Pro His Thr Gln 260 265 270Pro Thr Glu
Thr Thr His Ser Gln His Leu Gly Gln Gln Leu Pro Ile 275 280 285Gly
Ala Ile Ala Ala Glu His Leu Arg Ser Asp Pro Gly Leu Arg Gly 290 295
300Asn Ile Leu305102070DNAMus musculusmisc_feature(186)..(186)n is
a, t, g, or c 10gtgagaccct aggagcaatg gccgggcggc tggctggctt
cctgatgttg ctggggctcg 60cgtcgcaggg gcccgcgccg gcatgtgccg ggaagatgaa
ggtggtggag gagcctaaca 120cattcgggtg agcggatcac ggtcctgcgg
cttggggacc gagcctggct ggttcttctg 180accttntcaa ttccataggc
tgaataaccc gttcttgccc caggcaagcc gccttcagcc 240caagagagag
ccttcagctg tatcccgcaa attaagagaa attaatttca aacgatttag
300aaagtattct agccaggcga tgatggcgca cgcctttaat cccagcactt
gggaggcaga 360ggcaggcaga tttccgagtt caaggccatc agaactgact
gtacatctta gtacagttta 420gcatgtgatc agagatctga atcacaaagc
tgggcctgcg tggtaaagca ggtcctttct 480aataaggttg cagtttagat
tttctttctt aactctttta ttctttgaga cagggtttct 540caacagtggg
tgtcctggaa ctcacttttg taaaccaggc tgcccttaaa ctcacaaagc
600tctgtcagcc tctgcctcct gagtgctggg attaaaggtc cacaccctgt
tcattcattt 660ttaatttttg agactgggtc tcattatgtg gccctagaca
gatactgaga gcctcctcca 720caggaacaag catgggaatc ctgccacaga
caaccagttc tgtggtctgg agatgagttt 780gtcagtccct aggagttagg
tcagcctgcc tctgcattcc caataattta ggaaaggagc 840ttggggcgtt
ctggccttga tggttagtgc cctcctgcca accttagctt ccagctttag
900gggtagcaga gtttataccg atgctaaact gctgttgtgt tcttccccag
ggcccctgca 960tctcttcaga cttgctggca agtgctttag cctagtggag
tccacgtgag tgccaggctg 1020gtgggtggag tgggcggagt ctgcagagct
cctgatgtgc ctgtgtttcc caggtacaag 1080tatgaattct gccctttcca
caacgtcacc cagcacgagc agaccttccg ctggaatgcc 1140tacagcggga
tccttggcat ctggcatgag tgggaaatca tcaacaatac cttcaagggc
1200atgtggatga ctgatgggga ctcctgccac tcccggagcc ggcagagcaa
ggtggagctc 1260acctgtggaa agatcaaccg actggcccac gtgtctgagc
caagcacctg tgtctatgca 1320ttgacattcg agacccctct tgtttgccat
ccccactctt tgttagtgta tccaactctg 1380tcagaagccc tgcagcagcc
cttggaccag gtggaacagg acctggcaga tgaactgatc 1440acaccacagg
gctatgagaa gttgctaagg gtactttttg aggatgctgg ctacttaaag
1500gtcccaggag aaacccatcc cacccagctg gcaggaggtt ccaagggcct
ggggcttgag 1560actctggaca actgtagaaa ggcacatgca gagctgtcac
aggaggtaca aagactgacg 1620agtctgctgc aacagcatgg aatcccccac
actcagccca caggtcagtc tgcctgccct 1680ggtcagctgc cagccactcc
ggggcctgca gcactggggc agatctttat tgctacccat 1740tctggcagaa
accactcact ctcagcacct gggtcagcag ctccccatag gtgcaatcgc
1800agcagagcat ctgcggagtg acccaggact acgtgggaac atcctgtgag
caaggtggcc 1860acgaagaata gaaatatcct gagctttgag tgtcctttca
cagagtgaac aaaactggtg 1920tggtgtagac acggcttctt ttggcatatt
ctagatcaga cagtgtcact gacaaacaag 1980agggacctgc tggccagcct
ttgttgtgcc caaagatcca gacaaaataa agattcaaag 2040ttttaattaa
aaaaaaaaaa aaaggaattc 207011113PRTRattus rattus 11Phe Pro Pro Thr
Phe Lys Glu Thr Ser Gln Leu Lys Thr Lys Leu Pro1 5 10 15Glu Asn Leu
Ser Ser Lys Ile Lys Leu Leu Gln Leu Tyr Ser Glu Ala 20 25 30Ser Val
Ala Leu Leu Lys Leu Asn Asn Pro Lys Gly Phe Pro Glu Leu 35 40 45Asn
Lys Gln Thr Lys Lys Asn Met Ser Ile Ser Gly Lys Glu Leu Ala 50 55
60Ile Ser Pro Ala Tyr Leu Leu Trp Asp Leu Ser Ala Ile Ser Gln Ser65
70 75 80Lys Gln Asp Glu Asp Val Ser Ala Ser Arg Phe Glu Asp Asn Glu
Glu 85 90 95Leu Arg Tyr Ser Leu Arg Ser Ile Glu Arg His Asp Ser Met
Ser Pro 100 105 110Leu12460DNARattus rattus 12attcccacca acattcaagg
agacgagtca gctgaagaca aaactgccag aaaatctttc 60ttctaaaata aaactgttgc
agctgtactc ggaggccagc gtcgctcttc tgaaattgaa 120taaccccaaa
ggtttccccg agctgaacaa gcagaccaag aagaacatga gcatcagtgg
180gaaggaactg gccatcagcc ctgcctatct gctgtgggac ctgagcgcca
tcagccagtc 240caagcaggat gaagatgtgt ctgccagccg cttcgaggat
aacgaagagc tgaggtactc 300actgagatct atcgagagac atgattccat
gagtccttta tgaattctgg ccatatcttc 360aatcatgatc tcagtagtat
tcctctgaaa tggcacacat ttttctaatg agaacttgaa 420atgtaaatat
tgtgtttgtg ctgtaaattt tgtgtatttc 46013502PRTDrosophila melanogaster
13Gly Thr Arg Arg Phe Asp Asp Lys Asn Glu Leu Arg Tyr Ser Leu Arg1
5 10 15Ser Leu Glu Lys His Ala Ala Trp Ile Arg His Val Tyr Ile Val
Thr 20 25 30Asn Gly Gln Ile Pro Ser Trp Leu Asp Leu Ser Tyr Glu Arg
Val Thr 35 40 45Val Val Pro His Glu Val Leu Ala Pro Asp Pro Asp Gln
Leu Pro Thr 50 55 60Phe Ser Ser Ser Ala Ile Glu Thr Phe Leu His Arg
Ile Pro Lys Leu65 70 75 80Ser Lys Arg Phe Leu Tyr Leu Asn Asp Asp
Ile Phe Leu Gly Ala Pro 85 90 95Leu Tyr Pro Glu Asp Leu Tyr Thr Glu
Ala Glu Gly Val Arg Val Tyr 100 105 110Gln Ala Trp Met Val Pro Gly
Cys Ala Leu Asp Cys Pro Trp Thr Tyr 115 120 125Ile Gly Asp Gly Ala
Cys Asp Arg His Cys Asn Ile Asp Ala Cys Gln 130 135 140Phe Asp Gly
Gly Asp Cys Ser Glu Thr Gly Pro Ala Ser Asp Ala His145
150 155 160Val Ile Pro Pro Ser Lys Glu Val Leu Glu Val Gln Pro Ala
Ala Val 165 170 175Pro Gln Ser Arg Val His Arg Phe Pro Gln Met Gly
Leu Gln Lys Leu 180 185 190Phe Arg Arg Ser Ser Ala Asn Phe Lys Asp
Val Met Arg His Arg Asn 195 200 205Val Ser Thr Leu Lys Glu Leu Arg
Arg Ile Val Glu Arg Phe Asn Lys 210 215 220Ala Lys Leu Met Ser Leu
Asn Pro Glu Leu Glu Thr Ser Ser Ser Glu225 230 235 240Pro Gln Thr
Thr Gln Arg His Gly Leu Arg Lys Glu Asp Phe Lys Ser 245 250 255Ser
Thr Asp Ile Tyr Ser His Ser Leu Ile Ala Thr Asn Met Leu Leu 260 265
270Asn Arg Ala Tyr Gly Phe Lys Ala Arg His Val Leu Ala His Val Gly
275 280 285Phe Leu Ile Asp Lys Asp Ile Val Glu Ala Met Gln Arg Arg
Phe His 290 295 300Gln Gln Ile Leu Asp Thr Ala His Gln Arg Phe Arg
Ala Pro Thr Asp305 310 315 320Leu Gln Tyr Ala Phe Ala Tyr Tyr Ser
Phe Leu Met Ser Glu Thr Lys 325 330 335Val Met Ser Val Glu Glu Ile
Phe Asp Glu Phe Asp Thr Asp Gly Ser 340 345 350Ala Thr Trp Ser Asp
Arg Glu Val Arg Thr Phe Leu Thr Arg Ile Tyr 355 360 365Gln Pro Pro
Leu Asp Trp Ser Ala Met Arg Tyr Phe Glu Glu Val Val 370 375 380Gln
Asn Cys Thr Arg Asn Leu Gly Met His Leu Lys Val Asp Thr Val385 390
395 400Glu His Ser Thr Leu Val Tyr Glu Arg Tyr Glu Asp Ser Asn Leu
Pro 405 410 415Thr Ile Thr Arg Asp Leu Val Val Arg Cys Pro Leu Leu
Ala Glu Ala 420 425 430Leu Ala Ala Asn Phe Ala Val Arg Pro Lys Tyr
Asn Phe His Val Ser 435 440 445Pro Lys Arg Thr Ser His Ser Asn Phe
