U.S. patent application number 09/809790 was filed with the patent office on 2002-06-13 for disintegrin homologs.
Invention is credited to Baindur, Nand, Bishop, Paul D., Deisher, Theresa A., Sheppard, Paul O..
Application Number | 20020072102 09/809790 |
Document ID | / |
Family ID | 26785602 |
Filed Date | 2002-06-13 |
United States Patent
Application |
20020072102 |
Kind Code |
A1 |
Sheppard, Paul O. ; et
al. |
June 13, 2002 |
Disintegrin homologs
Abstract
The present invention relates to polynucleotide and polypeptide
molecules for zdint1, a novel member of the Disintegrin Proteases.
The polypeptides, and polynucleotides encoding them, are believed
to be cell-cell interaction modulating and may be used for delivery
and therapeutics. The present invention also includes antibodies to
the zdint1 polypeptides.
Inventors: |
Sheppard, Paul O.; (Granite
Falls, WA) ; Baindur, Nand; (Longmont, CO) ;
Deisher, Theresa A.; (Seattle, WA) ; Bishop, Paul
D.; (Fall City, WA) |
Correspondence
Address: |
Robyn Adams
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Family ID: |
26785602 |
Appl. No.: |
09/809790 |
Filed: |
March 16, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09809790 |
Mar 16, 2001 |
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09351414 |
Jul 9, 1999 |
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6265199 |
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60092371 |
Jul 10, 1998 |
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Current U.S.
Class: |
435/183 ;
435/320.1; 435/325; 435/69.1 |
Current CPC
Class: |
C07K 14/47 20130101;
C12N 9/6489 20130101 |
Class at
Publication: |
435/183 ;
435/69.1; 435/325; 435/320.1 |
International
Class: |
C12P 021/02; C12N
005/06; C12N 009/00 |
Claims
What is claimed is:
1. An isolated polypeptide molecule comprising a contiguous
sequence of 14 amino acids of SEQ ID NO:2.
2. An isolated polypeptide molecule according to claim 1, wherein
the polypeptide molecule comprises residues 437 to 450 of SEQ ID
NO:2.
3. An isolated polypeptide molecule according to claim 1 wherein
the polypeptide molecule is between 82 and 232 amino acids in
length.
4. An isolated polypeptide molecule according to claim 3 wherein
the polypeptide molecule is residues 164 to 382 of SEQ ID NO:2.
5. An isolated polypeptide molecule according to claim 3 wherein
the polypeptide molecule is residues 383 to 464 of SEQ ID NO:2.
6. An isolated polypeptide molecule according to claim 3 wherein
the polypeptide molecule is residues 465 to 696 of SEQ ID NO:2.
7. A isolated polypeptide molecule selected from the group
consisting of: a) a polypeptide molecule comprising residues 164 to
382 of SEQ ID NO:2; b) a polypeptide molecule comprising residues
383 to 464 of SEQ ID NO:2; c) a polypeptide molecule comprising
residues 465 to 696 of SEQ ID NO:2; d) a polypeptide molecule
comprising residues 438 to 449 of SEQ ID NO:2; e) a polypeptide
molecule comprising residues 164 to 464 of SEQ ID NO:2; f) a
polypeptide molecule comprising residues 164 to 696 of SEQ ID NO:2;
g) a polypeptide molecule comprising residues 383 to 696 of SEQ ID
NO:2; h) a polypeptide molecule comprising residues 164 to 449 of
SEQ ID NO:2; i) a polypeptide molecule comprising residues 438 to
696 of SEQ ID NO:2; and j) a polypeptide molecule comprising
residues 1 to 696 of SEQ ID NO:2.
8. An isolated polynucleotide molecule encoding a polypeptide
molecule, wherein the polypeptide molecule comprises a contiguous
sequence of 14 amino acids of SEQ ID NO:2.
9. An isolated polynucleotide molecule according to claim 8,
wherein the polypeptide molecule comprises residues 437 to 450 of
SEQ ID NO:2.
10. An isolated nucleotide molecule according to claim 8, wherein
the polypeptide molecule is between 82 and 232 amino acids in
length.
11. An isolated polynucleotide molecule according to claim 10,
wherein the polypeptide molecule is residues 164 to 382 of SEQ ID
NO:2.
12. An isolated polynucleotide molecule according to claim 10,
wherein the polypeptide molecule is residues 383 to 464 of SEQ ID
NO:2.
13. An isolated polynucleotide molecule according to claim 10,
wherein the polypeptide molecule is residues 465 to 696 of SEQ ID
NO:2.
14. A isolated polynucleotide molecule encoding a polypeptide
molecule, wherein the polypeptide molecule is selected from the
group consisting of: a) a polypeptide molecule comprising residues
164 to 382 of SEQ ID NO:2; b) a polypeptide molecule comprising
residues 383 to 464 of SEQ ID NO:2; c) a polypeptide molecule
comprising residues 465 to 696 of SEQ ID NO:2; d) a polypeptide
molecule comprising residues 438 to 449 of SEQ ID NO:2; e) a
polypeptide molecule comprising residues 164 to 464 of SEQ ID NO:2;
f) a polypeptide molecule comprising residues 164 to 696 of SEQ ID
NO:2; g) a polypep tide molecule comprising residues 383 to 696 of
SEQ ID NO:2; h) a polypeptide molecule comprising residues 164 to
449 of SEQ ID NO:2; i) a polypeptide molecule comprising residues
438 to 696 of SEQ ID NO:2; and j) a polypeptide molecule comprising
residues 1 to 696 of SEQ ID NO:2.
15. An isolated polynucleotide encoding a fusion protein having a
first segment and a second segment, wherein the first segment
comprises a first polypeptide encoding a polypeptide having a
protease domain and the second segment comprises a second
polynucleotide encoding a polypeptide that has a contiguous
sequence of 14 amino acids between residues 383 and 464 of SEQ ID
NO:2, and wherein the first segment is positioned amino-terminally
to the second segment.
16. An isolated polynucleotide according to claim 15, wherein the
protease domain is selected from the group consisting of; a) a
protease domain that is a member of the Disintegrin Proteases; and
b) a protease domain that is at least 80% identical to amino acid
residues 164 to 382 of SEQ ID NO:2.
17. An isolated polynucleotide molecule encoding a polypeptide
molecule wherein the polynucleotide molecule is selected from the
group consisting of: a) a polynucleotide molecule that encodes a
polypeptide molecule that is at least 80% identical to residues 383
to 464 of SEQ ID NO:2; and b) a polynucleotide molecule that is
complementary to a).
18. An isolated polynucleotide molecule according to claim 17
wherein the polynucleotide molecule is selected from the group
consisting of: a) a polynucleotide molecule that encodes a
polypeptide molecule that is at least 80% identical to residues 383
to 696 of SEQ ID NO:2; and b) a polynucleotide -molecule that is
complementary to a).
19. An isolated polynucleotide molecule according to claim 17,
wherein the polynucleotide molecule is selected from the group
consisting of: a) a polynucleotide molecule that encodes a
polypeptide molecule that is at least 80% identical to residues 1
to 696 of SEQ ID NO:2; and b) a polynucleotide molecule that is
complementary to a).
20. An expression vector comprising the following operably linked
elements: a) a transcription promoter; b) a DNA segment encoding
the polypeptide of claim 1; and c) a transcription terminator.
21. An expression vector of claim 20 wherein the DNA segment
further encodes an affinity tag.
22. A cultured cell into which has been introduced an expression
vector according to claim 21, wherein said cell expresses the
polypeptide encoded by the DNA segment.
23. A method of producing a polypeptide comprising culturing a cell
according to claim 22, whereby said cell expresses the polypeptide
encoded by the DNA segment; and recovering the polypeptide.
24. A method for modulating cell-cell interactions by combining the
polypeptide according to claim 1, with cells in vivo and in
vitro.
25. A method for modulating cell-cell interactions according to
claim 24, whereby the cells are derived from tissues selected from
the group consisting of: a) tissues from heart; b) tissues from
brain; c) tissues from spinal cord; and d) tissues from skeletal
muscle.
26. An isolated polypeptide molecule comprising a contiguous
sequence of amino acids, wherein the contiguous sequence of amino
acids is selected from the group consisting of: a) SEQ ID NO:7; b)
SEQ ID NO:8; c) SEQ ID NO:9; d) SEQ ID NO:10; and e) SEQ ID
NO:11.
27. An isolated polynucleotide molecule encoding the isolated
polypeptide molecule of claim 26.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is related to Provisional Application No.
60/092,371 filed on Jul. 10, 1998. Under 35 U.S.C. .sctn. 119(e)
(1), this application claims benefit of said Provisional
Application.
BACKGROUND OF THE INVENTION
[0002] Disintegrins have been shown to bind cell surface molecules,
including integrins, on the surface of various cells, such as
platelets, fibroblasts, tumor, endothelial, muscle, neuronal, bone,
and sperm cells. Disintegrins are unique and potentially useful
tools for investigating cell-matrix and cell-cell interactions.
Additionally, they have been useful in the development of
antithrombotic and antimetastatic agents due to their
anti-adhesive, anti-migration of certain tumor cells, and
antiangiogenesis activities.
[0003] Families of proteins which have disintegrin domains include
ADAMs (A Metalloprotease and Disintegrin), MDCs
(Metalloprotease/Disintegrin/Cy- steine-rich) and SVMPs (Snake
Venom Metalloprotease).
[0004] For a review of ADAMs, see Wolfsberg and White,
Developmental Biology, 180:389-401, 1996. ADAMs have been shown to
exist as independent functional units or in conjunction with other
members of this family in heterodimeric complexes. Some members of
the family exist in multiple isoforms which may have resulted from
alternative splicing. ADAMs proteins have been shown to have
adhesive as well as anti-adhesive functions. Some members of the
ADAMs family have very specific tissue distribution while others
are widely distributed. Not all members of this family are capable
of manifesting all of the potential functions represented by the
domains common to their genetic structure.
[0005] The ADAMs are characterized by having a propeptide domain, a
metalloprotease-like domain, a disintegrin-like domain, a
cysteine-rich domain, an EGF-like domain, and a cytoplasmic
domain.
[0006] A prototypical example of this family is ADAM 12. ADAM 12,
also known as meltrin a, has a truncated isoform, as well as a
full-length isoform, and is involved in muscle cell fusion and
differentiation (Gilpin et al., J. Biol. Chem. 273:157-166,
1998).
[0007] Another prototypical example of this family is ADAM 1, which
forms a heterodimer with ADAM 2 and is involved in sperm/egg fusion
(Wolfsberg and White, supra).
[0008] The SVMP family is represented by three classes (P-I, P-II,
and P-III). All three classes contain propeptide and
metalloprotease domains. The P-II and P-III classes also contain a
disintegrin domain, and the P-III class further contains a
cysteine-rich domain. These domains are similar in sequence to
those found in the ADAMs. Some members of the SVMP family have a
conserved "RGD" amino acid sequence. This tripeptide has been shown
to form a hairpin loop whose conformation can disrupt the binding
of fibrinogen to activated platelets. This RGD sequence may be
substituted by RSE, MVD, MSE, and KGD in P-II SVMPs, and by MSEC,
RSEC, IDDC, and RDDC (a tripeptide along with a carboxy-terminal
cysteine residue) in P-III SVMPs. Thus, these sequences may be
responsible for integrin binding in the P-II and P-III SVMPs.
[0009] A prototypical example of a SVMP is jararhagin, which
mediates platelet aggregation by binding to the platelet
.alpha..sub.2 subunit (GPIa) via the disintegrin domain followed by
proteolysis of the P.beta..sub.1 subunit (GPIIA) (Huang and Liu, J.
Toxicol-Toxin Reviews 16: 135-161, 1997).
[0010] The proteins of the
Metalloprotease/Disintegrin/Cysteine-rich family are involved in
diverse tasks, ranging from roles in fertilization and muscle
fusion, TNF.alpha. release from plasma membranes, intracellular
protein cleavage, and essential functions in neuronal development
(Blobel, Cell 90:589-592, 1997). This family is also characterized
by the metalloprotease, disintegrin and cysteine-rich domains, as
described above.
[0011] The present invention provides a novel disintegrin homolog
and related compositions whose uses should be apparent to those
skilled in the art from the teachings herein.
SUMMARY OF THE INVENTION
[0012] Within one aspect, the present invention provides an
isolated polypeptide molecule comprising a contiguous sequence of
14 amino acids of SEQ ID NO:2. Within an embodiment the polypeptide
molecule comprises residues 437 to 450 of SEQ ID NO:2. Within
another embodiment, the polypeptide molecule is between 82 and 232
amino acids in length. Within further embodiments polypeptide
molecule is residues 164 to 382 of SEQ ID NO:2; residues 383 to 464
of SEQ ID NO:2; and/or residues 465 to 696 of SEQ ID NO:2.
[0013] Within another aspect, the invention provides an isolated
polypeptide molecule selected from the group consisting of: a) a
polypeptide molecule comprising residues 164 to 382 of SEQ ID NO:2;
b) a polypeptide molecule comprising residues 383 to 464 of SEQ ID
NO:2; c) a polypeptide molecule comprising residues 465 to 696 of
SEQ ID NO:2; d) a polypeptide molecule comprising residues 438 to
449 of SEQ ID NO:2; e) a polypeptide molecule comprising residues
164 to 464 of SEQ ID NO:2; f) a polypeptide molecule comprising
residues 164 to 696 of SEQ ID NO:2; g) a polypeptide molecule
comprising residues 383 to 696 of SEQ ID NO:2; h) a polypeptide
molecule comprising residues 164 to 449 of SEQ ID NO:2; i) a
polypeptide molecule comprising residues 438 to 696 of SEQ ID NO:2;
and j) a polypeptide molecule comprising residues 1 to 696 of SEQ
ID NO:2.
[0014] Within another aspect is provided an isolated polynucleotide
molecule encoding a polypeptide molecule, wherein the polypeptide
molecule comprises a contiguous sequence of 14 amino acids of SEQ
ID NO:2. Within an embodiment, the polypeptide molecule comprises
residues 437 to 450 of SEQ ID NO:2. Within a further embodiment,
the polypeptide molecule is between 82 and 232 amino acids in
length. Within further embodiments, the polypeptide molecule is
residues 164 to 382 of SEQ ID NO:2; residues 383 to 464 of SEQ ID
NO:2; and/or residues 465 to 696 of SEQ ID NO:2.
[0015] Within another aspect, the invention provides an isolated
polynucleotide molecule encoding a polypeptide molecule, wherein
the polypeptide molecule is selected from the group consisting of:
a) a polypeptide molecule comprising residues 164 to 382 of SEQ ID
NO:2; b) a polypeptide molecule comprising residues 383 to 464 of
SEQ ID NO:2; c) a polypeptide molecule comprising residues 465 to
696 of SEQ ID NO:2; d) a polypeptide molecule comprising residues
438 to 449 of SEQ ID NO:2; e) a polypeptide molecule comprising
residues 164 to 464 of SEQ ID NO:2; f) a polypeptide molecule
comprising residues 164 to 696 of SEQ ID NO:2; g) a polypeptide
molecule comprising residues 383 to 696 of SEQ ID NO:2; h) a
polypeptide molecule comprising residues 164 to 449 of SEQ ID NO:2;
i) a polypeptide molecule comprising residues 438 to 696 of SEQ ID
NO:2; and j) a polypeptide molecule comprising residues 1 to 696 of
SEQ ID NO:2.
[0016] Within another aspect is provided an isolated polynucleotide
encoding a fusion protein having a first segment and a second
segment, wherein the first segment comprises a first polypeptide
encoding a polypeptide having a protease domain and the second
segment comprises a second polynucleotide encoding a polypeptide
that has a contiguous sequence of 14 amino acids between residues
383 and 464 of SEQ ID NO:2, and wherein the first segment is
positioned amino-terminally to the second segment. Within an
embodiment, the protease domain is selected from the group
consisting of; a) a protease domain that is a member of the
Disintegrin Proteases; and b) a protease domain that is at least
80% identical to amino acid residues 164 to 382 of SEQ ID NO:2.
[0017] Within another aspect the invention provides an isolated
polynucleotide molecule encoding a polypeptide molecule wherein the
polynucleotide molecule is selected from the group consisting of:
a) a polynucleotide molecule that encodes a polypeptide molecule
that is at least 80% identical to residues 383 to 464 of SEQ ID
NO:2; and b) a polynucleotide molecule that is complementary to a).
Within an embodiment, the polynucleotide molecule is selected from
the group consisting of: a) a polynucleotide molecule that encodes
a polypeptide molecule that is at least 80% identical to residues
383 to 696 of SEQ ID NO:2; and b) a polynucleotide molecule that is
complementary to a). Within a further embodiment, the
polynucleotide molecule is selected from the group consisting of:
a) a polynucleotide molecule that encodes a polypeptide molecule
that is at least 80% identical to residues 1 to 696 of SEQ ID NO:2;
and b) a polynucleotide molecule that is complementary to a).
[0018] Within another aspect is provided an expression vector
comprising the following operably linked elements: a) a
transcription promoter; b) a DNA segment encoding the polypeptide
of claim 1; and c) a transcription terminator. Within an embodiment
the DNA segment further encodes an affinity tag.
[0019] Within another aspect, the invention provides a cultured
cell into which has been introduced said expression vector, wherein
the cell expresses the polypeptide encoded by the DNA segment.
[0020] Within another aspect, the invention provides a method of
producing a polypeptide comprising culturing the cell expressing
the polypeptide encoded by the DNA segment; and recovering the
polypeptide.
[0021] Within another aspect is provided a method for modulating
cell-cell interactions by combining the polypeptide comprising the
sequence of 14 contiguous amino acids, with cells in vivo and in
vitro. Within an embodiment, the cells are derived from tissues
selected from the group consisting of: a) tissues from heart; b)
tissues from brain; c) tissues from spinal cord; and d) tissues
from skeletal muscle.
