U.S. patent application number 11/947249 was filed with the patent office on 2008-07-17 for heparan sulfate glycosaminoglycan lyase and uses thereof.
This patent application is currently assigned to MOMENTA PHARMACEUTICALS, INC., a Delaware corporation. Invention is credited to James Myette.
Application Number | 20080171375 11/947249 |
Document ID | / |
Family ID | 37996602 |
Filed Date | 2008-07-17 |
United States Patent
Application |
20080171375 |
Kind Code |
A1 |
Myette; James |
July 17, 2008 |
Heparan Sulfate Glycosaminoglycan Lyase and Uses Thereof
Abstract
The invention provides recombinant B. thetaiotaomicron HSGAG
lyase polypeptides. The invention also provides nucleic acid
molecules encoding such polypeptides, recombinant expression
vectors containing B. thetaiotaomicron HSGAG lyase nucleic acid
molecules, and host cells into which the expression vectors have
been introduced. Characterization, diagnostic and therapeutic
methods utilizing compositions of the invention are also
provided.
Inventors: |
Myette; James; (Waltham,
MA) |
Correspondence
Address: |
LOWRIE, LANDO & ANASTASI, LLP
ONE MAIN STREET, SUITE 1100
CAMBRIDGE
MA
02142
US
|
Assignee: |
MOMENTA PHARMACEUTICALS, INC., a
Delaware corporation
|
Family ID: |
37996602 |
Appl. No.: |
11/947249 |
Filed: |
November 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11265908 |
Nov 3, 2005 |
|
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11947249 |
|
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Current U.S.
Class: |
435/232 |
Current CPC
Class: |
C08B 37/0081 20130101;
C12P 19/26 20130101; A61P 19/02 20180101; C12N 9/1051 20130101;
A61P 9/00 20180101; C08B 37/0078 20130101; A61P 7/02 20180101; A61P
35/00 20180101; A61P 3/10 20180101; A61P 43/00 20180101; A61P 9/10
20180101; A61P 27/02 20180101 |
Class at
Publication: |
435/232 |
International
Class: |
C12N 9/88 20060101
C12N009/88 |
Claims
1. A method of specifically cleaving heparin or heparan sulfate,
comprising: contacting heparin or heparan sulfate with a B.
thetaiotaomicron HSGAG lyase polypeptide II, or functional
fragments thereof, to thereby cleave the heparin or heparan
sulfate.
2.-3. (canceled)
4. The method of claim 1, further comprising contacting the heparin
or heparan sulfate with B. thetaiotaomicron HSGAG lyase I
polypeptide, or functional fragment thereof.
5.-6. (canceled)
7. The method of claim 4, wherein the B. thetaiotaomicron HSGAG
lyase I cleaves a heparin at one or more glycosidic linkages of
sulfated uronic acids.
8. The method of claim 1, wherein the B. thetaiotaomicron HSGAG
lyase II polypeptide, or functional fragment thereof, comprises a)
an amino acid sequence which is at least 90% identical to the amino
acid sequence of SEQ ID NO:6, 8 or 10; b) an amino acid sequence of
at least 600 contiguous amino acid residues of SEQ ID NO:6, 8, 10;
c) an amino acid sequence of SEQ ID NO:6, 8, or 10; and d) an amino
acid sequence which differs by at least 1 amino acid but not more
than 30 amino acids from the amino acid sequence of SEQ ID NO:6, 8,
10.
9. The method of claim 8, wherein the B. thetaiotaomicron HSGAG
lyase II polypeptide, or functional fragment thereof, is encoded by
a nucleotide sequence selected from the group consisting of: a) a
nucleic acid molecule comprising a nucleotide sequence which is at
least 90% identical to the nucleotide sequence of SEQ ID NO:5, 7 or
9; b) a nucleic acid molecule comprising a fragment of at least
1700 nucleotides of the nucleotide sequence of SEQ ID NO: 5, 7 or
9; c) a nucleic acid molecule which encodes a polypeptide
comprising the amino acid sequence of SEQ ID NO:6, 8 or 10; d) a
nucleic acid molecule which encodes a fragment of a polypeptide
comprising the amino acid sequence of SEQ ID NO:6, 8 or 10, wherein
the fragment comprises at least 600 contiguous amino acids of SEQ
ID NO: 6, 8 or 10; and e) a nucleic acid molecule which encodes a
naturally occurring allelic variant of a polypeptide comprising the
amino acid sequence of SEQ ID NO:6, 8 or 10, wherein the nucleic
acid molecule hybridizes to a nucleic acid molecule comprising SEQ
ID NO: 5, 7 or 9, or a complement thereof, under stringent
conditions.
10. The method of claim 1, wherein the B. thetaiotaomicron HSGAG
lyase II cleaves a heparin at one or more glycosidic linkages of
sulfated or undersulfated uronic acids.
11. The method of claim 1, wherein the heparin or heparan sulfate
is cleaved into di-, tri-, tetra-, penta-, hexa-, octa-, and/or
deca-saccharides.
12. The method of claim 1, further comprising determining the
sequence of the cleaved heparin or heparan sulfate.
13. The method of claim 12, further comprising contacting the
heparin or heparan sulfate with one or more HLGAG degrading enzyme
other than the B. thetaiotaomicron HSGAG lyase II polypeptide.
14. The method of claim 13, wherein the HLGAG degrading enzyme is
selected from Flavobacterium heparinum heparinase I, Flavobacterium
heparinum heparinase II, Flavobacterium heparinum heparinase III,
Flavobacterium heparinum heparinase IV, heparanase, sulfatase,
delta 4,5 glucuronidase and functional fragments and variants
thereof.
15-80. (canceled)
Description
BACKGROUND OF THE INVENTION
[0001] Heparin and heparan sulfate represent a class of
glycosaminoglycans characterized by a linear polysaccharide of
D-glucosamine linked to hexuronic acid (Linhardt, R. J. (1991)
Chem. Ind. 2, 45-50; Casu, B. (1985) Adv. Carbohydr. Chem. Biochem.
43, 51-134). Heparin and heparan sulfate are complex carbohydrates
that play an important functional role in the extracellular matrix
of mammals. These polysaccharides modulate and regulate critical
biochemical signaling pathways which impinge on normal
physiological processes such as cell and tissue morphogenesis,
cell-cell interactions, and growth and differentiation. These
polysaccharides also play a critical role in various pathologies
including wound healing, tumor growth and metastasis, certain
neurodegenerative disorders and microbial pathogenesis, to name a
few.
[0002] Much of the current understanding of heparin and heparan
sulfate sequence has relied on studies of their biosynthesis
(Linhardt, R. J., Wang, H. M., Loganathan, D., and Bae, J. H.
(1992) Biol. Chem. 267, 2380-2387; Lindahl, U., Feingold, D., and
Roden, L. (1986) Trends Biochem. Sci. 11, 221-225; Jacobson, I.,
and Lindahl U. (1980) J. Biol. Chem. 255, 5094-5100; Lindahl, U.,
and Kjellen, L. (1987) in The Biology of Extracellular Matrix
Proteoglycans (Wight, T. N., and Mecham R., eds) pp. 59-104,
Academic Press, New York).
[0003] Heparan sulfate, which is chemically related to heparin, is
present on the cell surface and within the extracellular matrix
(ECM) of virtually every mammalian cell type. These heparin-like
glycosaminoglycans (HLGAGs) are present in this extracellular
environment as protein-polysaccharide conjugates known as
proteoglycans. It is increasingly recognized that HLGAGs play much
more than a mere structural role as they interact in a functional
manner with numerous proteins of the extracellular matrix, such as
laminin, fibronectin, integrins, and collagen. As such, HLGAGs (as
part of proteolycans) help to define the biological properties of
the matrix. These HLGAGs also interact with an array of
cytokine-like growth factors and morphogens present within the
extracellular matrix by facilitating their biochemical interaction
with receptors and by protecting them from proteolytic degradation.
For example, heparin potentiates the biological activity of aFGF,
as reported by Thornton, et al., Science 222, 623-625 (1983),
possibly by potentiating the affinity of aFGF for its cell surface
receptors, as reported by Schreiber, et al., Proc. Natl. Acad. Sci.
USA 82, 6138-6142 (1985). Heparin protects aFGF and bFGF from
degradation by heat, acid and proteases, as reported by
Gospodarowicz and Cheng, J. Cell Physiol. 128, 475-484 (1986);
Rosengart, et al., Biochem. Biophys. Res. Commun. 152, 432-440
(1988); and Lobb Biochem. 27, 2572-2578 (1988). bFGF is stored in
the extracellular matrix and can be mobilized in a biologically
active form by the hydrolyzing activity of enzymes such as
heparanase as reported by Vlodavsky, et al., Proc. Natl. Acad. Sci.
USA 84, 2292-2296 (1987) and Folkman, et al., Am. J. Pathol. 130,
393-400 (1988) and Emerson et. al. Proc. Natl. Acad. Sci. USA
101(14): 4833-8 (2004).
[0004] The binding of FGF to heparan sulfate is a prerequisite for
the binding of FGF to its high affinity receptor on the cell
surface, as reported by Yayon, et al., Cell 64, 841-848 (1991) and
Papraeger, et al., Science 252, 1705-1708 (1991). A specific
heparan sulfate proteoglycan has been found to mediate the binding
of bFGF to the cell surface, as described by Kiefer, et al., Proc.
Natl. Acad. Sci. USA 87, 6985-6989 (1990).
[0005] Heparin lyases, such as heparinases, are a general class of
enzymes that are capable of specifically cleaving the major
glycosidic linkages in heparin and heparan sulfate. Three
heparinases have been identified in Flavobacterium heparinum, a
GAG-utilizing organism that also produces exoglycoronidases,
glycosidases, sulfoesterases, and sulfamidases and other enzymes
which further act on the lyase-generated oligosaccharide products
(Yang, et al. J. Biol. Chem. 260, 1849-1857 (1987); Galliher, et
al. Eur. J. Appl. Microbiol. Biotechnol. 15, 252-257 (1982). These
lyases are designated as heparinase I (heparinase, EC 4.2.2.7),
heparinase II (heparinase II, no EC number) and heparinase III
(heparinase EC 4.2.2.8). The three purified heparinases differ in
their capacity to cleave heparin and heparan sulfate: Heparinase I
primarily cleaves heparin, heparinase III specifically cleaves
heparan sulfate, and heparinase II acts on both heparin and heparan
sulfate. Several Bacteroides species (Saylers, et al. Appl.
Environ. Microbiol. 33, 319-322 (1977); Nakamura, et al. J. Clin.
Microbiol. 26, 1070-1071 (1988)) also produce heparin lyases. A
heparin lyase has also been purified to apparent homogeneity from
an unidentified soil bacterium by Bohmer, et al. J. Biol. Chem.
265, 13609-13617 (1990).
SUMMARY OF THE INVENTION
[0006] The invention is based, in part, on the discovery and
recombinant expression of heparin sulfate glucosaminoglycan (HSGAG)
lyases from Bacteroides thetaiotaomicron, hereafter referred to as
"B. thetaiotaomicron HSGAG lyases", e.g., B. thetaiotaomicron HSGAG
lyase I and B. thetaiotaomicron HSGAG lyase II, useful, inter alia,
in the structure-specific cleavage of heparin and/or heparan
sulfate. Thus, the invention includes methods, compositions and
kits with a B. thetaiotaomicron HSGAG lyase or functional fragments
thereof and combinations of B. thetaiotaomicron HSGAG lyases or
functional fragments thereof, for, e.g., characterization or
modification of glycoaminoglycans (GAGs) such as heparin-like
glycoaminoglycans (HLGAGs), e.g., heparin and heparan sulfate. For
example, the methods, compositions and kits can be used to analyze
and monitor heterogeneous populations of GAGs, e.g., HLGAGs. In
other aspects, the methods, compositions and kits can be used to
modify the structure and/or activity of GAGs, e.g., HLGAGs.
[0007] According, in one aspect, the invention features B.
thetaiotaomicron HSGAG lyase polypeptides, or functional fragments
thereof, e.g., B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragments thereof, having the amino acid sequence shown
in SEQ ID NOs:2, 4, 6, 8, 10 or 23; an amino acid substantially
identical to the amino acid sequence shown in SEQ ID NOs:2, 4, 6,
8, 10 or 23; or an amino acid encoded by a nucleic acid molecule
that hybridizes under stringent hybridization conditions to a
nucleic acid molecule comprising the nucleic acid sequence of SEQ
ID NOs:1, 3, 5, 7, 9, 22, wherein the nucleic acid encodes a full
length B. thetaiotaomicron HSGAG lyase protein, or functional
fragments thereof.
[0008] In another aspect, the invention features a composition that
includes a B. thetaiotaomicron HSGAG lyase polypeptide, B.
thetaiotaomicron HSGAG lyase polypeptides, or functional fragments
thereof, e.g., a B. thetaiotaomicron HSGAG lyase polypeptides, B.
thetaiotaomicron HSGAG lyase polypeptides, or functional fragments
thereof, described herein. In one embodiment, the composition
further comprises one or more HLGAG degrading enzyme, e.g., one or
more heparinase and/or one or more HSGAG lyase polypeptide other
than a B. thetaiotaomicron HSGAG lyase polypeptides. For example,
the composition can further include one or more of: an unsaturated
glucuronyl hydrolase (e.g., F. heparinum .DELTA.4,5 glycuronidase;
B. thetaiotaomicron .DELTA.4,5 glycuronidase); a glucuronyl
hydrolase (e.g., mammalian .alpha.-iduronidase,
.beta.-glucuronidase); a sulfohydrolase (e.g., F. heparinum
2-O-sulfatase, 6-O-sulfatase, 3-O-sulfatase: B. thetaiotaomicron
6-O-sulfatase; mucin desulfating enzymes; mammalian
N-acetylglucosamine-6-sulfatase; mammalian iduronic
acid-2-sulfatase); a N-sulfamidase (e.g., F. heparinum
N-sulfamidase; mammalian heparan-N-sulfatase); an arylsulfatase; a
hexosaminidase; a glycosyl hydrolase (e.g., endo-N-acetyl
glucosaminidase); a heparinase (e.g., Flavobacterum heparinum
heparinase I, Flavobacterum heparinum heparinase II, Flavobacterum
heparinum heparinase III, Flavobacterum heparinum heparinase IV,
mammalian heparanase); and functional fragments and variants
thereof. Such compositions can be used, e.g., to cleave a HLGAG
such as heparin and/or heparan sulfate, e.g., to characterize a
preparation of HLGAGs such as heparin and/or heparan sulfate.
[0009] In another aspect, the invention features a method of
specifically cleaving an HLGAG, e.g., heparin or heparan sulfate,
that includes contacting an HLGAG with a B. thetaiotaomicron HSGAG
lyase polypeptide, B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragments thereof, e.g., a B. thetaiotaomicron HSGAG
lyase polypeptide, B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragments thereof, described herein. In one embodiment,
the HLGAG is cleaved into di-, tri-, tetra-, penta-, hexa-, octa-,
and/or deca-saccharides and, e.g., the method further includes
determining the sequence of the di-, tri-, tetra-, penta-, hexa-,
octa-, deca- and/or longer saccharides of the HLGAG. In one
embodiment, the method further includes contacting the HLGAG with
one or more HLGAG degrading enzyme, e.g., a heparinase polypeptide
or a HSGAG lyase polypeptide other than a B. thetaiotaomicron HSGAG
lyase polypeptide. For example, the HLGAG degrading enzyme can be
one or more of: an unsaturated glucuronyl hydrolase (e.g., F.
heparinum .DELTA.4,5 glycuronidase; B. thetaiotaomicron .DELTA.4,5
glycuronidase); a glucuronyl hydrolase (e.g., mammalian
.alpha.-iduronidase, .beta.-glucuronidase); a sulfohydrolase (e.g.,
F. heparinum 2-O-sulfatase, 6-O-sulfatase, 3-O-sulfatase: B.
thetaiotaomicron 6-O-sulfatase; mucin desulfating enzymes;
mammalian N-acetylglucosamine-6-sulfatase; mammalian iduronic
acid-2-sulfatase); a N-sulfamidase (e.g., F. heparinum
N-sulfamidase; mammalian heparan-N-sulfatase); an arylsulfatase; a
hexosaminidase; a glycosyl hydrolase (e.g., endo-N-acetyl
glucosaminidase); a heparinase (e.g., Flavobacterum heparinum
heparinase I, Flavobacterum heparinum heparinase II, Flavobacterum
heparinum heparinase III, Flavobacterum heparinum heparinase IV,
mammalian heparanase); and functional fragments and variants
thereof.
[0010] In another aspect, the invention features methods for
analyzing heterogeneous populations of HLGAGs, e.g., heparin (e.g.,
UFH, LMWH, and synthetic heparins), and heparan sulfate, that
include contacting the population with a B. thetaiotaomicron HSGAG
lyase polypeptide, B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragments thereof, e.g., a B. thetaiotaomicron HSGAG
lyase polypeptide, B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragments thereof, described herein. Thus, in some
aspects, the invention relates to methods and products associated
with analyzing and monitoring heterogeneous populations of HLGAGs,
e.g., to thus defining the structural signature and activity of
heterogeneous populations of HLGAGs, using a B. thetaiotaomicron
HSGAG lyase polypeptide, B. thetaiotaomicron HSGAG lyase
polypeptides, or functional fragments thereof, e.g., a B.
thetaiotaomicron HSGAG lyase polypeptide, or functional fragment
thereof, described herein.
[0011] In some embodiments, the method includes determining the
structural signature of one or more batches of an HLGAG product
that has been contacted with a B. thetaiotaomicron HSGAG lyase
polypeptide, B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragments thereof, e.g., a B. thetaiotaomicron HSGAG
lyase polypeptide, B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragments thereof, described herein. In some
embodiments, the method further includes selecting a batch as a
result of the determination. In some embodiments, the method
further includes comparing the results of the determination to
preselected values, e.g., a reference standard. The preselected
value can be, e.g., the presence or absence or a set value (e.g.,
mole % or area under the curve) of one or more di-, tri-, tetra-,
penta-, hexa-, octa-, and/or deca-saccharide associated with
cleavage of the HLGAG with a B. thetaiotaomicron HSGAG lyase
polypeptide, B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragments thereof, e.g., a B. thetaiotaomicron HSGAG
lyase polypeptide, B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragment thereof, described herein.
[0012] For any of the methods described herein, a completely or
partially B. thetaiotaomicron HSGAG lyase polypeptide (or
polypeptides) digested sample can be analyzed to determine the
structural signature by, e.g., one or more of mass spectroscopy,
NMR spectroscopy, gel electrophoresis, capillary electrophoresis,
reverse-phase column chromatography (e.g., HPLC, e.g., HPLC with a
stationary phase dynamically coated with a quanternary ammonium
salt), ion-pair HPLC. The methods described herein can further
include digesting the sample with one or more HLGAG degrading
enzyme, e.g., a heparinase or a heparin lyase polypeptide other
than a B. thetaiotaomicron HSGAG lyase polypeptide. For example,
the HLGAG degrading enzyme can be one or more of: an unsaturated
glucuronyl hydrolase (e.g., F. heparinum .DELTA.4,5 glycuronidase;
B. thetaiotaomicron .DELTA.4,5 glycuronidase); a glucuronyl
hydrolase (e.g., mammalian .alpha.-iduronidase,
.beta.-glucuronidase); a sulfohydrolase (e.g., F. heparinum
2-O-sulfatase, 6-O-sulfatase, 3-O-sulfatase: B. thetaiotaomicron
6-O-sulfatase; mucin desulfating enzymes; mammalian
N-acetylglucosamine-6-sulfatase; mammalian iduronic
acid-2-sulfatase); a N-sulfamidase (e.g., F. heparinum
N-sulfamidase; mammalian heparan-N-sulfatase); an arylsulfatase; a
hexosaminidase; a glycosyl hydrolase (e.g., endo-N-acetyl
glucosaminidase); a heparinase (e.g., Flavobacterum heparinum
heparinase I, Flavobacterum heparinum heparinase II, Flavobacterum
heparinum heparinase III, Flavobacterum heparinum heparinase IV,
mammalian heparanase); and functional fragments and variants
thereof.
[0013] In another aspect, the invention features an HLGAG
preparation (e.g., a heparin or heparan sulfate preparation)
produced by contacting an HLGAG preparation with a B.
thetaiotaomicron HSGAG lyase polypeptide, B. thetaiotaomicron HSGAG
lyase polypeptides, or functional fragments thereof, e.g., a B.
thetaiotaomicron HSGAG lyase polypeptide, B. thetaiotaomicron HSGAG
lyase polypeptides, or functional fragments thereof, described
herein. In one embodiment, the HLGAG preparation (e.g., the heparin
or heparan sulfate preparation) has one or more of reduced anti-Xa
activity and anti-IIa activity, e.g., as compared to a reference
standard, e.g., as compared to a commercially available heparin or
heparan sulfate or as compared to the heparin or heparan sulfate
preparation prior to contacting with a B. thetaiotaomicron HSGAG
lyase polypeptide. In some embodiments, anti-Xa activity is reduced
while anti-IIa activity is maintained or increased. In other
embodiments, anti-IIa activity is reduced while anti-Xa activity is
maintained or enhanced. In other embodiments, anti-Xa activity and
anti-IIa activity is reduced. Such preparation can be useful, e.g.,
for applications where reduced anti-Xa activity and/or anti-IIa
activity is desirable, e.g., such as the use of heparin or heparan
sulfate as a carrier for another agent, e.g., a therapeutic agent,
prophylactic or diagnostic agent. Thus, in some embodiments, the
HLGAG preparation can further include a second agent other than the
HLGAG, e.g., the preparation can further include one or more
therapeutic, prophylactic or diagnostic agents. In another
embodiment, the HLGAG preparation (e.g., the heparin or heparan
sulfate preparation) has one or more of increased anti-Xa activity
and anti-IIa activity, e.g., as compared to a reference standard,
e.g., as compared to a commercially available heparin or heparan
sulfate or as compared to the heparin or heparan sulfate
preparation prior to contacting with a B. thetaiotaomicron HSGAG
lyase polypeptide. Such preparation can be useful, e.g., for
applications were increased anti-Xa activity and/or anti-IIa
activity is desirable, e.g., as an anti-coagulant and/or
anti-thrombotic agent.
[0014] In another aspect, the invention features a method of
neutralizing one or more activities of an HLGAG (e.g., a heparin or
heparan sulfate). The method includes contacting the HLGAG with a
B. thetaiotaomicron HSGAG lyase polypeptide, or functional fragment
thereof, e.g., a B. thetaiotaomicron HSGAG lyase I polypeptide, or
functional fragment thereof, described herein. When the HLGAG is
heparin or heparan sulfate, the activity to be neutralized can be
one or more of anti-Xa activity and anti-IIa activity. In some
embodiments, anti-Xa activity is reduced while anti-IIa activity is
maintained or increased. In other embodiments, anti-IIa activity is
reduced while anti-Xa activity is maintained or enhanced. In other
embodiments, anti-Xa activity and anti-IIa activity is reduced. The
HLGAG can be, e.g., contacted ex vivo or in vivo. Thus, in some
embodiments, the method can include administering the B.
thetaiotaomicron HSGAG lyase polypeptide, or functional fragment
thereof, to a subject in an amount effective to neutralize anti-Xa
activity and/or anti-IIa activity in the subject, e.g., a subject
that has been administered an HLGAG such as heparin or heparan
sulfate, e.g., a subject that has been administered heparin or
heparan sulfate to inhibit coagulation and/or thrombosis.
[0015] In another aspect, the invention features a method of
inhibiting angiogenesis in a subject. The method includes
administering to the subject an effect amount of a B.
thetaiotaomicron HSGAG lyase polypeptide, B. thetaiotaomicron HSGAG
lyase polypeptides, or functional fragments thereof, e.g., a B.
thetaiotaomicron HSGAG lyase polypeptide, B. thetaiotaomicron HSGAG
lyase polypeptides, or functional fragments thereof, described
herein, to thereby inhibit angiogenesis. In one embodiment, the
subject has a disease or disorder associated with unwanted
angiogenesis. Such disorders include, but are not limited to,
arthritis (e.g., rheumatoid arthritis), various eye disorders
(e.g., diabetic retinopathy, neovascular glaucoma, inflammatory
disorders, ocular tumors (e.g., retinoblastoma), retrolental
fibroplasias, uveitis as well as disorders associated with
choroidal neovascularization and iris neovascularization) and
cancer (e.g., tumor growth and metastases).
[0016] In another aspect, the invention features a method of
inhibiting unwanted cellular proliferation and/or differentiation
in a subject. The method includes administering to the subject an
effect amount of a B. thetaiotaomicron HSGAG lyase polypeptide, B.
thetaiotaomicron HSGAG lyase polypeptides, or functional fragments
thereof, e.g., a B. thetaiotaomicron HSGAG lyase polypeptide, B.
thetaiotaomicron HSGAG lyase polypeptides, or functional fragments
thereof, described herein, to thereby inhibit cellular
proliferation and/or differentiation. In one embodiment, the
subject has cancer.