Met Met Leu Thr Ser Asn Leu 450 455 460Thr Glu Val Val Glu Ser Leu
Asp Arg Leu Arg Arg Asn Pro Arg Lys465 470 475 480Phe Asn Cys Ile
Asn Asp Asn Leu Asp Ala Asn Arg Gly Glu Asp Asn 485 490 495Glu Asp
Gly Ala Pro Ser 500149792DNAMus musculus 14caggctcggg acttactata
acacaggaca cttgtcacct gaaagcttga gtcagtcagt 60tattatggtc tgtgtgtgag
atacaagtgg gtgcataggc agtggtgcac acatgtagat 120cagactttct
acagccaatt ctcttcttcc tcctctccat gggttcaggg tcttcatctc
180aggttgcaca gcgagttcat ttatgtgctg tgccatctcg ccagtcgttc
ctatatccta 240gaggaaaact agtttcttct ggtcaagagg aggaaagagt
ggagacctgt cattctaaga 300tacccaaaac agggccaggt tggggacctg
tgcctttaat cccatcactt ggggattagg 360tagaagcaag aggctctaga
ccagtctaca cactgaattt caagccagcc tacctataaa 420tcagagaccc
tgcttcaaaa ataaaattaa acaaaaacga agataaacca agctacccaa
480aacacaagag ttaatccagt cagacaggtc tagcaaatgc taggatgaaa
ggtgtgcacc 540accacgagtg ggctgcaagc ctctctctct ctctctctct
ctctctctct ctcgtttgtt 600ttgtttttcg agacaaggtt tctctgtgta
gccctggctg tcctggaact cactctgtag 660accaggctgg cctcgagctt
cactcttaaa agttcctctt cctcctcctc catcttttcc 720tcctcttacc
ccctaggctc cttttcctct tcttgtcttt cagataaagt ctcaagtagt
780ccagactggt ctcaaactaa ctaactagcc aagaatagcc aacctcttaa
cttccgattc 840tcctgcctct gctgaatgct ggggttgtgg cgtgggccac
cacttctggt ttgtgcaaca 900cagaaggaac tagggcttta agcacgagaa
gcaagttctg tacagactta cacaggccca 960gcatctgttc ttgcaatttt
ctgtaagttt gacataatat gagaataaaa agctatctat 1020ctcccttcca
gccttaccct ctctgatgga attcgaatgc gtaatcaaag cacccaacag
1080cctggcctga aatcacgtgg ggcaagccca cgtgaccgga gcaccaatcc
aatatggcgg 1140cgcccagggg gcccgggctg ttcctcatac ccgcgctgct
cggcttactc ggggtggcgt 1200ggtgcagctt aagcttcggg tgagtgcaag
ccgccggggc cagcctggct ggggtccacc 1260tttcctgagc gctctcaggc
acagccctcc gacctcacga tcgccccgtc cctgcagggt 1320ttcccgcgac
gatgacctgc tgctgcctta cccactagcg cgcagacgtc cctcgcgaga
1380ctgcgcccgg gtgcgctcag gtagcccaga gcaggagagc tggcctccgc
cacctctggc 1440cacccacgaa ccccgggcgc caagccacca cgcggccgtg
cgcaccttcg tgtcgcactt 1500cgaggggcgc gcggtggccg gccacctgac
gcgggtcgcc gatcccctac gcactttctc 1560ggtgctggag cccggaggag
ccgggggctg cggcggcaga agcgccgcgg ctactgtgga 1620ggacacagcc
gtccgggccg gttgccgcat cgctcagaac ggtggcttct tccgcatgag
1680cactggcgag tgcttgggga acgtggtgag cgacgggcgg ctggtgagca
gctcaggggg 1740actgcagaac gcgcagttcg gtatccgacg cgatggaacc
atagtcaccg ggtgaggagg 1800cagggagccc cggggctgta gagggcaaag
ggtctctgat gttctttcag agccatgcct 1860ccgagtccag gtccctaacc
aaacttcctg tctttcttct tccgagtaat gacgctgaca 1920ccttccttcc
tttaagttta ttcatgtgcc actgaataat ctgtgatcag gccgtgtgtg
1980gggacttggg gaggcgaccg tgagcctgaa cacagtttgt gccctagtga
actttgtgta 2040gtattagaga aacatttcgt gttcaacgaa gccatggaac
caattggaaa tagtgtagag 2100tttatggagc agtcccagac agctagctgg
aggccttttg ctgtcctgat aaaaatccag 2160gttagacaag gagcttgttg
agggcagcct ttggaagttt ctgtgtttct tgaaatttga 2220cagcagccag
agttgacagc aggcaggcag gagtagaagg tagcgccatc tggtgttcca
2280gttctcttcc aaggttccgt tttttgccaa ggctgggaag tgggctttcc
ccaactcttc 2340tcagcccttg gttgcaattt ctgggcctgc ccatgtatct
ggttcttcat ccttcaacat 2400cagccagtgt caccactgtt gatcttaggt
tttcacagat cctaaaactt ctgccagtga 2460ccagcgcctg cagtttctct
tccctggctc tgtccttcaa cctctctaca ttccagccat 2520ctccctagct
cctctcttgg actccctttc agacttgttg tcatgatcac tgtctcagaa
2580cccctattgc tcctttacaa tggtccactg acctgctcac ctcctacttt
ttttttttaa 2640atgtgtgtgc atctgtgtgt gcctgagggg agaccagagt
ttgatttcaa atgtcttcta 2700ttctcttttc ctccatctta ttttctaaca
caaaatctga atctagagat cactggttca 2760gttaacctgg ctggccggta
aaccccaggg ccctcctgct tccctctgtc caccccaccc 2820cagcactaag
gctacagtgt gtgctgttcc agccagcttt ctcatgggtg ctgaggatct
2880gaacgcaggt tcacatgtgt ggtgggaagg cttttaccca atgctctgtc
tttccagccc 2940atcctccctt gttaactgcc aaacagctgc ctatcctgtc
catgtgtagc tcactgctac 3000ttcttttatt atgaggtcag cacatgttac
taaagatggc aagagaagaa ggttctttca 3060ttgtgtcata gctatagctc
aggaggaatt ttatttcctg tgtaggcaca caggagagca 3120tcttccagct
cacactccaa ctgaactaac tgaacacctg cctatatatc caaagaaggg
3180gtgtcagtgc caatcacagc acacctccag tgcaaatgaa ggtttgtgtt
tgcaccaatc 3240acagccttgc ctcttttagc atgcatcaca acaaagtcct
cctagactat caggggatat 3300gctctcttgg ccaaggtagg aatagttgca
gtgtcatctg gcacaaacca tttcaaacgg 3360cctggctgag gttatgcctt
cgggaacctg aagtctttgt gtggttgtct ccaagtgtct 3420gtggagctcc
aggcggctgg tgctgacaga cgctttgtct agttggctgt ttgacttttg
3480cttaagcagc cagggcagta gagtctaaca gatgctaatt tcaggatcag
gaagactgta 3540gaaaaatgag catcaagaag cccctggtac ccaaagctgc
tcttgccaat gagtgaacct 3600ctgccttccc gcttccaggt cctgtcttga
agaagaggtt ctggatcccg tgaatccgtt 3660cgtgcagctg ctgagcggag
tcgtgtggct catccgcaat ggaaacatct acatcaacga 3720gagccaagcc
atcgagtgtg acgagacaca ggagacaggt caggaagcac aggtgttctg
3780ttttatttgt attaggtttt gatttgttta ttttgtgcat gcagcgggtg
catgcatgct 3840cctttccttt cgccatgtga gtcctgagta ttgaactcag
actgttaagt gtgatgggag 3900gcactttacc cactgagcca ctttcccagc
cctcagcatc agctttcttc agacccagga 3960acagtgtgag tgggttattc
tttagtgttc ccaaacattt actgagcagc tatttactgt 4020ttagcactat
ggtgagagtc ctagggattc agtcttatgt agaatataga aggagaatcc
4080ttggcaataa gctggaaaat tgtgacaagt gccaagaaag aaacaggaga
aaggggaccg 4140gtggggacca gaagcacagg tatgaggaaa gtgcctgcag
atttgctgta tggtggcctc 4200cacatggcct aggagtttgt cataaatgca
gagccatgag tccaccctcc ctatacctcc 4260catccagaaa ccactggtta
aatcctaaca acttgggtgt gcaggcactc ccttggtgac 4320tctgatggac
actcaaggtc aagggccact tggggatggg ctgatgagtt ggcttggtca
4380gtaaagtatt tgccttgaaa gtgtgaggac ctgagttgga gccccagaaa
gaaacattaa 4440aagccaagtg ctgggatgca cacttgcatt cccagggatg
gagctggaag gcagggatag 4500gcagatccac ggccacacgg tgatattcta
agctaacaag agacctgtct cacacagaaa 4560gtgggtggca cctgaggacc
aacacccagg gttatcctct gacgtacctc cagagtggaa 4620aatactgggg
tggtggaaaa ggacactttg gtcctgggaa tctggctatt cagggtatag
4680tgtagaggga gagggagact caagaggctg tctttgagtc aaaggaacaa
gctatcagaa 4740gaactcaggg cagaggcctg tggttcccag gctcagggca
gccttcaagg ccctaggcag 4800agagtagctg ctgggtgaac aagtacagaa
gtgaggcctg gggcctcagg caaggcctgt 4860gaaatccttc caccaacata
gaagtttctg gagactgaga tcacatgaag tgcttctggc 4920tgtggcatgg