[0022] Within another aspect, the invention provides an isolated
polypeptide molecule comprising a contiguous sequence of amino
acids, wherein the contiguous sequence of amino acids is selected
from the group consisting of: a) SEQ ID NO:7; b) SEQ ID NO:8; c)
SEQ ID NO:9; d) SEQ ID NO:10; and e) SEQ ID NO:11.
[0023] Within another aspect is provide an isolated polynucleotide
molecule encoding an isolated polypeptide molecule, wherein the
polypeptide comprises a contiguous sequence of amino acids and is
selected from the group consisting of: a) SEQ ID NO:7; b) SEQ ID
NO:8; c) SEQ ID NO:9; d) SEQ ID NO:10; and e) SEQ ID NO:11.
[0024] These and other aspects of the invention will become evident
upon reference to the following detailed description of the
invention and attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a Hopp/Woods hydrophilicity profile of the zdint1
protein sequence shown in SEQ ID NO:2. The profile is based on a
sliding six-residue window. Buried G, S, and T residues and exposed
H, Y, and W residues were ignored. These residues are indicated in
the figure by lower case letters.
[0026] FIG. 2 schematically shows a domain level alignment of
members of ADAMs, MDCs, and SVMPs. DISA TRIGA is a SVMP. MS2 HUMAN
is an ADAM. HSUTSP1 (TACE) is a MDC. And HSU52370.sub.--1 is
fertilin-.beta., ADAM 2. "sig" denotes the secretory signal
peptide; "propep" denotes the propeptide domain; "Metal-protease"
denotes the metalloprotease domain; "disint" denotes the
disintegrin domain; "cys" denotes the cysteine-rich domain; "RGD"
denotes a tripeptide, Arginine-Glycine-Asparagine; and "TMD"
denotes the transmembrane domain.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0028] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection of the second polypeptide or
provide sites for attachment of the second polypeptide to a
substrate. In principal, any peptide or protein for which an
antibody or other specific binding agent is available can be used
as an affinity tag. Affinity tags include a poly-histidine tract,
protein A (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al.,
Methods Enzymol. 198:3, 1991), glutathione S transferase (Smith and
Johnson, Gene 67:31, 1988), Glu-Glu affinity tag (Grussenmeyer et
al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985), substance P,
Flag.TM. peptide (Hopp et al., Biotechnology 6:1204-10, 1988),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2: 95-107, 1991. DNAs encoding affinity tags are
available from commercial suppliers (e.g., Pharmacia Biotech,
Piscataway, N.J.).
[0029] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0030] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0031] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complem- ent pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0032] The term "complements of a polynucleotide molecule" is a
polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to 5.degree.
CCCGTGCAT 3'.
[0033] The term "contig" denotes a polynucleotide that has a
contiguous stretch of identical or complementary sequence to
another polynucleotide. Contiguous sequences are said to "overlap"
a given stretch of polynucleotide sequence either in their entirety
or along a partial stretch of the polynucleotide. For example,
representative contigs to the polynucleotide sequence
5'-ATGGAGCTT-3' are 5'-AGCTTgagt-3' and 3'-tcgacTACC-5'.
[0034] The term "corresponding to", when applied to positions of
amino acid residues in sequences, means corresponding positions in
a plurality of sequences when the sequences are optimally
aligned.
[0035] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide) Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0036] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0037] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0038] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a
highly-purified form, i.e. greater than 95% pure, more preferably
greater than 99% pure. When used in this context, the term
"isolated" does not exclude the presence of the same polypeptide in
alternative physical forms, such as dimers or alternatively
glycosylated or derivatized forms.
[0039] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0040] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0041] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, a-globin, b-globin, and myoglobin are
paralogs of each other.
[0042] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from-the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules, it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus, all nucleotides within a double-stranded
polynucleotide molecule may not be paired. Such unpaired ends will
in general not exceed 20 nt in length.
[0043] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0044] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0045] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0046] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-domain or multi-peptide structure
comprising an extracellular ligand-binding domain and an
intracellular effector domain that is typically involved in signal
transduction. Binding of ligand to receptor results in a
conformational change in the receptor that causes an interaction
between the effector domain and other molecule(s) in the cell. This
interaction in turn leads to an alteration in the metabolism of the
cell. Metabolic events that are linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, increases in cyclic AMP production, mobilization
of cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids. In general, receptors can be membrane bound,
cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone
receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF
receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor,
G-CSF receptor, erythropoietin receptor and IL-6 receptor).
[0047] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0048] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a protein encoded by a splice
variant of an mRNA transcribed from a gene.
[0049] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or "approximately" X, the stated value of X will be
understood to be accurate to .+-.10%.
[0050] All references cited herein are incorporated by reference in
their entirety.
[0051] The present invention is based upon the discovery of a novel
cDNA sequence (SEQ ID NO:1) and corresponding polypeptide (SEQ ID
NO:2) having homology to disintegrin-like family members (ADAMs,
SVMPs and MDCs; referred to herein as Disintegrin Proteases, or
"DPs"). See, for example, Blobel, Cell 90:589-592, 1997, and
Wolfsberg and White, Developmental Biology 180:389-401, 1996.
Disintegrins can be involved in, for example, anticoagulation,
fertilization, muscle fusion, connective tissue disorders,
chondrogenesis, arthritis, metastasis and neurogenesis.
[0052] The secretory signal (also known as a leader sequence,
prepro sequence or pre sequence) domain of these polypeptides
directs the polypeptide through a secretory pathway of a cell in
which it is synthesized. The secretory signal and propeptide domain
are cleaved from the full length molecule, resulting in the mature
form of the zdint1 polypeptide. The protease domain may be active
or inactive. Some members of the disintegrin family have "active"
zinc catalytic sites which may be regulated by a "cysteine-switch"
in the cysteine-rich domain. Examples of family members which have
"active" protease domains are ADAM 1 and ADAM 10, which are
involved in sperm/egg fusion and degradation of myelin basic sheath
protein, respectively. Other members of this family do not have
such a catalytic site and are "inactive". An example of a family
member which contains an inactive protease domain is ADAM 11, which
may be involved in tumor suppression. Other protein families which
are known to have inactive protease domains are the serine
proteases.
[0053] The adhesion (disintegrin) domain of this protein binds
integrin domains on the surface a multitude of cells, depending on
the specificity of the disintegrin. The predicted binding site
within this disintegrin domain is often an amino acid loop
comprising about 13 amino acids. The conformation of this sequence
upon folding results in a hairpin loop presenting an amino acid
sequence at its tip. This sequence is often "RGD", but may be
substituted by a variety of other amino acid residues (Wolfsberg
and White, supra; and Jia, J. Biol. Chem. 272:13094-13102 1997).
The diversity of these sequences may reflect that: 1) not all
disintegrin domains serve as ligands for integrins (or other cell
surface receptors); 2) disintegrin domains with different sequences
bind to different types of cell surface receptors; or 3) the
important part of the disintegrin structure loop is its structure,
not its sequence, and thus, that the receptors for the specific
classes of disintegrin domains can recognize a multitude of
disintegrin binding loop sequences. Disintegrin domains have been
shown to be responsible for cell-cell interactions, including
inhibition of platelet aggregation by binding GPIIb/IIIa
(fibronectin receptor) and/or GPIa/IIa (collagen receptor) as well
as cell fusion.
[0054] Many disintegrin family members have a fusion domain, a
relatively hydrophobic domain of about 23 amino acids. This domain
is present within some of the ADAM family members, and has been
shown to be involved in cell-cell fusion, and particularly in
sperm/egg fusion, and muscle fusion.
[0055] The cysteine-rich domain varies in the DP family members and
is believed to be involved in structurally presenting the
integrin-binding region to integrins.
[0056] Many DP family members have a transmembrane domain, which
acts to anchor the polypeptide to the cell membrane.
[0057] The signaling domain of disintegrin family members tends to
be conserved in length and sites for phosphorylation. However,
beyond that they tend to be unique in amino acid composition. Some
disintegrin family members may signal by binding to the SH3 domain
of Abl, Src, and/or Src-related SH3 domains.
[0058] The zdint1 polypeptides of the present invention are a novel
member of the DP family. The presence of isoforms of zdint1 which
also comprise a transmembrane domain would suggest that zdint1 will
have an alternatively spliced variant with a signaling domain.
[0059] The novel zdint1 polypeptide-encoding polynucleotides of the
present invention were initially identified by performing a Blast
similarity search. An expressed sequence tag corresponding to
nucleotides 1097 to 1415 of SEQ ID NO:1 was used to obtain a clone
that had been isolated from an infant brain plasmid library.
[0060] Examination of the zdint1 deduced amino acid sequence (SEQ
ID NO:2) permitted identification of the following domains: a
propeptide sequence, ending at residue 163 of SEQ ID NO: 2; a
protease sequence, residues 164 to 382 of SEQ ID NO: 2; a
disintegrin sequence, residues 383 to 464 of SEQ ID NO: 2; and a
cysteine-rich sequence, residues 465 to 696 of SEQ ID NO:2. Within
the disintegrin domain, there is a "disintegrin loop" sequence,
residues 438 to 449 of SEQ ID NO:2. The amino acid sequence, ECD,
which corresponds to residues 443 to 445 of SEQ ID NO: 2, is
analogous to the "RGD binding loop" of some other members of the
DPs.
[0061] Analysis of tissue distribution of zdint1 was performed by
the Northern blotting technique using Human Multiple Tissue, Master
Dot, and human vascular blots. Strong signals of three transcript
sizes, approximately 3.0 kb, 4.4 kb, and 7.5 kb, were observed in
heart on the multiple tissue Northern blots. Faint signals of the
same transcript sizes were observed in brain and spinal cord.
Fainter signals of the three transcript sizes were observed in
skeletal muscle. The Master Dot Blot showed strong signals in
brain, heart, fetal brain, and fetal heart. The human vascular blot
showed a strong signal at 3-3.Skb in human aortic endothelial cells
and weaker signals in aortic smooth muscle cells and normal human
lung fibroblast cells.
[0062] The protease domain of zdint1 has 49.5% identity to the
protease domain of the nearest family neighbor, ADAM 11, at the
polypeptide level, and 58% identity at the polynucleotide level.
The disintegrin domain of zdint1 has 66.7% identity to the
disintegrin domain of the nearest family neighbor, ADAM 11, at the
polypeptide level, and 64.3% identity at the polynucleotide level.
The expression of ADAM 11 has been shown to decrease in breast
cancer tissues and, thus, is suggested to act as a tumor suppresser
in breast cancer (Emi et al., Nature Gen. 5:151-157, 1993).
Additionally, ADAM 11 has been shown to have multiple isoforms as a
result of alternative splicing.
[0063] Another protein which is an example of alternative splicing
in the DPs is ADAM 12, meltrin a. The truncated form of this
molecule, which lacks the propeptide and metalloprotease domains,
is associated with ectopic muscle formation in vivo, but not in
vitro, indicating that cells expressing this gene produce a growth
factor that acts on neighboring progenitor cells.
[0064] Other ADAMs have been considered for treating angioplasty,
acute coronary syndrome, prevention of restenosis on stents, and
prevention of excess adhesion following surgical procedures,
prevention of metastasis, as well as for degradation of specific
proteins, such as, for example, amyloid precursor protein.
[0065] Polynucleotides:
[0066] The highly conserved amino acids in the disintegrin domain
of zdint1 can be used as a tool to identify new family members. For
instance, reverse transcription-polymerase chain reaction (RT-PCR)
can be used to amplify sequences encoding the conserved disintegrin
domain from RNA obtained from a variety of tissue sources or cell
lines. In particular, highly degenerate primers designed from the
zdint1 sequences are useful for this purpose.
[0067] The present invention also provides polynucleotide
molecules, including DNA and RNA molecules, that encode the zdint1
polypeptides disclosed herein. Those skilled in the art will
readily recognize that, in view of the degeneracy of the genetic
code, considerable sequence variation is possible among these
polynucleotide molecules. SEQ ID NO:3 is a degenerate DNA sequence
that encompasses all DNAs that encode the zdint1 polypeptide of SEQ
ID NO:2. Those skilled in the art will recognize that the
degenerate sequence of SEQ ID NO:3 also provides all RNA sequences
encoding SEQ ID NO:2 by substituting U for T. Thus, zdint1
polypeptide-encoding polynucleotides comprising nucleotide 1 to
nucleotide 2088 of SEQ ID NO:3 and their RNA equivalents are
contemplated by the present invention. Table 1 sets forth the
one-letter codes used within SEQ ID NO:3 to denote degenerate
nucleotide positions. "Resolutions" are the nucleotides denoted by
a code letter. "Complement" indicates the code for the
complementary nucleotide(s). For example, the code Y denotes either
C or T, and its complement R denotes A or G, A being complementary
to T, and G being complementary to C.
1TABLE 1 Nucleotide Resolution Nucleotide Complement A A T T C C G
G G G C C T T A A R A .vertline. G Y C .vertline. T Y C .vertline.
T R A .vertline. G M A .vertline. C K G .vertline. T K G .vertline.
T M A .vertline. C S C .vertline. G S C .vertline. G W A .vertline.
T W A .vertline. T H A .vertline. C .vertline. T D A .vertline. G
.vertline. T B C .vertline. G .vertline. T V A .vertline. C
.vertline. G V A .vertline. C .vertline. G B C .vertline. G
.vertline. T D A .vertline. G .vertline. T H A .vertline. C
.vertline. T N A .vertline. C .vertline. G .vertline. T N A
.vertline. C .vertline. G .vertline. T
[0068] The degenerate codons used in SEQ ID NO:3, encompassing all
possible codons for a given amino acid, are set forth in Table
2.
2TABLE 2 One De- Amino Letter generate Acid Code Codons Codon Cys C
TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT
ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA
GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG
GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG
CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L
CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT
TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR Asn
.vertline. Asp B RAY Glu .vertline. Gln Z SAR Any X NNN
[0069] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding each amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequence of SEQ ID NO:2.
Variant sequences can be readily tested for functionality as
described herein.
[0070] One of ordinary skill in the art will also appreciate that
different species can exhibit "preferential codon usage." In
general, see, Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980;
Haas, et al. Curr. Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene
13:355-64, 1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm,
Nuc. Acids Res. 14:3075-87, 1986; Ikemura, J. Mol. Biol.
158:573-97, 1982. As used herein, the term "preferential codon
usage" or "preferential codons" is a term of art referring to
protein translation codons that are most frequently used in cells
of a certain species, thus favoring one or a few representatives of
the possible codons encoding each amino acid (See Table 2). For
example, the amino acid Threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequence
disclosed in SEQ ID NO:3 serves as a template for optimizing
expression of polynucleotides in various cell types and species
commonly used in the art and disclosed herein. Sequences containing
preferential codons can be tested and optimized for expression in
various species, and tested for functionality as disclosed
herein.
[0071] Within preferred embodiments of the invention the isolated
polynucleotides will hybridize to similar sized regions of SEQ ID
NO:1, or a sequence complementary thereto, under stringent
conditions. In general, stringent conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence at a defined ionic strength and pH. The
T.sub.m is the temperature (under defined ionic strength and pH) at
which 50% of the target sequence hybridizes to a perfectly matched
probe. Typical stringent conditions are those in which the salt
concentration is up to about 0.03 M at pH 7 and the temperature is
at least about 60.degree. C.
[0072] The isolated polynucleotides of the present invention
include DNA and RNA. Methods for preparing DNA and RNA are well
known in the art. In general, RNA is isolated from a tissue or cell
that produces large amounts of zdint1 RNA. Such tissues and cells
are identified by Northern blotting (Thomas, Proc. Natl. Acad. Sci.
USA 77:5201, 1980), and include heart, brain, skeletal muscle,
spinal cord, fetal heart, and fetal brain. Total RNA can be
prepared using guanidine HCl extraction followed by isolation by
centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry
18:52-94, 1979). Poly (A).sup.+ RNA is prepared from total RNA
using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA
69:1408-12, 1972). Complementary DNA (cDNA) is prepared from
poly(A).sup.+ RNA using known methods. In the alternative, genomic
DNA can be isolated. Polynucleotides encoding zdint1 polypeptides
are then identified and isolated by, for example, hybridization or
PCR.
[0073] A full-length clone encoding zdint1 can be obtained by
conventional cloning procedures. Complementary DNA (cDNA) clones
are preferred, although for some applications (e.g., expression in
transgenic animals) it may be preferable to use a genomic clone, or
to modify a cDNA clone to include at least one genomic intron.
Methods for preparing cDNA and genomic clones are well known and
within the level of ordinary skill in the art, and include the use
of the sequence disclosed herein, or parts thereof, for probing or
priming a library. Expression libraries can be probed with
antibodies to zdint1 or other specific binding partners.
[0074] Zdint1 polynucleotide sequences disclosed herein can also be
used as probes or primers to clone 5' non-coding regions of a
zdint1 gene. In view of the tissue-specific expression observed for
zdint1 by Northern blotting, this gene region is expected to
provide for heart-, brain-, spinal cord-, and skeletal
muscle-specific expression. Promoter elements from a zdint1 gene
could thus be used to direct the tissue-specific expression of
heterologous genes in, for example, transgenic animals or patients
treated with gene therapy. Cloning of 5' flanking sequences also
facilitates production of zdint1 proteins by "gene activation" as
disclosed in U.S. Pat. No. 5,641,670. Briefly, expression of an
endogenous zdint1 gene in a cell is altered by introducing into the
zdint1 locus a DNA construct comprising at least a targeting
sequence, a regulatory sequence, an exon, and an unpaired splice
donor site. The targeting sequence is a zdint1 5' non-coding
sequence that permits homologous recombination of the construct
with the endogenous zdint1 locus, whereby the sequences within the
construct become operably linked with the endogenous zdint1 coding
sequence. In this way, an endogenous zdint1 promoter can be
replaced or supplemented with other regulatory sequences to provide
enhanced, tissue-specific, or otherwise regulated expression.