[0017] In another aspect, the invention features a pharmaceutical
composition that includes a B. thetaiotaomicron HSGAG lyase
polypeptide, B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragments thereof, e.g., a B. thetaiotaomicron HSGAG
lyase polypeptide, B. thetaiotaomicron HSGAG lyase polypeptides, or
functional fragments thereof, described herein, and a
pharmaceutically acceptable carrier. In one embodiment, the B.
thetaiotaomicron HSGAG lyase polypeptide, B. thetaiotaomicron HSGAG
lyase polypeptides, or functional fragments thereof, is present in
an amount effective to neutralize one or more activity of an HLGAG.
Preferably, the HLGAG is heparin or heparan sulfate and the B.
thetaiotaomicron HSGAG lyase polypeptide, or functional fragment
thereof, is present in an amount effective to neutralize one or
more of anti-Xa activity and anti-IIa activity. In some
embodiments, anti-Xa activity is reduced while anti-IIa activity is
maintained or increased. In other embodiments, anti-IIa activity is
reduced while anti-Xa activity is maintained or enhanced. In other
embodiments, anti-Xa activity and anti-IIa activity is reduced. In
another embodiment, the B. thetaiotaomicron HSGAG lyase
polypeptide, or functional fragment thereof, is present in an
amount effective to inhibit angiogenesis.
[0018] In another aspect, the invention features a kit comprising a
composition of B. thetaiotaomicron HSGAG lyase polypeptide, B.
thetaiotaomicron HSGAG lyase polypeptides, or functional fragments
thereof. In one embodiment, the kit further includes one or more
HLGAG degrading enzyme, e.g., one or more heparinase polypeptide
and/or one or more HSGAG lyase polypeptide other than B.
thetaiotaomicron HSGAG lyase polypeptide. For example, the kit can
further comprise one or more of: an unsaturated glucuronyl
hydrolase (e.g., F. heparinum .DELTA.4,5 glycuronidase; B.
thetaiotaomicron .DELTA.4,5 glycuronidase); a glucuronyl hydrolase
(e.g., mammalian .alpha.-iduronidase, .beta.-glucuronidase); a
sulfohydrolase (e.g., F. heparinum 2-O-sulfatase, 6-O-sulfatase,
3-O-sulfatase: B. thetaiotaomicron 6-O-sulfatase; mucin desulfating
enzymes; mammalian N-acetylglucosamine-6-sulfatase; mammalian
iduronic acid-2-sulfatase); a N-sulfamidase (e.g., F. heparinum
N-sulfamidase; mammalian heparan-N-sulfatase); an arylsulfatase; a
hexosaminidase; a glycosyl hydrolase (e.g., endo-N-acetyl
glucosaminidase); a heparinase (e.g., Flavobacterum heparinum
heparinase I, Flavobacterum heparinum heparinase II, Flavobacterum
heparinum heparinase III, Flavobacterum heparinum heparinase IV,
mammalian heparanase); and functional fragments and variants
thereof. In one embodiment, the B. thetaiotaomicron HSGAG lyase
polypeptide, or functional fragment thereof, and one or more of the
other HLGAG degrading enzymes are in the same composition. In
another embodiment, the B. thetaiotaomicron HSGAG lyase
polypeptide, or functional fragment thereof, and the other HLGAG
degrading enzyme are in different compositions. In another
embodiment, the B. thetaiotaomicron HSGAG lyase polypeptide, or
functional fragment thereof, is in a pharmaceutical composition
with a pharmaceutically effective carrier. The kits can further
include an HLGAG, e.g., heparin and/or heparan sulfate. In one
embodiment, when the kit includes a pharmaceutical composition of a
B. thetaiotaomicron HSGAG lyase polypeptide, or functional fragment
thereof, the HLGAG, e.g., heparin and/or heparan sulfate, is also
in a pharmaceutical composition and, e.g., the kit further includes
instructional material for neutralizing one or more activity of the
HLGAG.
[0019] In another aspect, the invention features a nucleic acid
molecule which encodes a B. thetaiotaomicron HSGAG lyase
polypeptides, or functional fragments thereof. In one embodiment,
the isolated nucleic acid molecule encodes a polypeptide having the
amino acid sequence of SEQ ID NOs:2, 4, 6, 8, 10 or 23. In other
embodiments, the invention provides isolated B. thetaiotaomicron
HSGAG lyase nucleic acid molecules having the nucleotide sequence
shown in SEQ ID NOs:1, 3, 5, 7, 9 or 22. In another embodiment, the
invention provides nucleic acid molecules that are substantially
identical to (e.g., naturally occurring allelic variants) to the
nucleotide sequence shown in SEQ ID NOs:1 or 5 and nucleic acid
molecules that hybridize under stringent hybridization conditions
to a nucleic acid molecule comprising the nucleotide sequence of
SEQ ID NOs:1, 3, 5, 7, 9 or 22 wherein the nucleic acid encodes a
full length B. thetaiotaomicron HSGAG lyase protein, or functional
fragments thereof.
[0020] In a related aspect, the invention further provides nucleic
acid constructs which include a B. thetaiotaomicron HSGAG lyase
nucleic acid molecule described herein. In certain embodiments, the
nucleic acid molecules of the invention are operatively linked to
native or heterogenous regulatory sequences. Also included are
vectors and host cells containing the B. thetaiotaomicron HSGAG
lyase nucleic acid molecules of the invention, e.g., vectors and
host cells suitable for producing B. thetaiotaomicron HSGAG lyase
nucleic acid molecules and polypeptides.
[0021] In another aspect, the invention features antibodies and
antigen-binding fragments thereof, that react with, or more
preferably, specifically bind B. thetaiotaomicron HSGAG lyase
polypeptides.
[0022] In another aspect, the invention provides methods of
screening for compounds that modulate the expression or activity of
the B. thetaiotaomicron HSGAG lyase polypeptides or nucleic
acids.
[0023] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A depicts a DNA sequence (SEQ ID NO: 1) encoding B.
thetaiotaomicron HSGAG lyase I. Initiating methinione codon (ATG)
is underlined and a second, internal methinione codon is doubled
unlined. FIG. 1B depicts its predicted amino acid sequence (SEQ ID
NO:2) as well as indicating in bold the N-terminal amino acid
residues of two variants of B. thetaiotaomicron HSGAG lyase I
referred to as the M17 variant (SEQ ID NO:4) and the Q26 variant
(SEQ ID NO:23).
[0025] FIG. 2 depicts a BLAST alignment of B. thetaiotaomicron
HSGAG lyase I with a heparinase I from Flavobacterium
heparinum.
[0026] FIG. 3A depicts a DNA sequence (SEQ ID NO:3) encoding the
M17 variant of B. thetaiotaomicron HSGAG lyase I. FIG. 3B depicts
its predicted amino acid sequence (SEQ ID NO:4).
[0027] FIG. 4A depicts a DNA sequence (SEQ ID NO:22) encoding the
Q26 variant of B. thetaiotaomicron HSGAG lyase I. FIG. 4B depicts
its predicted amino acid sequence (SEQ ID NO:23).
[0028] FIG. 5A depicts a DNA sequence (SEQ ID NO:5) encoding B.
thetaiotaomicron HSGAG lyase II. FIG. 5A also depicts portions of
the nucleotide sequence encoding B. thetaiotaomicron HSGAG lyase II
that are not present in to variants of B. thetaiotaomicron HSGAG
lyase II, namely the "Q23 variant" (SEQ ID NO:7) the deleted
portion indicated by underlining, and the "K169 variant" (SEQ ID
NO:9) the deleted portion indicated by shading. FIG. 5B depicts the
predicted amino acid sequence B. thetaiotaomicron HSGAG lyase II
(SEQ ID NO:6) as well as indicating the portions deleted from the
amino acid sequence of the Q23 variant (SEQ ID NO:8) and the K169
variant (SEQ ID NO: 10).
[0029] FIG. 6 depicts a BLAST alignment of B. thetaiotaomicron
HSGAG lyase II with a heparinase III from Flavobacterium
heparinum.
[0030] FIG. 7 is a representation of an SAX-HPLC chromatogram.
Trace A depicts the digestion products of porcine mucosa treated
with recombinant B. thetaiotaomicron HSGAG lyase I. Trace B depicts
the digestion products of porcine mucosa treated with recombinant
Flavobacterium heparinum heparinase I. Trace C depicts the
digestion products of porcine mucosa treated with recombinant
Flavobacterium heparinum heparinase II.
[0031] FIG. 8 is a representation of a MALDI-MS mass spectrum.
Panel A depicts the peaks of untreated ATIII pentasaccharide
ARIXTRA.RTM., the structure of which is also shown. Panel B depicts
the peaks produced after ARIXTRA.RTM. was digested with recombinant
B. thetaiotaomicron HSGAG lyase I. A pentasulfated trisaccharide
product, the structure of which is shown, results after
digestion.
DETAILED DESCRIPTION
[0032] Overview
[0033] This disclosure describes recombinant expression of active
B. thetaiotaomicron HSGAG lyases from B. thetaiotaomicron, that are
useful, inter alia, in the modification and characterization of
GAGs such as heparin and/or heparan sulfate glycosaminoglycans and
derivatives thereof.
[0034] For example, the B. thetaiotaomicron HSGAG lyases described
herein can be a complementary tool to existing chemo-enzymatic
methods for cleaving GAGs such as heparin and heparan sulfate
polysaccharides in a structure-specific fashion. Structure specific
cleavage of a GAG, e.g., heparin and/or heparan sulfate, can be
used, e.g., to determine the structure of GAGs in a heterogenous
GAG preparation. In addition, cleavage can be used, e.g., to
produce lower molecular weight oligosaccharides from the GAG. Thus,
the B. thetaiotaomicron HSGAG lyases can be used to generate, e.g.,
heparin- and heparan sulfate-derived oligosaccharides. Such
heparin- and heparan sulfate-derived oligosaccharides may have
diagnostic, prophylactic and therapeutic potential.
[0035] In addition, the B. thetaiotaomicron HSGAG lyases described
herein may also have prophylactic and therapeutic potential, e.g.,
in disorders associated with angiogenesis.
[0036] The B. thetaiotaomicron HSGAG lyases further can be used in
the ex vivo and/or in vivo to neutralize an anti-coagulant and/or
anti-thrombotic activity of heparin and/or heparan sulfate.
[0037] The B. thetaiotaomicron HSGAG lyase I sequence (FIG. 1; SEQ
ID NO: 1), which is approximately 1130 nucleotides long including
untranslated regions, contains a predicted methionine-initiated
coding sequence of about 1128 nucleotides, including the
termination codon (nucleotides indicated as coding of SEQ ID NO: 1
in FIG. 1). The coding sequence encodes a 376 amino acid protein
(SEQ ID NO:2).
[0038] A variant in which the amino terminus begins at the
methinione at residue 17 (M17) can also be used to produce
recombinant protein. The amino acid sequence and nucleotide
sequence encoding the M17 variant of B. thetaiotaomicron HSGAG
lyase I are depicted in FIGS. 3B (SEQ ID NO:4) and 3A (SEQ ID
NO:3), respectively.
[0039] Another variant in which the amino terminus begins at the
glutamine at residue Q26 can also be used to produce recombinant
protein. The amino acid sequence and nucleotide sequence encoding
the Q26 variant of B. thetaiotaomicron HSGAG lyase I are depicted
in FIGS. 4B (SEQ ID NO:23) and 4A (SEQ ID NO:22), respectively.
[0040] The B. thetaiotaomicron HSGAG lyase I protein contains a
significant number of structural characteristics in common with
heparinase I obtained from Flavobacterium heparinum.
[0041] The B. thetaiotaomicron HSGAG lyase II sequence (FIG. 5; SEQ
ID NO:5), which is approximately 2001 nucleotides long including
untranslated regions including the termination codon (nucleotides
indicated as coding of SEQ ID NO:5 in FIG. 5). The coding sequence
encodes a 666 amino acid protein (SEQ ID NO:6).
[0042] A variant in which the amino terminus begins at the
glutamine at residue 23 (Q23) can also be used to produce
recombinant protein. The amino acid sequence and nucleotide
sequence encoding the Q23 variant of B. thetaiotaomicron HSGAG
lyase II are depicted in FIGS. 5B (SEQ ID NO:8) and 5A (SEQ ID
NO:7), respectively.
[0043] Another variant in which is a deletion beginning at the
lysine at residue 169 (K169) and ending at the glutamic acid at
residue 186 can also be used to produce recombinant protein. The
amino acid sequence and nucleotide sequence encoding the K169
variant of B. thetaiotaomicron HSGAG lyase II are depicted in FIGS.
5B (SEQ ID NO: 10) and 5A (SEQ ID NO:9), respectively.
[0044] The B. thetaiotaomicron HSGAG lyase II protein contains a
significant number of structural characteristics in common with
heparinase III obtained from Flavobacterium heparinum.
[0045] As the B. thetaiotaomicron HSGAG lyase polypeptides of the
invention may modulate heparin- and/or heparan sulfate-mediated
activities, they may be useful in various prophylactic and
therapeutic applications as well as for developing novel
prophylactic and diagnostic agents for heparin- or heparan
sulfate-mediated or related disorders.
[0046] As used herein, a "HSGAG lyase activity", "biological
activity of HSGAG lyase" or "functional activity of HSGAG lyase",
refers to an activity exerted by a B. thetaiotaomicron HSGAG lyase
protein, polypeptide or nucleic acid molecule in a physiological
milieu. For example, a HSGAG lyase activity can be an activity
exerted by B. thetaiotaomicron HSGAG lyase on e.g., on a HSGAG
lyase substrate, e.g., glycosidic linkages in heparin or heparan
sulfate. A HSGAG lyase activity can be determined in vivo or in
vitro.
[0047] The B. thetaiotaomicron HSGAG lyase molecules of the present
invention are predicted to have similar biological activities to
various heparinases obtained from Flavobacterium heparinum. For
example, the B. thetaiotaomicron HSGAG lyase proteins of the
present invention can have one or more of the following activities:
(1) binds a heparin and/or a heparan sulfate; (2) cleaves one or
more glycosidic linkages of a heparin and/or a heparan sulfate; (3)
modulates, e.g., increases or reduces, anti-Xa activity and/or
anti-IIa activity of a heparin and/or a heparan sulfate; and (4)
reduces or eliminates angiogenesis.
[0048] In some aspects, the B. thetaiotaomicron HSGAG lyase I has
biological activity similar to, but not identical with, heparinase
I obtained from Flavobacterium heparinum. For example, the B.
thetaiotaomicron HSGAG lyase I can have one or more of the
following activities: (1) binds a heparin and/or heparan sulfate;
(2) cleaves one or more glycosidic linkages of heparin and/or
heparan sulfate, e.g., cleaves one or more glycosidic linkages of
sulfated uronic acids, e.g., 2-O and/or 3-O sulfated uronic acids;
(3) reduces anti-Xa activity and/or anti-IIa activity of a heparin
and/or a heparan sulfate, e.g., as compared to a reference
standard, e.g., the anti-Xa activity and/or anti-IIa activity of a
commercially available heparin or heparan sulfate or of the heparin
or heparan sulfate prior to cleavage. In some embodiments, anti-Xa
activity is reduced while anti-IIa activity is maintained or
increased. In other embodiments, anti-IIa activity is reduced while
anti-Xa activity is maintained or enhanced. In other embodiments,
anti-Xa activity and anti-IIa activity is reduced.
[0049] Thus, the B. thetaiotaomicron HSGAG lyase molecules, e.g.,
the B. thetaiotaomicron HSGAG lyase 1 molecules can act as novel
therapeutic agents for controlling heparin-associated disorders.
Examples of such disorders include heparin-induced anticoagulation
and/or angiogenesis. For example, the B. thetaiotaomicron HSGAG
lyase molecules, e.g., the B. thetaiotaomicron HSGAG lyase 1
molecules, can be used to reduce or eliminate (e.g., neutralize)
one or more anticoagulation properties of a heparin and/or a
heparan sulfate, e.g., during or after surgery. In other
embodiments, the B. thetaiotaomicron HSGAG lyase molecules, e.g.,
B. thetaiotaomicron HSGAG lyase 1 molecules, can be used to
deheparinize blood, e.g., in a bioreactor, e.g., a bioreactor used
in heart-lung and/or kidney dialysis.
[0050] In some aspects, the B. thetaiotaomicron HSGAG lyase II has
biological activity similar to, but not identical with, heparinase
II obtained from Flavobacterium heparinum. For example, the B.
thetaiotaomicron HSGAG lyase II can have one or more of the
following activities: (1) binds a heparin and/or heparan sulfate;
(2) cleaves one or more glycosidic linkages of heparin and/or
heparan sulfate, e.g., cleaves one or more glycosidic linkages of
sulfated and undersulfated uronic acids; (3) increases anti-Xa
activity and/or anti-IIa activity of a heparin and/or a heparan
sulfate, e.g., as compared to a reference standard, e.g., the
anti-Xa activity and/or anti-IIa activity of a commercially
available heparin or heparan sulfate or of the heparin or heparan
sulfate prior to cleavage. In some embodiments, anti-Xa activity is
increased while anti-IIa activity is maintained or reduced. In
other embodiments, anti-IIa activity is increased while anti-Xa
activity is maintained or reduced. In other embodiments, anti-Xa
activity and anti-IIa activity are reduced.
[0051] Thus, such B. thetaiotaomicron HSGAG lyase molecules, e.g.,
B. thetaiotaomicron HSGAG lyase II molecules, can be used to
prepare a heparin and/or heparan sulfate preparation useful for
treatment of coagulation and/or thrombosis. Examples of such
disorders include dissolving or inhibiting formation of thromboses,
treatment and prevention of conditions resulting from infarction of
cardiac and central nervous system vessels, atherosclerosis,
thrombosis, myocardial infarction, arrythmias, atrial fibrillation,
angina, unstable angina, refractory angina, congestive heart
failure, disseminated intravascular coagulation, percutaneous
coronary intervention (PCI), coronary artery bypass graft surgery
(CABG), reocclusion or restenosis of reperfused coronary arteries,
rheumatic fever, stroke, transient ischemic attacks, thrombotic
stroke, embolic stroke, deep venous thrombosi, pulmonary embolism,
migraine, allergy, asthma, emphysema, adult respiratory stress
syndrome (ARDS), cystic fibrosis, neovascularization of the ocular
space, osteoporosis, psoriasis, arthritis (rheumatoid or
osteogenic), Alzheimer's disease, bone fractures, major
surgery/trauma, burns, surgical procedures, transplantation such as
bone marrow transplantation, hip replacement, knee replacement,
sepsis, septic shock, pregnancy, hereditary disorders such as
hemophilias.
[0052] In other embodiments, the B. thetaiotaomicron HSGAG lyase
molecules, e.g., B. thetaiotaomicron HSGAG lyase II molecules, can
be used to treat or prevent cellular proliferative or
differentiative disorders, e.g., by preventing or inhibiting
angiogenesis of cells exhibiting or otherwise associated with
unwanted proliferation and/or differentiation. Examples of cellular
proliferative and/or differentiative disorders include diabetes;
arthritis, e.g., rheumatoid arthritis; ocular disorders, e.g.,
ocular neovascularization, diabetic retinopathy, neovascular
glaucoma, retrolental fibroplasia, uevitis, eye disease associated
with choroidal neovascularization, eye disorders associated with
iris neovascularization; cancer, e.g., carcinoma, sarcoma,
metastatic disorders or hematopoietic neoplastic disorders, e.g.,
leukemias.
[0053] The B. thetaiotaomicron HSGAG lyase proteins, fragments
thereof, and derivatives and other variants of the sequence in SEQ
ID NO:2 or SEQ ID NO:6 thereof are collectively referred to as
"polypeptides or proteins of the invention" or "B. thetaiotaomicron
HSGAG lyase polypeptides or proteins". Nucleic acid molecules
encoding such polypeptides or proteins are collectively referred to
as "nucleic acids of the invention" or "B. thetaiotaomicron HSGAG
lyase nucleic acids." "B. thetaiotaomicron HSGAG lyase molecules"
refer to B. thetaiotaomicron HSGAG lyase nucleic acids,
polypeptides, and antibodies.
[0054] As used herein, the term "nucleic acid molecule" includes
DNA molecules (e.g., a cDNA or genomic DNA), RNA molecules (e.g.,
an mRNA) and analogs of the DNA or RNA. A DNA or RNA analog can be
synthesized from nucleotide analogs. A DNA molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0055] The term "isolated nucleic acid molecule" or "purified
nucleic acid molecule" includes nucleic acid molecules that are
separated from other nucleic acid molecules present in the natural
source of the nucleic acid. For example, with regards to genomic
DNA, the term "isolated" includes nucleic acid molecules which are
separated from the chromosome with which the genomic DNA is
naturally associated. Preferably, an "isolated" nucleic acid is
free of sequences which naturally flank the nucleic acid (i.e.,
sequences located at the 5' and/or 3' ends of the nucleic acid) in
the genomic DNA of the organism from which the nucleic acid is
derived. For example, in various embodiments, the isolated nucleic
acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1
kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which
naturally flank the nucleic acid molecule in genomic DNA of the
cell from which the nucleic acid is derived. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular material, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically
synthesized.
[0056] As used herein, the term "hybridizes under stringent
conditions" describes conditions for hybridization and washing.
Stringent hybridization conditions are hybridization in 6.times.
sodium chloride/sodium citrate (SSC) at about 45.degree. C.,
followed by two washes in 0.2.times.SSC, 0.1% SDS at 65.degree. C.
Hybridization conditions are known to those skilled in the art and
can be found in Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueous and nonaqueous
methods are described in that reference and either can be used.
Additional examples of hybridization conditions are as follows: 1)
low stringency, hybridization in 6.times.SSC at about 45.degree.
C., followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
55.degree. C.; 2) medium stringency, hybridization in 6.times.SSC
at about 45.degree. C., followed by one or more washes in
0.2.times.SSC, 0.1% SDS at 60.degree. C.; and preferably, 3) high
stringency, hybridization in 6.times.SSC at about 45.degree. C.,
followed by one or more washes in 0.2.times.SSC, 0.1% SDS at
65.degree. C. Particularly preferred stringency conditions (and the
conditions that should be used if the practitioner is uncertain
about what conditions should be applied to determine if a molecule
is within a hybridization limitation of the invention) are 0.5M
sodium phosphate, 7% SDS at 65.degree. C., followed by one or more
washes at 0.2.times.SSC, 1% SDS at 65.degree. C. Preferably, an
isolated nucleic acid molecule of the invention that hybridizes
under stringent conditions to the sequence of SEQ ID NO: 1
corresponds to a naturally-occurring nucleic acid molecule.
[0057] As used herein, a "naturally-occurring" nucleic acid
molecule refers to an RNA or DNA molecule having a nucleotide
sequence that occurs in nature. For example a naturally occurring
nucleic acid molecule can encode a natural protein.
[0058] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules which include at least an open
reading frame encoding a B. thetaiotaomicron HSGAG lyase protein.
The gene can optionally further include non-coding sequences, e.g.,
regulatory sequences and introns.
[0059] An "isolated" or "purified" polypeptide or protein is
substantially free of cellular material or other contaminating
proteins from the cell or tissue source from which the protein is
derived, or substantially free from chemical precursors or other
chemicals when chemically synthesized. "Substantially free" means
that a preparation of B. thetaiotaomicron HSGAG lyase protein is at
least 10% pure. In a preferred embodiment, the preparation of B.
thetaiotaomicron HSGAG lyase protein has less than about 30%, 20%,
10% and more preferably 5% (by dry weight), of non-B.
thetaiotaomicron HSGAG lyase protein (also referred to herein as a
"contaminating protein"), or of chemical precursors or non-B.
thetaiotaomicron HSGAG lyase chemicals. When the B.
thetaiotaomicron HSGAG lyase protein or biologically active portion
thereof is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, more preferably less than about
10%, and most preferably less than about 5% of the volume of the
protein preparation. The invention includes isolated or purified
preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry
weight.
[0060] A "non-essential" amino acid residue is a residue that can
be altered from the wild-type sequence of B. thetaiotaomicron HSGAG
lyase without abolishing or substantially altering a HSGAG lyase
activity. Preferably, the alteration does not substantially alter
the HSGAG lyase activity, e.g., the activity is at least 20%, 40%,
60%, 70% or 80% of wild-type. An "essential" amino acid residue is
a residue that, when altered from the wild-type sequence of B.
thetaiotaomicron HSGAG lyase, results in abolishing a HSGAG lyase
activity such that less than 20% of the wild-type activity is
present. For example, conserved amino acid residues in B.
thetaiotaomicron HSGAG lyase are predicted to be particularly
unamenable to alteration.
[0061] A "conservative amino acid substitution" is one in which the
amino acid residue is replaced with an amino acid residue having a
similar side chain. Families of amino acid residues having similar
side chains have been defined in the art. These families include
amino acids with basic side chains (e.g., lysine, arginine,
histidine), acidic side chains (e.g., aspartic acid, glutamic
acid), uncharged polar side chains (e.g., glycine, asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan), beta-branched side chains
(e.g., threonine, valine, isoleucine) and aromatic side chains
(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted nonessential amino acid residue in a B. thetaiotaomicron
HSGAG lyase protein is preferably replaced with another amino acid
residue from the same side chain family. Alternatively, in another
embodiment, mutations can be introduced randomly along all or part
of a B. thetaiotaomicron HSGAG lyase coding sequence, such as by
saturation mutagenesis, and the resultant mutants can be screened
for HSGAG lyase biological activity to identify mutants that retain
activity. Following mutagenesis of SEQ ID NO: 1 or SEQ ID NO:5, the
encoded protein can be expressed recombinantly and the activity of
the protein can be determined.