aagctcactg gaggtggagc tgggatgtgg ctcagtgatc cagtgcttgc
4980cacacgtgca cgagggaagg agccatcaaa agagagaaag tcgggagacc
tgaggggtcc 5040cctggagagc tgggtaacca ccccgggccc ttctccttta
ggttctttta gcaaatttgt 5100gaatgtgatg tcagccagga cagccgtggg
tcatgaccgt gaggggcagc ttatcctctt 5160ccatgctgat ggacagacgg
aacagcgtgg tgagtcccag gaaccttggg gctgtttgca 5220cttcagccac
cctacctttc cagtcggttc tggggtattg gtgggacaag acagctttcc
5280ggccattttg gaagtttcat ctggaggcaa tagcatttac ctactagtga
aagaagccag 5340ttaagccaga gaccacaggg gctcaagctg cataccccct
ctgcacagcc ttaacctatg 5400ggagatggca gagttcctgc gtcaacaaga
tgtcgtcaat gccatcaacc tggatggagg 5460cggttctgct acttttgtgc
tcaatgggac cctggccagt tacccttcag atcactggta 5520agaacccttg
agccaccttt gtggctctct cagactgtct cactcagtca atactgagac
5580cctgttgtgt gccaggccct gggtatccaa aagtgagcag aagagccgag
atctcttccc 5640tcagggtgct gcacagccca tccctggaaa cctgagacag
gtcaggaaag gcctccctga 5700ggacagtgaa gtaagacctg aggagatggc
tggccggggt tgagagagcc tttaccggaa 5760gacaaactgt acgcaatggg
gaaatccgct aagtggccca gggagaggct ggagctatag 5820ctcaggagga
aaagtacttg cctcgcaagc gaaggacctg agtttaaact ccaaaaccca
5880tataaaaagc cagatacgag caagtggcac atgcttgcag tcccagcctt
gttgaggaag 5940agtcaggtga atcctgaccc tctggccagc cagcctagcc
tactttttgg caaggtccag 6000gccagcgaga aagataaata aaataaagtt
ttaaatgaca tgtatctaag gttgtcctga 6060ctccatatgc gcacgcacgc
atgcacgcac gcacaactgg cagaatggaa agggaggcaa 6120actggacagc
ctttataggc tgcggcaggg accagcacca aggcctagac ctcgtctcac
6180agtgaatccc ccacagccag gacaacatgt ggcgctgtcc ccgccaagtg
tccactgtgg 6240tgtgtgtgca tgaaccgcgc tgccagccac ccgactgcag
tggccatggg acctgtgtgg 6300atggccactg tgaatgcacc agccacttct
ggcggggcga ggcctgcagc gagctggact 6360gtggcccctc caactgcagc
cagcatgggc tgtgcacaga gagtgagtgg ggagcccaca 6420ggagggtggt
gctctggcgg gaccccagct cgcccatgct agactcccgc ctgtgtcctt
6480acccagcctc tgtggtcttg ctttggtagc tggctgccac tgtgatgctg
ggtggacagg 6540atccaactgc agtgaaggtg agagctgcct gcaaacactc
ctggagaggg tggcctggct 6600gcacgcagct ggtatgacgc cttcgtccct
ccttctggct tggaacttac cttcagagcc 6660ttttctcatt tcgcatgtgg
atacccgatg ttctacctac tgaaagagcc cacaagtagg 6720aagccagatt
ttcagtattg tcactcaact ctaaggacca atagcaaaaa aacaaagtgg
6780ccacgcccct gagggagatc caccaaagtc cttaactcct ggaaagcagc
tcctggtgat 6840cctaggcatg ggtagggtgg tttcagcatc agctcagtgg
agttcccatt cataatttct 6900tcatcctttt aaggtcataa gttctagagc
ccaccttaaa tctaggcagt attcttggtg 6960tttatctgag acaaagtctt
atacagccca cgcagttctc taacttagta tgtaaccgag 7020aatggcctca
agcaacctgc ttcctccttt caagcgctgg gattataggc atagcaccaa
7080cttatagggt gctagaagtc aaacccaggg ccctatgtat atgcagcaag
cactctagaa 7140actggaacac agccctgttt gcagcccggt taccttggag
ggttgggtcc cagggatctg 7200agggcatctc cttcagcatg gccatgtgca
cacccaggag ccaggctgtc tgtgacagga 7260gaccatgcca cccaaggtga
gacctccctg ccaccatctc ctctccacag agtgtcctct 7320gggctggtat
gggccaggtt gccagaggcc ctgccagtgt gagcaccagt gtttctgtga
7380cccgcagact ggcaactgca gcatctccca aggtatgcgg ccttaaaggt
tcttgagctg 7440ggagcccttg gggcaggtct ggggtaggtg gactctcccc
agcccttctt tctggtgtct 7500tgcagtgagg cagtgtctcc agccaactga
ggctacgccg agggcaggag agctggcctc 7560tttcaccagg taagtgtttt
agcaggcact gagcccctat gtctcatccg tgaggcacta 7620gccaggccag
gaggtcacag gttaccctct actttgcaag ctcagggaca gtcacaggta
7680aaactggcat ccaggaaaga ccctgagcta cccagtggaa ctcaaaggta
gcaggctatg 7740ggtgtcatgc ctctggctgc agagactcca cttagatgct
ggagcagggc catagagaca 7800ggaaggactc accttatttc tgaactcttc
cgtgtgttca ggctttgtgt tgttgttgct 7860tcctttctgc tgtttcctgg
gtttccagct ccatccccac agggctcatg gaaagaattg 7920tgaagcaggg
ggtgtggctc aattggcaga ttgattgcct ggcatgcaga aagccctagg
7980ttcaatcccc agcatttcat atcataaccc aggcatggtg gcatcatgtg
cctgtaagtc 8040cagcacttgg gaggtagaag cagaaaagcc acgagtttaa
gaatgttagg gagtcttagg 8100ccaacctggg atacctaaga caagagatag
atgtagggag atagattgac agacagacag 8160acagacagac agacagacag
atcttgagct ggaccttctg gcacaagcct gtcatcctag 8220ctattccagg
aagctgaagc aggaagatag caaattcaag gccagcttaa gccacagatt
8280gagttcaaga tcaacctgag caactttatg aaatcctatt ataacataaa
aagtaggggt 8340gggaggttag gctgtagctc agtggtagag tgattgccta
gcacgcacaa gacccaggtt 8400caattcccag tactgcaaaa aatatattag
gaacccccta aaagcagtaa cattcacatt 8460agatgtgtgt gtgtgtgtgt
gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgttttg 8520ttgggtattt
atttcattta catttccaat gctatcccaa aagtccccca catcctcccc
8580cacccaccac cttgtttttt tttttttttt tttttttttt tttgacctga
aactcacagg 8640ttaggttaga caagctgact ggtgagctcc aacttccaac
gtaccatcat gcctggcttt 8700tgttttggtg tctctgtgta accctggatg
tcctggagct ctctctgtag accagcctgg 8760ccttaaactc acagaaaccc
acctgtttct gcctcccatg tgctgggatt aaaggcgtgt 8820gccacctcac
ccagccctgc tggacttaaa ttgggtcttc attttataag acaagcatga
8880gctaattccc cagttcctaa aatgttttta acatccttaa acatcagaga
ctgtctgtgg 8940tattccctcc atgtgtcttc agtataccta ctcccctccc
tgcctactgg gttcaacatg 9000cccagtttgg gttctggctg cctgccccca
ctcaagactc tcttttccat ctcaggacca 9060cctggctagc cctcaccctg
acactaattt tcctgctgct gatcagcact ggggtcaacg 9120tgtccttgtt
cctgggctcc agggccgaga ggaaccggca cctcgacggg gactatgtgt
9180atcacccact gcaggaggtg aacggggaag cgctgactgc agagaaggag
cacatggagg 9240aaactagcaa ccccttcaag gactgaagag ctgccccaac
ggcatgctcc agataatctt 9300gtccctgctc ctcacttcca caggggacat
tgtgaggcca ctggcatgga tgctatgcac 9360cccacccttt gctggccata
ttcctcctgt ccccatgctg tggctcatgc caacctagca 9420ataaggagct
ctggagagcc tgcacctgcc tcccgctcgc ctatatctgc tgcccagagg
9480cctgtctcgc acaggggtct cgccactgcc aaagactccc aggaagtcaa
agactcccag 9540taatccacta gcaaatggaa ctctgtaacg ccatcataac
aagagtggcc actctccgcg 9600tgcacaggta tgaaatataa atccttacac
acacacacac acacaccctc ggctcagcca 9660cggcactcgc cttttataca
gcgtcatcgc tggacagcca actagaactc tgcatcctgt 9720cacaggaagc
acctcataag aaggaatggg gagggaaggc agtcgccttg ttttcagacc
9780ttagccgaat tc 979215908PRTMus musculus 15Met Leu Phe Lys Leu
Leu Gln Arg Gln Thr Tyr Thr Cys Leu Ser His1 5 10 15Arg Tyr Gly Leu
Tyr Val Cys Phe Val Gly Val Val Val Thr Ile Val 20 25 30Ser Ala Phe
Gln Phe Gly Glu Val Val Leu Glu Trp Ser Arg Asp Gln 35 40 45Tyr His