[0075] The polynucleotides of the present invention can also be
synthesized using DNA synthesizers. Currently the method of choice
is the phosphoramidite method. If chemically synthesized double
stranded DNA is required for an application such as the synthesis
of a gene or a gene fragment, then each complementary strand is
made separately. The production of short genes (60 to 80 bp) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 bp), however, special
strategies must be invoked, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. To overcome
this problem, synthetic genes (double-stranded) are assembled in
modular form from single-stranded fragments that are from 20 to 100
nucleotides in length. See Glick and Pasternak, Molecular
Biotechnology, Principles and Applications of Recombinant DNA, (ASM
Press, Washington, D.C. 1994); Itakura et al., Annu. Rev. Biochem.
53: 323-356 (1984) and Climie et al., Proc. Natl. Acad. Sci. USA
87:633-637 (1990).
[0076] The present invention further provides counterpart
polypeptides and polynucleotides from other species (orthologs).
These species include, but are not limited to mammalian, avian,
amphibian, reptile, fish, insect and other vertebrate and
invertebrate species. Of particular interest are zdint1
polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human zdint1 can be cloned using
information and compositions provided by the present invention in
combination with conventional cloning techniques. For example, a
cDNA can be cloned using mRNA obtained from a tissue or cell type
that expresses zdint1 as disclosed herein. Such tissue or cell type
would include, for example, heart, brain, spinal cord, and skeletal
muscle. Suitable sources of mRNA can be identified by probing
Northern blots with probes designed from the sequences disclosed
herein. A library is then prepared from mRNA of a positive tissue
or cell line. A zdint1-encoding cDNA can then be isolated by a
variety of methods, such as by probing with a complete or partial
human cDNA or with one or more sets of degenerate probes based on
the disclosed sequences. A cDNA can also be cloned using the
polymerase chain reaction, or PCR (Mullis, U.S. Pat. No.
4,683,202), using primers designed from the representative human
zdint1 sequence disclosed herein. Within an additional method, the
cDNA library can be used to transform or transfect host cells, and
expression of the cDNA of interest can be detected with an antibody
to zdint1 polypeptide. Similar techniques can also be applied to
the isolation of genomic clones.
[0077] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human zdint1
and that allelic variation and alternative splicing are expected to
occur. Allelic variants of this sequence can be cloned by probing
cDNA or genomic libraries from different individuals according to
standard procedures. Allelic variants of the DNA sequence shown in
SEQ ID NO:1, including those containing silent mutations and those
in which mutations result in amino acid sequence changes, are
within the scope of the present invention, as are proteins which
are allelic variants of SEQ ID NO:2. cDNAs generated from
alternatively spliced mRNAs, which retain the properties of the
zdint1 polypeptide are included within the scope of the present
invention, as are polypeptides encoded by such cDNAs and mRNAs.
Allelic variants and splice variants of these sequences can be
cloned by probing cDNA or genomic libraries from different
individuals or tissues according to standard procedures known in
the art.
[0078] The present invention also provides isolated zdint1
polypeptides that are substantially homologous to the polypeptides
of SEQ ID NO:2 and their orthologs. The term "substantially
homologous" is used herein to denote polypeptides having about 50%,
preferably 60% more preferably at least 70%, and even more
preferably 80% sequence identity to the sequences shown in SEQ ID
NO:2 or their orthologs. Such polypeptides will more preferably be
at least 90% identical, and most preferably 95% or more identical
to SEQ ID NO:2 or its orthologs.) Percent sequence identity is
determined by conventional methods. See, for example, Altschul et
al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff,
Proc. Natl. Acad. Sci. USA 89:10915-9, 1992. Briefly, two amino
acid sequences are aligned to optimize the alignment scores using a
gap opening penalty of 10, a gap extension penalty of 1, and the
"blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as
shown in Table 3 (amino acids are indicated by the standard
one-letter codes). The percent identity is then calculated as: 1
Total number of identical matches [ length of the longer sequence
plus the number of gaps introduced into the longer sequence in
order to align the two sequences ] .times. 100
3 TABLE 3 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0
6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0
-2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3
-4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1
-3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3
-3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1
-1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1
-1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3
-2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3
-3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0079] Sequence identity of polynucleotide molecules is determined
by similar methods using a ratio as disclosed above.
[0080] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative variant zdint1. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990).
[0081] Briefly, FASTA first characterizes sequence similarity by
identifying regions shared by the query sequence (e.g., SEQ ID
NO:2) and a test sequence that have either the highest density of
-identities (if the ktup variable is 1) or pairs of identities (if
ktup=2), without considering conservative amino acid substitutions,
insertions, or deletions. The ten regions with the highest density
of identities are then rescored by comparing the similarity of all
paired amino acids using an amino acid substitution matrix, and the
ends of the regions are "trimmed" to include only those residues
that contribute to the highest score. If there are several regions
with scores greater than the "cutoff" value (calculated by a
predetermined formula based upon the length of the sequence and the
ktup value), then the trimmed initial regions are examined to
determine whether the regions can be joined to form an approximate
alignment with gaps. Finally, the highest scoring regions of the
two amino acid sequences are aligned using a modification of the
Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.
Biol. 48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)),
which allows for amino acid insertions and deletions. Preferred
parameters for FASTA analysis are: ktup=1, gap opening penalty=10,
gap extension penalty=1, and substitution matrix=BLOSUM62. These
parameters can be introduced into a FASTA program by modifying the
scoring matrix file ("SMATRIX"), as explained in Appendix 2 of
Pearson, Meth. Enzymol. 183:63 (1990).
[0082] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from three to six, most preferably three,
with other parameters set as default.
[0083] The present invention includes nucleic acid molecules that
encode a polypeptide having one or more conservative amino acid
changes, compared with the amino acid sequence of SEQ ID NO:2. The
BLOSUM62 table is an amino acid substitution matrix derived from
about 2,000 local multiple alignments of protein sequence segments,
representing highly conserved regions of more than 500 groups of
related proteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA
89:10915 (1992)). Accordingly, the BLOSUM62 substitution
frequencies can be used to define conservative amino acid
substitutions that may be introduced into the amino acid sequences
of the present invention. As used herein, the language
"conservative amino acid substitution" refers to a substitution
represented by a BLOSUM62 value of greater than -1. For example, an
amino acid substitution is conservative if the substitution is
characterized by a BLOSUM62 value of 0, 1, 2, or 3. Preferred
conservative amino acid substitutions are characterized by a
BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
[0084] Variant zdint1 polypeptides or substantially homologous
zdint1 polypeptides are characterized as having one or more amino
acid substitutions, deletions or additions. These changes are
preferably of a minor nature, that is conservative amino acid
substitutions (see Table 4) and other substitutions that do not
significantly affect the folding or activity of the polypeptide;
small deletions, typically of one to about 30 amino acids; and
small amino- or carboxyl-terminal extensions, such as an
amino-terminal methionine residue, a small linker peptide of up to
about 20-25 residues, or an affinity tag. The present invention
thus includes polypeptides of from 383 to 464 amino acid residues
that comprise a sequence that is at least 50%, preferably at least
60%, and more preferably 80% or more identical to the corresponding
region of SEQ ID NO:2. Polypeptides comprising affinity tags can
further comprise a proteolytic cleavage site between the zdint1
polypeptide and the affinity tag. Preferred such sites include
thrombin cleavage sites and factor Xa cleavage sites.
4TABLE 4 Conservative amino acid substitutions Basic: arginine
lysine histidine Acidic: glutamic acid aspartic acid Polar:
glutamirie asparagine Hydrophobic: leucine isoleucine valine
Aromatic: phenylalanine trypt ophan tyrosine Small: glycine alanine
serine threonine methionine
[0085] The present invention further provides a variety of
polypeptide fusions and related multimeric proteins comprising one
or more polypeptide fusions. For example, a disintegrin polypeptide
domain can be prepared as a fusion to a dimerizing protein, as
disclosed in U.S. Pat. Nos. 5,155,027 and 5,567,584. Preferred
dimerizing proteins in this regard include other disintegrin
polypeptide domains or disintegrin polypeptide domain fragments.
Disintegrin polypeptide domain fusions, or disintegrin polypeptide
domain fragment fusions, can be expressed in genetically engineered
cells to produce a variety of multimeric disintegrin-like analogs.
Auxiliary domain polypeptides can be fused to disintegrin domain
polypeptides to target them to specific cells, tissues, or
macromolecules (e.g., heart, brain, spinal cord, skeletal muscle,
platelets). For example, a protease polypeptide domain, or protease
polypeptide fragment or protein, could be targeted to a
predetermined cell type by fusing it to a disintegrin polypeptide
domain or fragment that specifically binds to an integrin
polypeptide or integrin-like polypeptide on the surface of the
target cell. In this way, polypeptides, polypeptide fragments and
proteins can be targeted for therapeutic or diagnostic purposes.
Such disintegrins or protease polypeptide domains or fragments can
be fused to two or more moieties, such as an affinity tag for
purification and a targeting-disintegrin domain. Polypeptide
fusions can also comprise one or more cleavage sites, particularly
between domains. See Tuan et al., Connective Tissue Research
34:1-9, 1996.
[0086] Polypeptide fusions of the present invention will generally
contain not more than about 1,500 amino acid residues, preferably
not more than about 1,200 residues, more preferably not more than
about 1,000 residues, and will in many cases be considerably
smaller. For example, residues of zdint1 polypeptide can be fused
to E. coli .beta.-galactosidase (1,021 residues; see Casadaban et
al., J. Bacteriol. 143:971-980, 1980), a 10-residue spacer, and a
4-residue factor Xa cleavage site. In a second example, residues of
zdint1 polypeptide can be fused to maltose binding protein
(approximately 370 residues), a 4-residue cleavage site, and a
6-residue polyhistidine tag.
[0087] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3- and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vitro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are known in the art. Transcription and translation of plasmids
containing nonsense mutations is carried out in a cell-free system
comprising an E. coil S30 extract and commercially available
enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991;
Chung et al., Science 259:806-9, 1993; and Chung et al., Proc.
Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method,
translation is carried out in Xenopus oocytes by microinjection of
mutated mRNA and chemically aminoacylated suppressor tRNAs
(Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third
method, E. coli cells are cultured in the absence of a natural
amino acid that is to be replaced (e.g., phenylalanine) and in the
presence of the desired non-naturally occurring amino acid(s)
(e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine,
or 4-fluorophenylalanine). The non-naturally occurring amino acid
is incorporated into the protein in place of its natural
counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally
occurring amino acid residues can be converted to non-naturally
occurring species by in vitro chemical modification. Chemical
modification can be combined with site-directed mutagenesis to
further expand the range of substitutions (Wynn and Richards,
Protein Sci. 2:395-403, 1993).
[0088] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for zdint1 amino acid residues.
[0089] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989; Bass
et al., Proc. Natl. Acad. Sci. USA 88:4498-502, 1991). In the
latter technique, single alanine mutations are introduced at every
residue in the molecule, and the resultant mutant molecules are
tested for biological activity as disclosed below to identify amino
acid residues that are critical to the activity of the molecule.
See also, Hilton et al., J. Biol. Chem. 271:4699-708, 1996. Sites
of disintegrin-integrin, or protease interaction can also be
determined by physical analysis of structure, as determined by such
techniques as nuclear magnetic resonance, crystallography, electron
diffraction or photoaffinity labeling, in conjunction with mutation
of putative contact site amino acids. See, for example, de Vos et
al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol.
224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The
identities of essential amino acids can also be inferred from
analysis of homologies with related disintegrin-like molecules.
[0090] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or
Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et
al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204)
and region-directed mutagenesis (Derbyshire et al., Gene 46:145,
1986; Ner et al., DNA 7:127, 1988).
[0091] Variants of the disclosed zdint1 DNA and polypeptide
sequences can be generated through DNA shuffling, as disclosed by
Stemmer, Nature 370:389-91, 1994, Stemmer, Proc. Natl. Acad. Sci.
USA 91:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly,
variant DNAs are generated by in vitro homologous recombination by
random fragmentation of a parent DNA followed by reassembly using
PCR, resulting in randomly introduced point mutations. This
technique can be modified by using a family of parent DNAs, such as
allelic variants or DNAs from different species, to introduce
additional variability into the process. Selection or screening for
the-desired activity, followed by additional iterations of
mutagenesis and assay provides for rapid "evolution" of sequences
by selecting for desirable mutations while simultaneously selecting
against detrimental changes.
[0092] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode active polypeptides (e.g., disintegrin-cell
surface binding or protease activity) can be recovered from the
host cells and rapidly sequenced using modern equipment. These
methods allow the rapid determination of the importance of
individual amino acid residues in a polypeptide of interest, and
can be applied to polypeptides of unknown structure.
[0093] Using the methods discussed herein, one of ordinary skill in
the art can identify and/or prepare a variety of polypeptide
fragments or variants of SEQ ID NO:2 or that retain the disintegrin
and or protease activity of the wild-type zdint1 protein. Such
polypeptides may include additional amino acids from, for example,
a secretory domain, a propeptide domain, a protease domain, part or
all of a transmembrane and intracellular domains, including amino
acids responsible for intracellular signaling; a fusion domains;
affinity tags; and the like.
[0094] For any zdint1 polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above.
[0095] Protein Production
[0096] The zdint1 polypeptides of the present invention, including
full-length polypeptides, biologically active fragments, and fusion
polypeptides, can be produced in genetically engineered host cells
according to conventional techniques. Suitable host cells are those
cell types that can be transformed or transfected with exogenous
DNA and grown in culture, and include bacteria, fungal cells, and
cultured higher eukaryotic cells. Eukaryotic cells, particularly
cultured cells of multicellular organisms, are preferred.
Techniques for manipulating cloned DNA molecules and introducing
exogenous DNA into a variety of host cells are disclosed by
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
1989, and Ausubel et al., eds., Current Protocols in Molecular
Biology, John Wiley and Sons, Inc., NY, 1987.
[0097] In general, a DNA sequence encoding a zdint1 polypeptide is
operably linked to other genetic elements required for its
expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0098] To direct a zdint1 polypeptide into the secretory pathway of
a host cell, a secretory signal sequence (also known as a leader
sequence, prepro sequence or pre sequence) is provided in the
expression vector. The secretory signal sequence may be derived
from another secreted protein (e.g., t-PA) or synthesized de novo.
The secretory signal sequence is operably linked to the zdint1 DNA
sequence, i.e., the two sequences are joined in the correct reading
frame and positioned to direct the newly synthesized polypeptide
into the secretory pathway of the host cell. Secretory signal
sequences are commonly positioned 5' to the DNA sequence encoding
the polypeptide of interest, although certain secretory signal
sequences may be positioned elsewhere in the DNA sequence of
interest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland
et al., U.S. Pat. No. 5,143,830). Polypeptides and peptide
fragments of the present invention are considered biologically
active in the absence of the native signal sequence.
[0099] The protease domain of zdint1 can be substituted by a
heterologous sequence providing a different protease domain. In
this case, the fusion product can be secreted, and the disintegrin
domain of zdint1 can direct the protease domain to a specific
tissue described above. This substituted protease domain can be
chosen from the protease domains represented by the DP protein
families, or domains from other known proteases.
[0100] Cultured mammalian cells are suitable hosts within the
present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-5, 1982), DEAE-dextran mediated transfection (Ausubel et al.,
ibid.), and liposome-mediated transfection (Hawley-Nelson et al.,
Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993, and viral
vectors (Miller and Rosman, BioTechniques 7:980-90, 1989; Wang and
Finer, Nature Med. 2:714-6, 1996). The production of recombinant
polypeptides in cultured mammalian cells is disclosed, for example,
by Levinson et al., U.S. Patent No. 4,713,339; Hagen et al., U.S.
Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; and
Ringold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cells
include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651),
BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC
No. CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and
Chinese hamster ovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines.
Additional suitable cell lines are known in the art and available
from public depositories such as the American Type Culture
Collection, Rockville, Md. In general, strong transcription
promoters are preferred, such as promoters from SV-40 or
cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other suitable
promoters include those from metallothionein genes (U.S. Pat. Nos.
4,579,821 and 4,601,978) and the adenovirus major late
promoter.
[0101] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. Selection is
carried out in the presence of a neomycin-type drug, such as G-418
or the like. Selection systems can also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate. Other drug
resistance genes (e.g., hygromycin resistance, multi-drug
resistance, puromycin acetyltransferase) can also be used.
Alternative markers that introduce an altered phenotype, such as
green fluorescent protein, or cell surface proteins, such as CD4,
CD8, Class I MHC, or placental alkaline phosphatase, may be used to
sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0102] Other higher eukaryotic cells can also be used as hosts,
including plant cells, insect cells and avian cells. The use of
Agrobacterium rhizogenes as a vector for expressing genes in plant
cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore)
11:47-58, 1987. Transformation of insect cells and production of
foreign polypeptides therein is disclosed by Guarino et al., U.S.
Pat. No. 5,162,222 and WIPO publication WO 94/06463. Insect cells
can be infected with recombinant baculovirus, commonly derived from
Autographa californica nuclear polyhedrosis virus (AcNPV). See,
King, L. A. and Possee, R. D., The Baculovirus Expression System: A
Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. et
al., Baculovirus Expression Vectors: A Laboratory Manual, New York,
Oxford University Press., 1994; and, Richardson, C. D., Ed.,
Baculovirus Expression Protocols. Methods in Molecular Biology,
Totowa, N.J., Humana Press, 1995. A second method of making
recombinant zdint1 baculovirus utilizes a transposon-based system
described by Luckow (Luckow, V. A, et al., J Virol 67:4566-79,
1993). This system, which utilizes transfer vectors, is sold in the
Bac-to-Bac.TM. kit (Life Technologies, Rockville, Md.). This system
utilizes a transfer vector, pFastBac1.TM. (Life Technologies)
containing a Tn7 transposon to move the DNA encoding the zdint1
polypeptide into a baculovirus genome maintained in E. coli as a
large plasmid called a "bacmid." See, Hill-Perkins, M. S. and
Possee, R. D., J Gen Virol 71:971-6, 1990; Bonning, B. C. et al., J
Gen Virol 75:1551-6, 1994; and, Chazenbalk, G. D., and Rapoport,
B., J Biol Chem 270:1543-9, 1995. In addition, transfer vectors can
include an in-frame fusion with DNA encoding an epitope tag at the
C- or N-terminus of the expressed zdint1 polypeptide, for example,
a Glu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad.