[0062] As used herein, a "biologically active portion" of a B.
thetaiotaomicron HSGAG lyase protein includes a fragment of a B.
thetaiotaomicron HSGAG lyase protein which participates in an
interaction, e.g., an inter-molecular interaction. An
inter-molecular interaction can be a binding interaction or an
enzymatic interaction (e.g., the interaction can be transient and a
covalent bond is formed or broken). An inter-molecular interaction
can be between a HSGAG lyase B. thetaiotaomicron molecule and a
non-B. thetaiotaomicron HSGAG lyase molecule, e.g., heparin,
heparan sulfate, and fragments thereof. Biologically active
portions of a B. thetaiotaomicron HSGAG lyase protein include
peptides comprising amino acid sequences sufficiently homologous to
or derived from the amino acid sequence of the B. thetaiotaomicron
HSGAG lyase protein, e.g., the amino acid sequence shown in SEQ ID
NO:2 and SEQ ID NO:6, which include less amino acids than the full
length B. thetaiotaomicron HSGAG lyase proteins, and exhibit at
least one activity of a HSGAG lyase protein. Typically,
biologically active portions comprise a domain or motif with at
least one activity of the HSGAG lyase protein, e.g.,
depolymerization of heparin, heparan sulfate, and fragments thereof
(e.g., in a site specific manor); cleavage of a glycosidic linkage
of heparin, heparan sulfate, and fragments thereof; reduce or
eliminate an anticoagulant activity, e.g., an anticoagulant
activity of heparin, heparan sulfate, and fragments thereof. A
biologically active portion of a B. thetaiotaomicron HSGAG lyase
protein can be a polypeptide which is, for example, 10, 25, 50,
100, 200, 300, 400, 500 or more amino acids in length.
[0063] Calculations of homology or sequence identity between
sequences (the terms are used interchangeably herein) are performed
as follows.
[0064] To determine the percent identity of two amino acid
sequences, or of two nucleic acid sequences, the sequences are
aligned for optimal comparison purposes (e.g., gaps can be
introduced in one or both of a first and a second amino acid or
nucleic acid sequence for optimal alignment and non-homologous
sequences can be disregarded for comparison purposes). The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences, taking into account
the number of gaps, and the length of each gap needed to be
introduced for optimal alignment of the two sequences. For the
purposes of determining if a molecule is within a sequence identity
or a homology limitation herein, percent identity is determined by
the mathematical algorithm of Needleman and Wunsch ((1970) J. Mol.
Biol. 48:444-453) as implemented in the GAP program of the GCG
software package (available at http://www.gcg.com) with the
following parameters: a Blossum 62 scoring matrix with a gap
penalty of 12, a gap extend penalty of 4, and a frameshift gap
penalty of 5.
[0065] In a preferred embodiment, the length of a reference
sequence aligned for comparison purposes is at least 30%,
preferably at least 40%, more preferably at least 50%, 60%, and
even more preferably at least 70%, 80%, 90%, 100% of the length of
the reference sequence. The amino acid residues or nucleotides at
corresponding amino acid positions or nucleotide positions are then
compared. When a position in the first sequence is occupied by the
same amino acid residue or nucleotide as the corresponding position
in the second sequence, then the molecules are identical at that
position (as used herein amino acid or nucleic acid "identity" is
equivalent to amino acid or nucleic acid "homology").
[0066] For the purposes of analyzing a biological sequence with
reference to B. thetaiotaomicron HSGAG lyase molecules, the
following alignment procedures can be used in addition to the
aforementioned Needleman and Wunsch algorithm. The percent identity
between two amino acid or nucleotide sequences can be determined
using the algorithm of E. Meyers and W. Miller ((1989) CABIOS,
4:11-17) which has been incorporated into the ALIGN program
(version 2.0), using a PAM120 weight residue table, a gap length
penalty of 12 and a gap penalty of 4.
[0067] The nucleic acid and protein sequences described herein can
be used as a "query sequence" to perform a search against public
databases to, for example, identify other family members or related
sequences. Such searches can be performed using the NBLAST and
XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol.
Biol. 215:403-10. BLAST nucleotide searches can be performed with
the NBLAST program, score=100, wordlength=12 to obtain nucleotide
sequences homologous to B. thetaiotaomicron HSGAG lyase nucleic
acid molecules of the invention. BLAST protein searches can be
performed with the XBLAST program, score=50, wordlength=3 to obtain
amino acid sequences homologous to B. thetaiotaomicron HSGAG lyase
protein molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs (e.g., XBLAST and NBLAST) can be used.
See http://www.ncbi.nlm.nih.gov.
[0068] Particular B. thetaiotaomicron HSGAG lyase polypeptides of
the present invention have an amino acid sequence sufficiently
identical to the amino acid sequence of SEQ ID Nos:2, 4, 6, 8, 10
or 23. In the context of an amino acid sequence, the term
"sufficiently identical" or "substantially identical" is used
herein to refer to a first amino acid that contains a sufficient or
minimum number of amino acid residues that are i) identical to or
ii) conservative substitutions of to aligned amino acid residues in
a second amino acid sequence such that the first and second amino
acid sequences have a common structural fold and/or a common
functional activity. For example, amino acid sequences that contain
a common structural domain having at least about 60%, or 65%
identity, likely 75% identity, more likely 85%, 90%. 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98% or 99% identity are termed sufficiently or
substantially identical. In the context of nucleotide sequence, the
term "sufficiently identical" or "substantially identical" is used
herein to refer to a first nucleic acid sequence that contains a
sufficient or minimum number of nucleotides that are identical to
aligned nucleotides in a second nucleic acid sequence such that the
first and second nucleotide sequences have a common functional
activity or encode a common structural polypeptide fold or a common
functional polypeptide activity.
[0069] The methods taught herein are sometimes described with
reference to heparin-like glycoaminoglycans (HLGAGs) but the
properties taught herein can be extended to other polysaccharides.
As used herein the terms "HLGAG" and "glycosaminoglycans" (GAGs)
are used interchangeably to refer to a family of molecules having
heparin like structures and properties, generally referred to
herein as "heparin". These molecules include but are not limited to
low molecular weight heparin (LMWH), unfractioned heparin,
biotechnologically prepared heparin, chemically modified heparin,
synthetic heparin such as pentasaccharides (e.g., ARIXTRA.TM.),
heparin mimetics and heparan sulfate. The term "biotechnological
heparin" encompasses heparin that is prepared from natural sources
of polysaccharides which have been chemically modified and is
described in Razi et al., Bioche. J. 1995 Jul. 15; 309 (Pt 2):
465-72. Chemically modified heparin is described in Yates et al.,
Carbohydrate Res 1996 Nov. 20; 294:15-27, and is known to those of
skill in the art. Synthetic heparin is well known to those of skill
in the art and is described in Petitou, M. et al., Bioorg Med Chem.
Lett. 1999 Apr. 19; 9(8): 1161-6 and Vlodavsky et al., Int. J.
Cancer, 1999, 83:424-431. An example of a synthetic heparin is
fondaparinux. Fondaparinux (ARIXTRA.TM.) is a 5 unit synthetic
glycoaminoglycan corresponding to the AT-III binding site. Heparan
Sulfate refers to a glycoaminoglycan containing a disaccharide
repeat unit similar to heparin, but which has more N-acetyl groups
and fewer N- and O-sulfate groups. Heparin mimetics are
monosaccharides (e.g., sucralfate), oligosaccharides, or
polysaccharides having at least one biological activity of heparin
(i.e., anticoagulation, inhibition of cancer, treatment of lung
disorders, etc.). Preferably these molecules are highly sulfated.
Heparin mimetics may be naturally occurring, synthetic or
chemically modified. (Barchi, J. J., Curr. Pharm. Des., 2000 Mar.
6(4):485-501). The term "HLGAG" also encompasses functional
variants of the above-described HLGAG molecules. These functional
variants have a similar structure but include slight modifications
to the structure which allow the molecule to retain most of its
biological activity or have increased biological activity.
[0070] "LMWH" as used herein refers to a preparation of sulfated
glycosaminoglycans (GAGs) having an average molecular weight of
less than 8000 Da, with about at least 60% of the oligosaccharide
chains of a LMWH preparation having a molecular weight of less than
8000 Da. Several LMWH preparations are commercially available, but,
LMWHs can also be prepared from heparin, using e.g., HLGAG
degrading enzymes. HLGAG degrading enzymes include but are not
limited to heparinase-I, heparinase-II, heparinase-III, heparinase
IV, heparanase, D-glucuronidase and L-iduronidase. The three
heparinases from Flavobacterium heparinum are enzymatic tools that
have been used for the generation of LMWH (5,000-8,000 Da) and
ultra-low molecular weight heparin (.about.3,000 Da). In addition,
LMWHs can be prepared using, e.g., the B. thetaiotaomicron HSGAG
lyase polypeptides described herein. Commercially available LMWH
include, but are not limited to, enoxaparin (brand name Lovenox;
Aventis Pharmaceuticals), dalteparin (Fragmin, Pharmacia and
Upjohn), certoparin (Sandobarin, Novartis), ardeparin (Normiflo,
Wyeth Lederle), nadroparin (Fraxiparine, Sanofi-Winthrop),
pamaparin (Fluxum, Wassermann), reviparin (Clivarin, Knoll AG), and
tinzaparin (Innohep, Leo Laboratories, Logiparin, Novo Nordisk).
Some preferred forms of LMWH include enoxaparin (Lovenox) and
dalteparin (Fragmin). The term "Arixtra" as used herein refers to a
composition which includes a synthetic pentasaccharide of methyl
O-2-deoxy-6-O-sulfo-2-(sulfoamino)-.alpha.-D-glucopyranosyl-(1.fwdarw.4)--
O-.beta.-D-glucopyranosyl-(1.fwdarw.4)-O-2-deoxy-3,6-di-O-sulfo-2-(sulfoam-
ino)-.alpha.-D-glucopyranosyl-(1.fwdarw.4)-O-2-O-sulfo-.alpha.-L-idopyranu-
ronosyl-(1.fwdarw.4)-2-deoxy-6-O-sulfo-2-(sulfoamino)-.alpha.-D-glucopyran-
oside, decasodium salt and derivatives thereof. A "synthetic
heparin" or "synthetic HLGAG" as used herein refers to HLGAGs are
synthesized compounds and are not derived by fragmentation of
heparin. Methods of preparing synthetic heparins are provided, for
example, in Petitou et al. (1999) Nature 398:417, the contents of
which is incorporated herein by reference.
[0071] "Misexpression or aberrant expression", as used herein,
refers to a non-wildtype pattern of gene expression at the RNA or
protein level. It includes: expression at non-wild type levels,
i.e., over- or under-expression; a pattern of expression that
differs from wild type in terms of the time or stage at which the
gene is expressed, e.g., increased or decreased expression (as
compared with wild type) at a predetermined developmental period or
stage; a pattern of expression that differs from wild type in terms
of altered expression (as compared with wild type) in a
predetermined cell type or tissue type; a pattern of expression
that differs from wild type in terms of the splicing size,
translated amino acid sequence, post-transitional modification, or
biological activity of the expressed polypeptide; a pattern of
expression that differs from wild type in terms of the effect of an
environmental stimulus or extracellular stimulus on expression of
the gene, e.g., a pattern of increased or decreased expression (as
compared with wild type) in the presence of an increase or decrease
in the strength of the stimulus.
[0072] "Subject", as used herein, refers to a mammal organism. In a
preferred embodiment, the subject is a human. In another
embodiment, the subject is an experimental animal or animal
suitable as a disease model. Non-limiting examples of such subjects
include mice, rats, and rabbits. The subject can also be a
non-human animal, e.g., a horse, cow, goat, or other domestic
animal.
[0073] Various aspects of the invention are described in further
detail below.
Isolated Nucleic Acid Molecules
[0074] In one aspect, the invention provides, an isolated or
purified, nucleic acid molecule that encodes a B. thetaiotaomicron
HSGAG lyase polypeptide described herein, e.g., a full length B.
thetaiotaomicron HSGAG lyase protein or a fragment thereof, e.g., a
biologically active portion of B. thetaiotaomicron HSGAG lyase
protein. Also included is a nucleic acid fragment suitable for use
as a hybridization probe, which can be used, e.g., to identify a
nucleic acid molecule encoding a polypeptide of the invention, B.
thetaiotaomicron HSGAG lyase mRNA, and fragments suitable for use
as primers, e.g., PCR primers for the amplification or mutation of
nucleic acid molecules.
[0075] In one embodiment, an isolated nucleic acid molecule of the
invention includes the nucleotide sequence shown in SEQ ID NO: 1 or
SEQ ID NO:5, or a portion of any of these nucleotide sequences. In
one embodiment, the nucleic acid molecule includes sequences
encoding the B. thetaiotaomicron HSGAG lyase protein (i.e., "the
coding region" of SEQ ID NO: 1 or SEQ ID NO:5), as well as 5'
untranslated sequences. Alternatively, the nucleic acid molecule
can include no flanking sequences which normally accompany the
subject sequence. In another embodiment, an isolated nucleic acid
molecule of the invention includes a nucleic acid molecule which is
a complement of the nucleotide sequence shown in SEQ ID NO: 1 or
SEQ ID NO:5, or a portion of any of these nucleotide sequences. In
other embodiments, the nucleic acid molecule of the invention is
sufficiently complementary to the nucleotide sequence shown in SEQ
ID NO: 1 or SEQ ID NO:5, such that it can hybridize to the
nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:5, thereby
forming a stable duplex.
[0076] In one embodiment, an isolated nucleic acid molecule of the
present invention includes a nucleotide sequence which is at least
about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, or more homologous to the entire length of the
nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO:5, or a
portion, preferably of the same length, of any of these nucleotide
sequences.
[0077] A nucleic acid molecule of the invention can include only a
portion of the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID
NO:5. A nucleic acid includes a nucleotide sequence that includes
part, or all, of the coding region and extends into either (or
both) the 5' or 3' noncoding region. Other embodiments include a
fragment which includes a nucleotide sequence encoding an amino
acid fragment described herein, particularly fragments thereof
which are at least 100 amino acids in length. Fragments also
include nucleic acid sequences corresponding to specific amino acid
sequences described above or fragments thereof. Nucleic acid
fragments should not to be construed as encompassing those
fragments that may have been disclosed prior to the invention.
[0078] A nucleic acid fragment encoding a "biologically active
portion of a B. thetaiotaomicron HSGAG lyase polypeptide" can be
prepared by isolating a portion of the nucleotide sequence of SEQ
ID NO: 1, 3, 5, 7, 9 or 22, which encodes a polypeptide having a
HSGAG lyase biological activity (e.g., the biological activities of
HSGAG lyase proteins are described herein), expressing the encoded
portion of the B. thetaiotaomicron HSGAG lyase protein (e.g., by
recombinant expression in vitro) and assessing the activity of the
encoded portion of the B. thetaiotaomicron HSGAG lyase protein. A
nucleic acid fragment encoding a biologically active portion of a
B. thetaiotaomicron HSGAG lyase polypeptide, may comprise a
nucleotide sequence which is greater than 300 or more nucleotides
in length.
[0079] In preferred embodiments, a nucleic acid includes a
nucleotide sequence which is about 300, 400, 500, 600, 700, 800,
900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or
more nucleotides in length and hybridizes under stringent
hybridization conditions to a nucleic acid molecule of SEQ ID NO:
1, 3, 5, 7, 9 or 22.
[0080] The invention encompasses nucleic acid molecules that differ
from the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9 or
22. Such differences can be due to degeneracy of the genetic code
(and result in a nucleic acid which encodes the same B.
thetaiotaomicron HSGAG lyase proteins as those encoded by the
nucleotide sequence disclosed herein. In another embodiment, an
isolated nucleic acid molecule of the invention has a nucleotide
sequence encoding a protein having an amino acid sequence which
differs, by at least 1, but less than 5, 10, 20, 50, or 100 amino
acid residues that shown in SEQ ID NO:2, 4, 6, 8, 10 or 23. If
alignment is needed for this comparison the sequences should be
aligned for maximum homology. "Looped" out sequences from deletions
or insertions, or mismatches, are considered differences.
[0081] Nucleic acids of the inventor can be chosen for having
codons, which are preferred, or non-preferred, for a particular
expression system. E.g., the nucleic acid can be one in which at
least one codon, at preferably at least 10%, or 20% of the codons
has been altered such that the sequence is optimized for expression
in E. coli, yeast, human, insect, or CHO cells.
[0082] Nucleic acid variants can be naturally occurring, such as
allelic variants (same locus) and homologs (different locus), or
can be non naturally occurring. Non-naturally occurring variants
can be made by mutagenesis techniques, including those applied to
polynucleotides, cells, or organisms. The variants can contain
nucleotide substitutions, deletions, inversions and insertions.
Variation can occur in either or both the coding and non-coding
regions. The variations can produce both conservative and
non-conservative amino acid substitutions (as compared in the
encoded product).
[0083] In a preferred embodiment, the nucleic acid differs from
that of SEQ ID NO: 1, 3, 5, 7, 9 or 23, e.g., as follows: by at
least one but less than 10, 20, 30, or 40 nucleotides; at least one
but less than 1%, 5%, 10% or 20% of the nucleotides in the subject
nucleic acid. If necessary for this analysis the sequences should
be aligned for maximum homology. "Looped" out sequences from
deletions or insertions, or mismatches, are considered
differences.
[0084] Homologs and allelic variants can be identified using
methods known in the art. These variants comprise a nucleotide
sequence encoding a polypeptide that is 50%, at least about 55%,
typically at least about 70-75%, more typically at least about
80-85%, and most typically at least about 90-95% or more identical
to the nucleotide sequence shown in SEQ ID NO:2, 4, 6, 8, 10 or 23,
or a fragment of these sequences. Such nucleic acid molecules can
readily be identified as being able to hybridize under stringent
conditions, to the nucleotide sequence shown in SEQ ID NO 2, SEQ ID
NO:6, or a fragment of the sequence. Nucleic acid molecules
corresponding to homologs and allelic variants of the B.
thetaiotaomicron HSGAG lyase DNAs of the invention can further be
isolated by mapping to the same chromosome or locus as the B.
thetaiotaomicron HSGAG lyase gene.
[0085] Preferred variants include those that are correlated with a
HSGAG lyase activity described herein.
[0086] Allelic variants of B. thetaiotaomicron HSGAG lyase include
both functional and non-functional proteins. Functional allelic
variants are naturally occurring amino acid sequence variants of
the B. thetaiotaomicron HSGAG lyase protein within a population
that maintain one or more HSGAG lyase activity. Functional allelic
variants will typically contain only conservative substitution of
one or more amino acids of SEQ ID NO:2, or substitution, deletion
or insertion of non-critical residues in non-critical regions of
the protein. Examples of functional variants include the M17, Q26,
Q23 and K169 variants described herein (i.e., SEQ ID NOs:4, 23, 8
and 10, respectively). Non-functional allelic variants are
naturally-occurring amino acid sequence variants of the B.
thetaiotaomicron HSGAG lyase protein within a population that do
not have one or more of the HSGAG lyase activities described
herein. Non-functional allelic variants will typically contain a
non-conservative substitution, a deletion, or insertion, or
premature truncation of the amino acid sequence of SEQ ID NO:2 or
SEQ ID NO:6, or a substitution, insertion, or deletion in critical
residues or critical regions of the protein.
[0087] Moreover, nucleic acid molecules encoding other B.
thetaiotaomicron HSGAG lyase family members and, thus, which have a
nucleotide sequence which differs from the B. thetaiotaomicron
HSGAG lyase sequences of SEQ ID NO: 1 or SEQ ID NO:5 are intended
to be within the scope of the invention.
Isolated B. thetaiotaomicron HSGAG Lyase Polypeptides
[0088] In another aspect, the invention features, isolated B.
thetaiotaomicron HSGAG lyase proteins, and fragments thereof, e.g.,
biologically active portions thereof. B. thetaiotaomicron HSGAG
lyase protein can be isolated from cells or tissue sources using
standard protein purification techniques. B. thetaiotaomicron HSGAG
lyase protein or fragments thereof can be produced by recombinant
DNA techniques or synthesized chemically.
[0089] Polypeptides of the invention include those which arise as a
result of the existence of multiple genes, alternative
transcription events, alternative RNA splicing events, and
alternative translational and post-translational events. The
polypeptide can be expressed in systems, e.g., cultured cells,
which result in substantially the same post-translational
modifications present when expressed the polypeptide is expressed
in a native cell, or in systems which result in the alteration or
omission of post-translational modifications, e.g., glycosylation
or cleavage, present when expressed in a native cell.
[0090] In a preferred embodiment, a B. thetaiotaomicron HSGAG lyase
polypeptide has one or more of the following characteristics: (1)
binds a heparin and/or a heparan sulfate; (2) cleaves one or more
glycosidic linkages of a heparin and/or a heparan sulfate; (3)
modulates, e.g., increases or reduces, anti-Xa activity and/or
anti-IIa activity of a heparin and/or a heparan sulfate; and (4)
reduces or eliminates angiogenesis.
[0091] In some embodiments, the B. thetaiotaomicron HSGAG lyase is
B. thetaiotaomicron HSGAG lyase I and the B. thetaiotaomicron HSGAG
lyase I can have one or more of the following activities: (1) binds
a heparin and/or heparan sulfate; (2) cleaves one or more
glycosidic linkages of heparin and/or heparan sulfate, e.g.,
cleaves one or more glycosidic linkages of sulfated uronic acids,
e.g., 2-O and/or 3-O sulfated uronic acids; (3) reduces anti-Xa
activity and/or anti-IIa activity of a heparin and/or a heparan
sulfate, e.g., as compared to a reference standard, e.g., the
anti-Xa activity and/or anti-IIa activity of a commercially
available heparin or heparan sulfate or of the heparin or heparan
sulfate prior to cleavage. In some embodiments, anti-Xa activity is
reduced while anti-IIa activity is maintained or increased. In
other embodiments, anti-IIa activity is reduced while anti-Xa
activity is maintained or enhanced. In other embodiments, anti-Xa
activity and anti-IIa activity are reduced.
[0092] In some embodiments, the B. thetaiotaomicron HSGAG lyase is
B. thetaiotaomicron HSGAG lyase II and the B. thetaiotaomicron
HSGAG lyase II can have one or more of the following activities:
(1) binds a heparin and/or heparan sulfate; (2) cleaves one or more
glycosidic linkages of heparin and/or heparan sulfate, e.g.,
cleaves one or more glycosidic linkages of sulfated and
undersulfated uronic acids; (3) increases anti-Xa activity and/or
anti-IIa activity of a heparin and/or a heparan sulfate, e.g., as
compared to a reference standard, e.g., the anti-Xa activity and/or
anti-IIa activity of a commercially available heparin or heparan
sulfate or of the heparin or heparan sulfate prior to cleavage. In
some embodiments, anti-Xa activity is increased while anti-IIa
activity is maintained or reduced. In other embodiments, anti-IIa
activity is increased while anti-Xa activity is maintained or
reduced. In other embodiments, anti-Xa activity and anti-IIa
activity are increased.
[0093] In a preferred embodiment, the B. thetaiotaomicron HSGAG
lyase protein, or fragment thereof, differs from the corresponding
sequence in SEQ ID NO:2, 4, 6, 8, 10 or 23. In one embodiment, it
differs by at least one but by less than 15, 10 or 5 amino acid
residues. In another, it differs from the corresponding sequence in
SEQ ID NO:2, 4, 6, 8, 10 or 23 by at least one residue but less
than 20%, 15%, 10% or 5% of the residues in it differ from the
corresponding sequence in SEQ ID NO:2, 4, 6, 8, 10 or 23. (If this
comparison requires alignment the sequences should be aligned for
maximum homology. "Looped" out sequences from deletions or
insertions, or mismatches, are considered differences.) The
differences are, preferably, differences or changes at a non
essential residue or a conservative substitution.
[0094] Other embodiments include a protein that contain one or more
changes in amino acid sequence, e.g., a change in an amino acid
residue which is not essential for activity. Such B.
thetaiotaomicron HSGAG lyase proteins differ in amino acid sequence
from SEQ ID NO:2, 4, 6, 8, 10 or 23, yet retain biological
activity.
[0095] In one embodiment, the protein includes an amino acid
sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, 98% or more homologous to SEQ ID NO:2, 4, 6, 8, 10 or
23.
[0096] Biologically active portions, in which regions of the
protein are deleted, can be prepared by recombinant techniques and
evaluated for one or more of the functional activities of a native
B. thetaiotaomicron HSGAG lyase protein.
[0097] In a preferred embodiment, the B. thetaiotaomicron HSGAG
lyase protein has an amino acid sequence shown in SEQ ID NOs:2, 4,
6, 8, 10 or 23. In other embodiments, the B. thetaiotaomicron HSGAG
lyase protein is substantially identical to SEQ ID NOs:2, 4, 6, 8,
10 or 23. In yet another embodiment, the B. thetaiotaomicron HSGAG
lyase protein is substantially identical to SEQ ID NOs:2, 4, 6, 8,
10 or 23 and retains the functional activity of the protein of SEQ
ID NO:2, 4, 6, 8, 10 or 23, as described in detail in the
subsections above.