Val Leu Phe Asp Ser Tyr Arg Asp Asn Ile Ala Gly Lys Ser 50 55 60Phe
Gln Asn Arg Leu Cys Leu Pro Met Pro Ile Asp Val Val Tyr Thr65 70 75
80Trp Val Asn Gly Thr Asp Leu Glu Leu Leu Lys Glu Leu Gln Gln Val
85 90 95Arg Glu His Met Glu Glu Glu Gln Arg Ala Met Arg Glu Thr Leu
Gly 100 105 110Lys Asn Thr Thr Glu Pro Thr Lys Lys Ser Glu Lys Gln
Leu Glu Cys 115 120 125Leu Leu Thr His Cys Ile Lys Val Pro Met Leu
Val Leu Asp Pro Ala 130 135 140Leu Pro Ala Thr Ile Thr Leu Lys Asp
Leu Pro Thr Leu Tyr Pro Ser145 150 155 160Phe His Ala Ser Ser Asp
Met Phe Asn Val Ala Lys Pro Lys Asn Pro 165 170 175Ser Thr Asn Val
Pro Val Val Val Phe Asp Thr Thr Lys Asp Val Glu 180 185 190Asp Ala
His Ala Gly Pro Phe Lys Gly Gly Gln Gln Thr Asp Val Trp 195 200
205Arg Ala Tyr Leu Thr Thr Asp Lys Asp Ala Pro Gly Leu Val Leu Ile
210 215 220Gln Gly Leu Ala Phe Leu Ser Gly Phe Pro Pro Thr Phe Lys
Glu Thr225 230 235 240Ser Gln Leu Lys Thr Lys Leu Pro Arg Lys Ala
Phe Pro Leu Lys Ile 245 250 255Lys Leu Leu Arg Leu Tyr Ser Glu Ala
Ser Val Ala Leu Leu Lys Leu 260 265 270Asn Asn Pro Lys Gly Phe Gln
Glu Leu Asn Lys Gln Thr Lys Lys Asn 275 280 285Met Thr Ile Asp Gly
Lys Glu Leu Thr Ile Ser Pro Ala Tyr Leu Leu 290 295 300Trp Asp Leu
Ser Ala Ile Ser Gln Ser Lys Gln Asp Glu Asp Ala Ser305 310 315
320Ala Ser Arg Phe Glu Asp Asn Glu Glu Leu Arg Tyr Ser Leu Arg Ser
325 330 335Ile Glu Arg His Ala Pro Trp Val Arg Asn Ile Phe Ile Val
Thr Asn 340 345 350Gly Gln Ile Pro Ser Trp Leu Asn Leu Asp Asn Pro
Arg Val Thr Ile 355 360 365Val Thr His Gln Asp Ile Phe Gln Asn Leu
Ser His Leu Pro Thr Phe 370 375 380Ser Ser Pro Ala Ile Glu Ser His
Ile His Arg Ile Glu Gly Leu Ser385 390 395 400Gln Lys Phe Ile Tyr
Leu Asn Asp Asp Val Met Phe Gly Lys Asp Val 405 410 415Trp Pro Asp
Asp Phe Tyr Ser His Ser Lys Gly Gln Lys Val Tyr Leu 420 425 430Thr
Trp Pro Val Pro Asn Cys Ala Glu Gly Cys Pro Gly Ser Trp Ile 435 440
445Lys Asp Gly Tyr Cys Asp Lys Ala Cys Asn Thr Ser Pro Cys Asp Trp
450 455 460Asp Gly Gly Asn Cys Ser Gly Asn Thr Ala Gly Asn Arg Phe
Val Ala465 470 475 480Arg Gly Gly Gly Thr Gly Asn Ile Gly Ala Gly
Gln His Trp Gln Phe 485 490 495Gly Gly Gly Ile Asn Thr Ile Ser Tyr
Cys Asn Gln
Gly Cys Ala Asn 500 505 510Ser Trp Leu Ala Asp Lys Phe Cys Asp Gln
Ala Cys Asn Val Leu Ser 515 520 525Cys Gly Phe Asp Ala Gly Asp Cys
Gly Gln Asp His Phe His Glu Leu 530 535 540Tyr Lys Val Thr Leu Leu
Pro Asn Gln Thr His Tyr Val Val Pro Lys545 550 555 560Gly Glu Tyr
Leu Ser Tyr Phe Ser Phe Ala Asn Ile Ala Arg Lys Arg 565 570 575Ile
Glu Gly Thr Tyr Ser Asp Asn Pro Ile Ile Arg His Ala Ser Ile 580 585
590Ala Asn Lys Trp Lys Thr Leu His Leu Ile Met Pro Gly Gly Met Asn
595 600 605Ala Thr Thr Ile Tyr Phe Asn Leu Thr Leu Gln Asn Ala Asn
Asp Glu 610 615 620Glu Phe Lys Ile Gln Ile Ala Val Glu Val Asp Thr
Arg Glu Ala Pro625 630 635 640Lys Leu Asn Ser Thr Thr Gln Lys Ala
Tyr Glu Ser Leu Val Ser Pro 645 650 655Val Thr Pro Leu Pro Gln Ala
Asp Val Pro Phe Glu Asp Val Pro Lys 660 665 670Glu Lys Arg Phe Pro
Lys Ile Arg Arg His Asp Val Asn Ala Thr Gly 675 680 685Arg Phe Gln
Glu Glu Val Lys Ile Pro Arg Val Asn Ile Ser Leu Leu 690 695 700Pro
Lys Glu Ala Gln Val Arg Leu Ser Asn Leu Asp Leu Gln Leu Glu705 710
715 720Arg Gly Asp Ile Thr Leu Lys Gly Tyr Asn Leu Ser Lys Ser Ala
Leu 725 730 735Leu Arg Ser Phe Leu Gly Asn Ser Leu Asp Thr Lys Ile
Lys Pro Gln 740 745 750Ala Arg Thr Asp Glu Thr Lys Gly Asn Leu Glu
Val Pro Gln Glu Asn 755 760 765Pro Ser His Arg Arg Pro His Gly Phe
Ala Gly Glu His Arg Ser Glu 770 775 780Arg Trp Thr Ala Pro Ala Glu
Thr Val Thr Val Lys Gly Arg Asp His785 790 795 800Ala Leu Asn Pro
Pro Pro Val Leu Glu Thr Asn Ala Arg Leu Ala Gln 805 810 815Pro Thr
Leu Gly Val Thr Val Ser Lys Glu Asn Leu Ser Pro Leu Ile 820 825
830Val Pro Pro Glu Ser His Leu Pro Lys Glu Glu Glu Ser Asp Arg Ala
835 840 845Glu Gly Asn Ala Val Pro Val Lys Glu Leu Val Pro Gly Arg
Arg Leu 850 855 860Gln Gln Asn Tyr Pro Gly Phe Leu Pro Trp Glu Lys
Lys Lys Tyr Phe865 870 875 880Gln Asp Leu Leu Asp Glu Glu Glu Ser
Leu Lys Thr Gln Leu Ala Tyr 885 890 895Phe Thr Asp Arg Lys His Thr
Gly Arg Gln Leu Lys 900 905165229DNAMus musculus 16ggcggtgaag
gggtgatgct gttcaagctc ctgcagagac agacctatac ctgcctatcc 60cacaggtatg
ggctctacgt ctgcttcgtg ggcgtcgttg tcaccatcgt ctcggctttc
120cagttcggag aggtggttct ggaatggagc cgagatcagt accatgtttt
gtttgattcc 180tacagagaca acattgctgg gaaatccttt cagaatcggc
tctgtctgcc catgccaatc 240gacgtggttt acacctgggt gaatggcact
gaccttgaac tgctaaagga gctacagcag 300gtccgagagc acatggagga
agagcagaga gccatgcggg aaaccctcgg gaagaacaca 360accgaaccga
caaagaagag tgagaagcag ctggaatgtc tgctgacgca ctgcattaag
420gtgcccatgc ttgttctgga cccggccctg ccagccacca tcaccctgaa
ggatctgcca 480accctttacc catctttcca cgcgtccagc gacatgttca
atgttgcgaa accaaaaaat 540ccgtctacaa atgtccccgt tgtcgttttt
gacactacta aggatgttga agacgcccat 600gctggaccgt ttaagggagg
ccagcaaaca gatgtttgga gagcctactt gacaacagac 660aaagacgccc
ctggcttagt gctgatacaa ggcttggcgt tcctgagtgg attcccaccg
720accttcaagg agacgagtca actgaagaca aagctgccaa gaaaagcttt
ccctctaaaa 780ataaagctgt tgcggctgta ctcggaggcc agtgtcgctc
ttctgaaatt gaataatccc 840aagggtttcc aagagctgaa caagcagacc
aagaagaaca tgaccatcga tgggaaggaa 900ctgaccatca gccctgcgta
tctgctgtgg gacctgagtg ccatcagcca gtccaagcag 960gatgaggacg
cgtctgccag ccgctttgag gataatgaag agctgaggta ctcgctgcga
1020tctatcgaga gacacgcgcc atgggtacgg aatattttca ttgtcaccaa
cgggcagatt 1080ccatcctggc tgaaccttga caaccctcga gtgaccatag
tgacccacca ggacattttc 1140caaaatctga gccacttgcc tactttcagt
tcccctgcta ttgaaagtca cattcaccgc 1200atcgaagggc tgtcccagaa
gtttatttat ctaaatgacg atgtcatgtt cggtaaggac 1260gtctggccgg
acgattttta cagccactcc aaaggtcaaa aggtttattt gacatggcct
1320gtgccaaact gtgcagaggg ctgcccgggc tcctggataa aggacggcta
ttgtgataag 1380gcctgtaata cctcaccctg tgactgggat ggcggaaact
gctctggtaa tactgcaggg 1440aaccggtttg ttgcaagagg tgggggtacc
gggaatattg gagctggaca gcactggcag 1500tttggtggag gaataaacac
catctcttac tgtaaccaag gatgtgcaaa ctcctggctg 1560gctgacaagt
tctgtgacca agcctgtaac gtcttatcct gcgggtttga tgctggtgac