Sci. 82:7952-4, 1985). Using a technique known in the art, a
transfer vector containing zdint1 is transformed into E. Coli, and
screened for bacmids which contain an interrupted lacZ gene
indicative of recombinant baculovirus. The bacmid DNA containing
the recombinant baculovirus genome is isolated, using common
techniques, and used to transfect Spodoptera frugiperda cells, e.g.
Sf9 cells. Recombinant virus that expresses zdint1 is subsequently
produced. Recombinant viral stocks are made by methods commonly
used the art.
[0103] The recombinant virus is used to infect host cells,
typically a cell line derived from the fall armyworm, Spodoptera
frugiperda. See, in general, Glick and Pasternak, Molecular
Biotechnology: Principles and Applications of Recombinant DNA, ASM
Press, Washington, D.C., 1994. Another suitable cell line is the
High FiveO.TM. cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
are used to grow and maintain the cells. Suitable media are Sf900
II.TM. (Life Technologies) or ESF 921.TM. (Expression Systems) for
the Sf9 cells; and Ex-cellO405.TM. (JRH Biosciences, Lenexa, Kans.)
or Express FiveO.TM. (Life Technologies) for the T. ni cells. The
cells are grown up from an inoculation density of approximately
2-5.times.10.sup.5 cells to a density of 1-2.times.10.sup.6 cells
at which time a recombinant viral stock is added at a multiplicity
of infection (MOI) of 0.1 to 10, more typically near 3. Procedures
used are generally described in available laboratory manuals (King,
L. A. and Possee, R. D., ibid.; O'Reilly, D. R. et al., ibid.;
Richardson, C. D., ibid.). Subsequent purification of the zdint1
polypeptide from the supernatant can be achieved using methods
described herein.
[0104] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Pichia pastoris, and
Pichia methanolica. Methods for transforming S. cerevisiae cells
with exogenous DNA and producing recombinant polypeptides therefrom
are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311;
Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No.
4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et
al., U.S. Pat. No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient (e.g., leucine). A preferred vector system for use in
Saccharomyces cerevisiae is the POT1 vector system disclosed by
Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed
cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those
from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No.
4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter,
U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also
U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533.
[0105] The use of Pichia methanolica as host for the production of
recombinant proteins is disclosed in WIPO Publications WO 97/17450,
WO 97/17451, WO 98/02536, and WO 98/02565. DNA molecules for use in
transforming P. methanolica will commonly be prepared as
double-stranded, circular plasmids, which are preferably linearized
prior to transformation. For polypeptide production in P.
methanolica, it is preferred that the promoter and terminator in
the plasmid be that of a P. methanolica gene, such as a P.
methanolica alcohol utilization gene (AUG1 or AUG2). Other useful
promoters include those of the dihydroxyacetone synthase (DHAS),
formate dehydrogenase (FMD), and catalase (CAT) genes. To
facilitate integration of the DNA into the host chromosome, it is
preferred to have the entire expression segment of the plasmid
flanked at both ends by host DNA sequences. A preferred selectable
marker for use in Pichia methanolica is a P. methanolica ADE2 gene,
which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC
4.1.1.21), which allows ade2 host cells to grow in the absence of
adenine. For large-scale, industrial processes where it is
desirable to minimize the use of methanol, it is preferred to use
host cells in which both methanol utilization genes (AUG1 and AUG2)
are deleted. For production of secreted proteins, host cells
deficient in vacuolar protease genes (PEP4 and PRB1) are preferred.
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. It is preferred to transform P. methanolica
cells by electroporation using an exponentially decaying, pulsed
electric field having a field strength of from 2.5 to 4.5 kV/cm,
preferably about 3.75 kV/cm, and a time constant (t) of from 1 to
40 milliseconds, most preferably about 20 milliseconds.
[0106] Prokaryotic host cells, including strains of the bacteria
Escherichia coli, Bacillus and other genera are also useful host
cells within the present invention. Techniques for transforming
these hosts and expressing foreign DNA sequences cloned therein are
well known in the art (see, e.g., Sambrook et al., ibid.). When
expressing a zdint1 polypeptide in bacteria such as E. coli, the
polypeptide may be retained in the cytoplasm, typically as
insoluble granules, or may be directed to the periplasmic space by
a bacterial secretion sequence. In the former case, the cells are
lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0107] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell. P. methanolica cells
are cultured in a medium comprising adequate sources of carbon,
nitrogen and trace nutrients at a temperature of about 25.degree.
C. to 35.degree. C. Liquid cultures are provided with sufficient
aeration by conventional means, such as shaking of small flasks or
sparging of fermentors. A preferred culture medium for P.
methanolica is YEPD (2% D-glucose, 2% Bacto.TM. Peptone (Difco
Laboratories, Detroit, Mich.), 1% Bacto.TM. yeast extract (Difco
Laboratories), 0.004% adenine and 0.006% L-leucine).
[0108] Protein Isolation
[0109] It is preferred to purify the polypeptides of the present
invention to .gtoreq.80% purity, more preferably to .gtoreq.90%
purity, even more preferably .gtoreq.95% purity, and particularly
preferred is a pharmaceutically pure state, that is greater than
99.9% pure with respect to contaminating macromolecules,
particularly other proteins and nucleic acids, and free of
infectious and pyrogenic agents. Preferably, a purified polypeptide
is substantially free of other polypeptides, particularly other
polypeptides of animal origin.
[0110] Expressed recombinant zdint1 polypeptides (or chimeric
zdint1 polypeptides) can be purified using fractionation and/or
conventional purification methods and media. Ammonium sulfate
precipitation and acid or chaotrope extraction may be used for
fractionation of samples. Exemplary purification steps may include
hydroxyapatite, size exclusion, FPLC and reverse-phase high
performance liquid chromatography. Suitable chromatographic media
include derivatized dextrans, agarose, cellulose, polyacrylamide,
specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives
are preferred. Exemplary chromatographic media include those media
derivatized with phenyl, butyl, or octyl groups, such as
Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,
Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; or
polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the
like. Suitable solid supports include glass beads, silica-based
resins, cellulosic resins, agarose beads, cross-linked agarose
beads, polystyrene beads, cross-linked polyacrylamide resins and
the like that are insoluble under the conditions in which they are
to be used. These supports may be modified with reactive groups
that allow attachment of proteins by amino groups, carboxyl groups,
sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Methods for binding receptor
polypeptides to support media are well known in the art. Selection
of a particular method is a matter of routine design and is
determined in part by the properties of the chosen support. See,
for example, Affinity Chromatography: Principles & Methods,
Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
[0111] The polypeptides of the present invention can be isolated by
a combination of procedures including, but not limited to, anion
and cation exchange chromatography, size exclusion, and affinity
chromography. See Example 3 for a procedure. For example,
immobilized metal ion adsorption (IMAC) chromatography can be used
to purify histidine-rich proteins, including those comprising
polyhistidine tags. Briefly, a gel is first charged with divalent
metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1-7,
1985). Histidine-rich proteins will be adsorbed to this matrix with
differing affinities, depending upon the metal ion used, and will
be eluted by competitive elution, lowering the pH, or use of strong
chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (Methods in
Enzymol., Vol. 182, "Guide to Protein Purification", M. Deutscher,
(ed.), Acad. Press, San Diego, 1990, pp.529-39). Within additional
embodiments of the invention, a fusion of the polypeptide of
interest and an affinity tag (e.g., maltose-binding protein, an
immunoglobulin domain) may be constructed to facilitate
purification.
[0112] Fragments/Fusion Proteins
[0113] To direct the export of a zdint1 polypeptide from the host
cell, the zdint1 DNA is linked to a second DNA segment encoding a
secretory peptide, such as a t-PA secretory peptide. To facilitate
purification of the secreted receptor polypeptide, a C-terminal
extension, such as a poly-histidine tag, substance P, Flag peptide
(Hopp et al., Bio/Technology 6:1204-1210, 1988; available from
Eastman Kodak Co., New Haven, Conn.) or another polypeptide or
protein for which an antibody or other specific binding agent is
available, can be fused to the zdint1 polypeptide.
[0114] Moreover, using methods described in the art, polypeptide
fusions, or hybrid zdint1 proteins, are constructed using regions
or domains of the inventive zdint1 in combination with those of
other disintegrin-like molecules. (e.g. ADAM, MDC, and SVMP), or
heterologous proteins (Sambrook et al., ibid., Altschul et al.,
ibid., Picard, Cur. Opin. Biology, 5:511-5, 1994, and references
therein). These methods allow the determination of the biological
importance of larger domains or regions in a polypeptide of
interest. Such hybrids may alter reaction kinetics, binding,
constrict or expand the substrate specificity, or alter tissue and
cellular localization of a polypeptide, and can be applied to
polypeptides of unknown structure.
[0115] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. For example, part or
all of a domains conferring a biological function may be swapped
between zdint1 of the present invention with the functionally
equivalent domains from another family member, such as ADAM, MDC,
and SVMP. Such domains include, but are not limited to, conserved
motifs such as the secretory signal sequence, protease, RGD,
cysteine, and disintegrin domains. Such fusion proteins would be
expected to have a biological functional profile that is the same
or similar to polypeptides of the present invention or other known
disintegrin-like family proteins (e.g. ADAMs, MDCs, and. SVMPs),
depending on the fusion constructed. Moreover, such fusion proteins
may exhibit other properties as disclosed herein.
[0116] zdint1 polypeptides or fragments thereof may also be
prepared through chemical synthesis. zdint1 polypeptides may be
monomers or multimers; glycosylated or non-glycosylated; pegylated
or non-pegylated; and may or may not include an initial methionine
amino acid residue.
[0117] Chemical Synthesis of Polypeptides
[0118] Zdint1 polypeptides, peptides, variants and or fragments
thereof may also be prepared through chemical synthesis. TML
polypeptides may be monomers or multimers; glycosylated or
non-glycosylated; pegylated or non-pegylated; amidated or
non-amidated; sulfated or non-sulfated; and may or may not include
an initial methionine amino acid residue. For example, TML
polypeptides can also be synthesized by exclusive solid phase
synthesis, partial solid phase methods, fragment condensation or
classical solution synthesis. The polypeptides are preferably
prepared by solid phase peptide synthesis, for example as described
by Merrifield, J. Am. Chem. Soc. 85:2149, 1963. The synthesis is
carried out with amino acids that are protected at the alpha-amino
terminus. Trifunctional amino acids with labile side-chains are
also protected with suitable groups to prevent undesired chemical
reactions from occurring during the assembly of the polypeptides.
The alpha-amino protecting group is selectively removed to allow
subsequent reaction to take place at the amino-terminus. The
conditions for the removal of the alpha-amino protecting group do
not remove the side-chain protecting groups.
[0119] The alpha-amino protecting groups are those known to be
useful in the art of stepwise polypeptide synthesis. Included are
acyl type protecting groups (e.g., formyl, trifluoroacetyl,
acetyl), aryl type protecting groups (e.g., biotinyl), aromatic
urethane type protecting groups [e.g., benzyloxycarbonyl (Cbz),
substituted benzyloxycarbonyl and 9-fluorenylmethyloxy-carbonyl
(Fmoc)], aliphatic urethane protecting groups [e.g.,
t-butyloxycarbonyl (tBoc), isopropyloxycarbonyl,
cyclohexloxycarbonyl] and alkyl type protecting groups (e.g.,
benzyl, triphenylmethyl). The preferred protecting groups are tBoc
and Fmoc.
[0120] The side-chain protecting groups selected must remain intact
during coupling and not be removed during the deprotection of the
amino-terminus protecting group or during coupling conditions. The
side-chain protecting groups must also be removable upon the
completion of synthesis using reaction conditions that will not
alter the finished polypeptide. In tBoc chemistry, the side-chain
protecting groups for trifunctional amino acids are mostly benzyl
based. In Fmoc chemistry, they are mostly tert-butyl or trityl
based.
[0121] In tBoc chemistry, the preferred side-chain protecting
groups are tosyl for arginine, cyclohexyl for aspartic acid,
4-methylbenzyl (and acetamidomethyl) for cysteine, benzyl for
glutamic acid, serine and threonine, benzyloxymethyl (and
dinitrophenyl) for histidine, 2-Cl-benzyloxycarbonyl for lysine,
formyl for tryptophan and 2-bromobenzyl for tyrosine. In Fmoc
chemistry, the preferred side-chain protecting groups are
2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) or
2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for
arginine, trityl for asparagine, cysteine, glutamine and histidine,
tert-butyl for aspartic acid, glutamic acid, serine, threonine and
tyrosine, tBoc for lysine and tryptophan.
[0122] For the synthesis of phosphopeptides, either direct or
post-assembly incorporation of the phosphate group is used. In the
direct incorporation strategy, the phosphate group on serine,
threonine or tyrosine may be protected by methyl, benzyl, or
tert-butyl in Fmoc chemistry or by methyl, benzyl or phenyl in tBoc
chemistry. Direct incorporation of phosphotyrosine without
phosphate protection can also be used in Fmoc chemistry. In the
post-assembly incorporation strategy, the unprotected hydroxyl
groups of serine, threonine or tyrosine are derivatized on solid
phase with di-tert-butyl-, dibenzyl- or
dimethyl-N,N'-diisopropylphosphoramidite and then oxidized by
tert-butylhydroperoxide.
[0123] Solid phase synthesis is usually carried out from the
carboxyl-terminus by coupling the alpha-amino protected (side-chain
protected) amino acid to a suitable solid support. An ester linkage
is formed when the attachment is made to a chloromethyl,
chlortrityl or hydroxymethyl resin, and the resulting polypeptide
will have a free carboxyl group at the C-terminus. Alternatively,
when an amide resin such as benzhydrylamine or
p-methylbenzhydrylamine resin (for tBoc chemistry) and Rink amide
or PAL resin (for Fmoc chemistry) are used, an amide bond is formed
and the resulting polypeptide will have a carboxamide group at the
C-terminus. These resins, whether polystyrene- or polyamide-based
or polyethyleneglycol-grafted, with or without a handle or linker,
with or without the first amino acid attached, are commercially
available, and their preparations have been described by Stewart et
al., "Solid Phase Peptide Synthesis" (2nd Edition), (Pierce
Chemical Co., Rockford, IL, 1984) and Bayer & Rapp Chem. Pept.
Prot. 3:3 (1986); and Atherton et al., Solid Phase Peptide
Synthesis: A Practical Approach, IRL Press, Oxford, 1989.
[0124] The C-terminal amino acid, protected at the side chain if
necessary, and at the alpha-amino group, is attached to a
hydroxylmethyl resin using various activating agents including
dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide
(DIPCDI) and carbonyldiimidazole (CDI). It can be attached to
chloromethyl or chlorotrityl resin directly in its cesium
tetramethylammonium salt form or in the presence of triethylamine
(TEA) or diisopropylethylamine (DIEA). First amino acid attachment
to an amide resin is the same as amide bond formation during
coupling reactions.
[0125] Following the attachment to the resin support, the
alpha-amino protecting group is removed using various reagents
depending on the protecting chemistry (e.g., tBoc, Fmoc). The
extent of Fmoc removal can be monitored at 300-320 nm or by-a
conductivity cell. After removal of the alpha-amino protecting
group, the remaining protected amino acids are coupled stepwise in
the required order to obtain the desired sequence.
[0126] Various activating agents can be used for the coupling
reactions including DCC, DIPCDI, 2-chloro-1,3-dimethylimidium
hexafluorophosphate (CIP),
benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium
hexafluoro-phosphate (BOP) and its pyrrolidine analog (PyBOP),
bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP),
O-(benzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate (HBTU) and its tetrafluoroborate analog (TBTU)
or its pyrrolidine analog (HBPyU),
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium
hexafluorophosphate (HATU) and its tetrafluoroborate analog (TATU)
or its pyrrolidine analog (HAPyU). The most common catalytic
additives used in coupling reactions include
4-dimethylaminopyridine (DMAP),
3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine (HODhbt),
N-hydroxybenzotriazole (HOBt) and 1-hydroxy-7-azabenzotriazole
(HOAt). Each protected amino acid is used in excess (>2.0
equivalents), and the couplings are usually carried out in
N-methylpyrrolidone (NMP) or in DMF, CH2Cl2 or mixtures thereof.
The extent of completion of the coupling reaction can be monitored
at each stage, e.g., by the ninhydrin reaction as described by
Kaiser et al., Anal. Biochem. 34:595, 1970.
[0127] After the entire assembly of the desired peptide, the
peptide-resin is cleaved with a reagent with proper scavengers. The
Fmoc peptides are usually cleaved and deprotected by TFA with
scavengers (e.g., H2O, ethanedithiol, phenol and thioanisole). The
tBoc peptides are usually cleaved and deprotected with liquid HF
for 1-2 hours at -5 to 0.degree. C., which cleaves the polypeptide
from the resin and removes most of the side-chain protecting
groups. Scavengers such as anisole, dimethylsulfide and
p-thiocresol are usually used with the liquid HF to prevent cations
formed during the cleavage from alkylating and acylating the amino
acid residues present in the polypeptide. The formyl group of
tryptophan and the dinitrophenyl group of histidine need to be
removed, respectively by piperidine and thiophenyl in DMF prior to
the HF cleavage. The acetamidomethyl group of cysteine can be
removed by mercury(II)acetate and alternatively by iodine,
thallium(III)trifluoroacetate or silver tetrafluoroborate which
simultaneously oxidize cysteine to cystine. Other strong acids used
for tBoc peptide cleavage and deprotection include
trifluoromethanesulfonic acid (TFMSA) and
trimethylsilyltrifluoroacetate (TMSOTf).
[0128] The disintegrin loop (residue 438 to residue 449 of SEQ ID
NO:2) is of particular interest for use in assays and treatment of
disorders of the heart, brain, spinal cord, and skeletal muscle.