[0098] In another aspect, the invention provides B.
thetaiotaomicron HSGAG lyase chimeric or fusion proteins. As used
herein, a B. thetaiotaomicron HSGAG lyase "chimeric protein" or
"fusion protein" includes a B. thetaiotaomicron HSGAG lyase
polypeptide linked to a non-B. thetaiotaomicron HSGAG lyase
polypeptide. A "non-B. thetaiotaomicron HSGAG lyase polypeptide"
refers to a polypeptide having an amino acid sequence corresponding
to a protein which is not substantially homologous to the B.
thetaiotaomicron HSGAG lyase protein, e.g., a protein which is
different from the B. thetaiotaomicron HSGAG lyase protein and
which is derived from the same or a different organism. The B.
thetaiotaomicron HSGAG lyase polypeptide of the fusion protein can
correspond to all or a portion e.g., a fragment described herein,
of a B. thetaiotaomicron HSGAG lyase amino acid sequence. In a
preferred embodiment, a B. thetaiotaomicron HSGAG lyase fusion
protein includes at least one (or two) biologically active portion
of a B. thetaiotaomicron HSGAG lyase protein. The non-B.
thetaiotaomicron HSGAG lyase polypeptide can be fused to the
N-terminus or C-terminus of the B. thetaiotaomicron HSGAG lyase
polypeptide.
[0099] The fusion protein can include a moiety which has a high
affinity for a ligand. For example, the fusion protein can be a
GST-B. thetaiotaomicron HSGAG lyase fusion protein in which the B.
thetaiotaomicron HSGAG lyase sequences are fused to the C-terminus
of the GST sequences. Such fusion proteins can facilitate the
purification of recombinant B. thetaiotaomicron HSGAG lyase.
Alternatively, the fusion protein can be a B. thetaiotaomicron
HSGAG lyase protein containing a heterologous signal sequence at
its N-terminus. In certain host cells (e.g., mammalian host cells),
expression and/or secretion of B. thetaiotaomicron HSGAG lyase can
be increased through use of a heterologous signal sequence.
[0100] Moreover, the B. thetaiotaomicron HSGAG lyase-fusion
proteins of the invention can be used as immunogens to produce
anti-B. thetaiotaomicron HSGAG lyase antibodies in a subject, to
purify B. thetaiotaomicron HSGAG lyase ligands and in screening
assays to identify molecules which inhibit the interaction of B.
thetaiotaomicron HSGAG lyase with a B. thetaiotaomicron HSGAG lyase
substrate.
[0101] Expression vectors are commercially available that already
encode a fusion moiety (e.g., a GST polypeptide). A B.
thetaiotaomicron HSGAG lyase-encoding nucleic acid can be cloned
into such an expression vector such that the fusion moiety is
linked in-frame to the B. thetaiotaomicron HSGAG lyase protein.
[0102] In another aspect, the invention also features a variant of
a B. thetaiotaomicron HSGAG lyase polypeptide, e.g., which
functions as an agonist (mimetics). Variants of the B.
thetaiotaomicron HSGAG lyase proteins can be generated by
mutagenesis, e.g., discrete point mutation, the insertion or
deletion of sequences or the truncation of a B. thetaiotaomicron
HSGAG lyase protein. An agonist of the B. thetaiotaomicron HSGAG
lyase proteins can retain substantially the same, or a subset, of
the biological activities of the naturally occurring form of a B.
thetaiotaomicron HSGAG lyase protein.
[0103] Variants of a B. thetaiotaomicron HSGAG lyase protein can be
identified by screening combinatorial libraries of mutants, e.g.,
truncation mutants, of a B. thetaiotaomicron HSGAG lyase protein
for agonist activity. Variants of a B. thetaiotaomicron HSGAG lyase
I include the M17 variant as shown in SEQ ID NO:4 and the Q26
variant as shown in SEQ ID NO:23. Variants of a B. thetaiotaomicron
HSGAG lyase II include the Q23 variant as shown in SEQ ID NO:8 and
the K169 variant as shown in SEQ ID NO:10.
[0104] Libraries of fragments e.g., N terminal, C terminal, or
internal fragments, of a B. thetaiotaomicron HSGAG lyase protein
coding sequence can be used to generate a variegated population of
fragments for screening and subsequent selection of variants of a
B. thetaiotaomicron HSGAG lyase protein. Variants in which a
cysteine residues is added or deleted or in which a residue which
is glycosylated is added or deleted are particularly preferred.
[0105] Methods for screening gene products of combinatorial
libraries made by point mutations or truncation, and for screening
cDNA libraries for gene products having a selected property are
known in the art. Such methods are adaptable for rapid screening of
the gene libraries generated by combinatorial mutagenesis of B.
thetaiotaomicron HSGAG lyase proteins. Recursive ensemble
mutagenesis (REM), a new technique which enhances the frequency of
functional mutants in the libraries, can be used in combination
with the screening assays to identify B. thetaiotaomicron HSGAG
lyase variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA
89:7811-7815; Delgrave et al. (1993) Protein Engineering
6:327-331).
[0106] Cell based assays can be exploited to analyze a variegated
B. thetaiotaomicron HSGAG lyase library. For example, a library of
expression vectors can be transfected into a cell line, e.g., a
cell line, which ordinarily responds to B. thetaiotaomicron HSGAG
lyase in a substrate-dependent manner. The transfected cells are
then contacted with B. thetaiotaomicron HSGAG lyase and the effect
of the expression of the mutant on the activity of the B.
thetaiotaomicron HSGAG lyase substrate can be detected, e.g., by
measuring cleavage of heparin or heparan sulfate. Plasmid DNA can
then be recovered from the cells which score for inhibition, or
alternatively, potentiation of signaling by the B. thetaiotaomicron
HSGAG lyase substrate, and the individual clones further
characterized.
[0107] In another aspect, the invention features a method of making
a fragment or analog of a naturally occurring B. thetaiotaomicron
HSGAG lyase polypeptide. The method includes: altering the
sequence, e.g., by substitution or deletion of one or more
residues, of a B. thetaiotaomicron HSGAG lyase polypeptide, e.g.,
altering the sequence of a non-conserved region, or a domain or
residue described herein, and testing the altered polypeptide for
the desired activity, e.g., as described above.
Recombinant Expression Vectors, Host Cells and Genetically
Engineered Cells
[0108] In another aspect, the invention includes, vectors,
preferably expression vectors, containing a nucleic acid encoding a
polypeptide described herein. As used herein, the term "vector"
refers to a nucleic acid molecule capable of transporting another
nucleic acid to which it has been linked and can include a plasmid,
cosmid or viral vector. The vector can be capable of autonomous
replication or it can integrate into a host DNA. Viral vectors
include, e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses.
[0109] A vector can include a B. thetaiotaomicron HSGAG lyase
nucleic acid in a form suitable for expression of the nucleic acid
in a host cell. Preferably the recombinant expression vector
includes one or more regulatory sequences operatively linked to the
nucleic acid sequence to be expressed. The term "regulatory
sequence" includes promoters, enhancers and other expression
control elements (e.g., polyadenylation signals). Regulatory
sequences include those which direct constitutive expression of a
nucleotide sequence, as well as tissue-specific regulatory and/or
inducible sequences. The design of the expression vector can depend
on such factors as the choice of the host cell to be transformed,
the level of expression of protein desired, and the like. The
expression vectors of the invention can be introduced into host
cells to thereby produce proteins or polypeptides, including fusion
proteins or polypeptides, encoded by nucleic acids as described
herein (e.g., B. thetaiotaomicron HSGAG lyase proteins, mutant
forms of B. thetaiotaomicron HSGAG lyase proteins, fusion proteins,
and the like).
[0110] The recombinant expression vectors of the invention can be
designed for expression of B. thetaiotaomicron HSGAG lyase proteins
in prokaryotic or eukaryotic cells. For example, polypeptides of
the invention can be expressed in E. coli, insect cells (e.g.,
using baculovirus expression vectors), yeast cells or mammalian
cells. Suitable host cells are discussed further in Goeddel, (1990)
Gene Expression Technology: Methods in Enzymology 185, Academic
Press, San Diego, Calif. Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0111] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, a proteolytic cleavage site is
introduced at the junction of the fusion moiety and the recombinant
protein to enable separation of the recombinant protein from the
fusion moiety subsequent to purification of the fusion protein.
Such enzymes, and their cognate recognition sequences, include
Factor Xa, thrombin and enterokinase. Typical fusion expression
vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and
Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs,
Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the target recombinant protein.
[0112] Purified fusion proteins can be used in B. thetaiotaomicron
HSGAG lyase activity assays, (e.g., direct assays or competitive
assays described in detail below), or to generate antibodies
specific for B. thetaiotaomicron HSGAG lyase proteins. In a
preferred embodiment, a fusion protein expressed in a retroviral
expression vector of the present invention can be used to infect
bone marrow cells which are subsequently transplanted into
irradiated recipients. The pathology of the subject recipient is
then examined after sufficient time has passed (e.g., six
weeks).
[0113] To maximize recombinant protein expression in E. coli is to
express the protein in a host bacteria with an impaired capacity to
proteolytically cleave the recombinant protein (Gottesman, S.,
(1990) Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. 119-128). Another strategy is to
alter the nucleic acid sequence of the nucleic acid to be inserted
into an expression vector so that the individual codons for each
amino acid are those preferentially utilized in E. coli (Wada et
al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of
nucleic acid sequences of the invention can be carried out by
standard DNA synthesis techniques.
[0114] The B. thetaiotaomicron HSGAG lyase expression vector can be
a yeast expression vector, a vector for expression in insect cells,
e.g., a baculovirus expression vector or a vector suitable for
expression in mammalian cells.
[0115] When used in mammalian cells, the expression vector's
control functions can be provided by viral regulatory elements. For
example, commonly used promoters are derived from polyoma,
Adenovirus 2, cytomegalovirus and Simian Virus 40.
[0116] In another embodiment, the promoter is an inducible
promoter, e.g., a promoter regulated by a steroid hormone, by a
polypeptide hormone (e.g., by means of a signal transduction
pathway), or by a heterologous polypeptide (e.g., the
tetracycline-inducible systems, "Tet-On" and "Tet-Off"; see, e.g.,
Clontech Inc., CA, Gossen and Bujard (1992) Proc. Natl. Acad. Sci.
USA 89:5547, and Paillard (1989) Human Gene Therapy 9:983).
[0117] In still another embodiment, the recombinant mammalian
expression vector is capable of directing expression of the nucleic
acid preferentially in a particular cell type (e.g.,
tissue-specific regulatory elements are used to express the nucleic
acid). Non-limiting examples of suitable tissue-specific promoters
include the albumin promoter (liver-specific; Pinkert et al. (1987)
Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and
Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of
T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733)
and immunoglobulins (Baneiji et al. (1983) Cell 33:729-740; Queen
and Baltimore (1983) Cell 33:741-748), neuron-specific promoters
(e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc.
Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters
(Edlund et al. (1985) Science 230:912-916), and mammary
gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No.
4,873,316 and European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example, the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546).
[0118] The invention further provides a recombinant expression
vector comprising a DNA molecule of the invention cloned into the
expression vector in an antisense orientation. Regulatory sequences
(e.g., viral promoters and/or enhancers) operatively linked to a
nucleic acid cloned in the antisense orientation can be chosen
which direct the constitutive, tissue specific or cell type
specific expression of antisense RNA in a variety of cell types.
The antisense expression vector can be in the form of a recombinant
plasmid, phagemid or attenuated virus.
[0119] Another aspect the invention provides a host cell which
includes a nucleic acid molecule described herein, e.g., a B.
thetaiotaomicron HSGAG lyase nucleic acid molecule within a
recombinant expression vector or a B. thetaiotaomicron HSGAG lyase
nucleic acid molecule containing sequences which allow it to
homologously recombine into a specific site of the host cell's
genome. The terms "host cell" and "recombinant host cell" are used
interchangeably herein. Such terms refer not only to the particular
subject cell but to the progeny or potential progeny of such a
cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0120] A host cell can be any prokaryotic or eukaryotic cell. For
example, a B. thetaiotaomicron HSGAG lyase protein can be expressed
in bacterial cells (such as E. coli), insect cells, yeast or
mammalian cells (such as Chinese hamster ovary cells (CHO) or COS
cells). Other suitable host cells are known to those skilled in the
art.
[0121] Vector DNA can be introduced into host cells via
conventional transformation or transfection techniques. As used
herein, the terms "transformation" and "transfection" are intended
to refer to a variety of art-recognized techniques for introducing
foreign nucleic acid (e.g., DNA) into a host cell, including
calcium phosphate or calcium chloride coprecipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation.
[0122] A host cell of the invention can be used to produce (i.e.,
express) a B. thetaiotaomicron HSGAG lyase protein. Accordingly,
the invention further provides methods for producing a B.
thetaiotaomicron HSGAG lyase protein using the host cells of the
invention. In one embodiment, the method includes culturing the
host cell of the invention (into which a recombinant expression
vector encoding a B. thetaiotaomicron HSGAG lyase protein has been
introduced) in a suitable medium such that a B. thetaiotaomicron
HSGAG lyase protein is produced. In another embodiment, the method
further includes isolating a B. thetaiotaomicron HSGAG lyase
protein from the medium or the host cell.
[0123] In another aspect, the invention features, a cell or
purified preparation of cells which include a B. thetaiotaomicron
HSGAG lyase transgene, or which otherwise misexpress B.
thetaiotaomicron HSGAG lyase. The cell preparation can consist of
human or non-human cells, e.g., rodent cells, e.g., mouse or rat
cells, rabbit cells, or pig cells. In preferred embodiments, the
cell or cells include a B. thetaiotaomicron HSGAG lyase transgene,
e.g., a heterologous form of a B. thetaiotaomicron HSGAG lyase,
e.g., a gene derived from humans (in the case of a non-human cell).
The B. thetaiotaomicron HSGAG lyase transgene can be misexpressed,
e.g., overexpressed or underexpressed. In other preferred
embodiments, the cell or cells include a gene that mis-expresses an
endogenous B. thetaiotaomicron HSGAG lyase, e.g., a gene the
expression of which is disrupted, e.g., a knockout. Such cells can
serve as a model for studying disorders that are related to mutated
or mis-expressed B. thetaiotaomicron HSGAG lyase alleles or for use
in drug screening.
[0124] In another aspect, the invention features, a human cell,
e.g., a hematopoietic stem cell, transformed with nucleic acid
which encodes a subject B. thetaiotaomicron HSGAG lyase
polypeptide.
[0125] Also provided are cells in which a B. thetaiotaomicron HSGAG
lyase is under the control of a regulatory sequence that does not
normally control the expression of the endogenous B.
thetaiotaomicron HSGAG lyase gene. The expression characteristics
of an endogenous gene within a cell, e.g., a cell line or
microorganism, can be modified by inserting a heterologous DNA
regulatory element into the genome of the cell such that the
inserted regulatory element is operably linked to the endogenous B.
thetaiotaomicron HSGAG lyase gene. For example, an endogenous B.
thetaiotaomicron HSGAG lyase gene which is "transcriptionally
silent," e.g., not normally expressed, or expressed only at very
low levels, may be activated by inserting a regulatory element
which is capable of promoting the expression of a normally
expressed gene product in that cell. Techniques such as targeted
homologous recombinations, can be used to insert the heterologous
DNA as described in, e.g., Chappel, U.S. Pat. No. 5,272,071; WO
91/06667, published in May 16, 1991.
Uses
[0126] As described herein, the B. thetaiotaomicron HSGAG lyase
molecules of the invention are useful in many applications
including, but not limited to: 1) characterization of GAGs such as
heparins and heparan sulfates in terms of chemical composition
(di-, tri-, tetra-, penta-, hexa-, octa-, and/or
deca-oligosaccharides); 2) characterization of a pharmaceutical
formulation of GAGs such as a formulation of heparin or a heparan
sulfate; 3) fractionation of a GAG such as a heparin and a heparan
sulfate into both its chemical constituents as well as into smaller
fragments of defined length, sequence, and potential bioactivities;
4) in vitro neutralization of the anticoagulant activity (anti-Xa)
of a heparin or a heparan sulfate; 5) identification of the
presence and purity of a particular GAG such as a heparin or a
heparan sulfate in a sample; 6) determination of the composition of
a GAG in a sample; 7) determination of the sequence of di-, tetra-,
hexa-, octa- and deca-saccharide units in a particular heparin or
heparan sulfate; 8) use as an additional analytic tool for chemical
analysis using techniques such as mass spectrometry, NMR
spectroscopy, gel electrophoresis, capillary electrophoresis, HPLC,
and ion-pair HPLC; 9) for cleaving a particular GAG such as a
heparin or heparan sulfate that comprises at least two disaccharide
units; 10) for inhibiting angiogenesis, e.g., through
administration to a subject in need thereof an effective amount of
a composition (e.g., a pharmaceutical composition) containing B.
thetaiotaomicron HSGAG lyase molecules; 1) for treating cancer
through the administration to a subject a composition (e.g., a
pharmaceutical composition) containing B. thetaiotaomicron HSGAG
lyase molecules; 12) inhibiting cellular proliferation through the
administration to a subject in need thereof an effective amount of
a composition (e.g., a pharmaceutical composition) containing B.
thetaiotaomicron HSGAG lyase molecules for inhibiting cellular
proliferation; 13) for ex vivo neutralization of the anti-Xa
activity of a preparation (e.g., a pharmaceutical preparation) of a
heparin or a heparan sulfate previously administered to a subject
for the inhibition of coagulation; 14) for in vivo neutralization
of the anti-Xa activity of preparation (e.g., a pharmaceutical
preparation) of a heparin or a heparan sulfate through
administration to a subject in need of such neutralization (e.g., a
subject to whom a pharmaceutical preparation of a heparin or a
heparan sulfate had previously been administered); 15) for ex vivo
neutralization of the anti-IIa activity of a preparation (e.g.,
pharmaceutical preparation) of a heparin or heparan sulfate
previously administered to a subject for the inhibition of
thrombosis; or 16) for in vivo neutralization of the anti-IIa
activity of preparation (e.g., a pharmaceutical preparation) of a
heparin or a heparan sulfate through administration to a subject in
need in need of such neutralization (e.g., a subject to whom a
pharmaceutical preparation of a heparin or heparan sulfate had
previously been administered).
[0127] Characterization and Sequencing of GAGs
[0128] Methods described herein can be used, e.g., for analyzing
polysaccharides such as GAGs, (e.g., a mixed population of
polysaccharides), e.g., to define the structural signature and/or
activity of a polysaccharides (e.g., a mixed population of
polysaccharides), by contacting the polysaccharide with a B.
thetaiotaomicron HSGAG lyase molecule. A structural signature, as
used herein, refers to information regarding, e.g., the identity
and number the mono- and di-saccharide building blocks of a
polysaccharide, information regarding the physiochemical properties
such as the overall charge (also referred to as the "net charge" or
"total charge"), charge density, molecular size, charge to mass
ratio and the presence of iduronic and/or glucuronic acid content
as well as the relationships between the mono- and di-saccharide
building blocks, and active sites associated with these building
blocks, inter alia. The structural signature can be provided by
determining one or more primary outputs that include the following:
the presence or the amount of one or more component saccharides or
disaccharides; as used herein, "component saccharides" refers to
the saccharides that make up the polysaccharide. Component
saccharides can include monosaccharides, disaccharides,
trisaccharides, etc., and can also include sugars normally found in
nature as well as non-natural and modified sugars, e.g., that
result due to production, processing and/or purification; the
presence or the amount of one or more block components, wherein a
"block component" is made up of more than one saccharide or
polysaccharide; and the presence or amount of one or more modified
saccharides, wherein a modified saccharide is one present in a
starting material used to make a preparation but which is altered
in the production of the preparation, e.g., a saccharide modified
by cleavage. "Sequence" with respect to polysaccharides refers to
the linear arrangement of covalently linked component saccharides,
and can be determined by methods known in the art, e.g., the
methods disclosed herein and in PCT Publication Nos: WO 00/65521,
WO 02/23190, and WO 04/055491; U.S. Publication Nos: 20030191587
and 20040197933; Venkataraman (1999); Shriver et al. (2000a);
Shriver et al. (2000b); and Keiser et al. (2001); the entire
teachings of which are incorporated herein by reference.
"Positioning of the active site" refers to a correlation between a
certain component polysaccharide and a given activity.
[0129] In one embodiment, the invention provides, methods of
evaluating a polysaccharide mixture, e.g., a heterogeneous
population of HLGAGs, by evaluating one or more parameters related
to a structural signature species described herein. Such parameters
can include the presence, size distribution, or quantity of a
structural signature disclosed herein. The structural signature can
be one or more of the following:
##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005##
##STR00006##
[0130] In a preferred embodiment, the structural signature is
determined by one or more methods chosen from the group consisting
of MALDI-MS, ESI-MS, CE, HPLC, FPLC, fluorometry, ELISA,
chromogenic assays such as reverse phase column chromatography
(e.g., HPLC), colorimetric assays, NMR and other spectroscopic
techniques.
[0131] The polysaccharide composition is digested, incompletely or
completely digested, with one or more B. thetaiotaomicron HSGAG
lyase molecule. The composition can further be digested with one or
more HLGAG degrading enzyme. Examples of other HLGAG degrading
enzymes include: heparinase I, heparinase II, heparinase III,
heparinase IV, heparanase, D-glucuronidase, L-iduronidase and
functionally active variants and fragments thereof. Various HLGAG
degrading enzymes, and variants and fragments thereof, are known
and described, e.g., in U.S. Pat. Nos. 5,569,600; 5,389,539;
5,830,726; 5,714376; 5,919,693; 5,681,733 and 6,869,789; and U.S.
Patent Publications Nos: 20030099628; 20030303301; and 20010565375,
the contents of which are incorporated herein by reference.
[0132] The methods described herein can further include: providing
or determining a first structural signature by contacting a batch
of a polysaccharide (e.g., a heterogenous population of
polysaccharides) with a B. thetaiotaomicron HSGAG lyase molecule or
molecules; providing or determining a second structural signature
of a different batch of a polysaccharide (e.g., a heterogenous
population of polysaccharides) by contacting the batch with a B.
thetaiotaomicron HSGAG lyase molecule or molecules; and comparing
the first and second structural determinations to determine if one
or more of the batches has a structural determination associated
with a particular property. The methods can further include
selecting or discarding a batch of the polysaccharide depending on
its structural determination.
[0133] In other embodiments, a batch of a polysaccharide (e.g., a
heterogenous population of polysaccharides) can be analyzed by
comparing one or more structural signature of the polysaccharide
obtained by contacting the polysaccharide with one or more B.
thetaiotaomicron HSGAG lyase molecules to a reference standard. The
reference standard can be, e.g., a preselected range or level
and/or the absence or presence of a structural signature present in
a mixed population of polysaccharides, e.g., a commercially
available population of polysaccharides such as enoxaparin
(Lovenox.TM.); dalteparin (Fragmin.TM.); certoparin
(Sandobarin.TM.); ardeparin (Normiflo.TM.); nadroparin
(Fraxiparin.TM.); pamaparin (Fluxum.TM.); reviparin (Clivarin.TM.);
tinzaparin (Innohep.TM. or Logiparin.TM.), or Fondaparinux
(Arixtra.TM.), that has been digested with the B. thetaiotaomicron
HSGAG lyase molecule or molecules.
[0134] The B. thetaiotaomicron HSGAG lyase molecules can also be
used to determine a reference standard for a drug by analyzing a
composition contacted with a B. thetaiotaomicron HSGAG lyase
molecule or molecules and determining the bioequivalence and/or
bioavailability of one or more of the components in the mixture. As
used herein, "bioequivalence" means "the absence of a significant
difference in the rate and extent to which an active ingredient or
active moiety in pharmaceutical equivalents or pharmaceutical
alternatives becomes available at the site of drug action when
administered at the same molar dose under similar conditions."
[0135] Production of Fractionated HLGAG Preparations
[0136] The B. thetaiotaomicron HSGAG lyase molecules described
herein can be used to produce polysaccharides (e.g., fractionated
heparin or heparan sulfate), e.g., having desired properties, e.g.,
desired activities and/or reduced undesired properties, e.g.,
undesired side effects. As used herein, "desired activities" refers
to those activities that are beneficial for a given indication,
e.g., a positive patient reaction as defined herein, inter alia. An
"undesirable activity" may include those activities that are not
beneficial for a given indication, e.g., a negative patient
reaction, as defined herein, inter alia. A given activity may be a
desired activity for one indication, and an undesired activity for
another, such as anti-IIa activity, which while undesirable for
certain indications, is desirable in others, notably acute or
trauma situations. Thus, the invention relates to methods for
designing heparins, LMWHs or synthetic heparins with ideal product
profiles including, but not limited to such features as high
activity, e.g., high anti-Xa and/or anti-IIa activity, reduced
activity, e.g., reduced anti-Xa and/or anti-IIa activity, well
characterized, neutralizable, lower side effects including reduced
HIT, attractive pharmacokinetics, and/or reduced PF4 binding.