1620tgtggacaag atcattttca tgaattgtat aaagtaacac ttctcccaaa
ccagactcac 1680tatgttgtcc ccaaaggtga atacctgtct tatttcagct
ttgcaaacat agccagaaaa 1740agaattgaag ggacctacag cgacaacccc
atcatccgcc acgcgtccat tgcaaacaag 1800tggaaaaccc tacacctgat
aatgcccggg gggatgaacg ccaccacgat ctattttaac 1860ctcactcttc
aaaacgccaa cgacgaagag ttcaagatcc agatagcagt agaggtggac
1920acgagggagg cgcccaaact gaattctaca acccagaagg cctatgaaag
tttggttagc 1980ccagtgacac ctcttcctca ggctgacgtc ccttttgaag
atgtccccaa agagaaacgc 2040ttccccaaga tcaggagaca tgatgtaaat
gcaacaggga gattccaaga ggaggtgaaa 2100atcccccggg taaatatttc
actccttccc aaagaggccc aggtgaggct gagcaacttg 2160gatttgcaac
tagaacgtgg agacatcact ctgaaaggat ataacttgtc caagtcagcc
2220ctgctaaggt ctttcctggg gaattcacta gatactaaaa taaaacctca
agctaggacc 2280gatgaaacaa aaggcaacct ggaggtccca caggaaaacc
cttctcacag acgtccacat 2340ggctttgctg gtgaacacag atcagagaga
tggactgccc cagcagagac agtgaccgtg 2400aaaggccgtg accacgcttt
gaatccaccc ccggtgttgg agaccaatgc aagattggcc 2460cagcctacac
taggcgtgac tgtgtccaaa gagaaccttt caccgctgat cgttccccca
2520gaaagccact tgccaaaaga agaggagagt gacagggcag aaggcaatgc
tgtacctgta 2580aaggagttag tgcctggcag acggttgcag cagaattatc
caggcttttt gccctgggag 2640aaaaaaaagt atttccaaga ccttcttgat
gaggaagagt cattgaagac ccagttggcg 2700tactttacag accgcaaaca
taccgggagg caactaaaag atacatttgc agactccctc 2760cgatacgtca
ataaaattct caacagcaag tttggattca catccaggaa agtccctgca
2820cacatgccgc acatgattga caggatcgtt atgcaagaac tccaagatat
gttccctgaa 2880gaatttgaca agacttcatt tcacaaggtg cgtcactctg
aggacatgca gtttgccttc 2940tcctactttt attacctcat gagtgcagtt
cagcccctca atatttccca agtctttcat 3000gaagtagaca cagaccaatc
tggtgtcttg tctgataggg aaatccgaac wctggccacg 3060agaattcacg
acctaccttt aagcttgcag gatttgacag gtttggaaca catgttaata
3120aattgctcaa aaatgctccc cgctaatatc actcaactca acaacatccc
accgactcag 3180gaagcatact acgaccccaa cctgcctccg gtcactaaga
gtcttgtcac caactgtaag 3240ccagtaactg acaagatcca caaagcctat
aaagacaaga acaaatacag gtttgaaatc 3300atgggagagg aagaaatcgc
tttcaagatg atacgaacca atgtttctca tgtggttggt 3360cagttggatg
acatcagaaa aaaccccagg aagttcgttt gtctgaatga caacattgac
3420cacaaccata aagatgcccg gacagtgaag gctgtcctca gggacttcta
tgagtccatg 3480tttcccatac cttcccagtt tgagctgcca agagagtatc
ggaaccgctt tctgcacatg 3540catgagctcc aagaatggcg ggcatatcga
gacaagctga agttttggac ccactgcgta 3600ctagcaacgt tgattatatt
tactatattc tcattttttg ctgaacagat aattgctctg 3660aagcgaaaga
tatttcccag gaggaggata cacaaagaag ctagtccaga ccgaatcagg
3720gtgtagaaga tcttcatttg aaagtcacct accttagcat ctgtgaacat
ctccctcctc 3780gacaccacag cggagtccct gtgatgtggc acagaggcag
cctcgtgggg agaagggaca 3840tcgtgcagac cgggttcttc tgcaatggga
agagagccca ctgacctgga attattcagc 3900acactaagaa cctgtgtcaa
tagcttgtac agcttgtact tttaaaggat ttgccgaagg 3960acctgtcggc
ttgttgacaa accctccctg acaagctgct ggtttcttcc cccagttact
4020gcagactgag aaaccagtcc atcttgaaag caagtgcgga ggggccccag
tctttgcatt 4080ccaaagcttt ccagcataat ttctggcttg tctcctcctt
tgatccattt cccatttttt 4140tttaaaaaac aataagtggc tactaagtta
gtcattctca cttctcaaaa taacaaatca 4200ggatgtcaaa acatttgtat
agatcttatt taaataatat agaacgatta cttctttagc 4260ctatctaaat
tattgatttt tattaacagt caagtggtct tgaaccgcta acaactactg
4320aagagctcga gattgacgtt gaaagtgctt tgagcttgtt taactcattc
cccaagaata 4380ctgtgacctc gtgtgcgggc ctgattgcga agggctagtg
tcacgtagca gtgctgctca 4440ccggatgtaa ttatgtcgtg gaaatgtaca
tacagacaaa agtgcctcac ttcagaaatg 4500agtagtgctg atggcaccag
cgagtgatgg tgtccatttg gaaacccatg ataccttcca 4560atgcccaccc
tgcttacttt atacagagca ggggttaacc aacttctgtc aaagaacagt
4620aaagaacttg agatacatcc atctttgtca aatagttttc cttgctaaca
tttattattg 4680ttggtgtttt gggaggttta ttttatttta ttgctttgtt
atttttcaag acggggattc 4740tctgtgtagc tctggctgtt tggtaattca
ctctaaagac caggctggcc ttgaacttag 4800agattcacct gcttctgctt
cctgaatggt aggacatgtg cccacattgc ctacccaccc 4860cccttttggg
gggggtgagc aactcaataa aaagatgaaa acctgcttta gtttgcagct
4920atacaaaagc agcaggcctc agccagactt gacccccggg gccattgttg
gcccacggga 4980gaatcatttt tgacgtgggt aagcaaaccc tgatattggt
catgctgtgt tatgtcatta 5040tgtggtggtt ttgaattttg gaagatattt
tcagtcatga tttcagtagt attcctccaa 5100aatggcacac atttttgtaa
taagaacttg aaatgtaaat attgtgtttg tgctgtaaat 5160tttgtgtatt
tcaaaaactg aagtttcata aaaaaacaca cttattggaa aaaaaaaaaa
5220aaaaaaaaa 5229171105DNADrosophila
melanogastermisc_feature(903)..(903)n is a, g, c, or t 17ctgcaggaat
tcggcacgag gcggttcgat gacaagaatg agctgcggta ctctctgagg 60tccctggaaa
aacacgccgc atggatcagg catgtgtaca tagtaaccaa tggccagatt
120ccaagttggc tggatctcag ctacgaaagg gtcacggtgg tgccccacga
agtcctggct 180cccgatcccg accagctgcc caccttctcc agctcggcca
tcgagacatt tctgcaccgc 240ataccaaagc tgtccaagag gttcctctac
ctcaacgacg acatattcct gggagctccg 300ctgtatccgg aggacttgta
cactgaagcg gagggagttc gcgtgtacca ggcatggatg 360gtgcccggct
gcgccttgga ttgcccctgg acgtacatag gtgatggagc ttgcgatcgg
420cactgcaaca ttgatgcgtg ccaatttgat ggaggcgact gcagtgaaac
tgggccagcg 480agcgatgccc acgtcattcc accaagcaaa gaagtgctcg
aggtgcagcc tgccgctgtt 540ccacaatcaa gagtccaccg atttcctcag
atgggtctcc aaaagctgtt caggcgcagc 600tctgccaatt ttaaggatgt
tatgcggcac cgcaatgtgt ccacactcaa ggaactacgt 660cgcattgtgg
agcgttttaa caaggccaaa ctcatgtcgc tgaaccccga actggagacc
720tccagctccg agccacagac aactcagcgc cacgggctgc gcaaggagga
ttttaagtct 780tccaccgata tttactctca ctcgctgatt gccaccaata
tgttgctgaa tagagcctat 840ggctttaagg cacgccatgt cctggcgcac
gtgggcttcc taattgacaa ggatattgtg 900gangccatgc aacgacgttt
taccagcgaa ttctngacac tggccattaa cgctttccga 960gccccaacag
atttgcagta cgcattcgct tactacttct ttctaatgag cgaaatccaa
1020gtnatgagtg tagangaaat cttcgatgaa gtcgacaccg gacggtttgg
ncacctggtc 1080ggatccagaa gtgcgaaccn tttta 1105182005DNAMus
musculus 18gtttcccgcg acgatgacct gctgctgcct tacccactag cgcgcagacg
tccctcgcga 60gactgcgccc gggtgcgctc aggtagccca gagcaggaga gctggcctcc
gccacctctg 120gccacccacg aaccccgggc gccaagccac cacgcggccg
tgcgcacctt cgtgtcgcac 180ttcgaggggc gcgcggtggc cggccacctg
acgcgggtcg ccgatcccct acgcactttc 240tcggtgctgg agcccggagg
agccgggggc tgcggcggca gaagcgccgc ggctactgtg 300gaggacacag