For these purposes the disintegrin loop peptide synthesized
includes the terminal cysteine residues and thus, would be from
residue 437 to residue 450 of SEQ ID NO:2. This peptide can be
synthesized as a linear peptide or a disulfide linked peptide.
Peptides having disulfide bonds between residues can be 438, 444,
and 450 are of particular interest. See Jia, L. G., ibid for
additional description of peptide synthesis and disulfide
linkage.
[0129] One skilled in the art will recognize that it is useful to
design and synthesize new binding peptides using the integrin
binding peptides of zdint1 as a model. Methods for synthesizing
such peptides are described by P. L. Barker et al., J. Med. Chem.
35: 2040-2048, 1992, and L. Jia et al., J. Biol. Chem. 272:
13094-13102, 1997. As the structural conformation of the integrin
binding peptide is critical, it is recognized that although some
amino acid substitutions will not change the conformation of the
peptides, the cyclization of the peptide is advantageously
conserved. Synthetic peptides are useful as agonists or antagonsits
for zdint1 and could be assayed.
[0130] Assays
[0131] The activity of zdint1 polypeptides can be measured using a
variety of assays that measure, for example, cell-cell
interactions, proteolysis, extracellular matrix formation or
remodeling. Additionally, other biological functions associated
with disintegrin family members or with integrin/disintegrin
interactions, apoptosis, proliferation or differentiation can also
be measured. Of particular interest is a change in platelet
aggregation. Assays measuring platelet aggregation are well known
in the art. For a general reference, see Dennis, Proc. Natl. Acad.
Sci. 87: 2471-2475, 1989.
[0132] Another assay of interest measures or detects changes in
differentiation, development and/or and electrical coupling of
muscle cells or myocytes. Additionally, the effects of a zdint1
polypeptides on cell-cell interactions of fibroblasts, myoblasts,
nerve cells, white blood cells, endothelial cells and tumor cells
would be of interest to measure. Yet another assays examines
changes in protease activity and apoptosis.
[0133] The activity of molecules of the present invention can be
measured using a variety of assays that, for example, measure
neogenesis or hyperplasia (i.e., proliferation) of cardiac cells
based on the tissue specificity in adult heart. Additional
activities likely associated with the polypeptides of the present
invention include proliferation of endothelial cells,
cardiomyocytes, fibroblasts, skeletal myocytes directly or
indirectly through other growth factors; action as a chemotaxic
factor for endothelial cells, fibroblasts and/or phagocytic cells;
osteogenic factor; and factor for expanding mesenchymal stem cell
and precursor populations.
[0134] Proliferation can be measured using cultured cardiac cells
or in vivo by administering molecules of the claimed invention to
an appropriate animal model. Generally, proliferative effects are
observed as an increase in cell number and therefore, may include
inhibition of apoptosis, as well as mitogenesis. Cultured cells
include cardiac fibroblasts, cardiac myocytes, skeletal myocytes,
human umbilical vein endothelial cells from primary cultures.
Established cell lines include: NIH 3T3 fibroblast (ATCC No.
CRL-1658), CHH-1 chum heart cells (ATCC No. CRL-1680), H9c2 rat
heart myoblasts (ATCC No. CRL-1446), Shionogi mammary carcinoma
cells (Tanaka et al., Proc. Natl. Acad. Sci. 89:8928-8932, 1992)
and LNCap.FGC adenocarcinoma cells (ATCC No. CRL-1740). Assays
measuring cell proliferation are well known in the art. For
example, assays measuring proliferation include such assays as
chemosensitivity to neutral red dye (Cavanaugh et al.,
Investigational New Drugs 8:347-354, 1990), incorporation of
radiolabelled nucleotides (Cook et al., Analytical Biochem.
179:1-7, 1989), incorporation of 5-bromo-2'-deoxyuridine (BrdU) in
the DNA of proliferating cells (Porstmann et al., J. Immunol.
Methods 82:169-179, 1985), and use of tetrazolium salts (Mosmann,
J. Immunol. Methods 65:55-63, 1983; Alley et al., Cancer Res.
48:589-601, 1988; Marshall et al., Growth Reg. 5:69-84, 1995; and
Scudiero et al., Cancer Res. 48:4827-4833, 1988).
[0135] Differentiation is a progressive and dynamic process,
beginning with pluripotent stem cells and ending with terminally
differentiated cells. Pluripotent stem cells that can regenerate
without commitment to a lineage express a set of differentiation
markers that are lost when commitment to a cell lineage is made.
Progenitor cells express a set of differentiation markers that may
or may not continue to be expressed as the cells progress down the
cell lineage pathway toward maturation. Differentiation markers
that are expressed exclusively by mature cells are usually
functional properties such as cell products, enzymes to produce
cell products and receptors. The stage of a cell population's
differentiation is monitored by identification of markers present
in the cell population. Myocytes, osteoblasts, adipocytes,
chrondrocytes, fibroblasts and reticular cells are believed to
originate from a common mesenchymal stem cell (Owen et al., Ciba
Fdn. Symp. 136:42-46, 1988). Markers for mesenchymal stem cells
have not been well identified (Owen et al., J. of Cell Sci.
87:731-738, 1987), so identification is usually made at the
progenitor and mature cell stages. The existence of early stage
cardiac myocyte progenitor cells (often referred to as cardiac
myocyte stem cells) has been speculated, but not demonstrated, in
adult cardiac tissue. The novel polypeptides of the present
invention are useful for studies to isolate mesenchymal stem cells
and cardiac myocyte progenitor cells, both in vivo and ex vivo.
[0136] There is evidence to suggest that factors that stimulate
specific cell types down a pathway towards terminal differentiation
or dedifferentiation affect the entire cell population originating
from a common precursor or stem cell. Thus, zdint1 polypeptides may
stimulate inhibition or proliferation of myocytes, smooth muscle
cells, osteoblasts, adipocytes, chrondrocytes and endothelial
cells. Molecules of the present invention may, while stimulating
proliferation or differentiation of cardiac myocytes, inhibit
proliferation or differentiation of adipocytes, by virtue of their
effect on common precursor/stem cells. Thus, molecules of the
present invention have use in inhibiting chondrosarcomas,
atherosclerosis, restenosis and obesity.
[0137] Assays measuring differentiation include, for example,
measuring cell-surface markers associated with stage-specific
expression of a tissue, enzymatic activity, functional activity or
morphological changes (Watt, FASEB, 5:281-284, 1991; Francis,
Differentiation 57:63-75, 1994; Raes, Adv. Anim. Cell Biol.
Technol. Bioprocesses, 161-171, 1989; all incorporated herein by
reference).
[0138] In vivo assays for evaluating cardiac neogenesis or
hyperplasia include treating neonatal and mature rats with the
molecules of the present invention. The animals' cardiac function
is measured as heart rate, blood pressure, and cardiac output to
determine left ventricular function. Post-mortem methods for
assessing cardiac improvement include: increased cardiac weight,
nuclei/cytoplasmic volume, staining of cardiac histology sections
to determine proliferating cell nuclear antigen (PCNA) vs.
cytoplasmic actin levels (Quaini et al., Circulation Res.
75:1050-1063, 1994 and Reiss et al., Proc. Natl. Acad. Sci.
93:8630-8635, 1996.)
[0139] Assays measuring in vivo effects of synthetic zdint1
agonists include a Left Ventricular Hypertrophy model (A. M.
Feldman et al., Circ. Res. 73: 184-192, 1993), which measures
remodeling and repair after congestive heart failure and chronic
pressure overload.
[0140] Proteins, including alternatively spliced peptides, of the
present invention are useful for tumor suppression, and growth and
differentiation either working in isolation, or in conjunction with
other molecules (growth factors, cytokines, etc.) in brain, heart,
spinal column, and skeletal muscle cells. Alternative splicing of
zdint1 may be cell-type specific and confer activity to specific
tissues.
[0141] Proteins of the present invention are useful for delivery of
therapeutic agents such as, but not limited to, proteases,
radionuclides, chemotherapy agents, and small molecules. Effects of
these therapeutic agents can be measured in vitro using cultured
cells or in vivo by administering molecules of the claimed
invention to the appropriate animal model. For instance, zdint1
transfected expression host cells may be embedded in an alginate
environment and injected (implanted) into recipient animals.
Alginate-poly-L-lysine microencapsulation, permselective membrane
encapsulation and diffusion chambers have been described as a means
to entrap transfected mammalian cells or primary mammalian cells.
These types of non-immunogenic "encapsulations" or
microenvironments permit the transfer of nutrients into the
microenvironment, and also permit the diffusion of proteins and
other macromolecules secreted or released by the captured cells
across the environmental barrier to the recipient animal. Most
importantly, the capsules or microenvironments mask and shield the
foreign, embedded cells from the recipient animal's immune
response. Such microenvironments can extend the life of the
injected cells from a few hours or days (naked cells) to several
weeks (embedded cells).
[0142] Alginate threads provide a simple and quick means for
generating embedded cells. The materials needed to generate the
alginate threads are readily available and relatively inexpensive.
Once made, the alginate threads are relatively strong and durable,
both in vitro and, based on data obtained using the threads, in
vivo. The alginate threads are easily manipulable and the
methodology is scalable for preparation of numerous threads. In an
exemplary procedure, 3% alginate is prepared in sterile H.sub.2O,
and sterile filtered. Just prior to preparation of alginate
threads, the alginate solution is again filtered. An approximately
50% cell suspension (containing about 5.times.10.sup.5 to about
5.times.10.sup.7 cells/ml) is mixed with the 3% alginate solution.
One ml of the alginate/cell suspension is extruded into a 100 mM
sterile filtered CaCl.sub.2 solution over a time period of
.about.15 min, forming a "thread". The extruded thread is then
transferred into a solution of 50 mM CaCl2, and then into a
solution of 25 mM CaCl.sub.2. The thread is then rinsed with
deionized water before coating the thread by incubating in a 0.01%
solution of poly-L-lysine. Finally, the thread is rinsed with
Lactated Ringer's Solution and drawn from solution into a syringe
barrel (without needle attached) A large bore needle is then
attached to the syringe, and the thread is intraperitoneally
injected into a recipient in a minimal volume of the Lactated
Ringer's Solution.
[0143] An alternative in vivo approach for assaying proteins of the
present invention involves viral delivery systems. Exemplary
viruses for this purpose include adenovirus, herpesvirus,
lentivirus, vaccinia virus and adeno-associated virus (AAV).
Adenovirus, a double-stranded DNA virus, is currently the best
studied gene transfer vector for delivery of heterologous nucleic
acid (for a review, see T. C. Becker et al., Meth. Cell Biol.
43:161-89, 1994; and J. T. Douglas and D. T. Curiel, Science &
Medicine 4:44-53, 1997). The adenovirus system offers several
advantages: adenovirus can (i) accommodate relatively large DNA
inserts; (ii) be grown to high-titer; (iii) infect a broad range of
mammalian cell types; and (iv) be used with a large number of
available vectors containing different promoters. Also, because
adenoviruses are stable in the bloodstream, they can be
administered by intravenous injection.
[0144] By deleting portions of the adenovirus genome, larger
inserts (up to 7 kb) of heterologous DNA can be accommodated. These
inserts can be incorporated into the viral DNA by direct ligation
or by homologous recombination with a co-transfected plasmid. In an
exemplary system, the essential El gene has been deleted from the
viral vector, and the virus will not replicate unless the E1 gene
is provided by the host cell (the human 293 cell line is
exemplary). When intravenously administered to intact animals,
adenovirus primarily targets the liver. If the adenoviral delivery
system has an E1 gene deletion, the virus cannot replicate in the
host cells. However, the host's tissue (e.g., liver) will express
and process (and, if a secretory signal sequence is present,
secrete) the heterologous protein. Secreted proteins will enter the
circulation in the highly vascularized liver, and effects on the
infected animal can be determined.
[0145] The adenovirus system can also be used for protein
production in vitro. By culturing adenovirus-infected non-293 cells
under conditions where the cells are not rapidly dividing, the
cells can produce proteins for extended periods of time. For
instance, BHK cells are grown to confluence in cell factories, then
exposed to the adenoviral vector encoding the secreted protein of
interest. The cells are then grown under serum-free conditions,
which allows infected cells to survive for several weeks without
significant cell division. Alternatively, adenovirus vector
infected 293S cells can be grown in suspension culture at
relatively high cell density to produce significant amounts of
protein (see Garnier et al., Cytotechnol. 15:145-55, 1994). With
either protocol, an expressed, secreted heterologous protein can be
repeatedly isolated from the cell culture supernatant. Within the
infected 293S cell production protocol, non-secreted proteins may
also be effectively obtained.
[0146] Within yet another embodiment is provided an oligonucleotide
probe or primer comprising at least 14 contiguous nucleotides of a
polynucleotide of SEQ ID NO:1 or a sequence complementary to SEQ ID
NO:1.
[0147] Agonists and Antagonists
[0148] In view of the tissue distribution (heart, brain, spinal
cord and skeletal muscle) observed for zdint1 expression, agonists
(including the native disintegrin and protease domains) and
antagonists have enormous potential in both in vitro and in vivo
applications. Compounds identified as zdint1 agonists and
antagonists are useful for studying cell-cell interactions,
myogenesis, apoptosis, neurogenesis, connective tissue disorders,
chondrogenesis, arthritis, tumor proliferation and suppression,
extracellular matrix proteins, repair and remodeling of ischemia
reperfusion and inflammation in vitro and in vivo. For example,
zdint1 and agonist compounds are useful as components of defined
cell culture media, and may be used alone or in combination with
other cytokines and hormones to replace serum that is commonly used
in cell culture. Agonists are thus useful in specifically promoting
the growth and/or development of cells of the myeloid lineages in
culture. Additionally, zdint1 polypeptides and zdint1 agonists,
including small molecules are useful as a research reagent, such as
for the expansion, differentiation, and/or cell-cell interactions
of heart, brain, spinal cord, or skeletal muscle cells. zdint1
polypeptides are added to tissue culture media for these cell
types.
[0149] Antagonists
[0150] Antagonists are also useful as research reagents for
characterizing sites of complementary/anti-complementary
interaction. Inhibitors of zdint1 activity (zdint1 antagonists)
include anti-zdint1 antibodies and soluble zdint1 receptors, as
well as other peptidic and non-peptidic agents (including
ribozymes). zdint1 -can also be used to identify inhibitors
(antagonists) of its activity. Test compounds are added to the
assays disclosed herein to identify compounds that inhibit the
activity of zdint1. In addition to those assays disclosed herein,
samples can be tested for inhibition of zdint1 activity within a
variety of assays designed to measure disintegrin/integrin binding
or the stimulation/inhibition of zdint1-dependent cellular
responses. For example, zdint1-responsive cell lines can be
transfected with a reporter gene construct that is responsive to a
zdint1-stimulated cellular pathway. Reporter gene constructs of
this type are known in the art, and will generally comprise a DNA
response element operably linked to a gene encoding an assayable
protein, such as luciferase, or a metabolite. DNA response elements
can include, but are not limited to, cyclic AMP response elements
(CRE), hormone response elements (HRE), insulin response element
(IRE) (Nasrin et al., Proc. Natl. Acad. Sci. USA 87:5273-7, 1990)
and serum response elements (SRE) (Shaw et al. Cell 56: 563-72,
1989). Cyclic AMP response elements are reviewed in Roestler et
al., J. Biol. Chem. 263 (19):9063-6; 1988 and Habener, Molec.
Endocrinol. 4 (8):1087-94; 1990. Hormone response elements are
reviewed in Beato, Cell 56:335-44; 1989. The most likely reporter
gene construct would contain a disintegrin that, upon binding an
integrin, would signal intracellularly through, for example, a SRE
reporter. Candidate compounds, solutions, mixtures or extracts are
tested for the ability to inhibit the activity of zdint1 on the
target cells, as evidenced by a decrease in zdint1 stimulation of
reporter gene expression. Assays of this type will detect compounds
that directly block zdint1 binding to cell-surface receptors, i.e.,
integrin or the anti-complementary member of a
complementary/anti-complementary pair, as well as compounds that
block processes in the cellular pathway subsequent to
complement/anti-complement binding. In the alternative, compounds
or other samples can be tested for direct blocking of zdint1
binding to an integrin using zdint1 tagged with a detectable label
(e.g., .sup.125I, biotin, horseradish peroxidase, FITC, and the
like). Within assays of this type, the ability of a test sample to
inhibit the binding of labeled zdint1 to the integrin is indicative
of inhibitory activity, which can be confirmed through secondary
assays. Integrins used within binding assays may be cellular
integrins or isolated, immobilized integrins.
[0151] An amino acid sequence comprising the "ECD" integrin binding
component of zdint1, (residues 443 to 445 of SEQ ID NO: 2), which
is analogous to the "RGD", integrin binding loop, may also be used
as an inhibitor. Such an inhibitor would bind an integrin other
than its naturally occurring integrin by nature of its folding
structure. A particular interest in such an inhibitor would be to
mediate platelet aggregation. Assays measuring binding and
inhibition as well as platelet aggregation are known in the
art.
[0152] A zdint1 polypeptide can be expressed as a fusion with an
immunoglobulin heavy chain constant region, typically an Fc
fragment, which contains two constant region domains and lacks the
variable region. Methods for preparing such fusions are disclosed
in U.S. Pat. Nos. 5,155,027 and 5,567,584. Such fusions are
typically secreted as multimeric molecules wherein the Fc portions
are disulfide bonded to each other and two non-Ig polypeptides are
arrayed in closed proximity to each other. Fusions of this type can
be used to evaluate effects and potential of dimerization of zdint1
with itself or other disintegrin family members. Such fusions would
also be useful to isolate the corresponding integrin(s) that zdint1
binds. For use in assays, the chimeras are bound to a support via
the Fc region and used in an ELISA format.