[0137] Fractionated heparins can be designed, e.g., by contacting
composition that includes a mixed population of polysaccharides,
such as glycosaminoglycans (GAGs), HLGAGs, UFH, FH, LMWHs, or
synthetic heparins including but not limited to enoxaparin
(Lovenox.TM.); dalteparin (Fragmin.TM.); certoparin
(Sandobarin.TM.); ardeparin (Normiflo.TM.); nadroparin
(Fraxiparin.TM.); parnaparin (Fluxum.TM.); reviparin
(Clivarin.TM.); tinzaparin (Innohep.TM. or Logiparin.TM.), or
Fondaparinux (Arixtra.TM.) with a B. thetaiotaomicron HSGAG
lyase.
[0138] In some embodiments, a fractionated heparin preparation
having reduced anti-Xa and/or anti-IIa activity is prepared by
contacting a heparin with a B. thetaiotaomicron HSGAG lyase I
molecule. In some embodiments, anti-Xa activity is reduced while
anti-IIa activity is maintained or increased. In other embodiments,
anti-IIa activity is reduced while anti-Xa activity is maintained
or enhanced. In other embodiments, anti-Xa activity and anti-IIa
activity are reduced. Heparins having reduced anti-Xa and/or
anti-IIa activity can be used, e.g., as a carrier to deliver an
agent, e.g., a diagnostic, prophylactic or therapeutic agent. The
heparin molecule can be linked to the agent. Active agents can
include a therapeutic or prophylactic polypeptide, nucleic acid,
small molecule, lipid/glycolipids, etc. In one embodiment, the
active agent is a therapeutic polypeptide selected from the group
consisting of insulin, proinsulin, human growth hormone,
interferon, .alpha.-1 proteinase inhibitor, alkaline phosphotase,
angiogenin, cystic fibrosis transmembrane conductance regulator,
extracellular superoxide dismutase, fibrinogen, glucocerebrosidase,
glutamate decarboxylase, human serum albumin, myelin basic protein,
soluble CD4, lactoferrin, lactoglobulin, lysozyme, lactoalbumin,
erythropoietin, tissue plasminogen activator, antithrombin III,
prolactin, and .alpha.1-antitrypsin. The therapeutic or
prophylactic polypeptide can be an active derivative or fragment of
such polypeptides. The active agent can also be, but is not limited
to one or more of: parathyroid hormone and derivatives and
fragments thereof, erythropoietin, epoetin beta, gene activated
erythropoietin, second generation EPO, novel erythropoiesis
stimulating protein, insulin lispro, insulin (bovine), insulin,
insulin aspart, insulin analogue, Calcitonin, Theraccine,
becaplermin (recombinant human platelet derived growth factor-BB),
trafermin, human growth hormone-releasing factor, BMP-7, PEG
aspariginase, dornase alpha, alglucerase, agalsidase-beta, dornase
alpha, agalsidase-alfa, streptokinase, teneteplase, reteplase,
alteplase, pamiteplase, Rh factor VIII, Rh FVIIa, Factor IX
(Human), Factor IX (complex), HGH, Somatrem/somatropin,
anti-CD33-calicheamicin conjugate, Edrecolomab, rituxumab,
daclizumab, trastuzumab, sulesomab, abciximab, infliximab,
muromonab-CD3, palivizumab, alemtuzumab, basiliximab, oprelvekin,
gemtuzumab ozogamicin, ibritumomab tiuxetan, sulesomab,
palivizumab, interleukin-2, celmoleukin (rIL-2), interferon
alfacon-1, interferon alpha, interferon alpha+ribavirin, peg
interferon alpha-2a, interferon alpha-2b, interferon alpha 3n,
interferon beta-1a, interferon beta, interferon beta 1b, interferon
gamma, interferon gamma-1b, filgrastim, sargramostim, lenograstim,
molgramostim, mirimostim, nartograstim, oprelvekin, peptide
tyrosin-tyrosin (PYY), apolipoprotein A-IV, leptin, melanocortin,
amylin, orexin, adiponectin, and ghrelin. In one embodiment, the
active agent is an active polypeptide, e.g., a therapeutic or
prophylactic polypeptide, and the polypeptide has a molecular
weight of less than 150 kDa, more preferably less than 100 kDa, and
more preferably less than 50 kDa. In one embodiment, the active
agent is an active polypeptide, e.g., a therapeutic or prophylactic
polypeptide, and the polypeptide has a molecular weight of about
500 kDa-5 kDa, 5 to 10 kDa, 10 to 30 kDa, 18 to 35 kDa, 30 to 50
kDa, 50 to 100 kDa, 100 to 150 kDa. In one embodiment, the active
polypeptide is insulin or an active fragments or derivatives
thereof. In another embodiment, the active polypeptide is human
growth hormone or an active fragment or derivative thereof. In yet
another embodiment, the active polypeptide is interferon. In other
embodiments, the heparin molecule is linked to an inactive agent.
Examples of inactive agents include biological probes or contrast
agents for imaging. In another embodiment, the active agent can be
a small molecule drug, e.g., a small molecule drug currently
available for therapeutic, diagnostic, or prophylactic use, or a
drug in development. In some embodiments, the active agent is
linked to one or more heparin molecules in the formulation. As an
example, small molecule drugs, and protein-based drugs may be
linked to heparin molecule for delivery via known chemistries such
as EDC, CNBH.sub.4/DMSO/Acetic Acid, etc.
[0139] The invention also relates to fractionated heparin
preparations having increased anti-Xa and/or anti-IIa activity
prepared by contacting a heparin with a B. thetaiotaomicron HSGAG
lyase II molecule. Such preparation can be used, e.g., to treat or
prevent a disease associated with coagulation, such as thrombosis,
cardiovascular disease, vascular conditions or atrial fibrillation;
migraine, atherosclerosis; an inflammatory disorder, such as
autoimmune disease or atopic disorders; obesity or excess adipose,
an allergy; a respiratory disorder, such as asthma, emphysema,
adult respiratory distress syndrome (ARDS), cystic fibrosis, or
lung reperfusion injury; a cancer or metastatic disorder, e.g.,
lipomas; diabetes; an angiogenic disorder, such as neovascular
disorders of the eye, osteoporosis, psoriasis, arthritis,
Alzheimer's, a subject to undergo, undergoing or having undergone
surgical procedure, organ transplant, orthopedic surgery, treatment
for a fracture, e.g., a hip fracture, hip replacement, knee
replacement, percutaneous coronary intervention (PCI), stent
placement, angioplasty, coronary artery bypass graft surgery
(CABG).
Pharmaceutical Compositions
[0140] The B. thetaiotaomicron HSGAG lyase molecules, as well as
heparin molecules prepared by cleavage with the B. thetaiotaomicron
HSGAG lyase molecules can be incorporated into pharmaceutical
compositions. Such compositions typically include a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" includes solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents, and the like, compatible with
pharmaceutical administration. Supplementary active compounds can
also be incorporated into the compositions.
[0141] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0142] Alternatively, the pharmaceutical composition can be used to
treat a sample (e.g., blood in a bioreactor, e.g., to deheparinize
blood) before the sample is introduced into a subject.
[0143] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It should be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as manitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0144] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0145] Oral compositions generally include an inert diluent or an
edible carrier. For the purpose of oral therapeutic administration,
the active compound can be incorporated with excipients and used in
the form of tablets, troches, or capsules, e.g., gelatin capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash. Pharmaceutically compatible binding agents,
and/or adjuvant materials can be included as part of the
composition. The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring.
[0146] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0147] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0148] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0149] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0150] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active compound calculated to produce the desired therapeutic
effect in association with the required pharmaceutical carrier.
[0151] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
high therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0152] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. A dose may be formulated in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50 (i.e., the concentration of the test compound which
achieves a half-maximal inhibition of symptoms) as determined in
cell culture. Such information can be used to more accurately
determine useful doses in humans. Levels in plasma may be measured,
for example, by high performance liquid chromatography.
[0153] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to the severity of the disease
or disorder, previous treatments, the general health and/or age of
the subject, and other diseases present.
[0154] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0155] Therapeutic Applications
[0156] The B. thetaiotaomicron HSGAG lyase molecules can act as
novel diagnostic and therapeutic agents for controlling one or more
of cellular proliferative and/or differentiative disorders, e.g.,
by preventing or inhibiting angiogenesis of cells otherwise
exhibiting or otherwise associated with unwanted proliferation
and/or differentiation. Examples of cellular and/or differentiative
disorders include: diabetes; arthritis, e.g., rheumatoid arthritis;
ocular disorders, e.g., ocular neovascularization, diabetic
retinopathy, neovascular glaucoma, retinal fibroplasias, uevitis,
eye disorders associated with iris neovasculatization; and cancer,
e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic
neoplastic disorders, e.g., leukemias.
[0157] As used herein, the terms "cancer", "hyperproliferative" and
"neoplastic" refer to cells having the capacity for autonomous
growth. Examples of such cells include cells having an abnormal
state or condition characterized by rapidly proliferating cell
growth. Hyperproliferative and neoplastic disease states may be
categorized as pathologic, i.e., characterizing or constituting a
disease state, or may be categorized as non-pathologic, i.e., a
deviation from normal but not associated with a disease state. The
term is meant to include all types of cancerous growths or
oncogenic processes, metastatic tissues or malignantly transformed
cells, tissues, or organs, irrespective of histopathologic type or
stage of invasiveness. "Pathologic hyperproliferative" cells occur
in disease states characterized by malignant tumor growth. Examples
of non-pathologic hyperproliferative cells include proliferation of
cells associated with wound repair.
[0158] The terms "cancer" or "neoplasms" include malignancies of
the various organ systems, such as affecting lung, breast, thyroid,
lymphoid, gastrointestinal, and genitourinary tract, as well as
adenocarcinomas which include malignancies such as most colon
cancers, renal-cell carcinoma, prostate cancer and/or testicular
tumors, non-small cell carcinoma of the lung, cancer of the small
intestine and cancer of the esophagus.
[0159] The term "carcinoma" is art recognized and refers to
malignancies of epithelial or endocrine tissues including
respiratory system carcinomas, gastrointestinal system carcinomas,
genitourinary system carcinomas, testicular carcinomas, breast
carcinomas, prostatic carcinomas, endocrine system carcinomas, and
melanomas. Exemplary carcinomas include those forming from tissue
of the cervix, lung, prostate, breast, head and neck, colon and
ovary. The term also includes carcinosarcomas, e.g., which include
malignant tumors composed of carcinomatous and sarcomatous tissues.
An "adenocarcinoma" refers to a carcinoma derived from glandular
tissue or in which the tumor cells form recognizable glandular
structures.
[0160] The term "sarcoma" is art recognized and refers to malignant
tumors of mesenchymal derivation.
[0161] Additional examples of proliferative disorders include
hematopoietic neoplastic disorders. As used herein, the term
"hematopoietic neoplastic disorders" includes diseases involving
hyperplastic/neoplastic cells of hematopoietic origin. A
hematopoietic neoplastic disorder can arise from myeloid, lymphoid
or erythroid lineages, or precursor cells thereof. Preferably, the
diseases arise from poorly differentiated acute leukemias, e.g.,
erythroblastic leukemia and acute megakaryoblastic leukemia.
Additional exemplary myeloid disorders include, but are not limited
to, acute promyeloid leukemia (APML), acute myelogenous leukemia
(AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus,
L. (1991) Crit. Rev. in Oncol./Hemotol. 11:267-97); lymphoid
malignancies include, but are not limited to acute lymphoblastic
leukemia (ALL) which includes B-lineage ALL and T-lineage ALL,
chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),
hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).
Additional forms of malignant lymphomas include, but are not
limited to non-Hodgkin lymphoma and variants thereof, peripheral T
cell lymphomas, adult T cell leukemia/lymphoma (ATL), cutaneous
T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF),
Hodgkin's disease and Reed-Sternberg disease.
[0162] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography.
[0163] In another embodiment, the B. thetaiotaomicron HSGAG lyase
molecules, e.g., the B. thetaiotaomicron HSGAG lyase I molecules,
can act as prophylactic or therapeutic agents for controlling
heparin-associated disorders. Examples of such disorders include,
but are not limited to, heparin-induced anticoagulation and/or
angiogenesis. Thus, the B. thetaiotaomicron HSGAG lyase molecules,
e.g., the B. thetaiotaomicron HSGAG lyase I molecules, can be used
to reduce or eliminate (e.g., neutralize) one or more
anticoagulation and/or antithrombotic properties of heparin and/or
heparan sulfate, e.g., during or after surgery. In other
embodiments, the B. thetaiotaomicron HSGAG lyase molecules, e.g.,
the B. thetaiotaomicron HSGAG lyase 1 molecules, can be used to
deheparinized blood, e.g., in a bioreactor, e.g., a bioreactor used
in heart-lung and/or kidney dialysis.
[0164] The B. thetaiotaomicron HSGAG lyase molecules described
herein can also be used to design fractionated HLGAG preparations,
e.g., heparin and/or heparan sulfate preparations. Such
fractionated HLGAG preparations may have many therapeutic
utilities. For instance, it is known that HLGAG compositions are
useful for preventing and treating dementia, such as Alzheimer's
disease, coagulation, angiogenesis, thrombotic disorders,
cardiovascular disease, vascular conditions, atherosclerosis,
respiratory disorders, circulatory shock and related disorders, as
well as inhibiting cancer cell growth and metastasis. Each of these
disorders is well-known in the art and is described, for instance,
in Harrison's Principles of Internal Medicine (McGraw Hill, Inc.,
New York), which is incorporated by reference. The use of HLGAG
compositions in various therapeutic methods is described and
summarized in Huang, J. and Shimamura, A., Coagulation Disorders,
12, 1251-1281 (1998).
[0165] The fractionated HLGAG preparations can be used, e.g., to
treat or prevent a disorder where increased presence of active FGF,
e.g., aFGF and/or bFGF, is desirable.
[0166] The HLGAG preparations are useful for treating or preventing
disorders associated with coagulation. When an imbalance in the
coagulation pathway shifts towards excessive coagulation, the
result is the development of thrombotic tendencies, which are often
manifested as heart attacks, strokes, deep venous thrombosis, acute
coronary syndromes (ACS) such as unstable angina, and myocardial
infarcts. A "disease associated with coagulation" as used herein
refers to a condition characterized by local inflammation which can
result from an interruption or reduction in the blood supply to a
tissue which may occur, for instance, as a result of blockage of a
blood vessel responsible for supplying blood to the tissue such as
is seen for myocardial or cerebral infarction or peripheral
vascular disease, or as a result of emboli formation associated
with conditions such as atrial fibrillation or deep venous
thrombosis. Coagulation disorders include, but are not limited to,
cardiovascular disease and vascular conditions such as cerebral
ischemia. It is particularly useful to treat disorders such as
myocardial infarction and ACS with, e.g., a polysaccharide by
pulmonary delivery because of the fast absorption and action of
this delivery system.
[0167] The fractionated HLGAG preparations are useful for treating
cardiovascular disease. Cardiovascular diseases include, but are
not limited to, acute myocardial infarction, ACS, e.g., unstable
angina, and atrial fibrillation. Myocardial infarction is a disease
state which sometimes occurs with an abrupt decrease in coronary
blood flow that follows a thrombotic occlusion of a coronary artery
previously narrowed by atherosclerosis. Such injury may be produced
or facilitated by factors such as cigarette smoking, hypertension,
and lipid accumulation. Acute angina is due to transient myocardial
ischemia. This disorder is usually associated with a heaviness,
pressure, squeezing, smothering, or choking feeling below the
sternum. Episodes are usually caused by exertion or emotion, but
can occur at rest.
[0168] Atrial fibrillation is a common form of arrhythmia generally
arising as a result of emotional stress or following surgery,
exercise, or acute alcoholic intoxication. Persistent forms of
atrial fibrillation generally occur in patients with cardiovascular
disease. Atrial fibrillation is characterized by disorganized
atrial activity without discrete P waves on the surface ECG. This
disorganized activity can lead to improper blood flow in the atrium
and thrombus formation. These thrombi can embolize, resulting in
cerebral ischemia and other disorders.
[0169] Persons undergoing surgery, anesthesia and extended periods
of bed rest or other inactivity are often susceptible to a
condition known as deep venous thrombosis, or DVT, which is a
clotting of venous blood in the lower extremities and/or pelvis.
This clotting occurs due to the absence of muscular activity in the
lower extremities required to pump the venous blood (stasis), local
vascular injury or a hypercoaguble state. The condition can be
life-threatening if a blood clot migrates to the lung, resulting in
a "pulmonary embolus" or otherwise interferes with cardiovascular
circulation. One method of treatment involves administration of an
anti-coagulant.
[0170] The fractionated HLGAG preparations can be used for the
treatment of cardiovascular disorders alone or in combination with
other therapeutic agents for reducing the risk of a cardiovascular
disease or for treating the cardiovascular disease. Other
therapeutic agents include, but are not limited to,
anti-inflammatory agents, anti-thrombotic agents, anti-platelet
agents, fibrinolytic agents, lipid reducing agents, direct thrombin
inhibitors, anti-Xa inhibitors, anti-IIa inhibitors, glycoprotein
IIb/IIIa receptor inhibitors and direct thrombin inhibitors such as
hirudin, hirugen, Angiomax, agatroban, PPACK, thrombin
aptamers.
[0171] The HLGAG preparations are also useful for treating vascular
conditions. Vascular conditions include, but are not limited to,
disorders such as deep venous thrombosis, peripheral vascular
disease, cerebral ischemia, including stroke, and pulmonary
embolism. A cerebral ischemic attack or cerebral ischemia is a form
of ischemic condition in which the blood supply to the brain is
blocked. This interruption or reduction in the blood supply to the
brain may result from a variety of causes, including an intrinsic
blockage or occlusion of the blood vessel itself, a remotely
originated source of occlusion, decreased perfusion pressure or
increased blood viscosity resulting in inadequate cerebral blood
flow, or a ruptured blood vessel in the subarachnoid space or
intracerebral tissue.
[0172] The HLGAG preparations are useful for treating cerebral
ischemia. Cerebral ischemia may result in either transient or
permanent deficits and the seriousness of the neurological damage
in a patient who has experienced cerebral ischemia depends on the
intensity and duration of the ischemic event. A transient ischemic
attack is one in which the blood flow to the brain is interrupted
only briefly and causes temporary neurological deficits, which
often are clear in less than 24 hours. Symptoms of TIA include
numbness or weakness of face or limbs, loss of the ability to speak
clearly and/or to understand the speech of others, a loss of vision
or dimness of vision, and a feeling of dizziness. Permanent
cerebral ischemic attacks, also called stroke, are caused by a
longer interruption or reduction in blood flow to the brain
resulting from either a thrombus or embolism. A stroke causes a
loss of neurons typically resulting in a neurologic deficit that
may improve but that does not entirely resolve.
[0173] Thromboembolic stroke is due to the occlusion of an
extracranial or intracranial blood vessel by a thrombus or embolus.
Because it is often difficult to discern whether a stroke is caused
by a thrombosis or an embolism, the term "thromboembolism" is used
to cover strokes caused by either of these mechanisms.
[0174] The rapid absorption of HLGAGs, such as UFH or LMWH, after
inhalation can be very valuable in the treatment of venous
thromboembolism. Intravenous administration of UFH has been used
widely for treatment of venous thromboembolism in combination with
oral warfarin. Due to the improved efficacy and reduced risks,
however, LMWHs have been increasingly used as an alternative to
intravenous UFH in treatment of venous thromboembolism. It has been
established that efficacy of heparin therapy depends on achieving
critical therapeutic levels (e.g., of values of anti-factor Xa or
anti-factor IIa activity) within the first 24 hours of treatment.
Intrapulmonary delivery of heparin particles to achieve rapid
therapeutic levels of heparin in the early stage of
thromboembolism, could also be combined with other routes of
administration of LMWHs or heparin for prolonged
antithrombotic/anticoagulant effect such as oral
administration.
[0175] The HLGAG preparations can also be used to treat acute
thromboembolic stroke. An acute stroke is a medical syndrome
involving neurological injury resulting from an ischemic event,
which is an interruption or reduction in the blood supply to the
brain.
[0176] An effective amount of a HLGAG preparation alone or in
combination with another therapeutic for the treatment of stroke is
that amount sufficient to reduce in vivo brain injury resulting
from the stroke. A reduction of brain injury is any prevention of
injury to the brain which otherwise would have occurred in a
subject experiencing a thromboembolic stroke absent the treatment
described herein. Several physiological parameters may be used to
assess reduction of brain injury, including smaller infarct size,
improved regional cerebral blood flow, and decreased intracranial
pressure, for example, as compared to pretreatment patient
parameters, untreated stroke patients or stroke patients treated
with thrombolytic agents alone.
[0177] The pharmaceutical HLGAG preparation may be used alone or in
combination with a therapeutic agent for treating a disease
associated with coagulation. Examples of therapeutics useful in the
treatment of diseases associated with coagulation include
anticoagulation agents, antiplatelet agents, and thrombolytic
agents.
[0178] Anticoagulation agents prevent the coagulation of blood
components and thus prevent clot formation. Anticoagulants include,
but are not limited to, warfarin, Coumadin, dicumarol,
phenprocoumon, acenocoumarol, ethyl biscoumacetate, and indandione
derivatives. "Direct thrombin inhibitors" include hirudin, hirugen,
Angiomax, agatroban, PPACK, thrombin aptamers. Antiplatelet agents
inhibit platelet aggregation and are often used to prevent
thromboembolic stroke in patients who have experienced a transient
ischemic attack or stroke. Thrombolytic agents lyse clots which
cause the thromboembolic stroke. Thrombolytic agents have been used
in the treatment of acute venous thromboembolism and pulmonary
emboli and are well known in the art (e.g. see Hennekens et al, J
Am Coll Cardiol; v. 25 (7 supp), p. 18S-22S (1995); Holmes, et al,
J Am Coll Cardiol; v. 25 (7 suppl), p. 10S-17S(1995)).
[0179] Pulmonary embolism as used herein refers to a disorder
associated with the entrapment of a blood clot in the lumen of a
pulmonary artery, causing severe respiratory dysfunction. Pulmonary
emboli often originate in the veins of the lower extremities where
clots form in the deep leg veins and then travel to lungs via the
venous circulation. Thus, pulmonary embolism often arises as a
complication of deep venous thrombosis in the lower extremity
veins. Symptoms of pulmonary embolism include acute onset of
shortness of breath, chest pain (worse with breathing), and rapid
heart rate and respiratory rate. Some individuals may experience
haemoptysis.
[0180] The HLGAG preparations and methods are also useful for
treating or preventing atherosclerosis. Heparin has been shown to
be beneficial in prevention of atherosclerosis in various
experimental models. Due to the more direct access to the
endothelium of the vascular system, inhaled heparin can be useful
in prevention of atherosclerosis. Atherosclerosis is one form of
arteriosclerosis that is believed to be the cause of most coronary
artery disease, aortic aneurysm and atrial disease of the lower
extremities, as well as contributing to cerebrovascular
disease.
[0181] Due to its fast absorption and variable elimination rate,
HLGAG with or without excipients can be used as an alternative for
the intravenous heparin for surgical and dialysis procedures. For
example, HLGAG particles can be inhaled prior to surgery by
volunteer inhalation or passively inhaled via trachea tube during
the anesthesia prior to or during the surgery. Surgical patients,
especially those over the age of 40 years have an increased risk of
developing deep venous thrombosis. Thus, the use of HLGAG particles
for preventing the development of thrombosis associated with
surgical procedures is contemplated. In addition to general
surgical procedures such as percutaneous intervention (e.g.,
percutaneous coronary intervention (PCI)), PCTA, stents and other
similar approaches, hip or knee replacement, cardiac-pulmonary
by-pass surgery, coronary revascularization surgery, orthopedic
surgery, and prosthesis replacement surgery, the methods are also
useful in subjects undergoing a tissue or organ transplantation
procedure or treatment for fractures such as hip fractures.
[0182] In addition, pulmonary inhalation of heparin is valuable in
treatment of respiratory diseases such as cystic fibrosis, asthma,
allergy, emphysema, adult respiratory distress syndrome (ARDS),
lung reperfusion injury, and ischemia-reperfusion injury of the
lung, kidney, heart, and gut, and lung tumor growth and
metastasis.
[0183] Cystic fibrosis is a chronic progressive disease affecting
the respiratory system. One serious consequence of cystic fibrosis
is Pseudomonas aeruginosa lung infection, which by itself accounts
for almost 90% of the morbidity and mortality in cystic fibrosis.
Therapeutics for treating cystic fibrosis include antimicrobials
for treating the pathogenic infection.