ccgtccgggc cggttgccgc atcgctcaga acggtggctt cttccgcatg
360agcactggcg agtgcttggg gaacgtggtg agcgacgggc ggctggtgag
cagctcaggg 420ggactgcaga acgcgcagtt cggtatccga cgcgatggaa
ccatagtcac cgggtcctgt 480cttgaagaag aggttctgga tcccgtgaat
ccgttcgtgc agctgctgag cggagtcgtg 540tggctcatcc gcaatggaaa
catctacatc aacgagagcc aagccatcga gtgtgacgag 600acacaggaga
caggttcttt tagcaaattt gtgaatgtga tgtcagccag gacagccgtg
660ggtcatgacc gtgaggggca gcttatcctc ttccatgctg atggacagac
ggaacagcgt 720ggccttaacc tatgggagat ggcagagttc ctgcgtcaac
aagatgtcgt caatgccatc 780aacctggatg gaggcggttc tgctactttt
gtgctcaatg ggaccctggc cagttaccct 840tcagatcact gccaggacaa
catgtggcgc tgtccccgcc aagtgtccac tgtggtgtgt 900gtgcatgaac
cgcgctgcca gccacccgac tgcagtggcc atgggacctg tgtggatggc
960cactgtgaat gcaccagcca cttctggcgg ggcgaggcct gcagcgagct
ggactgtggc 1020ccctccaact gcagccagca tgggctgtgc acagctggct
gccactgtga tgctgggtgg 1080acaggatcca actgcagtga agagtgtcct
ctgggctggt atgggccagg ttgccagagg 1140ccctgccagt gtgagcacca
gtgtttctgt gacccgcaga ctggcaactg cagcatctcc 1200caagtgaggc
agtgtctcca gccaactgag gctacgccga gggcaggaga gctggcctct
1260ttcaccagga ccacctggct agccctcacc ctgacactaa ttttcctgct
gctgatcagc 1320actggggtca acgtgtcctt gttcctgggc tccagggccg
agaggaaccg gcacctcgac 1380ggggactatg tgtatcaccc actgcaggag
gtgaacgggg aagcgctgac tgcagagaag 1440gagcacatgg aggaaactag
caaccccttc aaggactgaa gagctgcccc aacggcatgc 1500tccagataat
cttgtccctg ctcctcactt ccacagggga cattgtgagg ccactggcat
1560ggatgctatg caccccaccc tttgctggcc atattcctcc tgtccccatg
ctgtggctca 1620tgccaaccta gcaataagga gctctggaga gcctgcacct
gcctcccgct cgcctatatc 1680tgctgcccag aggcctgtct cgcacagggg
tctcgccact gccaaagact cccaggaagt 1740caaagactcc cagtaatcca
ctagcaaatg gaactctgta acgccatcat aacaagagtg 1800gccactctcc
gcgtgcacag gtatgaaata taaatcctta cacacacaca cacacacacc
1860ctcggctcag ccacggcact cgccttttat acagcgtcat cgctggacag
ccaactagaa 1920ctctgcatcc tgtcacagga agcacctcat aagaaggaat
ggggagggaa ggcagtcgcc 1980ttgttttcag accttagccg aattc
200519492PRTMus musculus 19Val Ser Arg Asp Asp Asp Leu Leu Leu Pro
Tyr Pro Leu Ala Arg Arg1 5 10 15Arg Pro Ser Arg Asp Cys Ala Arg Val
Arg Ser Gly Ser Pro Glu Gln 20 25 30Glu Ser Trp Pro Pro Pro Pro Leu
Ala Thr His Glu Pro Arg Ala Pro 35 40 45Ser His His Ala Ala Val Arg
Thr Phe Val Ser His Phe Glu Gly Arg 50 55 60Ala Val Ala Gly His Leu
Thr Arg Val Ala Asp Pro Leu Arg Thr Phe65 70 75 80Ser Val Leu Glu
Pro Gly Gly Ala Gly Gly Cys Gly Gly Arg Ser Ala 85 90 95Ala Ala Thr
Val Glu Asp Thr Ala Val Arg Ala Gly Cys Arg Ile Ala 100 105 110Gln
Asn Gly Gly Phe Phe Arg Met Ser Thr Gly Glu Cys Leu Gly Asn 115 120
125Val Val Ser Asp Gly Arg Leu Val Ser Ser Ser Gly Gly Leu Gln Asn
130 135 140Ala Gln Phe Gly Ile Arg Arg Asp Gly Thr Ile Val Thr Gly
Ser Cys145 150 155 160Leu Glu Glu Glu Val Leu Asp Pro Val Asn Pro
Phe Val Gln Leu Leu 165 170 175Ser Gly Val Val Trp Leu Ile Arg Asn
Gly Asn Ile Tyr Ile Asn Glu 180 185 190Ser Gln Ala Ile Glu Cys Asp
Glu Thr Gln Glu Thr Gly Ser Phe Ser 195 200 205Lys Phe Val Asn Val
Met Ser Ala Arg Thr Ala Val Gly His Asp Arg 210 215 220Glu Gly Gln
Leu Ile Leu Phe His Ala Asp Gly Gln Thr Glu Gln Arg225 230 235
240Gly Leu Asn Leu Trp Glu Met Ala Glu Phe Leu Arg Gln Gln Asp Val
245 250 255Val Asn Ala Ile Asn Leu Asp Gly Gly Gly Ser Ala Thr Phe
Val Leu 260 265 270Asn Gly Thr Leu Ala Ser Tyr Pro Ser Asp His Cys
Gln Asp Asn Met 275 280 285Trp Arg Cys Pro Arg Gln Val Ser Thr Val
Val Cys Val His Glu Pro 290 295 300Arg Cys Gln Pro Pro Asp Cys Ser
Gly His Gly Thr Cys Val Asp Gly305 310 315 320His Cys Glu Cys Thr
Ser His Phe Trp Arg Gly Glu Ala Cys Ser Glu 325 330 335Leu Asp Cys
Gly Pro Ser Asn Cys Ser Gln His Gly Leu Cys Thr Ala 340 345 350Gly
Cys His Cys Asp Ala Gly Trp Thr Gly Ser Asn Cys Ser Glu Glu 355 360
365Cys Pro Leu Gly Trp Tyr Gly Pro Gly Cys Gln Arg Pro Cys Gln Cys
370 375 380Glu His Gln Cys Phe Cys Asp Pro Gln Thr Gly Asn Cys Ser
Ile Ser385 390 395 400Gln Val Arg Gln Cys Leu Gln Pro Thr Glu Ala
Thr Pro Arg Ala Gly 405 410 415Glu Leu Ala Ser Phe Thr Arg Thr Thr
Trp Leu Ala Leu Thr Leu Thr 420 425 430Leu Ile Phe Leu Leu Leu Ile
Ser Thr Gly Val Asn Val Ser Leu Phe 435 440 445Leu Gly Ser Arg Ala
Glu Arg Asn Arg His Leu Asp Gly Asp Tyr Val 450 455 460Tyr His Pro
Leu Gln Glu Val Asn Gly Glu Ala Leu Thr Ala Glu Lys465 470 475
480Glu His Met Glu Glu Thr Ser Asn Pro Phe Lys Asp 485
490203783DNAHomo sapiens 20gccaccatgg ggttcaagct cttgcagaga
caaacctata cctgcctgtc ccacaggtat 60gggctctacg tgtgcttctt gggcgtcgtt
gtcaccatcg tctccgcctt ccagttcgga 120gaggtggttc tggaatggag
ccgagatcaa taccatgttt tgtttgattc ctatagagac 180aatattgctg
gaaagtcctt tcagaatcgg ctttgtctgc ccatgccgat tgacgttgtt
240tacacctggg tgaatggcac agatcttgaa ctactgaagg aactacagca
ggtcagagaa 300cagatggagg aggagcagaa agcaatgaga gaaatccttg
ggaaaaacac aacggaacct 360actaagaaga gtgagaagca gttagagtgt
ttgctaacac actgcattaa ggtgccaatg 420cttgtcctgg acccagccct
gccagccaac atcaccctga aggacctgcc atctctttat 480ccttcttttc
attctgccag tgacattttc aatgttgcaa aaccaaaaaa cccttctacc
540aatgtctcag ttgttgtttt tgacagtact aaggatgttg aagatgccca
ctctggactg 600cttaaaggaa atagcagaca gacagtatgg aggggctact
tgacaacaga taaagaagtc 660cctggattag tgctaatgca agatttggct
ttcctgagtg gatttccacc aacattcaag 720gaaacaaatc aactaaaaac
aaaattgcca gaaaatcttt cctctaaagt caaactgttg 780cagttgtatt
cagaggccag tgtagcgctt ctaaaactga ataaccccaa ggattttcaa
840gaattgaata agcaaactaa gaagaacatg accattgatg gaaaagaact
gaccataagt 900cctgcatatt tattatggga tctgagcgcc atcagccagt
ctaagcagga tgaagacatc 960tctgccagtc gttttgaaga
taacgaagaa ctgaggtact cattgcgatc tatcgagagg 1020catgcaccat
gggttcggaa tattttcatt gtcaccaacg ggcagattcc atcctggctg
1080aaccttgaca atcctcgagt gacaatagta acacaccagg atgtttttcg
aaatttgagc 1140cacttgccta cctttagttc acctgctatt gaaagtcacg
ttcatcgcat cgaagggctg 1200tcccagaagt ttatttacct aaatgatgat
gtcatgtttg ggaaggatgt ctggccagat 1260gatttttaca gtcactccaa
aggccagaag gtttatttga catggcctgt gccaaactgt 1320gccgagggct
gcccaggttc ctggattaag gatggctatt gtgacaaggc ttgtaataat