[0153] A zdint1 integrin-binding polypeptide can also be used for
purification of integrin. The polypeptide is immobilized on a solid
support, such as beads of agarose, cross-linked agarose, glass,
cellulosic resins, silica-based resins, polystyrene, cross-linked
polyacrylamide, or like materials that are stable under the
conditions of use. Methods for linking polypeptides to solid
supports are known in the art, and include amine chemistry,
cyanogen bromide activation, N-hydroxysuccinimide activation,
epoxide activation, sulfhydryl activation, and hydrazide
activation. The resulting medium will generally be configured in
the form of a column, and fluids containing integrins are passed
through the column one or more times to allow integrins to bind to
the integrin binding loop polypeptide. The integrin is then eluted
using changes in salt concentration, chaotropic agents (guanidine
HCl), or pH to disrupt integrin-receptor binding.
[0154] An assay system that uses a ligand-binding receptor (or an
antibody, one member of a complementary/anti-complementary pair) or
a binding fragment thereof, and a commercially available biosensor
instrument (BIAcore, Pharmacia Biosensor, Piscataway, N.J.) may be
advantageously employed. Such receptor, antibody, member of a
complement/anti-complement pair or fragment is immobilized onto the
surface of a receptor chip. Use of this instrument is disclosed by
Karlsson, J. Immunol. Methods 145:229-40, 1991 and Cunningham and
Wells, J. Mol. Biol. 234:554-63, 1993. A receptor, antibody, member
or fragment is covalently attached, using amine or sulfhydryl
chemistry, to dextran fibers that are attached to gold film within
the flow cell. A test sample is passed through the cell. If a
ligand, epitope, or opposite member of the
complementary/anti-complementary pair is present in the sample, it
will bind to the immobilized receptor, antibody or member,
respectively, causing a change in the refractive index of the
medium, which is detected as a change in surface plasmon resonance
of the gold film. This system allows the determination of on- and
off-rates, from which binding affinity can be calculated, and
assessment of stoichiometry of binding.
[0155] Another method to assay cell-cell interactions caused by
zint1 polypeptides, peptides, or variants is with a silicon-based
biosensor microphysiometer which measures the extracellular
acidification rate or proton excretion associated with receptor
binding and subsequent physiologic cellular responses. An exemplary
device is the Cytosensor.TM. Microphysiometer manufactured by
Molecular Devices, Sunnyvale, Calif. A variety of cellular
responses, such as cell proliferation, ion transport, energy
production, inflammatory response, regulatory and receptor
activation, and the like, can be measured by this method. See, for
example, McConnell, H. M. et al., Science 257:1906-1912, 1992;
Pitchford, S. et al., Meth. Enzymol. 228:84-108, 1997; Arimilli, S.
et al., J. Immunol. Meth. 212:49-59, 1998; Van Liefde, I. et al.,
Eur. J. Pharmacol. 346:87-95, 1998. The microphysiometer can be
used for assaying adherent or non-adherent eukaryotic or
prokaryotic cells. By measuring extracellular acidification changes
in cell media over time, the microphysiometer directly measures
cellular responses to various stimuli, including zdint1
polypeptide, peptide, variant, agonists, or antagonists.
Preferably, the microphysiometer is used to measure responses of a
zdint1-responsive eukaryotic cell, compared to a control eukaryotic
cell that does not respond to zdint1 polypeptide, peptide, or
variant. Zdintl-responsive eukaryotic cells comprise cells into
which a receptor for zdint1 has been transfected creating a cell
that is responsive to zdint1 polypeptide, peptide, or variant; or
cells naturally responsive to zdint1 such as, for example, cells
derived from the kidney or small intestine. Differences, measured
by a change, for example, an increase or diminution in
extracellular acidification, in the response of cells exposed to
zdint1 polypeptide, peptide, or variant relative to a control not
exposed to zdint1 polypeptide, peptide, or variant, are a direct
measurement of zdint1-modulated cellular responses. Moreover, such
zdint1-modulated responses can be assayed under a variety of
stimuli. Using the microphysiometer, there is provided a method of
identifying agonists of zdint1 polypeptide, comprising providing
cells responsive to a zdint1 polypeptide, culturing a first portion
of the cells in the absence of a test compound, culturing a second
portion of the cells in the presence of a test compound, and
detecting a change, for example, an increase or diminution, in a
cellular response of the second portion of the cells as compared to
the first portion of the cells. The change in cellular response is
shown as a measurable change in extracellular acidification rate.
Moreover, culturing a third portion of the cells in the presence of
zdint1 polypeptide and the absence of a test compound can be used
as a positive control for the zdint1-responsive cells, and as a
control to compare the agonist activity of a test compound with
that of the zdint1 polypeptide. Moreover, using the
microphysiometer, there is provided a method of identifying
antagonists of zdint1 polypeptide, comprising providing cells
responsive to a zdint1 polypeptide, culturing a first portion of
the cells in the presence of zdint1 and the absence of a test
compound, culturing a second portion of the cells in the presence
of zdint1 and the presence of a test compound, and detecting a
change, for example, an increase or a diminution in a cellular
response of the second portion of the cells as compared to the
first portion of the cells. The change in cellular response is
shown as a measurable change in extracellular acidification rate.
Antagonists and agonists, for zdint1 polypeptide, can be rapidly
identified using this method.
[0156] Moreover, polypeptides, peptides and variants of zdint1 can
be used to identify cells, tissues, or cell lines which respond to
a zdint1-stimulated pathway. The microphysiometer, described above,
can be used to rapidly identify ligand-responsive cells, such as
cells responsive to zdint1 polypeptides peptides and variants of
the present invention. Cells can be cultured in the presence or
absence of zdint1 polypeptides, peptides and variants. Those cells
which elicit a measurable change in extracellular acidification in
the presence of zdint1 polypeptides, peptides and variants are
responsive to zdint1. Such cell lines, can be used to identify
antagonists and agonists of zdint1 polypeptide as described
above.
[0157] Integrin polypeptides and other receptor polypeptides which
bind disintegrin polypeptides, and variants thereof, can also be
used within other assay systems known in the art. Such systems
include Scatchard analysis for determination of binding affinity
(see Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949) and
calorimetric assays (Cunningham et al., Science 253:545-48, 1991;
Cunningham et al., Science 245:821-25, 1991).
[0158] A "soluble receptor" is a receptor polypeptide that is not
bound to a cell membrane. Soluble receptors are most commonly
ligand-binding receptor polypeptides that lack transmembrane and
cytoplasmic domains. Soluble receptors can comprise additional
amino acid residues, such as affinity tags that provide for
purification of the polypeptide or provide sites for attachment of
the polypeptide to a substrate, or immunoglobulin constant region
sequences. Many cell-surface receptors have naturally occurring,
soluble counterparts that are produced by proteolysis or translated
from alternatively spliced mRNAs. Receptor polypeptides are said to
be substantially. free of transmembrane and intracellular
polypeptide segments when they lack sufficient portions of these
segments to provide membrane anchoring or signal transduction,
respectively.
[0159] Soluble forms of zdint1 polypeptides may act as antagonsits
to zdint1 polypeptides, and would be useful to modulate the effects
of zdint1 in heart, brain, skeletal muscle and spinal cord.
Additionally, soluble zdint1 peptides and fragments can disrupt the
integrin-mediated attachment of a cell to the extracellular
matrix.
[0160] Antibodies:
[0161] zdint1 polypeptides can also be used to prepare antibodies
that specifically bind to zdint1 epitopes, peptides or
polypeptides. The zdint1 polypeptide or a fragment thereof serves
as an antigen (immunogen) to inoculate an animal and elicit an
immune response. Suitable antigens would include fragments of the
zdint1 polypeptide encoded by SEQ ID NO:2 which represent six or
more contiguous hydrophilic amino acids. Such antigenic regions
would be, for example, from amino acid residue 159 to 164 (SEQ ID
NO:7); amino acid residue 158 to 163 (SEQ ID NO:8); amino acid
residue 518 to 523 (SEQ ID NO:9); amino acid residue 658 to 663
(SEQ ID NO:10); and amino acid residue 190 to 195 (SEQ ID NO:11).
Antibodies generated from this immune response can be isolated and
purified as described herein. Methods for preparing and isolating
polyclonal and monoclonal antibodies are well known in the art.
See, for example, Current Protocols in Immunology, Cooligan, et al.
(eds.), National Institutes of Health, John Wiley and Sons, Inc.,
1995; Sambrook et al., Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor, N.Y., 1989; and Hurrell, J. G.
R., Ed., Monoclonal Hybridoma Antibodies: Techniques and
Applications, CRC Press, Inc., Boca Raton, Fla., 1982.
[0162] As would be evident to one of ordinary skill in the art,
polyclonal antibodies can be generated from inoculating a variety
of warm-blooded animals such as horses, cows, goats, sheep, dogs,
chickens, rabbits, mice, and rats with a zdint1 polypeptide or a
fragment thereof. The immunogenicity of a zdint1 polypeptide may be
increased through the use of an adjuvant, such as alum (aluminum
hydroxide) or Freund's complete or incomplete adjuvant.
Polypeptides useful for immunization also include fusion
polypeptides, such as fusions of zdint1 or a portion thereof with
an immunoglobulin polypeptide or with maltose binding protein. The
polypeptide immunogen may be a full-length molecule or a portion
thereof. If the polypeptide portion is "hapten-like", such portion
may be advantageously joined or linked to a macromolecular carrier
(such as keyhole limpet hemocyanin (KLH), bovine serum albumin
(BSA) or tetanus toxoid) for immunization.
[0163] As used herein, the term "antibodies" includes polyclonal
antibodies, affinity-purified polyclonal antibodies, monoclonal
antibodies, and antigen-binding fragments, such as F(ab').sub.2 and
Fab proteolytic fragments. Genetically engineered intact antibodies
or fragments, such as chimeric antibodies, Fv fragments, single
chain antibodies and the like, as well as synthetic antigen-binding
peptides and polypeptides, are also included. Non-human antibodies
may be humanized by grafting non-human CDRs onto human framework
and constant regions, or by incorporating the entire non-human
variable domains (optionally "cloaking" them with a human-like
surface by replacement of exposed residues, wherein the result is a
"veneered" antibody). In some instances, humanized antibodies may
retain non-human residues within the human variable region
framework domains to enhance proper binding characteristics.
Through humanizing antibodies, biological half-life may be
increased, and the potential for adverse immune reactions upon
administration to humans is reduced.
[0164] Alternative techniques for generating or selecting
antibodies useful herein include in vitro exposure of lymphocytes
to zdint1 protein or peptide, and selection of antibody display
libraries in phage or similar vectors (for instance, through use of
immobilized or labeled zdint1 protein or peptide). Genes encoding
polypeptides having potential zdint1 polypeptide binding domains
can be obtained by screening random peptide libraries displayed on
phage (phage display) or on bacteria, such as E. coli. Nucleotide
sequences encoding the polypeptides can be obtained in a number of
ways, such as through random mutagenesis and random polynucleotide
synthesis. These random peptide display libraries can be used to
screen for peptides which interact with a known target which can be
a protein or polypeptide, such as a ligand or receptor, a
biological or synthetic macromolecule, or organic or inorganic
substances. Techniques for creating and screening such random
peptide display libraries are known in the art (Ladner et al., U.S.
Pat. No. 5,223,409; Ladner et al., U.S. Pat. No. 4,946,778; Ladner
et al., U.S. Pat. No. 5,403,484 and Ladner et al., U.S. Pat. No.
5,571,698) and random peptide display libraries and kits for
screening such libraries are available commercially, for instance
from Clontech (Palo Alto, Calif.), Invitrogen Inc. (San Diego,
Calif.), New England Biolabs, Inc. (Beverly, Mass.) and Pharmacia
LKB Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the zdint1 sequences disclosed
herein to identify proteins which bind to zdint1. These "binding
proteins" which interact with zdint1 polypeptides can be used for
tagging cells; for isolating homolog polypeptides by affinity
purification; they can be directly or indirectly conjugated to
drugs, toxins, radionuclides and the like. These binding proteins
can also be used in analytical methods such as for screening
expression libraries and neutralizing activity. The binding
proteins can also be used for diagnostic assays for determining
circulating levels of polypeptides; for detecting or quantitating
soluble polypeptides as marker of underlying pathology or disease.
These binding proteins can also act as zdint1 "antagonists" to
block zdint1 binding and signal transduction in vitro and in vivo.
These anti-zdint1 binding proteins would be useful for inhibiting,
for example, platelet aggregation, apoptosis, neurogenesis,
myogenesis, tumor formation, and cell-cell interactions in
general.
[0165] Antibodies are determined to be specifically binding if: 1)
they exhibit a threshold level of binding activity, and/or 2) they
do not significantly cross-react with related polypeptide
molecules. First, antibodies herein specifically bind if they bind
to a zdint1 polypeptide, peptide or epitope with a binding affinity
(K.sub.a) of 10.sup.6 M.sup.-1 or greater, preferably 10.sup.7
M.sup.-1 or greater, more preferably 10.sup.8 M.sup.-1 or greater,
and most preferably 10.sup.9 M.sup.-1 or greater. The binding
affinity of an antibody can be readily determined by one of
ordinary skill in the art, for example, by Scatchard analysis
(Scatchard, G., Ann. NY Acad. Sci. 51: 660-672, 1949).
[0166] Second, antibodies are determined to specifically bind if
they do not significantly cross-react with related polypeptides.
Antibodies do not significantly cross-react with related
polypeptide molecules, for example, if they detect zdint1 but not
known related polypeptides using a standard Western blot analysis
(Ausubel et al., ibid.). Examples of known related polypeptides are
orthologs, proteins from the same species that are members of a
protein family, zdint1 polypeptides, and non-human zdint1.
Moreover, antibodies may, be "screened against" known related
polypeptides to isolate a population that specifically binds to the
inventive polypeptides. For example, antibodies raised to zdint1
are adsorbed to related polypeptides adhered to insoluble matrix;
antibodies specific to zdint1 will flow through the matrix under
the proper buffer conditions. Such screening allows isolation of
polyclonal and monoclonal antibodies non-crossreactive to closely
related polypeptides (Antibodies: A Laboratory Manual, Harlow and
Lane (eds.), Cold Spring Harbor Laboratory Press, 1988; Current
Protocols in Immunology, Cooligan, et al. (eds.), National
Institutes of Health, John Wiley and Sons, Inc., 1995). Screening
and isolation of specific antibodies is well known in the art. See,
Fundamental Immunology, Paul (eds.), Raven Press, 1993; Getzoff et
al., Adv. in Immunol. 43: 1-98, 1988; Monoclonal Antibodies:
Principles and Practice, Goding, J. W. (eds.), Academic Press Ltd.,
1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984.
[0167] A variety of assays known to those skilled in the art can be
utilized to detect antibodies which specifically bind to zdint1
proteins or peptides. Exemplary assays are described in detail in
Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold
Spring Harbor Laboratory Press, 1988. Representative examples of
such assays include: concurrent immunoelectrophoresis,
radioimmunoassay, radioimmuno-precipitation, enzyme-linked
immunosorbent assay (ELISA), dot blot or Western blot assay,
inhibition or competition assay, and sandwich assay. In addition,
antibodies can be screened for binding to wild-type versus mutant
zdint1 protein or polypeptide.
[0168] Antibodies to zdint1 may be used for tagging cells that
express zdint1; for isolating zdint1 by affinity purification; for
diagnostic assays for determining circulating levels of zdint1
polypeptides; for detecting or quantitating soluble zdint1 as
marker of underlying pathology or disease; in analytical methods
employing FACS; for screening expression libraries; for generating
anti-idiotypic antibodies; and as neutralizing antibodies or as
antagonists to block zdint1 in vitro and in vivo. Suitable direct
tags or labels include radionuclides, enzymes, substrates,
cofactors, inhibitors, fluorescent markers, chemiluminescent
markers, magnetic particles and the like; indirect tags or labels
may feature use of biotin-avidin or other
complement/anti-complement pairs as intermediates. Antibodies
herein may also be directly or indirectly conjugated to drugs,
toxins, radionuclides and the like, and these conjugates used for
in vivo diagnostic or therapeutic applications. Moreover,
antibodies to zdint1 or fragments thereof may be used in vitro to
detect denatured zdint1 or fragments thereof in assays, for
example, Western Blots or other assays known in the art.
[0169] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a zacrp2
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat. Acad. Sci. USA 81:3998, 1983).
[0170] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660, 1983). Accordingly,
antigenic epitope-bearing peptides and polypeptides of the present
invention are useful to raise antibodies that bind with the
polypeptides described herein.
[0171] Antigenic epitope-bearing peptides and polypeptides
preferably contain at least four to ten amino acids, at least ten
to fifteen amino acids, or about 15 to about 30 amino acids of SEQ
ID NO:2. Such epitope-bearing peptides and polypeptides can be
produced by fragmenting a zacrp2 polypeptide, or by chemical
peptide synthesis, as described herein. Moreover, epitopes can be
selected by phage display of random peptide libraries (see, for
example, Lane and Stephen, Curr. Opin. Immunol. 5:268, 1993, and
Cortese et al., Curr. Opin. Biotechnol. 7:616, 1996). Standard
methods for identifying epitopes and producing antibodies from
small peptides that comprise an epitope are described, for example,
by Mole, "Epitope Mapping," in Methods in Molecular Biology, Vol.
10, Manson (ed.), pages 105-16 (The Humana Press, Inc. 1992),
Price, "Production and Characterization of Synthetic
Peptide-Derived Antibodies," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 60-84 (Cambridge University Press 1995), and Coligan et al.
(eds.), Current Protocols in Immunology, pages 9.3.1-9.3.5 and
pages 9.4.1-9.4.11 (John Wiley & Sons 1997). Polypeptides, or
fragments thereof, of the present invention comprising sequences of
amino acids from, for example, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:8, SEQ ID NO:10, or SEQ ID NO:11 are epitope bearing.
[0172] Bioactive Conjugates:
[0173] Antibodies or polypeptides herein can also be directly or
indirectly conjugated to drugs, toxins, radionuclides and the like,
and these conjugates used for in vivo diagnostic or therapeutic
applications. For instance, polypeptides or antibodies of the
present invention can be used to identify or treat tissues or
organs that express a corresponding anti-complementary molecule
(integrin or antigen, respectively, for instance). More
specifically, zdint1 polypeptides or anti-zdint1 antibodies, or
bioactive fragments or portions thereof, can be coupled to
detectable or cytotoxic molecules and delivered to a mammal having
cells, tissues or organs that express the anti-complementary
molecule.