[0184] Heparin is also a well established inhibitor of elastase and
tumor growth and metastasis. The aerosolized heparin particles are
capable of inhibiting elastase induced lung injury in an acute lung
emphysema model. Asthma is a disorder of the respiratory system
characterized by inflammation, narrowing of the airways and
increased reactivity of the airways to inhaled agents. Asthma is
frequently, although not exclusively, associated with atopic or
allergic symptoms. Asthma may also include exercise induced asthma,
bronchoconstrictive response to bronchostimulants, delayed-type
hypersensitivity, auto immune encephalomyelitis and related
disorders. Allergies are generally caused by IgE antibody
generation against allergens. Emphysema is a distention of the air
spaces distal to the terminal bronchiole with destruction of
alveolar septa. Emphysema arises out of elastase induced lung
injury. Heparin is capable of inhibiting this elastase induced
injury. Adult respiratory distress syndrome is a term which
encompasses many acute defuse infiltrative lung lesions of diverse
ideologies which are accompanied by severe atrial hypoxemia. One of
the most frequent causes of ARDS is sepsis. Inflammatory diseases
include but are not limited to autoimmune diseases and atopic
disorders. Other types of inflammatory diseases which are treatable
with HLGAGs are refractory ulcerative colitis, Chrohn's disease,
multiple sclerosis, autoimmune disease, non-specific ulcerative
colitis and interstitial cystitis.
[0185] In one embodiment, the HLGAG preparations are used for
inhibiting angiogenesis. An effective amount for inhibiting
angiogenesis of the HLGAG preparation is administered to a subject
in need of treatment thereof. Angiogenesis as used herein is the
inappropriate formation of new blood vessels. "Angiogenesis" often
occurs in tumors when endothelial cells secrete a group of growth
factors that are mitogenic for endothelium causing the elongation
and proliferation of endothelial cells which results in the
generation of new blood vessels. Several of the angiogenic mitogens
are heparin binding peptides which are related to endothelial cell
growth factors. The inhibition of angiogenesis can cause tumor
regression in animal models, suggesting a use as a therapeutic
anticancer agent. An effective amount for inhibiting angiogenesis
is an amount of HLGAG preparation which is sufficient to diminish
the number of blood vessels growing into a tumor. This amount can
be assessed in an animal model of tumors and angiogenesis, many of
which are known in the art. Angiogenic disorders include, but are
not limited to, neovascular disorders of the eye, osteoporosis,
psoriasis, arthritis, cancer and cardiovascular disorders.
[0186] The HLGAG preparations are also useful for inhibiting
neovascularization associated with eye disease. In another
embodiment, the HLGAG preparation is administered to treat
psoriasis. Psoriasis is a common dermatologic disease caused by
chronic inflammation.
[0187] HLGAG containing compositions, may also inhibit cancer cell
growth and metastasis. Thus the methods are useful for treating
and/or preventing tumor cell proliferation or metastasis in a
subject. The cancer may be a malignant or non-malignant cancer.
Cancers or tumors include but are not limited to biliary tract
cancer; brain cancer; breast cancer; cervical cancer;
choriocarcinoma; colon cancer; endometrial cancer; esophageal
cancer; gastric cancer; intraepithelial neoplasms; leukemias,
lymphomas; liver cancer; lung cancer (e.g. small cell and non-small
cell); melanoma; neuroblastomas; oral cancer; ovarian cancer;
pancreatic cancer; prostate cancer; rectal cancer; sarcomas; skin
cancer; testicular cancer; thyroid cancer; and renal cancer, as
well as other carcinomas and sarcomas.
[0188] A subject in need of cancer treatment may be a subject who
has a high probability of developing cancer. These subjects
include, for instance, subjects having a genetic abnormality, the
presence of which has been demonstrated to have a correlative
relation to a higher likelihood of developing a cancer and subjects
exposed to cancer-causing agents such as tobacco, asbestos, or
other chemical toxins, or a subject who has previously been treated
for cancer and is in apparent remission.
OTHER EMBODIMENTS
[0189] This invention is further illustrated by the following
examples that should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are incorporated herein by
reference.
EXAMPLES
Example 1
Cloning and Recombinant Expression of B. thetaiotaomicron HSGAG
lyase I
[0190] The B. thetaiotaomicron HSGAG lyase I sequence (FIG. 1; SEQ
ID NO: 1), which is approximately 1130 nucleotides long contains a
predicted methionine-initiated coding sequence of about 1128
nucleotides, including the termination codon (SEQ ID NO: 1 in FIG.
1A). The coding sequence encodes a 376 amino acid protein (SEQ ID
NO:2 in FIG. 1B).
[0191] The B. thetaiotaomicron HSGAG lyase sequence is structurally
similar to the F. heparinum heparinase I sequence. A comparison of
the amino acid sequences of the two lyases is shown in FIG. 2.
[0192] The B. thetaiotaomicron HSGAG lyase gene was cloned by PCR
using genomic DNA from B. thetaiotaomicron obtained from the
American Type Culture Collection (ATCC), catalog no. 29148D. DNA
oligonucleotide primers for M17 variant were synthesized by
Integrated DNA technologies, Inc. (IDT) according to the following
nucleotide sequences: 1) 5' CATATGCTGACTGCTCAGACTAAAAATAC 3'
(forward primer) (SEQ ID NO: 11); 2) 5'
CTCGAGTTATCTTTCCGAATATCCTGCGAGAT 3' (reverse primer) (SEQ ID NO:
12). Primers were designed to introduce NdeI and XhoI endonuclease
restriction sites at the 5' and 3' ends, respectively. The
resulting gene sequence was cloned into pET28a bacterial expression
plasmid (EMD Biosciences) as an NdeI-XhoI fragment for subsequent
recombinant expression into E. coli strain BL21 (DE3), as an
engineered fusion protein containing the sequence
MGSSHHHHHHSSGLVPRGSH (SEQ ID NO: 13) fused to the amino terminus of
the B. thetaiotaomicron HSGAG lyase beginning at the methionine at
position 17 (M17).
[0193] A B. thetaiotaomicron HSGAG lyase variant with a modified
amino terminus that begins at position glutamine 26 (Q26) of the
protein sequence listed in SEQ ID NO:2, was cloned into pET28a for
recombinant expression as a fusion protein. The amino acid sequence
and nucleic acid sequence encoding the Q26 variant are provided in
SEQ ID NOs: 4 and 3, respectively DNA oligonucleotide primers for
Q26 variant were synthesized by Integrated DNA technologies, Inc.
(IDT) according to the following nucleotide sequences: 1) 5' CAT
ATG CAA ACA CTG ATG CCA CTC ACC GAA 3' (forward primer) (SEQ ID
NO:24) and 5' CTCGAGTTATCTTTCCGAATATCCTGCGAGAT 3' (reverse primer)
(SEQ ID NO: 12).
[0194] Both the full length, M17, and Q26 B. thetaiotaomicron HSGAG
lyase fusion proteins were recombinantly expressed in E. coli,
yielding soluble, highly active enzyme that was fully capable of
cleaving heparin and heparan sulfate (see Example 2 below).
[Sequence verified plasmid pET28 containing either the M17 coding
sequence or Q26 coding sequence was transformed into BL21 (DE3). 2
liter cultures were grown at room temperature (.about.20.degree.
C.) in LB media supplemented with 40 .mu.g/mL kanamycin. Protein
expression was induced with 500 .mu.M IPTG added at an A.sub.600 of
1.0. Induced cultures were allowed to grow for 15-18 hours at room
temperature.
[0195] Recombinant B. thetaiotaomicron HSGAG lyase purification.
Bacterial cells were harvested by centrifugation at 6000.times.g
for 15 minutes and resuspended in 30 mL of binding buffer (50 mM
Na.sub.2HPO.sub.4, pH 7.9, 0.5 M NaCl, and 5 mM imidazole). Lysis
was initiated by the addition of 0.1 mg/mL lysozyme (20 minutes at
room temperature) followed by intermittent sonication in an
ice-water bath using a Misonex XL sonicator at 40-50% output. The
crude lysate was fractionated by low-speed centrifugation
(20,000.times.g; 4.degree. C.; 30 minutes) and the supernatant was
filtered through a 0.45 micron filter. The 6.times.-His recombinant
B. thetaiotaomicron HSGAG lyase was purified by Ni.sup.+2 chelation
chromatography on a 5 mL Hi-Trap column (GE Healthcare) pre-charged
with 200 mM NiSO.sub.4 and subsequently equilibrated with binding
buffer. The column was run at a flow rate of approximately 3
ml/minute that included an intermediate wash step with 50 mM
imidazole. The lyase enzyme was eluted from the column in 5 mL
fractions using high imidazole elution buffer (50 mM
Na.sub.2HPO.sub.4, pH 7.9, 0.5 M NaCl, and 250 mM imidazole).
[0196] The resulting peak was buffer exchanged on a Sephadex G-25
column equilibrated with 20 mM Na.sub.2HPO.sub.4, pH 6.8, 150 mM
NaCl.
[0197] Protein concentrations were determined by the Bio-Rad
protein assay and confirmed by UV spectroscopy. Protein purity was
assessed by SDS-PAGE followed by Coomassie Brilliant Blue staining
and/or Sypro Ruby Red.
Example 2
Distinct Heparan Sulfate Substrate Specificities of B.
thetaiotaomicron HSGAG Lyase I and F. heparinum Heparinases I and
II
[0198] The cleavage patterns and thereby the substrate
specificities of recombinant B. thetaiotaomicron HSGAG lyase I and
F. heparinum heparinases I and II were compared using heparan
sulfate as a substrate. 200 .mu.g of "HI" heparan sulfate (Celsus
Labs) from porcine intestinal mucosa was digested with recombinant
B. thetaiotaomicron HSGAG lyase I under conditions favorable to
ensure a complete digestion. The HI was contacted with about 50
.mu.g B-thetaiotamicron HSGAG lyase 1,50 mM sodium phosphate, 100
mM NaCl, pH 8.0 at 37.degree. C. for 18 hours. The lyase digestion
products were analyzed by HPLC using strong anion chromatography
(SAX-HPLC). SAX-HPLC conditions were as follows: 50 .mu.g samples
was injected at 1 mg/ml into a 4.times.250 mm CarboPac PA1
analytical scale column (Dionex Corporation). The flow rate was 1
ml/min. The mobile phase was 0.2M to 2 M NaCl in water, pH 3.5,
gradient over 120 minutes. The column was preequilibrated with 0.2
M NaCl for 10 minutes. The results are shown in FIG. 6 as trace A.
FIG. 6 trace B shows the results of the same experiment except that
F. heparinum heparinase I was used to digest the heparan sulfate.
Briefly, The HI was contacted with about 50 .mu.g F. heparinum
heparinase I, 25 mM sodium acetate, 1 mM calcium acetate, 5%
glycine, pH 7.0 at 30.degree. C. for 18 hours. This digestion
profile is very similar to the profile in trace A, except that
novel peaks are present in trace A (depicted by arrows) that are
not present in trace B, demonstrating that the lyases have
different substrate specificities. FIG. 6 trace C shows the results
of the same experiment except that F. heparinum heparinase II was
used to digest the heparan sulfate. Briefly, The HI was contacted
with about 50 .mu.g F. heparinum heparinase II, 25 mM sodium
acetate, 1 mM calcium acetate, pH 7.0 at 37.degree. C. for 18
hours. In this case, the digestion profile is very much distinct
from A (B. thetaiotaomicron HSGAG lyase) and B (F. heparinum
heparinase I). These data demonstrate that the B. thetaiotaomicron
HSGAG lyase substrate specificity is distinct from the
specificities of F. heparinum heparinases I and II, but is more
"heparin like" (e.g., more similar to F. heparinum heparinase I)
than "heparan sulfate-like" (e.g., it is less like F. heparinum
heparinase II).
Example 3
Depolymerization and Neutralization of ARIXTRA.RTM. by B.
thetaiotaomicron HSGAG Lyase I
[0199] Recombinant B. thetaiotaomicron HSGAG lyase I can cleave and
thereby neutralize the ATIII pentasaccharide ARIXTRA.RTM. into a
pentasulfated trisaccharide and an unsaturated disulfated
disaccharide. ARIXTRA.RTM. is an anti-thrombotic drug that acts as
a selective inhibitor of Factor Xa, a component of the coagulation
cascade. Depolymerization of ARIXTRA.RTM. is unequivocally
demonstrated by matrix assisted laser desorption ionization mass
spectrometry (MALDI-MS) (FIG. 8). Panel A shows the scan of
ARIXTRA.RTM. in the absence of a lyase. The structure of
ARIXTRA.RTM. is also shown. Panel B shows the scan after cleavage
of ARIXTRA.RTM. with B. thetaiotaomicron HSGAG lyase. Briefly, 1
mg/ml ARIXTRA.RTM. in a 20 .mu.L reaction volume was treated with 5
.mu.g B. thetaiotaomicron HSGAG lyase I, 25 mM sodium acetate, 1 mM
calcium acetate, pH 7.0 at 37.degree. C. for 2 hours. Note the
disappearance in panel B of mass 4723.7 Da (net mass=1506 Da)
present in panel A with concomitant appearance of mass 4133.2 Da
(net mass=915.6 Da). The latter mass represents the pentasulfated
trisaccharide cleavage product. In cleaving Arixtra into two
smaller fragments, the drug's anti-Xa activity is effectively
neutralized by the B. thetaiotaomicron HSGAG lyase.
Example 4
Cloning and Recombinant Expression of B. thetaiotaomicron HSGAG
Lyase II
[0200] The complete coding sequence of a B. thetaiotaomicron
heparin/heparan sulfate lyase II (herein described as "full-length
gene") as well as the two variants described herein were cloned by
PCR using genomic DNA from Bacteroides thetaiotaomicron as obtained
from American Type Culture Collection (ATCC), catalog no. 29148D.
DNA oligonucleotide primers were synthesized by Integrated DNA
technologies (IDT), Inc. according to the following nucleotide
sequences: 1) For the full-length gene: 5' CAT ATG AAT AAA ACC CTG
AAA TAT ATC GTC CTG 3' (forward primer) (SEQ ID NO: 14), 5' CTC GAG
TTA TAA TTT ATA TTT TAA TGA CTG TTT CTT GC 3' (reverse primer) (SEQ
ID NO: 15); 2) Gene encoding variant No. 1 (amino terminal
truncation to remove putative signal sequence): 5' CAT ATG CAA GAG
TTG AAA AGC GAG GTA TTC TCG 3' (forward primer) (SEQ ID NO: 16), 5'
CTC GAG TTA TAA TTT ATA TTT TAA TGA CTG TTT CTT GC 3' (note: same
reverse primer listed above as for full-length gene) (SEQ ID NO:
15). Primers were designed to introduce Nde 1 and Xho 1
endonuclease restriction sites at the 5' and 3' ends, respectively.
Cloning of described gene sequence into pET28b bacterial expression
plasmid (EMD Biosciences) as an Nde 1-Xho 1 fragment for subsequent
recombinant expression into E. coli strain BL21 (DE3) as engineered
fusion protein containing the sequence MGSSHHHHHHSSGLVPRGSHMNKTLKY
. . . KVNGKKQSLKYKL (SEQ ID NO: 17) or
MGSSHHHHHHSSGLVPRGSHMQELKSEVF . . . KVNGKKQSLKYKL (SEQ ID NO: 18)
for the full-length gene and variant 1 (the Q23 variant, SEQ ID NO:
8), respectively (Bacteroides thetaiotaomicron HSGAG lyase sequence
is denoted in bold). See FIG. 4 for complete sequence.
[0201] Another variant, the K169 variant (SEQ ID NO: 10) represents
an engineered deletion of 18 contiguous amino acids comprising an
internal region within the protein and possessing the following
linear sequence: KMDKKEYELVSDGKIKGE. (SEQ ID NO:19) Deletion of
this region in the gene sequence (FIG. 4A) and in the corresponding
protein sequence (FIG. 4B) is noted by grey shading. Deletion of
this region at the DNA level was accomplished by PCR-based
mutagenesis using the Quick-change kit (Stratagene) in accordance
with the manufacturer's instructions. Mutagenesis primers used to
make this deletion at the gene (DNA) level were of the following
sequence: 5' GG ATT AAA AAG AAT CCG TTG GTG GAA AAT GTA CGT TTC GC
3' (SEQ ID NO:20) and 5' CC TAA TTT TTC TTA GGC AAC CAC CTT TTA CAT
GCA AAG CG 3' (SEQ ID NO:21) corresponding to the sense and
anti-sense strands, respectively. Recombinant expression of this
described gene variant in E. coli likewise based on the pET-based
expression for recombinant expression was also achieved.
[0202] Preliminary biochemical characterization of this variant
indicates that deletion of described amino acids is not deleterious
to the soluble expression of the enzyme nor to its ability to
cleave both heparin and heparan sulfate. It does suggest, however a
potential difference in the substrate specificity of this enzyme
variant relative to the full-length protein.
EQUIVALENTS
[0203] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
Sequence CWU 1
1
2711251DNABacteroides thetaiotaomicron 1gacaaacgaa aggcagccgt
aagggttgcc tttcgtattt ttgcaccgtc gataaactta 60ataccggata gaatgaaaaa
atacattttg gttatttata tgatggcggc aggatgcacg 120atgctgactg
ctcagactaa aaatacgcaa acactgatgc cactcaccga acgggtaaac
180gtacaggctg actctgcacg tatcaaccag attattgacg gttgctgggt
agctgtcggg 240acgaataaac ctcatgccat tcagcgtgat tttaccaacc
tgtttgatgg caagccctcc 300tatcgctttg aactcaaaac tgaagacaat
acactggaag gttatgcgaa aggagaaacg 360aaaggacgtg ccgagttttc
atattgctat gcaacttccg acgatttcag gggattacct 420gccgacgttt
atcagaaagc acagatcaca aagacagttt atcatcacgg gaagggagct
480tgtccgcaag gaagttcccg cgactatgag ttttcggttt atattccttc
ttctttagac 540agcaatgtct ccaccatctt tgcccaatgg cacggaatgc
ccgaccggac gctggtccag 600actcctcagg gcgaggtgaa gaaactgact
gttgacgaat ttgtagaact ggaaaaaacg 660accttcttca aaaagaatgt
cggacacgaa aaagtggcca gactggataa acaaggtaat 720ccggtgaaag
ataaaaatgg aaaacctgta tataaggcag gaaaacccaa cggatggttg
780gttgaacagg gaggataccc gccattggca ttcggatttt ccggaggact
gttttatatc 840aaagcaaact ccgaccgtaa atggctgaca gacaaagatg
accgttgcaa tgcaaacccg 900ggaaagacgc ccgttatgaa accgctgact