1380tcagcctgcg attgggatgg tggggattgc tctggaaaca gtggagggag
tcgctatatt 1440gcaggaggtg gaggtactgg gagtattgga gttggacagc
cctggcagtt tggtggagga 1500ataaacagtg tctcttactg taatcaggga
tgtgcgaatt cctggctcgc tgataagttc 1560tgtgaccaag catgcaatgt
cttgtcctgt gggtttgatg ctggcgactg tgggcaagat 1620cattttcatg
aattgtataa agtgatcctt ctcccaaacc agactcacta tattattcca
1680aaaggtgaat gcctgcctta tttcagcttt gcagaagtag ccaaaagagg
agttgaaggt 1740gcctatagtg acaatccaat aattcgacat gcttctattg
ccaacaagtg gaaaaccatc 1800cacctcataa tgcacagtgg aatgaatgcc
accacaatac attttaatct cacgtttcaa 1860aatacaaacg atgaagagtt
caaaatgcag ataacagtgg aggtggacac aagggaggga 1920ccaaaactga
attctacggc ccagaagggt tacgaaaatt tagttagtcc cataacactt
1980cttccagagg cggaaatcct ttttgaggat attcccaaag aaaaacgctt
cccgaagttt 2040aagagacatg atgttaactc aacaaggaga gcccaggaag
aggtgaaaat tcccctggta 2100aatatttcac tccttccaaa agacgcccag
ttgagtctca ataccttgga tttgcaactg 2160gaacatggag acatcacttt
gaaaggatac aatttgtcca agtcagcctt gctgagatca 2220tttctgatga
actcacagca tgctaaaata aaaaatcaag ctataataac agatgaaaca
2280aatgacagtt tggtggctcc acaggaaaaa caggttcata aaagcatctt
gccaaacagc 2340ttaggagtgt ctgaaagatt gcagaggttg acttttcctg
cagtgagtgt aaaagtgaat 2400ggtcatgacc agggtcagaa tccacccctg
gacttggaga ccacagcaag atttagagtg 2460gaaactcaca cccaaaaaac
cataggcgga aatgtgacaa aagaaaagcc cccatctctg 2520attgttccac
tggaaagcca gatgacaaaa gaaaagaaaa tcacagggaa agaaaaagag
2580aacagtagaa tggaggaaaa tgctgaaaat cacataggcg ttactgaagt
gttacttgga 2640agaaagctgc agcattacac agatagttac ttgggctttt
tgccatggga gaaaaaaaag 2700tatttcctag atcttctcga cgaagaagag
tcattgaaga cacaattggc atacttcact 2760gatagcaaga atactgggag
gcaactaaaa gatacatttg cagattccct cagatatgta 2820aataaaattc
taaatagcaa gtttggattc acatcgcgga aagtccctgc tcacatgcct
2880cacatgattg accggattgt tatgcaagaa ctgcaagata tgttccctga
agaatttgac 2940aagacgtcat ttcacaaagt gcgccattct gaggatatgc
agtttgcctt ctcttatttt 3000tattatctca tgagtgcagt gcagccactg
aatatatctc aagtctttga tgaagttgat 3060acagatcaat ctggtgtctt
gtctgacaga gaaatccgaa cactggctac cagaattcac 3120gaactgccgt
taagtttgca ggatttgaca ggtctggaac acatgctaat aaattgctca
3180aaaatgcttc ctgctgatat cacgcagcta aataatattc caccaactca
ggaatcctac 3240tatgatccca acctgccacc ggtcactaaa agtctagtaa
caaactgtaa accagtaact 3300gacaaaatcc acaaagcata taaggacaaa
aacaaatata ggtttgaaat catgggagaa 3360gaagaaatcg cttttaaaat
gattcgtacc aacgtttctc atgtggttgg ccagttggat 3420gacataagaa
aaaaccctag gaagtttgtt tgcctgaatg acaacattga ccacaatcat
3480aaagatgctc agacagtgaa ggctgttctc agggacttct atgaatccat
gttccccata 3540ccttcccaat ttgaactgcc aagagagtat cgaaaccgtt
tccttcatat gcatgagctg 3600caggaatgga gggcttatcg agacaaattg
aagttttgga cccattgtgt actagcaaca 3660ttgattatgt ttactatatt
ctcatttttt gctgagcagt taattgcact taagcggaag 3720atatttccca
gaaggaggat acacaaagaa gctagtccca atcgaatcag agtatagaag 3780atc
3783213621DNAHomo sapiens 21ctagccgcca ccatggagac agacacactc
ctgctatggg tactgctgct ctgggttcca 60ggttccactg gtgacgaaga tcaggtagat
ccgcggttaa tcgacggtaa gcttagccga 120gatcaatacc atgttttgtt
tgattcctat agagacaata ttgctggaaa gtcctttcag 180aatcggcttt
gtctgcccat gccgattgac gttgtttaca cctgggtgaa tggcacagat
240cttgaactac tgaaggaact acagcaggtc agagaacaga tggaggagga
gcagaaagca 300atgagagaaa tccttgggaa aaacacaacg gaacctacta
agaagagtga gaagcagtta 360gagtgtttgc taacacactg cattaaggtg
ccaatgcttg tcctggaccc agccctgcca 420gccaacatca ccctgaagga
cctgccatct ctttatcctt cttttcattc tgccagtgac 480attttcaatg
ttgcaaaacc aaaaaaccct tctaccaatg tctcagttgt tgtttttgac
540agtactaagg atgttgaaga tgcccactct ggactgctta aaggaaatag
cagacagaca 600gtatggaggg gctacttgac aacagataaa gaagtccctg
gattagtgct aatgcaagat 660ttggctttcc tgagtggatt tccaccaaca
ttcaaggaaa caaatcaact aaaaacaaaa 720ttgccagaaa atctttcctc
taaagtcaaa ctgttgcagt tgtattcaga ggccagtgta 780gcgcttctaa
aactgaataa ccccaaggat tttcaagaat tgaataagca aactaagaag
840aacatgacca ttgatggaaa agaactgacc ataagtcctg catatttatt
atgggatctg 900agcgccatca gccagtctaa gcaggatgaa gacatctctg
ccagtcgttt tgaagataac 960gaagaactga ggtactcatt gcgatctatc
gagaggcatg caccatgggt tcggaatatt 1020ttcattgtca ccaacgggca
gattccatcc tggctgaacc ttgacaatcc tcgagtgaca 1080atagtaacac
accaggatgt ttttcgaaat ttgagccact tgcctacctt tagttcacct
1140gctattgaaa gtcacgttca tcgcatcgaa gggctgtccc agaagtttat
ttacctaaat 1200gatgatgtca tgtttgggaa ggatgtctgg ccagatgatt
tttacagtca ctccaaaggc 1260cagaaggttt atttgacatg gcctgtgcca
aactgtgccg agggctgccc aggttcctgg 1320attaaggatg gctattgtga
caaggcttgt aataattcag cctgcgattg ggatggtggg 1380gattgctctg
gaaacagtgg agggagtcgc tatattgcag gaggtggagg tactgggagt
1440attggagttg gacagccctg gcagtttggt ggaggaataa acagtgtctc
ttactgtaat 1500cagggatgtg cgaattcctg gctcgctgat aagttctgtg
accaagcatg caatgtcttg 1560tcctgtgggt ttgatgctgg cgactgtggg
caagatcatt ttcatgaatt gtataaagtg 1620atccttctcc caaaccagac
tcactatatt attccaaaag gtgaatgcct gccttatttc 1680agctttgcag
aagtagccaa aagaggagtt gaaggtgcct atagtgacaa tccaataatt
1740cgacatgctt ctattgccaa caagtggaaa accatccacc tcataatgca
cagtggaatg 1800aatgccacca caatacattt taatctcacg tttcaaaata
caaacgatga agagttcaaa 1860atgcagataa cagtggaggt ggacacaagg
gagggaccaa aactgaattc tacggcccag 1920aagggttacg aaaatttagt
tagtcccata acacttcttc cagaggcgga aatccttttt 1980gaggatattc
ccaaagaaaa acgcttcccg aagtttaaga gacatgatgt taactcaaca
2040aggagagccc aggaagaggt gaaaattccc ctggtaaata tttcactcct
tccaaaagac 2100gcccagttga gtctcaatac cttggatttg caactggaac
atggagacat cactttgaaa 2160ggatacaatt tgtccaagtc agccttgctg
agatcatttc tgatgaactc acagcatgct 2220aaaataaaaa atcaagctat
aataacagat gaaacaaatg acagtttggt ggctccacag 2280gaaaaacagg
ttcataaaag catcttgcca aacagcttag gagtgtctga aagattgcag
2340aggttgactt ttcctgcagt gagtgtaaaa gtgaatggtc atgaccaggg
tcagaatcca 2400cccctggact tggagaccac agcaagattt agagtggaaa
ctcacaccca aaaaaccata 2460ggcggaaatg tgacaaaaga aaagccccca
tctctgattg ttccactgga aagccagatg 2520acaaaagaaa agaaaatcac
agggaaagaa aaagagaaca gtagaatgga ggaaaatgct 2580gaaaatcaca
taggcgttac tgaagtgtta cttggaagaa agctgcagca ttacacagat
2640agttacttgg gctttttgcc atgggagaaa aaaaagtatt tcctagatct