[0174] Suitable detectable molecules may be directly or indirectly
attached to the polypeptide or antibody, and include radionuclides,
enzymes, substrates, cofactors, inhibitors, fluorescent markers,
chemiluminescent markers, magnetic particles and the like. Suitable
cytotoxic molecules may be directly or indirectly attached to the
polypeptide or antibody, and include bacterial or plant toxins (for
instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and
the like), as well as therapeutic radionuclides, such as
iodine-131, rhenium-188 or yttrium-90 (either directly attached to
the polypeptide or antibody, or indirectly attached through means
of a chelating moiety, for instance). Polypeptides or antibodies
may also be conjugated to cytotoxic drugs, such as adriamycin. For
indirect attachment of a detectable or cytotoxic molecule, the
detectable or cytotoxic molecule can be conjugated with a member of
a complementary/ anticomplementary pair, where the other member is
bound to the polypeptide or antibody portion. For these purposes,
biotin/streptavidin is an exemplary complementary/
anticomplementary pair.
[0175] In another embodiment, polypeptide-toxin fusion proteins or
antibody-toxin fusion proteins can be used for targeted cell or
tissue inhibition or ablation (for instance, to treat cancer cells
or tissues). Alternatively, if the polypeptide has multiple
functional domains (i.e., an activation domain or a ligand binding
domain, plus a targeting domain), a fusion protein including only
the targeting domain may be suitable for directing a detectable
molecule, a cytotoxic molecule or a complementary molecule to a
cell or tissue type of interest. In instances where the domain only
fusion protein includes a complementary molecule, the
anti-complementary molecule can be conjugated to a detectable or
cytotoxic molecule. Such domain-complementary molecule fusion
proteins thus represent a generic targeting vehicle for
cell/tissue-specific delivery of generic
anti-complementary-detectable/cytotoxic molecule conjugates.
[0176] In another embodiment, zdint1-cytokine fusion proteins or
antibody-cytokine fusion proteins can be used for enhancing in vivo
killing of target tissues (for example, brain, heart, spinal cord
and skeletal muscle malignancies), if the zdint1 polypeptide or
anti-zdint1 antibody targets hyperproliferative brain, heart,
spinal cord, or skeletal muscle cells. (See, generally, Hornick et
al., Blood 89:4437-47, 1997). They described fusion proteins that
enable targeting of a cytokine to a desired site of action, thereby
providing an elevated local concentration of cytokine. Suitable
zdint1 polypeptides or anti-zdint1 antibodies target an undesirable
cell or tissue (i.e., a tumor or a leukemia), and the fused
cytokine mediated improved target cell lysis by effector cells.
Suitable cytokines for this purpose include interleukin 2 and
granulocyte-macrophage colony-stimulating factor (GM-CSF), for
instance.
[0177] In yet another embodiment, if the zdint1 polypeptide or
anti-zdint1 antibody targets vascular cells or tissues, such
polypeptide or antibody may be conjugated with a radionuclide, and
particularly with a beta-emitting radionuclide, to reduce
restenosis. Such therapeutic approach poses less danger to
clinicians who administer the radioactive therapy. For instance,
iridium-192 impregnated ribbons placed into stented vessels of
patients until the required radiation dose was delivered showed
decreased tissue growth in the vessel and greater luminal diameter
than the control group, which received placebo ribbons. Further,
revascularisation and stent thrombosis were significantly lower in
the treatment group. Similar results are predicted with targeting
of a bioactive conjugate containing a radionuclide, as described
herein.
[0178] The bioactive polypeptide or antibody conjugates described
herein can be delivered intravenously, intraarterially or
intraductally, or may be introduced locally at the intended site of
action.
[0179] Uses of Polynucleotide/Polypeptide:
[0180] Molecules of the present invention can be used to identify
and isolate receptors and integrins involved in cell-cell
interactions. For example, proteins and peptides of the present
invention can be immobilized on a column and membrane preparations
run over the column (Immobilized Affinity Ligand Techniques,
Hermanson et al., eds., Academic Press, San Diego, Calif., 1992,
pp.195-202). Polypeptides and peptides which bind to the zdint1
polypeptides, peptides, and variants fo the present invention can
then be eluted and characterized using methods known in the art.
Proteins and peptides can also be radiolabeled (Methods in
Enzymol., vol. 182, "Guide to Protein Purification", M. Deutscher,
ed., Acad. Press, San Diego, 1990, 721-37) or photoaffinity labeled
(Brunner et al., Ann. Rev. Biochem. 62:483-514, 1993 and Fedan et
al., Biochem. Pharmacol. 33:1167-80, 1984) and specific
cell-surface proteins can be identified.
[0181] The molecules of the present invention will be useful in
repair and remodeling after an ischemic event, and/or inhibiting
platelet aggregation. The polypeptides, nucleic acid and/or
antibodies of the present invention can be used in treatment of
disorders associated with infarct in brain or heart tissue, and/or
platelet aggregation. The molecules of the present invention can be
used to modulate proteolysis, apoptosis, neurogenesis, myogenesis,
connective tissue disorders, arthritis, chondrogenesis, cell
adhesion, cell fusion, and signaling or to treat or prevent
development of pathological conditions in such diverse tissue as
heart, brain, spinal cord and skeletal muscle. In particular,
certain diseases may be amenable to such diagnosis, treatment or
prevention. The molecules of the present invention can be used to
modulate inhibition and proliferation of neurons and myocytes in
heart, brain, spinal cord and skeletal muscle tissues. Disorders
which may be amenable to diagnosis, treatment or prevention with
zdint1 polypeptides include, for example, Alzheimers's Disease,
tumor formation, Multiple Sclerosis, Congestive Heart Failure,
Ischemic Reperfusion or infarct, and degenerative diseases.
[0182] The zdint1 molecules of the present invention may be
particularly useful in the treatment of intimal hyperplasia or
restenosis due to acute vascular injury. Acute vascular injuries
are those which occur rapidly (i.e. over days to months), in
contrast to chronic vascular injuries (e.g. atherosclerosis) which
develop over a lifetime. Acute vascular injuries often result from
surgical procedures such as vascular reconstruction, wherein the
techniques of angioplasty, endarterectomy, atherectomy, vascular
graft emplacement or the like are employed. Hyperplasia may also
occur as a delayed response in response to, e.g., graft emplacement
or organ transplantation. The dose of zdint1 in the treatment for
restenosis will vary with each patient but will generally be in the
range of those suggested above.
[0183] Advances in the treatment of coronary vascular disease
include the use of mechanical interventions to either remove or
displace offending plaque material in order to re-establish
adequate blood flow through the coronary arteries. Despite the use
of multiple forms of mechanical interventions, including balloon
angioplasty, reduction atherectomy, placement of vascular stents,
laser therapy, or rotoblator, the effectiveness of these techniques
remains limited by an approximately 40% restenosis rate within 6
months after treatment.
[0184] Restenosis is thought to result from a complex interaction
of biological processes including platelet deposition and thrombus
formation, release of chemotactic and mitogenic factors, and the
migration and proliferation of vascular smooth muscle cells into
the intima of the dilated arterial segment.
[0185] The inhibition of platelet accumulation at sites of
mechanical injury can limit the rate of restenosis in human
subjects. Therapeutic use of a monoclonal antibody to platelet
GpIIb/IIIa is able to limit the level of restenosis in human
subjects (Califf et al., N. Engl. J. Med., 330: 956-961 (1994)).
The antibody is able to bind to the GpIIb/IIIa receptor on the
surfaces of platelets and thereby inhibit platelet accumulation.
This data suggests that inhibition of platelet accumulation at the
site of mechanical injury in human coronary arteries is beneficial
for the ultimate healing response that occurs.
[0186] Gene Therapy:
[0187] Polynucleotides encoding zdint1 polypeptides are useful
within gene therapy applications where it is desired to increase or
inhibit zdint1 activity. If a mammal has a mutated or absent zdint1
gene, the zdint1 gene can be introduced into the cells of the
mammal. In one embodiment, a gene encoding a zdint1 polypeptide is
introduced in vivo in a viral vector. Such vectors include an
attenuated or defective DNA virus, such as, but not limited to,
herpes simplex virus (HSV), papillomavirus, Epstein Barr virus
(EBV), adenovirus, adeno-associated virus (AAV), and the like.
Defective viruses, which entirely or almost entirely lack viral
genes, are preferred. A defective virus is not infective after
introduction into a cell. Use of defective viral vectors allows for
administration to cells in a specific, localized area, without
concern that the vector can infect other cells. Examples of
particular vectors include, but are not limited to, a defective
herpes simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell.
Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as
the vector described by Stratford-Perricaudet et al., J. Clin.
Invest. 90:626-30, 1992; and a defective adeno-associated virus
vector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et
al., J. Virol. 63:3822-8, 1989).
[0188] In another embodiment, a zdint1 gene can be introduced in a
retroviral vector, e.g., as described in Anderson et al., U.S. Pat.
No. 5,399,346; Mann et al. Cell 33:153, 1983; Temin et al., U.S.
Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289;
Markowitz et al., J. Virol. 62:1120, 1988; Temin et al., U.S. Pat.
No. 5,124,263; International Patent Publication No. WO 95/07358,
published Mar. 16, 1995 by Dougherty et al.; and Kuo et al., Blood
82:845, 1993. Alternatively, the vector can be introduced by
lipofection in vivo using liposomes. Synthetic cationic lipids can
be used to prepare liposomes for in vivo transfection of a gene
encoding a marker (Felgner et al., Proc. Natl. Acad. Sci. USA
84:7413-7, 1987; Mackey et al., Proc. Natl. Acad. Sci. USA
85:8027-31, 1988). The use of lipofection to introduce exogenous
genes into specific organs in vivo has certain practical
advantages. Molecular targeting of liposomes to specific cells
represents one area of benefit. More particularly, directing
transfection to particular cells represents one area of benefit.
For instance, directing transfection to particular cell types would
be particularly advantageous in a tissue with cellular
heterogeneity, such as the pancreas, liver, kidney, and brain.
Lipids may be chemically coupled to other molecules for the purpose
of targeting. Targeted peptides (e.g., hormones or
neurotransmitters), proteins such as antibodies, or non-peptide
molecules can be coupled to liposomes chemically.
[0189] It is possible to remove the target cells from the body; to
introduce the vector as a naked DNA plasmid; and then to re-implant
the transformed cells into the body. Naked DNA vectors for gene
therapy can be introduced into the desired host cells by methods
known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun or use of a DNA vector
transporter. See, e.g., Wu et al., J. Biol. Chem. 267:963-7, 1992;
Wu et al., J. Biol. Chem. 263:14621-4, 1988.
[0190] Antisense methodology can be used to inhibit zdint1 gene
transcription, such as to inhibit cell proliferation in vivo.
Polynucleotides that are complementary to a segment of a
zdint1-encoding polynucleotide (e.g., a polynucleotide as set froth
in SEQ ID NO:1) are designed to bind to zdint1-encoding mRNA and to
inhibit translation of such mRNA. Such antisense polynucleotides
are used to inhibit expression of zdint1 polypeptide-encoding genes
in cell culture or in a subject.
[0191] The present invention also provides reagents which will find
use in diagnostic applications. For example, the zdint1 gene, a
probe comprising zdint1 DNA or RNA or a subsequence thereof can be
used to determine if the zdint1 gene is present on chromosome 2q33
or if a mutation has occurred. Detectable chromosomal aberrations
at the zdint1 gene locus include, but are not limited to,
aneuploidy, gene copy number changes, insertions, deletions,
restriction site changes and rearrangements. Such aberrations can
be detected using polynucleotides of the present invention by
employing molecular genetic techniques, such as restriction
fragment length polymorphism (RFLP) analysis, short tandem repeat
(STR) analysis employing PCR techniques, and other genetic linkage
analysis techniques known in the art (Sambrook et al., ibid.;
Ausubel et. al., ibid.; Marian, Chest 108:255-65, 1995).
[0192] Transgenic mice, engineered to express the zdint1 gene, or
fragments thereof, and mice that exhibit a complete absence of
zdint1 gene function, referred to as "knockout mice" (Snouwaert et
al., Science 257:1083, 1992) can also be generated (Lowell et al.,
Nature 366:740-42, 1993) by one skilled in the art. These mice can
be employed to study the zdint1 gene, gene fragments, and the
protein encoded thereby in an in vivo system.
[0193] Chromosomal Localization:
[0194] Radiation hybrid mapping is a somatic cell genetic technique
developed for constructing high-resolution, contiguous maps of
mammalian chromosomes (Cox et al., Science 250:245-50, 1990).
Partial or full knowledge of a gene's sequence allows one to design
PCR primers suitable for use with chromosomal radiation hybrid
mapping panels. Radiation hybrid mapping panels are commercially
available which cover the entire human genome, such as the Stanford
G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc.,
Huntsville, Ala.). These panels enable rapid, PCR-based chromosomal
localizations and ordering of genes, sequence-tagged sites (STSs),
and other nonpolymorphic and polymorphic markers within a region of
interest. This includes establishing directly proportional physical
distances between newly discovered genes of interest and previously
mapped markers. The precise knowledge of a gene's position can be
useful for a number of purposes, including: 1) determining if a
sequence is part of an existing contig and obtaining additional
surrounding genetic sequences in various forms, such as YACs, BACs
or cDNA clones; 2) providing a possible candidate gene for an
inheritable disease which shows linkage to the same chromosomal
region; and 3) cross-referencing model organisms, such as mouse,
which may aid in determining what function a particular gene might
have.
[0195] Sequence tagged sites (STSs) can also be used independently
for chromosomal localization. An STS is a DNA sequence that is
unique in the human genome and can be used as a reference point for
a particular chromosome or region of a chromosome. An STS is
defined by a pair of oligonucleotide primers that are used in a
polymerase chain reaction to specifically detect this site in the
presence of all other genomic sequences. Since STSs are based
solely on DNA sequence they can be completely described within an
electronic database, for example, Database of Sequence Tagged Sites
(dbSTS), GenBank, (National Center for Biological Information,
National Institutes of Health, Bethesda, MD
http://www.ncbi.nlm.nih.gov), and can be searched with a gene
sequence of interest for the mapping data contained within these
short genomic landmark STS sequences.
[0196] For pharmaceutical use, the proteins of the present
invention can be administered orally, rectally, parenterally,
intracisternally, intravaginally, intraperitoneally, topically (as
powders, ointments, drops or transdermal patch) bucally, or as a
pulmonary or nasal inhalant. Intravenous administration will be by
bolus injection or infusion over a typical period of one to several
hours. In general, pharmaceutical formulations will include a
zdint1 protein, alone, or in conjunction with a dimeric partner, in
combination with a pharmaceutically acceptable vehicle, such as
saline, buffered saline, 5% dextrose in water or the like.
Formulations may further include one or more excipients,
preservatives, solubilizers, buffering agents, albumin to prevent
protein loss on vial surfaces, etc. Methods of formulation are well
known in the art and are disclosed, for example, in Remington: The
Science and Practice of Pharmacy, Gennaro, ed., Mack Publishing
Co., Easton, Pa., 19th ed., 1995. Therapeutic doses will generally
be in the range of 0.1 to 100 .mu.g/kg of patient weight per day,
preferably 0.5-20 mg/kg per day, with the exact dose determined by
the clinician according to accepted standards, taking into account
the nature and severity of the condition to be treated, patient
traits, etc. Determination of dose is within the level of ordinary
skill in the art. The proteins may be administered for acute
treatment, over one week or less, often over a period of one to
three days or may be used in chronic treatment, over several months
or years. In general, a therapeutically effective amount of zdint1
is an amount sufficient to produce a clinically significant change
in extracellular matrix remodeling, scar tissue formation, tumor
suppression, platelet aggregation, apoptosis, myogenesis,
neurogenesis, electrical coupling, blood flow and/or cell
proliferation in brain, heart, spinal cord, and skeletal
muscle.
[0197] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Extension of EST Sequence
[0198] The novel zdint1 polypeptide-encoding polynucleotides of the
present invention were initially identified by querying an EST
database. This query identified an expressed sequence tag (EST) to
nucleotide 1097 to nucleotide 1415 of SEQ ID NO: 1. A cDNA clone,
corresponding to this EST was obtained and the deduced amino acid
sequence was determined to be incomplete. Primers ZC17,991 (SEQ ID
NO:4) and ZC17,992 (SEQ ID NO:5) were used to screen an arrayed
fetal brain cDNA plasmid library to identify clones of zdint1.
Thermocycler conditions were as follows: one cycle at 94.degree. C.
for 1 minute 30 seconds; followed by thirty cycles at 94.degree. C.
for 10 seconds, 64.degree. C. for 20 seconds, 72.degree. C. for 30
seconds, followed by one cycle at 72.degree. C. for 5 minutes,
followed by a 4.degree. C. hold. A sample of the reaction contents
was electrophoresed on a 4% agarose gel to identify positive pools.
These pools were screened by polymerase chain reaction with
ZC17,992 (SEQ ID NO:5) and the vector primer ZC13,006 (SEQ ID
NO:6). Thermocycler conditions were as follows: one cycle at
94.degree. C. for 1 minute 30 seconds; followed by five cycles at
94.degree. C. for 10 seconds, 68.degree. C. for 2 minutes, followed
by twenty-five cycles at 94.degree. C. for 10 seconds, 62.degree.
C. for 20 seconds, 72.degree. C. for 2 minutes, followed by one
cycle at 72.degree. C. for 10 minutes, followed by a 4.degree. C.
hold. A sample of the reaction contents was electrophoresed on a 1%
agarose gel and a band of .about.1.5 kb was further electrophoresed
on a 1% preparative gel and the resulting band gel purified using
commercially available gel purification reagents and protocol
(QIAEX II Gel Extraction Kit; Qiagen, Inc., Santa Clarita, Calif.).