tctgaataca aggcatccac cattgcctac 960aaattacctt ttgccgattt
cccgaaagac tgctggatta ctttccgtgt ccatatcgac 1020tggacggtct
atggcaagga agcggaaacg attgtgaaac cgggcatgct ggatgtacgg
1080atggattatc aggagcaagg taagaaagtg agcaaacaca ttgtcgataa
tgagaagatt 1140ctgattggac gtaacgacga agacgggtat tactttaagt
tcggaattta ccgcgtaggt 1200gatagtaccg ttcccgtttg ctacaatctc
gcaggatatt cggaaagata a 12512392PRTBacteroides thetaiotaomicron
2Met Lys Lys Tyr Ile Leu Val Ile Tyr Met Met Ala Ala Gly Cys Thr 1
5 10 15Met Leu Thr Ala Gln Thr Lys Asn Thr Gln Thr Leu Met Pro Leu
Thr 20 25 30Glu Arg Val Asn Val Gln Ala Asp Ser Ala Arg Ile Asn Gln
Ile Ile 35 40 45Asp Gly Cys Trp Val Ala Val Gly Thr Asn Lys Pro His
Ala Ile Gln 50 55 60Arg Asp Phe Thr Asn Leu Phe Asp Gly Lys Pro Ser
Tyr Arg Phe Glu65 70 75 80Leu Lys Thr Glu Asp Asn Thr Leu Glu Gly
Tyr Ala Lys Gly Glu Thr 85 90 95Lys Gly Arg Ala Glu Phe Ser Tyr Cys
Tyr Ala Thr Ser Asp Asp Phe 100 105 110Arg Gly Leu Pro Ala Asp Val
Tyr Gln Lys Ala Gln Ile Thr Lys Thr 115 120 125Val Tyr His His Gly
Lys Gly Ala Cys Pro Gln Gly Ser Ser Arg Asp 130 135 140Tyr Glu Phe
Ser Val Tyr Ile Pro Ser Ser Leu Asp Ser Asn Val Ser145 150 155
160Thr Ile Phe Ala Gln Trp His Gly Met Pro Asp Arg Thr Leu Val Gln
165 170 175Thr Pro Gln Gly Glu Val Lys Lys Leu Thr Val Asp Glu Phe
Val Glu 180 185 190Leu Glu Lys Thr Thr Phe Phe Lys Lys Asn Val Gly
His Glu Lys Val 195 200 205Ala Arg Leu Asp Lys Gln Gly Asn Pro Val
Lys Asp Lys Asn Gly Lys 210 215 220Pro Val Tyr Lys Ala Gly Lys Pro
Asn Gly Trp Leu Val Glu Gln Gly225 230 235 240Gly Tyr Pro Pro Leu
Ala Phe Gly Phe Ser Gly Gly Leu Phe Tyr Ile 245 250 255Lys Ala Asn
Ser Asp Arg Lys Trp Leu Thr Asp Lys Asp Asp Arg Cys 260 265 270Asn
Ala Asn Pro Gly Lys Thr Pro Val Met Lys Pro Leu Thr Ser Glu 275 280
285Tyr Lys Ala Ser Thr Ile Ala Tyr Lys Leu Pro Phe Ala Asp Phe Pro
290 295 300Lys Asp Cys Trp Ile Thr Phe Arg Val His Ile Asp Trp Thr
Val Tyr305 310 315 320Gly Lys Glu Ala Glu Thr Ile Val Lys Pro Gly
Met Leu Asp Val Arg 325 330 335Met Asp Tyr Gln Glu Gln Gly Lys Lys
Val Ser Lys His Ile Val Asp 340 345 350Asn Glu Lys Ile Leu Ile Gly
Arg Asn Asp Glu Asp Gly Tyr Tyr Phe 355 360 365Lys Phe Gly Ile Tyr
Arg Val Gly Asp Ser Thr Val Pro Val Cys Tyr 370 375 380Asn Leu Ala
Gly Tyr Ser Glu Arg385 39031179DNABacteroides thetaiotaomicron
3atgaaaaaat acattttggt tatttatatg atggcggcag gatgcacgat gctgactgct
60cagactaaaa atacgcaaac actgatgcca ctcaccgaac gggtaaacgt acaggctgac
120tctgcacgta tcaaccagat tattgacggt tgctgggtag ctgtcgggac
gaataaacct 180catgccattc agcgtgattt taccaacctg tttgatggca
agccctccta tcgctttgaa 240ctcaaaactg aagacaatac actggaaggt
tatgcgaaag gagaaacgaa aggacgtgcc 300gagttttcat attgctatgc
aacttccgac gatttcaggg gattacctgc cgacgtttat 360cagaaagcac
agatcacaaa gacagtttat catcacggga agggagcttg tccgcaagga
420agttcccgcg actatgagtt ttcggtttat attccttctt ctttagacag
caatgtctcc 480accatctttg cccaatggca cggaatgccc gaccggacgc
tggtccagac tcctcagggc 540gaggtgaaga aactgactgt tgacgaattt
gtagaactgg aaaaaacgac cttcttcaaa 600aagaatgtcg gacacgaaaa
agtggccaga ctggataaac aaggtaatcc ggtgaaagat 660aaaaatggaa
aacctgtata taaggcagga aaacccaacg gatggttggt tgaacaggga
720ggatacccgc cattggcatt cggattttcc ggaggactgt tttatatcaa
agcaaactcc 780gaccgtaaat ggctgacaga caaagatgac cgttgcaatg
caaacccggg aaagacgccc 840gttatgaaac cgctgacttc tgaatacaag
gcatccacca ttgcctacaa attacctttt 900gccgatttcc cgaaagactg
ctggattact ttccgtgtcc atatcgactg gacggtctat 960ggcaaggaag
cggaaacgat tgtgaaaccg ggcatgctgg atgtacggat ggattatcag
1020gagcaaggta agaaagtgag caaacacatt gtcgataatg agaagattct
gattggacgt 1080aacgacgaag acgggtatta ctttaagttc ggaatttacc
gcgtaggtga tagtaccgtt 1140cccgtttgct acaatctcgc aggatattcg
gaaagataa 11794376PRTBacteroides thetaiotaomicron 4Met Leu Thr Ala
Gln Thr Lys Asn Thr Gln Thr Leu Met Pro Leu Thr 1 5 10 15Glu Arg
Val Asn Val Gln Ala Asp Ser Ala Arg Ile Asn Gln Ile Ile 20 25 30Asp
Gly Cys Trp Val Ala Val Gly Thr Asn Lys Pro His Ala Ile Gln 35 40
45Arg Asp Phe Thr Asn Leu Phe Asp Gly Lys Pro Ser Tyr Arg Phe Glu
50 55 60Leu Lys Thr Glu Asp Asn Thr Leu Glu Gly Tyr Ala Lys Gly Glu
Thr65 70 75 80Lys Gly Arg Ala Glu Phe Ser Tyr Cys Tyr Ala Thr Ser
Asp Asp Phe 85 90 95Arg Gly Leu Pro Ala Asp Val Tyr Gln Lys Ala Gln
Ile Thr Lys Thr 100 105 110Val Tyr His His Gly Lys Gly Ala Cys Pro
Gln Gly Ser Ser Arg Asp 115 120 125Tyr Glu Phe Ser Val Tyr Ile Pro
Ser Ser Leu Asp Ser Asn Val Ser 130 135 140Thr Ile Phe Ala Gln Trp
His Gly Met Pro Asp Arg Thr Leu Val Gln145 150 155 160Thr Pro Gln
Gly Glu Val Lys Lys Leu Thr Val Asp Glu Phe Val Glu 165 170 175Leu
Glu Lys Thr Thr Phe Phe Lys Lys Asn Val Gly His Glu Lys Val 180 185
190Ala Arg Leu Asp Lys Gln Gly Asn Pro Val Lys Asp Lys Asn Gly Lys
195 200 205Pro Val Tyr Lys Ala Gly Lys Pro Asn Gly Trp Leu Val Glu
Gln Gly 210 215 220Gly Tyr Pro Pro Leu Ala Phe Gly Phe Ser Gly Gly
Leu Phe Tyr Ile225 230 235 240Lys Ala Asn Ser Asp Arg Lys Trp Leu
Thr Asp Lys Asp Asp Arg Cys 245 250 255Asn Ala Asn Pro Gly Lys Thr
Pro Val Met Lys Pro Leu Thr Ser Glu 260 265 270Tyr Lys Ala Ser Thr
Ile Ala Tyr Lys Leu Pro Phe Ala Asp Phe Pro 275 280 285Lys Asp Cys
Trp Ile Thr Phe Arg Val His Ile Asp Trp Thr Val Tyr 290 295 300Gly
Lys Glu Ala Glu Thr Ile Val Lys Pro Gly Met Leu Asp Val Arg305 310
315 320Met Asp Tyr Gln Glu Gln Gly Lys Lys Val Ser Lys His Ile Val
Asp 325 330 335Asn Glu Lys Ile Leu Ile Gly Arg Asn Asp Glu Asp Gly
Tyr Tyr Phe 340 345 350Lys Phe Gly Ile Tyr Arg Val Gly Asp Ser Thr
Val Pro Val Cys Tyr 355 360 365Asn Leu Ala Gly Tyr Ser Glu Arg 370
37552001DNABacteroides thetaiotaomicron 5atgaataaaa ccctgaaata
tatcgtcctg ctgacatttg cttgtttcgt aggcaaaggc 60tatgcccaag agttgaaaag
cgaggtattc tcgcttctca acctggacta ccccggattg 120gagaaagtaa
aagccttaca tcaggaaggc aaagatgagg atgccgcaaa agcactgctc
180gactactacc gtgcacgtac gaatgtgaag acgccggata ttaatctgaa
aaagatcact 240atcggcaaag aagaacagca atgggcggat gacggattga
agcatacatt ctttgttcac 300aaaggctatc agccttctta caactacgga
gaagatatca actggcaata ctggccggtg 360aaagacaatg aactccgctg
gcagttgcac cgtcataaat ggtttactcc gatgggtaag 420gcataccgtg
tatcgggtga cgagaaatat gccaaagaat gggcatacca atacatcgac
480tggattaaaa agaatccgtt ggtgaagatg gacaagaaag aatacgaact
ggtaagtgac 540ggtaagatta aaggcgaagt ggaaaatgta cgtttcgcat
ggcgtccgct ggaagtcagt 600aatcgtctgc aggatcagac tacccagttc
cagttgttcc tcccctctcc ttctttcact 660ccggatttcc tgactgaatt
tctggtgaac tatcataaac atgccgtaca tattctggct 720aattactctg
atcagggtaa tcacttgttg ttcgaagccc agcgtatgat ttatgcaggt
780gcattcttcc cggaatttaa agaagctccg gcctggagaa aaagcggtat
cgacattctg 840aaccgtgaag taaacgtaca ggtttacaac gatggcggcc
agtttgaact tgacccgcat 900tatcatcttg ctgctatcaa tatcttctgc
aaggcattgg gtatcgcgga tgttaacgga 960ttccgtaatg agttcccaca
ggaatatctg gatactatcg aaaagatgat catgttctat 1020gccaatattt
ctttcccgga ttacacaaat ccgtgtttca gtgatgctaa aatcacagaa
1080aagaaagaaa tgctgaagaa ctatcgtgca tggagcaaac tgttcccgaa
aaacgaaact 1140atcaagtatt tggcaacaga cggcaaagaa ggcgcgttac
ccgattatat gtcgaaaggt 1200ttcctgaaat caggtttctt tgtgttccgc
aattcctggg gaatggatgc tacacaaatg 1260gtagtaaaag ccggtccgaa
aggtttctgg cactgtcagc cggataacgg tactttcgaa 1320atgtggttta
acggcaagaa cctgttccca gactccggtt cgtatgtgta tgccggtgaa
1380ggcgaagtga tggaacaacg caactggcat cgtcagactt ccgtacacaa
caccgtgact 1440ctggacaata agaatctgga aacaaccgaa tctgttacta
aactgtggca gccggaaggc 1500aatatccaga ccttggttac agaaaaccca
agctacaaga acttcaagca ccgccgttcg 1560gtcttcttcg tagacaatac
ctactttgtc attgtagatg aagtatcagg cagcgccaaa 1620ggttctgtca
acctgcacta tcagatgccg aaaggtgaga tagccaacag ccgtgaagac
1680atgacattcc tgactcaatt cgaagatgga agcaacatga aacttcaatg
cttcggccct 1740gaaggcatga gcatgaaaaa agagccggga tggtgttcta
ctgcatatcg caagcgctac 1800aaacgtatga atgtttcatt caacgtaaag
aaagacaatg agaatgcggt acgttacatc 1860acagttattt acccagtcaa
gaagagcgca gatgccccta aatttgacgc taagttcaag 1920aacaaaacgt
tcgatgaaaa cggactggaa atagaagtga aagtaaacgg caagaaacag
1980tcattaaaat ataaattata a 20016666PRTBacteroides thetaiotaomicron
6Met Asn Lys Thr Leu Lys Tyr Ile Val Leu Leu Thr Phe Ala Cys Phe 1
5 10 15Val Gly Lys Gly Tyr Ala Gln Glu Leu Lys Ser Glu Val Phe Ser
Leu 20 25 30Leu Asn Leu Asp Tyr Pro Gly Leu Glu Lys Val Lys Ala Leu
His Gln 35 40 45Glu Gly Lys Asp Glu Asp Ala Ala Lys Ala Leu Leu Asp
Tyr Tyr Arg 50 55 60Ala Arg Thr Asn Val Lys Thr Pro Asp Ile Asn Leu
Lys Lys Ile Thr65 70 75 80Ile Gly Lys Glu Glu Gln Gln Trp Ala Asp
Asp Gly Leu Lys His Thr 85 90 95Phe Phe Val His Lys Gly Tyr Gln Pro
Ser Tyr Asn Tyr Gly Glu Asp 100 105 110Ile Asn Trp Gln Tyr Trp Pro
Val Lys Asp Asn Glu Leu Arg Trp Gln 115 120 125Leu His Arg His Lys
Trp Phe Thr Pro Met Gly Lys Ala Tyr Arg Val 130 135 140Ser Gly Asp
Glu Lys Tyr Ala Lys Glu Trp Ala Tyr Gln Tyr Ile Asp145 150 155
160Trp Ile Lys Lys Asn Pro Leu Val Lys Met Asp Lys Lys Glu Tyr Glu
165 170 175Leu Val Ser Asp Gly Lys Ile Lys Gly Glu Val Glu Asn Val
Arg Phe 180 185 190Ala Trp Arg Pro Leu Glu Val Ser Asn Arg Leu Gln
Asp Gln Thr Thr 195 200 205Gln Phe Gln Leu Phe Leu Pro Ser Pro Ser
Phe Thr Pro Asp Phe Leu 210 215 220Thr Glu Phe Leu Val Asn Tyr His
Lys His Ala Val His Ile Leu Ala225 230 235 240Asn Tyr Ser Asp Gln
Gly Asn His Leu Leu Phe Glu Ala Gln Arg Met 245 250 255Ile Tyr Ala
Gly Ala Phe Phe Pro Glu Phe Lys Glu Ala Pro Ala Trp 260 265 270Arg
Lys Ser Gly Ile Asp Ile Leu Asn Arg Glu Val Asn Val Gln Val 275 280
285Tyr Asn Asp Gly Gly Gln Phe Glu Leu Asp Pro His Tyr His Leu Ala
290 295 300Ala Ile Asn Ile Phe Cys Lys Ala Leu Gly Ile Ala Asp Val
Asn Gly305 310 315 320Phe Arg Asn Glu Phe Pro Gln Glu Tyr Leu Asp
Thr Ile Glu Lys Met 325 330 335Ile Met Phe Tyr Ala Asn Ile Ser Phe
Pro Asp Tyr Thr Asn Pro Cys 340 345 350Phe Ser Asp Ala Lys Ile Thr
Glu Lys Lys Glu Met Leu Lys Asn Tyr 355 360 365Arg Ala Trp Ser Lys
Leu Phe Pro Lys Asn Glu Thr Ile Lys Tyr Leu 370 375 380Ala Thr Asp
Gly Lys Glu Gly Ala Leu Pro Asp Tyr Met Ser Lys Gly385 390 395
400Phe Leu Lys Ser Gly Phe Phe Val Phe Arg Asn Ser Trp Gly Met Asp
405 410 415Ala Thr Gln Met Val Val Lys Ala Gly Pro Lys Gly Phe Trp
His Cys 420 425 430Gln Pro Asp Asn Gly Thr Phe Glu Met Trp Phe Asn
Gly Lys Asn Leu 435 440 445Phe Pro Asp Ser Gly Ser Tyr Val Tyr Ala
Gly Glu Gly Glu Val Met 450 455 460Glu Gln Arg Asn Trp His Arg Gln
Thr Ser Val His Asn Thr Val Thr465 470 475 480Leu Asp Asn Lys Asn
Leu Glu Thr Thr Glu Ser Val Thr Lys Leu Trp 485 490 495Gln Pro Glu
Gly Asn Ile Gln Thr Leu Val Thr Glu Asn Pro Ser Tyr 500 505 510Lys
Asn Phe Lys His Arg Arg Ser Val Phe Phe Val Asp Asn Thr Tyr 515 520
525Phe Val Ile Val Asp Glu Val Ser Gly Ser Ala Lys Gly Ser Val Asn
530 535 540Leu His Tyr Gln Met Pro Lys Gly Glu Ile Ala Asn Ser Arg
Glu Asp545 550 555 560Met Thr Phe Leu Thr Gln Phe Glu Asp Gly Ser
Asn Met Lys Leu Gln 565 570 575Cys Phe Gly Pro Glu Gly Met Ser Met
Lys Lys Glu Pro Gly Trp Cys 580 585 590Ser Thr Ala Tyr Arg Lys Arg
Tyr Lys Arg Met Asn Val Ser Phe Asn 595 600 605Val Lys Lys Asp Asn
Glu Asn Ala Val Arg Tyr Ile Thr Val Ile Tyr 610 615 620Pro Val Lys
Lys Ser Ala Asp Ala Pro Lys Phe Asp Ala Lys Phe Lys625 630 635
640Asn Lys Thr Phe Asp Glu Asn Gly Leu Glu Ile Glu Val Lys Val Asn
645 650 655Gly Lys Lys Gln Ser Leu Lys Tyr Lys Leu 660
66571935DNABacteroides thetaiotaomicron 7caagagttga aaagcgaggt
attctcgctt ctcaacctgg actaccccgg attggagaaa 60gtaaaagcct tacatcagga
aggcaaagat gaggatgccg caaaagcact gctcgactac 120taccgtgcac
gtacgaatgt gaagacgccg gatattaatc tgaaaaagat cactatcggc
180aaagaagaac agcaatgggc ggatgacgga ttgaagcata cattctttgt
tcacaaaggc 240tatcagcctt cttacaacta cggagaagat atcaactggc
aatactggcc ggtgaaagac 300aatgaactcc gctggcagtt gcaccgtcat
aaatggttta ctccgatggg taaggcatac 360cgtgtatcgg gtgacgagaa
atatgccaaa gaatgggcat accaatacat cgactggatt 420aaaaagaatc
cgttggtgaa gatggacaag aaagaatacg aactggtaag tgacggtaag
480attaaaggcg aagtggaaaa tgtacgtttc gcatggcgtc cgctggaagt
cagtaatcgt 540ctgcaggatc agactaccca gttccagttg ttcctcccct
ctccttcttt cactccggat 600ttcctgactg aatttctggt gaactatcat
aaacatgccg tacatattct ggctaattac 660tctgatcagg gtaatcactt
gttgttcgaa gcccagcgta tgatttatgc aggtgcattc 720ttcccggaat
ttaaagaagc tccggcctgg agaaaaagcg gtatcgacat tctgaaccgt
780gaagtaaacg tacaggttta caacgatggc ggccagtttg aacttgaccc
gcattatcat 840cttgctgcta tcaatatctt ctgcaaggca ttgggtatcg
cggatgttaa cggattccgt 900aatgagttcc cacaggaata tctggatact
atcgaaaaga tgatcatgtt ctatgccaat 960atttctttcc cggattacac
aaatccgtgt ttcagtgatg ctaaaatcac agaaaagaaa 1020gaaatgctga
agaactatcg tgcatggagc aaactgttcc cgaaaaacga aactatcaag
1080tatttggcaa cagacggcaa agaaggcgcg ttacccgatt atatgtcgaa
aggtttcctg 1140aaatcaggtt tctttgtgtt ccgcaattcc tggggaatgg
atgctacaca aatggtagta 1200aaagccggtc cgaaaggttt ctggcactgt
cagccggata acggtacttt cgaaatgtgg 1260tttaacggca agaacctgtt
cccagactcc ggttcgtatg tgtatgccgg tgaaggcgaa 1320gtgatggaac
aacgcaactg gcatcgtcag acttccgtac acaacaccgt gactctggac
1380aataagaatc tggaaacaac cgaatctgtt actaaactgt ggcagccgga
aggcaatatc 1440cagaccttgg ttacagaaaa cccaagctac aagaacttca
agcaccgccg ttcggtcttc 1500ttcgtagaca atacctactt tgtcattgta
gatgaagtat caggcagcgc caaaggttct 1560gtcaacctgc actatcagat
gccgaaaggt
gagatagcca acagccgtga agacatgaca 1620ttcctgactc aattcgaaga
tggaagcaac atgaaacttc aatgcttcgg ccctgaaggc 1680atgagcatga
aaaaagagcc gggatggtgt tctactgcat atcgcaagcg ctacaaacgt
1740atgaatgttt cattcaacgt aaagaaagac aatgagaatg cggtacgtta
catcacagtt 1800atttacccag tcaagaagag cgcagatgcc cctaaatttg
acgctaagtt caagaacaaa 1860acgttcgatg aaaacggact ggaaatagaa
gtgaaagtaa acggcaagaa acagtcatta 1920aaatataaat tataa
19358644PRTBacteroides thetaiotaomicron 8Gln Glu Leu Lys Ser Glu
Val Phe Ser Leu Leu Asn Leu Asp Tyr Pro 1 5 10 15Gly Leu Glu Lys
Val Lys Ala Leu His Gln Glu Gly Lys Asp Glu Asp 20 25 30Ala Ala Lys
Ala Leu Leu Asp Tyr Tyr Arg Ala Arg Thr Asn Val Lys 35 40 45Thr Pro
Asp Ile Asn Leu Lys Lys Ile Thr Ile Gly Lys Glu Glu Gln 50 55 60Gln
Trp Ala Asp Asp Gly Leu Lys His Thr Phe Phe Val His Lys Gly65 70 75
80Tyr Gln Pro Ser Tyr Asn Tyr Gly Glu Asp Ile Asn Trp Gln Tyr Trp
85 90 95Pro Val Lys Asp Asn Glu Leu Arg Trp Gln Leu His Arg His Lys
Trp 100 105 110Phe Thr Pro Met Gly Lys Ala Tyr Arg Val Ser Gly Asp
Glu Lys Tyr 115 120 125Ala Lys Glu Trp Ala Tyr Gln Tyr Ile Asp Trp
Ile Lys Lys Asn Pro 130 135 140Leu Val Lys Met Asp Lys Lys Glu Tyr
Glu Leu Val Ser Asp Gly Lys145 150 155 160Ile Lys Gly Glu Val Glu
Asn Val Arg Phe Ala Trp Arg Pro Leu Glu 165 170 175Val Ser Asn Arg
Leu Gln Asp Gln Thr Thr Gln Phe Gln Leu Phe Leu 180 185 190Pro Ser
Pro Ser Phe Thr Pro Asp Phe Leu Thr Glu Phe Leu Val Asn 195 200
205Tyr His Lys His Ala Val His Ile Leu Ala Asn Tyr Ser Asp Gln Gly
210 215 220Asn His Leu Leu Phe Glu Ala Gln Arg Met Ile Tyr Ala Gly
Ala Phe225 230 235 240Phe Pro Glu Phe Lys Glu Ala Pro Ala Trp Arg
Lys Ser Gly Ile Asp 245 250 255Ile Leu Asn Arg Glu Val Asn Val Gln
Val Tyr Asn Asp Gly Gly Gln 260 265 270Phe Glu Leu Asp Pro His Tyr
His Leu Ala Ala Ile Asn Ile Phe Cys 275 280 285Lys Ala Leu Gly Ile
Ala Asp Val Asn Gly Phe Arg Asn Glu Phe Pro 290 295 300Gln Glu Tyr
Leu Asp Thr Ile Glu Lys Met Ile Met Phe Tyr Ala Asn305 310 315
320Ile Ser Phe Pro Asp Tyr Thr Asn Pro Cys Phe Ser Asp Ala Lys Ile
325 330 335Thr Glu Lys Lys Glu Met Leu Lys Asn Tyr Arg Ala Trp Ser
Lys Leu 340 345 350Phe Pro Lys Asn Glu Thr Ile Lys Tyr Leu Ala Thr
Asp Gly Lys Glu 355 360 365Gly Ala Leu Pro Asp Tyr Met Ser Lys Gly
Phe Leu Lys Ser Gly Phe 370 375 380Phe Val Phe Arg Asn Ser Trp Gly
Met Asp Ala Thr Gln Met Val Val385 390 395 400Lys Ala Gly Pro Lys
Gly Phe Trp His Cys Gln Pro Asp Asn Gly Thr 405 410 415Phe Glu Met
Trp Phe Asn Gly Lys Asn Leu Phe Pro Asp Ser Gly Ser 420 425 430Tyr
Val Tyr Ala Gly Glu Gly Glu Val Met Glu Gln Arg Asn Trp His 435 440
445Arg Gln Thr Ser Val His Asn Thr Val Thr Leu Asp Asn Lys Asn Leu
450 455 460Glu Thr Thr Glu Ser Val Thr Lys Leu Trp Gln Pro Glu Gly
Asn Ile465 470 475 480Gln Thr Leu Val Thr Glu Asn Pro Ser Tyr Lys
Asn Phe Lys His Arg 485 490 495Arg Ser Val Phe Phe Val Asp Asn Thr
Tyr Phe Val Ile Val Asp Glu 500 505 510Val Ser Gly Ser Ala Lys Gly
Ser Val Asn Leu His Tyr Gln Met Pro 515 520 525Lys Gly Glu Ile Ala
Asn Ser Arg Glu Asp Met Thr Phe Leu Thr Gln 530 535 540Phe Glu Asp
Gly Ser Asn Met Lys Leu Gln Cys Phe Gly Pro Glu Gly545 550 555
560Met Ser Met Lys Lys Glu Pro Gly Trp Cys Ser Thr Ala Tyr Arg Lys
565 570 575Arg Tyr Lys Arg Met Asn Val Ser Phe Asn Val Lys Lys Asp
Asn Glu 580 585 590Asn Ala Val Arg Tyr Ile Thr Val Ile Tyr Pro Val
Lys Lys Ser Ala 595 600 605Asp Ala Pro Lys Phe Asp Ala Lys Phe Lys
Asn Lys Thr Phe Asp Glu 610 615 620Asn Gly Leu Glu Ile Glu Val Lys
Val Asn Gly Lys Lys Gln Ser Leu625 630 635 640Lys Tyr Lys
Leu91947DNABacteroides thetaiotaomicron 9atgaataaaa ccctgaaata
tatcgtcctg ctgacatttg cttgtttcgt aggcaaaggc 60tatgcccaag agttgaaaag
cgaggtattc tcgcttctca acctggacta ccccggattg 120gagaaagtaa
aagccttaca tcaggaaggc aaagatgagg atgccgcaaa agcactgctc
180gactactacc gtgcacgtac gaatgtgaag acgccggata ttaatctgaa
aaagatcact 240atcggcaaag aagaacagca atgggcggat gacggattga
agcatacatt ctttgttcac 300aaaggctatc agccttctta caactacgga
gaagatatca actggcaata ctggccggtg 360aaagacaatg aactccgctg
gcagttgcac cgtcataaat ggtttactcc gatgggtaag 420gcataccgtg
tatcgggtga cgagaaatat gccaaagaat gggcatacca atacatcgac
480tggattaaaa agaatccgtt ggtggtggaa aatgtacgtt tcgcatggcg
tccgctggaa 540gtcagtaatc gtctgcagga tcagactacc cagttccagt
tgttcctccc ctctccttct 600ttcactccgg atttcctgac tgaatttctg
gtgaactatc ataaacatgc cgtacatatt 660ctggctaatt actctgatca