tctcgacgaa 2700gaagagtcat tgaagacaca attggcatac ttcactgata
gcaagaatac tgggaggcaa 2760ctaaaagata catttgcaga ttccctcaga
tatgtaaata aaattctaaa tagcaagttt 2820ggattcacat cgcggaaagt
ccctgctcac atgcctcaca tgattgaccg gattgttatg 2880caagaactgc
aagatatgtt ccctgaagaa tttgacaaga cgtcatttca caaagtgcgc
2940cattctgagg atatgcagtt tgccttctct tatttttatt atctcatgag
tgcagtgcag 3000ccactgaata tatctcaagt ctttgatgaa gttgatacag
atcaatctgg tgtcttgtct 3060gacagagaaa tccgaacact ggctaccaga
attcacgaac tgccgttaag tttgcaggat 3120ttgacaggtc tggaacacat
gctaataaat tgctcaaaaa tgcttcctgc tgatatcacg 3180cagctaaata
atattccacc aactcaggaa tcctactatg atcccaacct gccaccggtc
3240actaaaagtc tagtaacaaa ctgtaaacca gtaactgaca aaatccacaa
agcatataag 3300gacaaaaaca aatataggtt tgaaatcatg ggagaagaag
aaatcgcttt taaaatgatt 3360cgtaccaacg tttctcatgt ggttggccag
ttggatgaca taagaaaaaa ccctaggaag 3420tttgtttgcc tgaatgacaa
cattgaccac aatcataaag atgctcagac agtgaaggct 3480gttctcaggg
acttctatga atccatgttc cccatacctt cccaatttga actgccaaga
3540gagtatcgaa accgtttcct tcatatgcat gagctgcagg aatggagggc
ttatcgagac 3600aaattgaagt agtagtctag a 3621221383DNAHomo sapiens
22atggcgacct ccacgggtcg ctggcttctc ctccggcttg cactattcgg cttcctctgg
60gaagcgtccg gcggcctcga ctcgggggcc tcccgcgacg acgacttgct actgccctat
120ccacgcgcgc gcgcgcgcct cccccgggac tgcacacggg tgcgcgccgg
caaccgcgag 180cacgagagtt ggcctccgcc tcccgcgact cccggcgccg
gcggtctggc cgtgcgcacc 240ttcgtgtcgc acttcaggga ccgcgcggtg
gccggccacc tgacgcgggc cgttgagccc 300ctgcgcacct tctcggtgct
ggagcccggt ggacccggcg gctgcgcggc gagacgacgc 360gccaccgtgg
aggagacggc gcgggcggcc gactgccgtg tcgcccagaa cggcggcttc
420ttccgcatga actcgggcga gtgcctgggg aacgtggtga gcgacgagcg
gcgggtgagc 480agctccgggg ggctgcagaa cgcgcagttc gggatccgcc
gcgacgggac cctggtcacc 540gggtacctgt ctgaggagga ggtgctggac
actgagaacc catttgtgca gctgctgagt 600ggggtcgtgt ggctgattcg
taatggaagc atctacatca acgagagcca agccacagag 660tgtgacgaga
cacaggagac aggttccttt agcaaatttg tgaatgtgat atcagccagg
720acggccattg gccacgaccg gaaagggcag ctggtgctct ttcatgcaga
cggccatacg 780gagcagcgtg gcatcaacct gtgggaaatg gcggagttcc
tgctgaaaca ggacgtggtc 840aacgccatca acctggatgg gggtggctct
gccacctttg tgctcaacgg gaccttggcc 900agttacccgt cagatcactg
ccaggacaac atgtggcgct gtccccgcca agtgtccacc 960gtggtgtgtg
tgcacgaacc ccgctgccag ccgcctgact gccacggcca cgggacctgc
1020gtggacgggc actgccaatg caccgggcac ttctggcggg gtcccggctg
tgatgagctg 1080gactgtggcc cctctaactg cagccagcac ggactgtgca
cggagaccgg ctgccgctgt 1140gatgccggat ggaccgggtc caactgcagt
gaagagtgtc cccttggctg gcatgggccg 1200ggctgccaga ggccttgtaa
gtgtgagcac cattgtccct gtgaccccaa gactggcaac 1260tgcagcgtct
ccagagtaaa gcagtgtctc cagccacctg aagccaccct gagggcggga
1320gaactctcct ttttcaccag ggaggaccag gtggacccca ggctgatcga
cggcaaggat 1380tga 13832332PRTHomo sapiensmisc_feature(2)..(2)Xaa
is any amino acid 23Asp Xaa Thr Arg Val His Ala Gly Arg Leu Glu His
Glu Ser Trp Pro1 5 10 15Pro Ala Ala Gln Thr Ala Gly Ala His Arg Pro
Ser Val Arg Thr Phe 20 25 302420PRTBos taurus 24Arg Asp Gly Thr Leu
Val Thr Gly Tyr Leu Ser Glu Glu Glu Val Leu1 5 10 15Asp Thr Glu Asn
202513PRTBos taurus 25Gly Ile Asn Leu Trp Glu Met Ala Glu Phe Leu
Leu Lys1 5 102613PRTBos taurus 26Met Leu Leu Lys Leu Leu Gln Arg
Gln Arg Gln Thr Tyr1 5 102728PRTBos taurus 27Asp Thr Phe Ala Asp
Ser Leu Arg Tyr Val Asn Lys Ile Leu Asn Ser1 5 10 15Lys Phe Gly Phe
Thr Ser Arg Lys Val Pro Ala His 20 252821PRTBos taurus 28Ala Lys
Met Lys Val Val Glu Glu Pro Asn Thr Phe Gly Leu Asn Asn1 5 10 15Pro
Phe Leu Pro Gln 20295PRTBos taurus 29Ile Leu Asn Ser Lys1
5305PRTBos taurus 30Thr Ser Phe His Lys1 5316PRTBos taurus 31Phe
Gly Phe Thr Ser Arg1 53212PRTBos taurus 32Ser Leu Val Thr Asn Cys
Lys Pro Val Thr Asp Lys1 5 103312PRTBos taurus 33Leu Ala His Val
Ser Glu Pro Ser Thr Cys Val Tyr1 5 103413PRTBos taurus 34Asn Asn
Pro Phe Leu Pro Gln Thr Ser Arg Leu Gln Pro1 5 103517PRTBos
taurusmisc_feature(8)..(8)Xaa is any amino acid 35Val Pro Met Leu
Val Leu Asp Xaa Ala Xaa Pro Thr Xaa Val Xaa Leu1 5 10
15Lys3622PRTBos taurus 36Glu Leu Pro Ser Leu Tyr Pro Ser Phe Leu
Ser Ala Ser Asp Val Phe1 5 10 15Asn Val Ala Lys Pro Lys
203725DNAArtificial Sequencesynthetic DNA 37gcgaagatga aggtggtgga
ggacc 253824DNAArtificial Sequencesynthetic DNA 38tgcagagaca
gacctatacc tgcc 243923DNAArtificial Sequencesynthetic DNA
39actcacctct ccgaactgga aag 234029DNAArtificial Sequencesynthetic
DNA 40ctagccacca tggggttcaa gctcttgca 294121DNAArtificial
Sequencesynthetic DNA 41agagcttgaa ccccatggtg g 214260DNAArtificial
Sequencesynthetic DNA 42gaagacacaa ttggcatact tcactgatag caagaatact
gggaggcaac taaaagatac 604320DNAArtificial Sequencesynthetic DNA
43actgcatatc ctcagaatgg 204433DNAArtificial Sequencesynthetic DNA
44tggttctgaa gcttagccga gatcaatacc atg 334540DNAArtificial
Sequencesynthetic DNA 45tagtacactc tagactacta cttcaatttg tctcgataag
4046218DNAhybridmisc_featuremouse/human hybrid 46ctagccgcca
ccatggagac agacacactc ctgctatggg tactgctgct cggcggtggt 60acctctgtct
gtgtgaggac gatacccatg acgacgagtg ggttccaggt tccactggtg
120acgaagatca ggtagatccg cggttaatca cccaaggtcc aaggtgacca
ctgcttctag 180tccatctagg cgccaattag gacggtactg ccattcga
21847205DNAhybridmisc_featuremouse/human hybrid 47ctagcggtac
catgagatta gcagtaggcg ccttattagt atgcgcagta ctccgccatg 60gtactctaat
cgtcatccgc ggaataatca tacgcgtcat gagggattat gtctcgcaga
120agatcaggta gatccgcggt taatcgacgg taccttatac agagcgtctt
ctagtccatc 180taggcgccaa ttagctgcca ttcga
20548207DNAhybridmisc_featuremouse/human hybrid 48ctagccgcca
ccatgggatt agcagtaggc gccttattag tatgcgcagt cgccggtggt 60accctaatcg
tcatccgcgg aataatcata cgcgtcaact cggattatgt ctcgcagaag
120atcaggtaga tccgcggtta atcgacgtga gcctaataca gagcgtcttc
tagtccatct 180aggcgccaat tagctgcgta cattcga 2074931DNAArtificial
Sequencesynthetic DNA 49ggaattccac catggcgacc tccacgggtc g
315019DNAArtificial Sequencesynthetic DNA 50tgaccagggt cccgtcgcg
195139DNAArtificial Sequencesynthetic DNA 51gaggaccagg tggaccccag
gctgatccac ggcaaggat 395213PRTHomo sapiens 52Glu Asp Gln Val Asp
Pro Arg Leu Ile Asp Gly Lys Asp1 5 10
* * * * *