This fragment was sequenced and was determined to extend the amino
acid sequence of zdint1 in the 5' direction.
Example 2
Tissue Distribution
[0199] Analysis of tissue distribution was performed by the
Northern blotting technique using Human Multiple Tissue and Master
Dot Blots from Clontech (Palo Alto, Calif.), and a human vascular
tissue blot prepared in-house. The human vascular blot was prepared
from the following cell lines: Human Umbilical Vein Endothelial
Cells (Cascade Biologics, Inc., Portland, Oreg.), Human Pulmonary
Artery Endothelial Cells (Cascade Biologics, Inc., Portland,
Oreg.), Human Aortic Endothelail Cells, (Cascade Biologics, Inc.,
Portland, Oreg.), Aortic Smooth Muscle Cells (Clonetics, San Diego,
Calif.), Human Intestinal Smooth Muscle Cells (American Type
Culture Collectio, Manasas, Va.), Normal Human Lung Fibroblast,
Clonetics, San Diego, Calif.) and Normal Human Dermal
Fibroblast-Neonatal, Clonetics, San Diego, Calif.). Messenger RNA
was extracted and blots prepared by methods known in the art. The
probe was obtained by restriction digest of the original cDNA clone
with a restriction endonuclease, PstI. The reaction mixture was
electrophoresed on a preparative agarose gel and two bands,
corresponding to a 239 base pair fragment and a 223 base pair
fragment from the cDNA clone, were gel purified using commercially
available gel purification reagents and protocol from Qiagen, Inc.
A probe was made by pooling the purified DNA from both bands and
was random prime labeled with .sup.32P using a commercially
available kit (Rediprime DNA labeling system; Amersham Corp.,
Arlington Heights, Ill.). The probe was then purified using a
NUCTRAP push column (Stratagene Cloning Systems, La Jolla, Calif.)
EXPRESSHYB (Clontech) solution was used for pre-hybridization and
hybridization. The hybridization solution consisted of 8 ml
EXPRESSHYB, 80 .mu.l Sheared Salmon Sperm DNA (10 mg/ml, 5 Prime-3
Prime, Boulder, Colo.), 48 .mu.l Human Cot-1 DNA (1 mg/ml, Gibco
BRL), and 57 .mu.l labeled probe (2.3.times.10.sup.-5 CPM/.mu.l).
Hybridization took place overnight at 50.degree. C., and the blots
were then washed in 2.times. SSC and 0.1% SDS at ambient room
temperature, then 2.times. SSC and 0.1% SDS at 60.degree. C.,
followed by 0.1.times. SSC and 0.1% SDS at 60.degree. C. The blots
were exposed overnight and developed. Strong signals of three
transcript sizes, approximately 3.0 kb, 4.4 kb, and 7.5 kb, were
observed in heart on the multiple tissue Northern blots. Faint
signals of the same transcript sizes were observed in brain and
spinal cord. An fainter signal of the three transcript sizes was
observed in skeletal muscle. The Master Dot Blot showed strong
signals in brain, heart, fetal brain, and fetal heart. For the
human vascular blot, a strong signal at 3-3.5kb in human aortic
endothelial cells and weaker signals in aortic smooth muscle cells
and normal human lung fibroblast cells was observed.
Example 3
Protein Purification
[0200] Purification conditions for zdint1 with N- and C-terminal EE
tags:
[0201] E. coli, Pichia, CHO and BHK cells are transfected with
expression vectors containing the DNA sequence of SEQ ID NO:1, or a
portion thereof, operably linked to a polynucleotide encoding a
Glu-Glu tag. Zdintl protein is expressed in conditioned media of E.
coil, Pichia methanolica, and or chinese hamster ovary (CHO) and
baby hamster kidney (BHK) cells. For zdint1 expressed in E. coli
and Pichia, the media is not concentrated prior to purification.
Unless otherwise noted, all operations are carried out at 4.degree.
C. A total of 25 liters of conditioned medium from BHK cells is
sequentially sterile filtered through a 4 inch, 0.2 mM Millipore
(Bedford, Mass.) OptiCap capsule filter and a 0.2 mM Gelman (Ann
Arbor, MI) Supercap 50. The material is then concentrated to about
1.3 liters using a Millipore ProFlux A30 tangential flow
concentrator fitted with a 3000 kDa cutoff Amicon (Bedford, MA)
S10Y3 membrane. The concentrated material is again sterile-filtered
with the Gelman filter, as described above. A mixture of protease
inhibitors is added to the concentrated conditioned medium to final
concentrations of 2.5 mM ethylenediaminetetraacetic acid (EDTA,
Sigma Chemical Co. St. Louis, Mo.), 0.001 mM leupeptin
(Boehringer-Mannheim, Indianapolis, Ind.), 0.001 mM pepstatin
(Boehringer-Mannheim) and 0.4 mM Pefabloc (Boehringer-Mannheim). A
50.0 ml sample of anti-EE Sepharose, prepared as described below,
is added and the mixture gently agitated on a Wheaton (Millville,
N.J.) roller culture apparatus for 18.0 h at 4.degree. C.
[0202] The mixture is then poured into a 5.0.times.20.0 cm
Econo-Column (Bio-Rad, Laboratories, Hercules, Calif.), and the gel
is washed with 30 column volumes of phosphate buffered saline
(PBS). The unretained flow-through fraction is discarded. Once the
absorbance of the effluent at 280 nM is less than 0.05, flow
through the column is reduced to zero, and the anti-EE Sepharose
gel is washed with 2.0 column volumes of PBS containing 0.2 mg/ml
of EE peptide (AnaSpec, San Jose, Calif.). The peptide that is used
has the sequence GluTyrMetProValAsp. After 1.0 h at 4.degree. C.,
flow is resumed and the eluted protein collected. This fraction is
referred to as the peptide elution. The anti-EE Sepharose gel is
then washed with 2.0 column volumes of 0.1 M glycine, pH 2.5, and
the glycine wash is collected separately. The pH of the
glycine-eluted fraction is adjusted to 7.0 by the addition of a
small volume of 10.times. PBS and stored at 4.degree. C. for future
analysis, if needed.
[0203] The peptide elution is concentrated to 5.0 ml using a 15,000
molecular weight cutoff membrane concentrator (Millipore, Bedford,
Mass.), according to the manufacturer's instructions. The
concentrated peptide elution is then separated from free peptide by
chromatography on a 1.5.times.50 cm Sephadex G-50 (Pharmacia,
Piscataway, N.J.) column equilibrated in PBS at a flow rate of 1.0
ml/min using a BioCad Sprint HPLC (PerSeptive BioSystems,
Framingham, Mass.). Two-ml fractions are collected and the
absorbance at 280 nM monitored. The first peak of material
absorbing at 280 nM and eluting near the void volume of the column
is collected. This fraction is pure zdint1 NEE or zdint1 CEE. The
pure material is concentrated as described above, analyzed by
SDS-PAGE and Western blotting with anti-EE antibodies, aliquoted,
and stored at -80.degree. C. according to standard procedures.
[0204] Preparation of anti-EE Sepharose:
[0205] A 100 ml bed volume of protein G-Sepharose (Pharmacia,
Piscataway, N.J.) is washed 3 times with 100 ml of PBS containing
0.02% sodium azide using a 500 ml Nalgene 0.45 micron filter unit.
The gel is washed with 6.0 volumes of 200 mM triethanolamine, pH
8.2 (TEA, Sigma, St. Louis, Mo.). and an equal volume of EE
antibody solution containing 900 mg of antibody is added. After an
overnight incubation at 4.degree. C., unbound antibody is removed
by washing the resin with 5 volumes of 200 mM TEA as described
above. The resin is resuspended in 2 volumes of TEA, transferred to
a suitable container, and dimethylpimilimidate-2HCl (Pierce,
Rockford, Ill.), dissolved in TEA, is added to a final
concentration of 36 mg/ml of gel. The gel is rocked at room
temperature for 45 min and the liquid is removed using the filter
unit as described above. Nonspecific sites on the gel are then
blocked by incubating for 10 min at room temperature with 5 volumes
of 20 mM ethanolamine in 200 mM TEA. The gel is then washed with 5
volumes of PBS containing 0.02% sodium azide and stored in this
solution at 4.degree. C.
[0206] Purification of Untagged zdint1
[0207] E. coli, Pichia, CHO and BHK cells are transfected with
expression vectors containing the DNA sequence of SEQ ID NO:1, or a
portion thereof. The procedure described below is used for protein
expressed in conditioned medium of E. coli, Pichia methanolica, and
Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells.
For zdint1 expressed in E. coli and Pichia, however, the medium is
not be concentrated prior to purification. Unless otherwise noted,
all operations are carried out at 4.degree. C. A total of 25 liters
of conditioned medium from BHK cells is sequentially sterile
filtered through a 4 inch, 0.2 mM Millipore (Bedford, Mass.)
OptiCap capsule filter and a 0.2 mM Gelman (Ann Arbor, Mich.)
Supercap 50. The material is then be concentrated to about 1.3
liters using a Millipore ProFlux A30 tangential flow concentrator
fitted with a 3000 kDa cutoff Amicon (Bedford, Mass.) S10Y3
membrane. The concentrated material is again be sterile-filtered
with the Gelman filter as described above. A mixture of protease
inhibitors is added to the concentrated conditioned medium to final
concentrations of 2.5 mM ethylenediaminetetraacetic acid (EDTA,
Sigma Chemical Co. St. Louis, Mo.), 0.001 mm leupeptin
(Boehringer-Mannheim, Indianapolis, Ind.), 0.001 mM pepstatin
(Boehringer-Mannheim) and 0.4 mM Pefabloc
(Boehringer-Mannheim).
[0208] The procedures outlined below are adaptations of those used
to purify metalloprotease/disintegrins from Crotalus viridus and
Crotalus atrox venom (Liu et al., Toxicol. 33: 1289-1298, 1995;
Shimokawa et al., Arch Biochem Biophys 343: 35-43, 1997). A
combination of procedures including, but not limited to, anion and
cation exchange chromatography, size exclusion, and affinity
chromography is used to purify untagged zdint1.
[0209] Concentrated conditioned medium is diluted {fraction (1/10)}
in line with 10 mM borate buffer, pH 9.0, 0.1 M NaCl, and 2.0 mM
CaCl.sub.2 using the BioCad Sprint HPLC (PerSeptive BioSystems,
Framingham, MA). The material is pumped onto a 3.5.times.20 cm
Poros HQ (PerSeptive BioSystems, Framingham, Mass.) column at 5
ml/min. The column is washed with loading buffer, and when the
absorbance of the effluent is less than 0.05, the column is
developed with a linear gradient of NaCl from 0.1 M to 1.0 M NaCl.
Fractions containing zdint1 are identified by SDS-PAGE and Western
blotting with anti-zdint1 peptide antibodies. zdint1-containing
fractions are pooled together, and concentrated using an Amicon
stirred cell concentrator fitted with a YM-10 membrane. The Poros
HQ pool is then chromatographed on a Sephadex G-75 column
equilibrated in 10 mM sodium phosphate, pH 7.0. Fractions
containing zdint1 are identified and pooled together, as described
above, and applied to a 1.0.times.5 cm Poros HA hydroxyapatite
column at 1.0 ml/min using the BioCad Sprint HPLC. The column is
washed with loading buffer and developed with a linear gradient
from 10 mM to 500 mM sodium phosphate. Fractions contained pure
zdint1 are identified by SDS-PAGE and Western blotting, as
described above. The purified material is aliquoted and stored as
described above.
Example 4
Chromosomal Assignment and Placement of Zdint1
[0210] Zdint1 was mapped to chromosome 2 using the commercially
available version of the "Stanford G3 Radiation Hybrid Mapping
Panel" (Research Genetics, Inc., Huntsville, Ala.). The "Stanford
G3 RH Panel" contains PCRable DNAs from each of 83 radiation hybrid
clones of the whole human genome, plus two control DNAs (the RM
donor and the A3 recipient). A publicly available WWW server
(http://shgc-www.stanford.edu) allows chromosomal localization of
markers.
[0211] For the mapping of zdint1 with the "Stanford G3 RH Panel",
20 .mu.l reactions were set up in a PCRable 96-well microtiter
plate (Stratagene, La Jolla, Calif.) and used in a "RoboCycler
Gradient 96" thermal cycler (Stratagene). Each of the 85 PCR
reactions consisted of 2 .mu.l 10.times. KlenTaq PCR reaction
buffer (CLONTECH Laboratories, Inc., Palo Alto, Calif.), 1.6 .mu.l
dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City, Calif.), 1 .mu.l
sense primer, ZC20,843 (SEQ ID NO:12), 1 .mu.l antisense primer,
ZC20,844 (SEQ ID NO:13), 2 .mu.l "RediLoad" (Research Genetics,
Inc., Huntsville, Ala.), 0.4 .mu.l 50.times. Advantage KlenTaq
Polymerase Mix (Clontech Laboratories, Inc.), 25 ng of DNA from an
individual hybrid clone or control and distilled water for a total
volume of 20 .mu.l. The reactions were overlaid with an equal
amount of mineral oil and sealed. The PCR cycler conditions were as
follows: an initial 1 cycle 5 minute denaturation at 94.degree. C.,
35 cycles of a 45 seconds denaturation at 94.degree. C., 45 seconds
annealing at 66.degree. C. and 1 minute and 15 seconds extension at
72.degree. C., followed by a final 1 cycle extension of 7 minutes
at 72.degree. C. The reactions were separated by electrophoresis on
a 2% agarose gel (Life Technologies, Gaithersburg, MD).
[0212] The results showed linkage of Zdint1 to the framework marker
SHGC-56733 with a LOD score of >12 and at a distance of 0
cR.sub.--10000 from the marker. The use of surrounding markers
positions Zdint1 in the 2q33 region on the integrated LDB
chromosome 2 map (The Genetic Location Database, University of
Southhampton, WWW server: http://cedar.genetics.
soton.ac.uk/public_html/).
Example 5
Synthesis of Peptides
[0213] Zdint1-1, a peptide corresponding to amino acid residue 437
(Cys) to amino acid residue 450 (Cys) of SEQ ID NO: 2, is
synthesized by solid phase peptide synthesis using a model 431A
Peptide Synthesizer (Applied Biosystems/Perkin Elmer, Foster City,
Calif.). Fmoc-Glutamine resin (0.63 mmol/g; Advanced Chemtech,
Louisville, Ky.) is used as the initial support resin. 1 mmol amino
acid cartridges (Anaspec, Inc. San Jose, Calif.) are used for
synthesis. A mixture of 2(1-Hbenzotriazol-y-yl
1,1,3,3-tetrahmethylhyluronium hexafluorophosphate (HBTU),
1-hydroxybenzotriazol (HOBt), 2m N,N-Diisolpropylethylamine,
N-Methylpyrrolidone, Dichloromethane (all from Applied
Biosystems/Perkin Elmer) and piperidine (Aldrich Chemical Co., St.
Louis, Mo.), are used for synthesis reagents.
[0214] The Peptide Companion software (Peptides International,
Louisville, Ky.) is used to predict the aggregation potential and
difficulty level for synthesis for the zdint-1 peptide. Synthesis
is performed using single coupling programs, according to the
manufacturer's specifications.
[0215] The peptide is cleaved from the solid phase following
standard TFA cleavage procedure (according to Peptide Cleavage
manual, Applied Biosystems/Perkin Elmer). Purification of the
peptide is done by RP-HPLC using a C18, 10 .mu.m semi-peparative
column (Vydac, Hesperial, Calif.) Eluted fractions from the column
are collected and analyzed for correct mass and purity by
electrospray mass spectrometry. Pools of the eluted material are
collected. If pure, the pools are combined, frozen and
lyophilized.
Example 6
Anticoagulant Activity of zdint1
[0216] The ability of the zdint1 protein to inhibit clotting is
measured in a one-stage clotting assay using wild-type zdint1 as a
control. Recombinant proteins are prepared essentially as described
above from cells cultured in media containing 5mg/ml vitamin K.
Varying amounts of the zdint1 or recombinant wild-type zdint1 are
diluted in 50 mM Tris pH 7.5, 0.1% BSA to 100 ml. The mixtures are
incubated with 100 ml of zdint1-deficient plasma and 200 ml of
thromboplastin C (Dade, Miami, Fla.; contains rabbit brain
thromboplastin and 11.8 mM Ca.sup.++). The clotting assay is
performed in an automatic coagulation timer (MLA Electra 800,
Medical Laboratory Automation Inc., Pleasantville, N.Y.), and
clotting times are converted to units of zdint1 activity using a
standard curve constructed with 1:5 to 1:640 dilutions of normal
pooled human plasma (assumed to contain one unit per ml zdint1
activity; prepared by pooling citrated serum from healthy
donors).
[0217] Zdint1 activity is seen as a reduction in clotting time over
control samples.
Example 7
Inhibition of Platelet Accumulation with zdint1
[0218] Zdint1 is analyzed for its ability to inhibit platelet
accumulation at sites of arterial thrombosis due to mechanical
injury in non-human primates. A model of aortic endarterectomy is
utilized in baboons, essentially as described by Lumsden et al.
(Blood 81: 1762-1770 (1993)). A section of baboon aorta 1-2 cm in
length is removed, inverted and scraped to remove the intima of the
artery and approximately 50% of the media. The artery is reverted
back to its correct orientation, cannulated on both ends and placed
into an extracorporeal shunt in a baboon, thereby exposing the
mechanically injured artery to baboon blood via the shunt. Just
prior to opening of the shunt to the circulating blood, In-labeled
autologous platelets are injected intravenously into the animal.
The level of platelet accumulation at the site of the injured
artery is determined by real-time gamma camera imaging.
[0219] Evaluation of zdint1 for inhibition of platelet accumulation
is done using bolus injections of zdint1 or saline control and are
given just prior to the opening of the shunt. The injured arteries
are measured continuously for 60 minutes.
[0220] Zdint1 activity is seen as an inhibition of platelet
accumulation.
[0221] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 0
0
* * * * *
References