gggtaatcac ttgttgttcg aagcccagcg tatgatttat 720gcaggtgcat
tcttcccgga atttaaagaa gctccggcct ggagaaaaag cggtatcgac
780attctgaacc gtgaagtaaa cgtacaggtt tacaacgatg gcggccagtt
tgaacttgac 840ccgcattatc atcttgctgc tatcaatatc ttctgcaagg
cattgggtat cgcggatgtt 900aacggattcc gtaatgagtt cccacaggaa
tatctggata ctatcgaaaa gatgatcatg 960ttctatgcca atatttcttt
cccggattac acaaatccgt gtttcagtga tgctaaaatc 1020acagaaaaga
aagaaatgct gaagaactat cgtgcatgga gcaaactgtt cccgaaaaac
1080gaaactatca agtatttggc aacagacggc aaagaaggcg cgttacccga
ttatatgtcg 1140aaaggtttcc tgaaatcagg tttctttgtg ttccgcaatt
cctggggaat ggatgctaca 1200caaatggtag taaaagccgg tccgaaaggt
ttctggcact gtcagccgga taacggtact 1260ttcgaaatgt ggtttaacgg
caagaacctg ttcccagact ccggttcgta tgtgtatgcc 1320ggtgaaggcg
aagtgatgga acaacgcaac tggcatcgtc agacttccgt acacaacacc
1380gtgactctgg acaataagaa tctggaaaca accgaatctg ttactaaact
gtggcagccg 1440gaaggcaata tccagacctt ggttacagaa aacccaagct
acaagaactt caagcaccgc 1500cgttcggtct tcttcgtaga caatacctac
tttgtcattg tagatgaagt atcaggcagc 1560gccaaaggtt ctgtcaacct
gcactatcag atgccgaaag gtgagatagc caacagccgt 1620gaagacatga
cattcctgac tcaattcgaa gatggaagca acatgaaact tcaatgcttc
1680ggccctgaag gcatgagcat gaaaaaagag ccgggatggt gttctactgc
atatcgcaag 1740cgctacaaac gtatgaatgt ttcattcaac gtaaagaaag
acaatgagaa tgcggtacgt 1800tacatcacag ttatttaccc agtcaagaag
agcgcagatg cccctaaatt tgacgctaag 1860ttcaagaaca aaacgttcga
tgaaaacgga ctggaaatag aagtgaaagt aaacggcaag 1920aaacagtcat
taaaatataa attataa 194710649PRTBacteroides thetaiotaomicron 10Met
Asn Lys Thr Leu Lys Tyr Ile Val Leu Leu Thr Phe Ala Cys Phe 1 5 10
15Val Gly Lys Gly Tyr Ala Gln Glu Leu Lys Ser Glu Val Phe Ser Leu
20 25 30Leu Asn Leu Asp Tyr Pro Gly Leu Glu Lys Val Lys Ala Leu His
Gln 35 40 45Glu Gly Lys Asp Glu Asp Ala Ala Lys Ala Leu Leu Asp Tyr
Tyr Arg 50 55 60Ala Arg Thr Asn Val Lys Thr Pro Asp Ile Asn Leu Lys
Lys Ile Thr65 70 75 80Ile Gly Lys Glu Glu Gln Gln Trp Ala Asp Asp
Gly Leu Lys His Thr 85 90 95Phe Phe Val His Lys Gly Tyr Gln Pro Ser
Tyr Asn Tyr Gly Glu Asp 100 105 110Ile Asn Trp Gln Tyr Trp Pro Val
Lys Asp Asn Glu Leu Arg Trp Gln 115 120 125Leu His Arg His Lys Trp
Phe Thr Pro Met Gly Lys Ala Tyr Arg Val 130 135 140Ser Gly Asp Glu
Lys Tyr Ala Lys Glu Trp Ala Tyr Gln Tyr Ile Asp145 150 155 160Trp
Ile Lys Lys Asn Pro Leu Val Val Glu Asn Val Arg Phe Ala Trp 165 170
175Arg Pro Leu Glu Val Ser Asn Arg Leu Gln Asp Gln Thr Thr Gln Phe
180 185 190Gln Leu Phe Leu Pro Ser Pro Ser Phe Thr Pro Asp Phe Leu
Thr Glu 195 200 205Phe Leu Val Asn Tyr His Lys His Ala Val His Ile
Leu Ala Asn Tyr 210 215 220Ser Asp Gln Gly Asn His Leu Leu Phe Glu
Ala Gln Arg Met Ile Tyr225 230 235 240Ala Gly Ala Phe Phe Pro Glu
Phe Lys Glu Ala Pro Ala Trp Arg Lys 245 250 255Ser Gly Ile Asp Ile
Leu Asn Arg Glu Val Asn Val Gln Val Tyr Asn 260 265 270Asp Gly Gly
Gln Phe Glu Leu Asp Pro His Tyr His Leu Ala Ala Ile 275 280 285Asn
Ile Phe Cys Lys Ala Leu Gly Ile Ala Asp Val Asn Gly Phe Arg 290 295
300Asn Glu Phe Pro Gln Glu Tyr Leu Asp Thr Ile Glu Lys Met Ile
Met305 310 315 320Phe Tyr Ala Asn Ile Ser Phe Pro Asp Tyr Thr Asn
Pro Cys Phe Ser 325 330 335Asp Ala Lys Ile Thr Glu Lys Lys Glu Met
Leu Lys Asn Tyr Arg Ala 340 345 350Trp Ser Lys Leu Phe Pro Lys Asn
Glu Thr Ile Lys Tyr Leu Ala Thr 355 360 365Asp Gly Lys Glu Gly Ala
Leu Pro Asp Tyr Met Ser Lys Gly Phe Leu 370 375 380Lys Ser Gly Phe
Phe Val Phe Arg Asn Ser Trp Gly Met Asp Ala Thr385 390 395 400Gln
Met Val Val Lys Ala Gly Pro Lys Gly Phe Trp His Cys Gln Pro 405 410
415Asp Asn Gly Thr Phe Glu Met Trp Phe Asn Gly Lys Asn Leu Phe Pro
420 425 430Asp Ser Gly Ser Tyr Val Tyr Ala Gly Glu Gly Glu Val Met
Glu Gln 435 440 445Arg Asn Trp His Arg Gln Thr Ser Val His Asn Thr
Val Thr Leu Asp 450 455 460Asn Lys Asn Leu Glu Thr Thr Glu Ser Val
Thr Lys Leu Trp Gln Pro465 470 475 480Glu Gly Asn Ile Gln Thr Leu
Val Thr Glu Asn Pro Ser Tyr Lys Asn 485 490 495Phe Lys His Arg Arg
Ser Val Phe Phe Val Asp Asn Thr Tyr Phe Val 500 505 510Ile Val Asp
Glu Val Ser Gly Ser Ala Lys Gly Ser Val Asn Leu His 515 520 525Tyr
Gln Met Pro Lys Gly Glu Ile Ala Asn Ser Arg Glu Asp Met Thr 530 535
540Phe Leu Thr Gln Phe Glu Asp Gly Ser Asn Met Lys Leu Gln Cys
Phe545 550 555 560Gly Pro Glu Gly Met Ser Met Lys Lys Glu Pro Gly
Trp Cys Ser Thr 565 570 575Ala Tyr Arg Lys Arg Tyr Lys Arg Met Asn
Val Ser Phe Asn Val Lys 580 585 590Lys Asp Asn Glu Asn Ala Val Arg
Tyr Ile Thr Val Ile Tyr Pro Val 595 600 605Lys Lys Ser Ala Asp Ala
Pro Lys Phe Asp Ala Lys Phe Lys Asn Lys 610 615 620Thr Phe Asp Glu
Asn Gly Leu Glu Ile Glu Val Lys Val Asn Gly Lys625 630 635 640Lys
Gln Ser Leu Lys Tyr Lys Leu Leu 6451129DNAArtificial SequencePrimer
11catatgctga ctgctcagac taaaaatac 291232DNAArtificial
SequencePrimer 12ctcgagttat ctttccgaat atcctgcgag at
321320PRTArtificial SequenceSynthetically generated peptide 13Met
Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10
15Arg Gly Ser His 201433DNAArtificial SequencePrimer 14catatgaata
aaaccctgaa atatatcgtc ctg 331538DNAArtificial SequencePrimer
15ctcgagttat aatttatatt ttaatgactg tttcttgc 381633DNAArtificial
SequencePrimer 16catatgcaag agttgaaaag cgaggtattc tcg
331740PRTArtificial SequenceSynthetically generated peptide 17Met
Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10
15Arg Gly Ser His Met Asn Lys Thr Leu Lys Tyr Lys Val Asn Gly Lys
20 25 30Lys Gln Ser Leu Lys Tyr Lys Leu 35 401842PRTArtificial
SequenceSynthetically generated peptide 18Met Gly Ser Ser His His
His His His His Ser Ser Gly Leu Val Pro 1 5 10 15Arg Gly Ser His
Met Gln Glu Leu Lys Ser Glu Val Phe Lys Val Asn 20 25 30Gly Lys Lys
Gln Ser Leu Lys Tyr Lys Leu 35 401918PRTBacteroides
thetaiotaomicron 19Lys Met Asp Lys Lys Glu Tyr Glu Leu Val Ser Asp
Gly Lys Ile Lys 1 5 10 15Gly Glu2040DNAArtificial SequencePrimer
20ggattaaaaa gaatccgttg gtggaaaatg tacgtttcgc 402140DNAArtificial
SequencePrimer 21cctaattttt cttaggcaac caccttttac atgcaaagcg
40221104DNABacteroides thetaiotaomicron 22caaacactga tgccactcac
cgaacgggta aacgtacagg ctgactctgc acgtatcaac 60cagattattg acggttgctg
ggtagctgtc gggacgaata aacctcatgc cattcagcgt 120gattttacca
acctgtttga tggcaagccc tcctatcgct ttgaactcaa aactgaagac
180aatacactgg aaggttatgc gaaaggagaa acgaaaggac gtgccgagtt
ttcatattgc 240tatgcaactt ccgacgattt caggggatta cctgccgacg
tttatcagaa agcacagatc 300acaaagacag tttatcatca cgggaaggga
gcttgtccgc aaggaagttc ccgcgactat 360gagttttcgg tttatattcc
ttcttcttta gacagcaatg tctccaccat ctttgcccaa 420tggcacggaa
tgcccgaccg gacgctggtc cagactcctc agggcgaggt gaagaaactg
480actgttgacg aatttgtaga actggaaaaa acgaccttct tcaaaaagaa
tgtcggacac 540gaaaaagtgg ccagactgga taaacaaggt aatccggtga
aagataaaaa tggaaaacct 600gtatataagg caggaaaacc caacggatgg
ttggttgaac agggaggata cccgccattg 660gcattcggat tttccggagg
actgttttat atcaaagcaa actccgaccg taaatggctg 720acagacaaag
atgaccgttg caatgcaaac ccgggaaaga cgcccgttat gaaaccgctg
780acttctgaat acaaggcatc caccattgcc tacaaattac cttttgccga
tttcccgaaa 840gactgctgga ttactttccg tgtccatatc gactggacgg
tctatggcaa ggaagcggaa 900acgattgtga aaccgggcat gctggatgta
cggatggatt atcaggagca aggtaagaaa 960gtgagcaaac acattgtcga
taatgagaag attctgattg gacgtaacga cgaagacggg 1020tattacttta
agttcggaat ttaccgcgta ggtgatagta ccgttcccgt ttgctacaat
1080ctcgcaggat attcggaaag ataa 110423367PRTBacteroides
thetaiotaomicron 23Gln Thr Leu Met Pro Leu Thr Glu Arg Val Asn Val
Gln Ala Asp Ser 1 5 10 15Ala Arg Ile Asn Gln Ile Ile Asp Gly Cys
Trp Val Ala Val Gly Thr 20 25 30Asn Lys Pro His Ala Ile Gln Arg Asp
Phe Thr Asn Leu Phe Asp Gly 35 40 45Lys Pro Ser Tyr Arg Phe Glu Leu
Lys Thr Glu Asp Asn Thr Leu Glu 50 55 60Gly Tyr Ala Lys Gly Glu Thr
Lys Gly Arg Ala Glu Phe Ser Tyr Cys65 70 75 80Tyr Ala Thr Ser Asp
Asp Phe Arg Gly Leu Pro Ala Asp Val Tyr Gln 85 90 95Lys Ala Gln Ile
Thr Lys Thr Val Tyr His His Gly Lys Gly Ala Cys 100 105 110Pro Gln
Gly Ser Ser Arg Asp Tyr Glu Phe Ser Val Tyr Ile Pro Ser 115 120
125Ser Leu Asp Ser Asn Val Ser Thr Ile Phe Ala Gln Trp His Gly Met
130 135 140Pro Asp Arg Thr Leu Val Gln Thr Pro Gln Gly Glu Val Lys
Lys Leu145 150 155 160Thr Val Asp Glu Phe Val Glu Leu Glu Lys Thr
Thr Phe Phe Lys Lys 165 170 175Asn Val Gly His Glu Lys Val Ala Arg
Leu Asp Lys Gln Gly Asn Pro 180 185 190Val Lys Asp Lys Asn Gly Lys
Pro Val Tyr Lys Ala Gly Lys Pro Asn 195 200 205Gly Trp Leu Val Glu
Gln Gly Gly Tyr Pro Pro Leu Ala Phe Gly Phe 210 215 220Ser Gly Gly
Leu Phe Tyr Ile Lys Ala Asn Ser Asp Arg Lys Trp Leu225 230 235
240Thr Asp Lys Asp Asp Arg Cys Asn Ala Asn Pro Gly Lys Thr Pro Val
245 250 255Met Lys Pro Leu Thr Ser Glu Tyr Lys Ala Ser Thr Ile Ala
Tyr Lys 260 265 270Leu Pro Phe Ala Asp Phe Pro Lys Asp Cys Trp Ile
Thr Phe Arg Val 275 280 285His Ile Asp Trp Thr Val Tyr Gly Lys Glu
Ala Glu Thr Ile Val Lys 290 295 300Pro Gly Met Leu Asp Val Arg Met
Asp Tyr Gln Glu Gln Gly Lys Lys305 310 315
320Val Ser Lys His Ile Val Asp Asn Glu Lys Ile Leu Ile Gly Arg Asn
325 330 335Asp Glu Asp Gly Tyr Tyr Phe Lys Phe Gly Ile Tyr Arg Val
Gly Asp 340 345 350Ser Thr Val Pro Val Cys Tyr Asn Leu Ala Gly Tyr
Ser Glu Arg 355 360 36524384PRTPedobacter heparinus 24Met Lys Lys
Gln Ile Leu Tyr Leu Ile Val Leu Gln Gln Leu Phe Leu 1 5 10 15Cys
Ser Ala Tyr Ala Gln Gln Lys Lys Ser Gly Asn Ile Pro Tyr Arg 20 25
30Val Asn Val Gln Ala Asp Ser Ala Lys Gln Lys Ala Ile Ile Asp Asn
35 40 45Lys Trp Val Ala Val Gly Ile Asn Lys Pro Tyr Ala Leu Gln Tyr
Asp 50 55 60Asp Lys Leu Arg Phe Asn Gly Lys Pro Ser Tyr Arg Phe Glu
Leu Lys65 70 75 80Ala Glu Asp Asn Ser Leu Glu Gly Tyr Ala Ala Gly
Glu Thr Lys Gly 85 90 95Arg Thr Glu Leu Ser Tyr Ser Tyr Ala Thr Thr
Asn Asp Phe Lys Lys 100 105 110Phe Pro Pro Ser Val Tyr Gln Asn Ala
Gln Lys Leu Lys Thr Val Tyr 115 120 125His Tyr Gly Lys Gly Ile Cys
Glu Gln Gly Ser Ser Arg Ser Tyr Thr 130 135 140Phe Ser Val Tyr Ile
Pro Ser Ser Phe Pro Asp Asn Ala Thr Thr Ile145 150 155 160Phe Ala
Gln Trp His Gly Ala Pro Ser Arg Thr Leu Val Ala Thr Pro 165 170
175Glu Gly Glu Ile Lys Thr Leu Ser Ile Glu Glu Phe Leu Ala Leu Tyr
180 185 190Asp Arg Met Ile Phe Lys Lys Asn Ile Ala His Asp Lys Val
Glu Lys 195 200 205Lys Asp Lys Asp Gly Lys Ile Thr Tyr Val Ala Gly
Lys Pro Asn Gly 210 215 220Trp Lys Val Glu Gln Gly Gly Tyr Pro Thr
Leu Ala Phe Gly Phe Ser225 230 235 240Lys Gly Tyr Phe Tyr Ile Lys
Ala Asn Ser Asp Arg Gln Trp Leu Thr 245 250 255Asp Lys Ala Asp Arg
Asn Asn Ala Asn Pro Glu Asn Ser Glu Val Met 260 265 270Lys Pro Tyr
Ser Ser Glu Tyr Lys Thr Ser Thr Ile Ala Tyr Lys Met 275 280 285Pro
Phe Ala Gln Phe Pro Lys Asp Cys Trp Ile Thr Phe Asp Val Ala 290 295
300Ile Asp Trp Thr Lys Tyr Gly Lys Glu Ala Asn Thr Ile Leu Lys
Pro305 310 315 320Gly Lys Leu Asp Val Met Met Thr Tyr Thr Lys Asn
Lys Lys Pro Gln 325 330 335Lys Ala His Ile Val Asn Gln Gln Glu Ile
Leu Ile Gly Arg Asn Asp 340 345 350Asp Asp Gly Tyr Tyr Phe Lys Phe
Gly Ile Tyr Arg Val Gly Asn Ser 355 360 365Thr Val Pro Val Thr Tyr
Asn Leu Ser Gly Tyr Ser Glu Thr Ala Arg 370 375
38025659PRTPedobacter heparinus 25Met Thr Thr Lys Ile Phe Lys Arg
Ile Ile Val Phe Ala Val Ile Ala 1 5 10 15Leu Ser Ser Gly Asn Ile
Leu Ala Gln Ser Ser Ser Ile Thr Arg Lys 20 25 30Asp Phe Asp His Ile
Asn Leu Glu Tyr Ser Gly Leu Glu Lys Val Asn 35 40 45Lys Ala Val Ala
Ala Gly Asn Tyr Asp Asp Ala Ala Lys Ala Leu Leu 50 55 60Ala Tyr Tyr
Arg Glu Lys Ser Lys Ala Arg Glu Pro Asp Phe Ser Asn65 70 75 80Ala
Glu Lys Pro Ala Asp Ile Arg Gln Pro Ile Asp Lys Val Thr Arg 85 90
95Glu Met Ala Asp Lys Ala Leu Val His Gln Phe Gln Pro His Lys Gly
100 105 110Tyr Gly Tyr Phe Asp Tyr Gly Lys Asp Ile Asn Trp Gln Met
Trp Pro 115 120 125Val Lys Asp Asn Glu Val Arg Trp Gln Leu His Arg
Val Lys Trp Trp 130 135 140Gln Ala Met Ala Leu Val Tyr His Ala Thr
Gly Asp Glu Lys Tyr Ala145 150 155 160Arg Glu Trp Val Tyr Gln Tyr
Ser Asp Trp Ala Arg Lys Asn Pro Leu 165 170 175Gly Leu Ser Gln Asp
Asn Asp Lys Phe Val Trp Arg Pro Leu Glu Val 180 185 190Ser Asp Arg
Val Gln Ser Leu Pro Pro Thr Phe Ser Leu Phe Val Asn 195 200 205Ser
Pro Ala Phe Thr Pro Ala Phe Leu Met Glu Phe Leu Asn Ser Tyr 210 215
220His Gln Gln Ala Asp Tyr Leu Ser Thr His Tyr Ala Glu Gln Gly
Asn225 230 235 240His Arg Leu Phe Glu Ala Gln Arg Asn Leu Phe Ala
Gly Val Ser Phe 245 250 255Pro Glu Phe Lys Asp Ser Pro Arg Trp Arg
Gln Thr Gly Ile Ser Val 260 265 270Leu Asn Thr Glu Ile Lys Lys Gln
Val Tyr Ala Asp Gly Met Gln Phe 275 280 285Glu Leu Ser Pro Ile Tyr
His Val Ala Ala Ile Asp Ile Phe Leu Lys 290 295 300Ala Tyr Gly Ser
Ala Lys Arg Val Asn Leu Glu Lys Glu Phe Pro Gln305 310 315 320Ser
Tyr Val Gln Thr Val Glu Asn Met Ile Met Ala Leu Ile Ser Ile 325 330
335Ser Leu Pro Asp Tyr Asn Thr Pro Met Phe Gly Asp Ser Trp Ile Thr
340 345 350Asp Lys Asn Phe Arg Met Ala Gln Phe Ala Ser Trp Ala Arg
Val Phe 355 360 365Pro Ala Asn Gln Ala Ile Lys Tyr Phe Ala Thr Asp
Gly Lys Gln Gly 370 375 380Lys Ala Pro Asn Phe Leu Ser Lys Ala Leu
Ser Asn Ala Gly Phe Tyr385 390 395 400Thr Phe Arg Ser Gly Trp Asp
Lys Asn Ala Thr Val Met Val Leu Lys 405 410 415Ala Ser Pro Pro Gly
Glu Phe His Ala Gln Pro Asp Asn Gly Thr Phe 420 425 430Glu Leu Phe
Ile Lys Gly Arg Asn Phe Thr Pro Asp Ala Gly Val Phe 435 440 445Val
Tyr Ser Gly Asp Glu Ala Ile Met Lys Leu Arg Asn Trp Tyr Arg 450 455
460Gln Thr Arg Ile His Ser Thr Leu Thr Leu Asp Asn Gln Asn Met
Val465 470 475 480Ile Thr Lys Ala Arg Gln Asn Lys Trp Glu Thr Gly
Asn Asn Leu Asp 485 490 495Val Leu Thr Tyr Thr Asn Pro Ser Tyr Pro
Asn Leu Asp His Gln Arg 500 505 510Ser Val Leu Phe Ile Asn Lys Lys
Tyr Phe Leu Val Ile Asp Arg Ala 515 520 525Ile Gly Glu Ala Thr Gly
Asn Leu Gly Val His Trp Gln Leu Lys Glu 530 535 540Asp Ser Asn Pro
Val Phe Asp Lys Thr Lys Asn Arg Val Tyr Thr Thr545 550 555 560Tyr
Arg Asp Gly Asn Asn Leu Met Ile Gln Ser Leu Asn Ala Asp Arg 565 570
575Thr Ser Leu Asn Glu Glu Glu Gly Lys Val Ser Tyr Val Tyr Asn Lys
580 585 590Glu Leu Lys Arg Pro Ala Phe Val Phe Glu Lys Pro Lys Lys
Asn Ala 595 600 605Gly Thr Gln Asn Phe Val Ser Ile Val Tyr Pro Tyr
Asp Gly Gln Lys 610 615 620Ala Pro Glu Ile Ser Ile Arg Glu Asn Lys
Gly Asn Asp Phe Glu Lys625 630 635 640Gly Lys Leu Asn Leu Thr Leu
Thr Ile Asn Gly Lys Gln Gln Leu Val 645 650 655Leu Val
Pro26286PRTArtificial SequenceConsensus sequence 26Met Lys Lys Ile
Leu Ile Met Met Gly Cys Arg Val Asn Val Gln Ala 1 5 10 15Asp Ser
Ala Arg Ile Ile Asp Trp Val Ala Val Gly Asn Lys Pro Ala 20 25 30Ile
Gln Asp Phe Gly Lys Pro Ser Tyr Arg Phe Glu Leu Lys Glu Asp 35 40
45Asn Thr Leu Glu Gly Tyr Ala Gly Glu Thr Lys Gly Arg Glu Ser Tyr
50 55 60Tyr Ala Thr Ser Asp Phe Arg Pro Val Tyr Gln Ala Gln Lys Thr
Val65 70 75 80Tyr His Gly Lys Gly Cys Gln Gly Ser Ser Arg Tyr Phe
Ser Val Tyr 85 90 95Ile Pro Ser Ser Asn Ser Thr Ile Phe Ala Gln Trp
His Gly Pro Arg 100 105 110Thr Leu Val Thr Pro Gly Glu Val Lys Leu
Thr Val Asp Glu Phe Val 115 120 125Leu Phe Lys Lys Asn Val Gly His
Glu Lys Val Arg Leu Asp Lys Gln 130 135 140Gly Asn Pro Val Lys Asp
Lys Gly Lys Tyr Ala Gly Lys Pro Asn Gly145 150 155 160Trp Val Glu
Gln Gly Gly Tyr Pro Leu Ala Phe Gly Phe Ser Gly Phe 165 170 175Tyr
Ile Lys Ala Asn Ser Asp Arg Trp Leu Thr Asp Lys Asp Arg Asn 180 185
190Ala Asn Pro Thr Val Met Lys Pro Thr Ser Glu Tyr Lys Ser Thr Ile
195 200 205Ala Tyr Lys Ile Pro Phe Ala Phe Pro Lys Asp Cys Trp Ile
Thr Phe 210 215 220Val Ile Asp Trp Thr Tyr Gly Lys Glu Ala Thr Ile
Val Lys Pro Gly225 230 235 240Leu Asp Val Met Tyr Gln Lys His Ile
Val Asn Ile Leu Ile Gly Arg 245 250 255Asn Asp Glu Asp Gly Tyr Tyr
Phe Lys Phe Gly Ile Tyr Arg Val Gly 260 265 270Ser Thr Val Pro Val
Tyr Asn Leu Gly Tyr Ser Glu Ala Arg 275 280 28527388PRTArtificial
SequenceConsensus sequence 27Met Lys Lys Ile Ile Val Gly Asn Ile
Ala Ile Phe Ile Asn Leu Glu 1 5 10 15Tyr Gly Leu Glu Lys Val Gly
Asp Asp Ala Ala Lys Ala Leu Leu Tyr 20 25 30Tyr Arg Lys Ser Arg Pro
Asp Asn Ala Glu Lys Pro Ala Arg Ile Lys 35 40 45Ala Asp Ala Leu His
Phe His Lys Gly Tyr Pro Phe Tyr Gly Asp Ile 50 55 60Asn Trp Gln Trp
Pro Val Lys Asp Asn Glu Val Arg Trp Gln Leu His65 70 75 80Arg Lys
Trp Trp Met Ala Tyr Thr Gly Asp Glu Lys Tyr Ala Arg Glu 85 90 95Trp
Tyr Gln Tyr Asp Trp Arg Lys Asn Pro Leu Lys Lys Glu Tyr Glu 100 105
110Leu Val Ser Asp Gly Lys Ile Lys Gly Glu Val Asp Asn Lys Phe Trp
115 120 125Arg Pro Leu Glu Val Ser Arg Val Gln Phe Leu Phe Val Ser
Pro Phe 130 135 140Thr Pro Phe Leu Glu Phe Leu Tyr His Ala Leu Tyr
Glu Gln Gly Asn145 150 155 160His Leu Phe Glu Ala Gln Arg Leu Phe
Ala Gly Phe Pro Glu Phe Lys 165 170 175Asp Pro Trp Arg Thr Gly Ile
Val Leu Asn Glu Ile Gln Val Tyr Asp 180 185 190Gly Gln Phe Glu Leu
Pro Tyr His Val Ala Ala Ile Ile Phe Lys Ala 195 200 205Gly Ala Glu
Phe Pro Gln Tyr Val Thr Val Glu Met Ile Met Ile Ser 210 215 220Pro
Asp Tyr Pro Phe Asp Ile Thr Asp Lys Met Gln Phe Trp Arg Val225 230
235 240Phe Pro Asn Ile Lys Tyr Ala Thr Asp Gly Lys Gly Pro Phe Leu
Ser 245 250 255Lys Ala Gly Phe Tyr Phe Arg Trp Ala Thr Met Val Ile
Lys Ala Pro 260 265 270Gly Phe His Gln Pro Asp Asn Gly Thr Phe Glu
Leu Phe Gly Arg Asn 275 280 285Pro Asp Gly Phe Val Tyr Gly Asp Ile
Met Arg Asn Trp Arg Gln Thr 290 295 300Ile His Thr Ile Thr Leu Asp
Asn Asn Met Thr Trp Asn Leu Leu Asn305 310 315 320Pro Ser Tyr Asn
His Arg Ser Val Phe Ile Tyr Phe Leu Val Ile Asp 325 330 335Gly Ala
Gly Leu Val His Trp Gln Leu Asp Lys Thr Tyr Asp Gly Asn 340 345
350Leu Ile Gln Asp Ser Leu Glu Gly Ser Tyr Lys Lys Arg Phe Lys Asn
355 360 365Phe Val Ser Ile Val Tyr Pro Lys Phe Glu Leu Leu Leu Ile
Asn Gly 370 375 380Lys Gln Val Leu385
* * * * *
References