U.S. patent application number 11/256802 was filed with the patent office on 2006-02-16 for adipocyte complement related protein homolog zacrp3.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Paul D. Bishop, Christopher S. Piddington.
Application Number | 20060034866 11/256802 |
Document ID | / |
Family ID | 26828242 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060034866 |
Kind Code |
A1 |
Piddington; Christopher S. ;
et al. |
February 16, 2006 |
Adipocyte complement related protein homolog zacrp3
Abstract
The present invention relates to polynucleotide and polypeptide
molecules for zacrp3, a novel member of the family of proteins
bearing a collagen-like domain and a C1q domain. The polypeptides
and polynucleotides encoding them, are involved in dimerization or
oligomerization and may be used in the study thereof. The present
invention also includes antibodies to the zacrp3 polypeptides.
Inventors: |
Piddington; Christopher S.;
(Thousand Oaks, CA) ; Bishop; Paul D.; (Fall City,
WA) |
Correspondence
Address: |
Brian J. Walsh;Patent Department
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
26828242 |
Appl. No.: |
11/256802 |
Filed: |
October 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10321164 |
Dec 17, 2002 |
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11256802 |
Oct 24, 2005 |
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09552225 |
Apr 19, 2000 |
6521233 |
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10321164 |
Dec 17, 2002 |
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60130199 |
Apr 20, 1999 |
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Current U.S.
Class: |
424/192.1 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 2319/02 20130101; C07K 14/472 20130101; C07K 14/4702
20130101 |
Class at
Publication: |
424/192.1 ;
530/350; 435/069.1; 435/320.1; 435/325; 536/023.5 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C07K 14/705 20060101 C07K014/705 |
Claims
1. An isolated polynucleotide encoding a polypeptide comprising
amino acid residues 51-246 of SEQ ID NO:2.
2. The isolated polynucleotide of claim 1 wherein the polypeptide
comprises amino acid residues 23-246 SEQ ID NO:2.
3. The isolated polynucleotide of claim 1 wherein the polypeptide
comprises Gly-Xaa-Xaa repeats and Gly-Xaa-Pro repeats forming a
collagen domain.
4. The isolated polynucleotide of claim 3 wherein the collagen
domain consists of 15 Gly-Xaa-Xaa repeats and 6 Gly-Xaa-Pro
repeats.
5. The isolated polynucleotide of claim 3 wherein the collagen
domain comprises amino acid residues 51-113 of SEQ ID NO:2.
6. The isolated polynucleotide of claim 1 wherein the polypeptide
comprises a carboxyl-terminal C1q domain which consists of 10 beta
strands.
7. The isolated polynucleotide of claim 6 wherein the
carboxyl-terminal C1q domain comprises an amino acid sequence of
SEQ ID NO:5.
8. The isolated polynucleotide of claim 6 wherein the
carboxy-terminal C1q domain comprises amino acid residues 119-123,
140-142, 148-151, 155-158, 161-173, 175-182, 190-197, 200-212,
217-222 and 236-241 of SEQ ID NO:2.
9. The isolated polynucleotide of claim 6 wherein the
carboxy-terminal C1q domain consists of amino acid residues 114-246
of SEQ ID NO:2.
10. The isolated polynucleotide of claim 1 wherein the polypeptide
specifically binds with an antibody that specifically binds with a
polypeptide consisting of the amino acid sequence of SEQ ID
NO:2.
11. An isolated polynucleotide encoding a polypeptide comprising
amino acid residues 23-246 of SEQ ID NO:2.
12. An isolated polynucleotide encoding a polypeptide comprising an
amino acid sequence of SEQ ID NO:2.
13. An isolated polynucleotide comprising a nucleotide sequence
selected from the group consisting of: a) nucleotide 1 to
nucleotide 1696 of SEQ ID NO:1; b) nucleotide 69 to nucleotide 806
of SEQ ID NO:1; c) nucleotide 135 to nucleotide 806 of SEQ ID NO:1;
d) nucleotide 219 to nucleotide 806 of SEQ ID NO:1; e) nucleotide
408 to nucleotide 806 of SEQ ID NO:1; f) nucleotide 69 to
nucleotide 407 of SEQ ID NO:1; g) nucleotide 135 to nucleotide 407
of SEQ ID NO:1; and h) nucleotide 219 to nucleotide 407 of SEQ ID
NO:1.
14. An isolated polynucleotide encoding a fusion protein comprising
a first portion and a second portion joined by a peptide bond,
wherein the first portion is selected from the group consisting of:
a) a polypeptide comprising amino acid residues 51 to 246 of SEQ ID
NO:2; b) a polypeptide comprising amino acid residues 1 to 246 of
SEQ ID NO:2; and c) a polypeptide comprising amino acid residues 23
to 246 of SEQ ID NO:2; and wherein the second portion comprises
another polypeptide.
15. An isolated polynucleotide consisting of a nucleotide sequence
of nucleotide 1 to nucleotide 738 of SEQ ID NO:10.
16. An expression vector comprising the following operably linked
elements: a transcription promoter; a DNA segment encoding a
polypeptide of claim 1; and a transcription terminator.
17. The expression vector of claim 16 wherein the DNA segment
encodes a polypeptide comprising amino acid residues 23-246 of SEQ
ID NO:2.
18. The expression vector of claim 16 wherein the DNA segment
encodes a polypeptide covalently linked at the amino or carboxyl
terminus to an affinity tag.
19. The expression vector of claim 16 wherein the DNA segment
further encodes a secretory signal sequence operably linked to the
polypeptide.
20. The expression vector of claim 19 wherein the secretory signal
sequence comprises amino acid residues 1-22 of SEQ ID NO:2.
21. A cultured cell into which has been introduced an expression
vector of claim 16 wherein the cell expresses the polypeptide
encoded by the DNA segment.
22. A method of producing a polypeptide comprising: culturing a
cell into which has been introduced the expression vector of claim
16; whereby the cell expresses the polypeptide encoded by the DNA
segment; and recovering the expressed polypeptide.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 10/321,164, filed Dec. 17, 2002, which is a
divisional application of U.S. patent application Ser. No.
09/552,225, filed Apr. 19, 2000, now U.S. Pat. No. 6,521,233, and
claims the benefit of U.S. Patent Application Ser. No. 60/130,199,
all of which are herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Energy balance (involving energy metabolism, nutritional
state, lipid storage and the like) is an important criteria for
health. This energy homeostasis involves food intake and metabolism
of carbohydrates and lipids to generate energy necessary for
voluntary and involuntary functions. Metabolism of proteins can
lead to energy generation, but preferably leads to muscle formation
or repair. Among other consequences, a lack of energy homeostasis
lead to over or under formation of adipose tissue.
[0003] Formation and storage of fat is insulin-modulated. For
example, insulin stimulates the transport of glucose into cells,
where it is metabolized into .alpha.-glycerophosphate which is used
in the esterification of fatty acids to permit storage thereof as
triglycerides. In addition, adipocytes (fat cells) express a
specific transport protein that enhances the transfer of free fatty
acids into adipocytes.
[0004] Adipocytes also secrete several proteins believed to
modulate homeostatic control of glucose and lipid metabolism. These
additional adipocyte-secreted proteins include adipsin, complement
factors C3 and B, tumor necrosis factor .alpha., the ob gene
product and Acrp30. Evidence also exists suggesting the existence
of an insulin-regulated secretory pathway in adipocytes. Scherer et
al., J. Biol. Chem. 270(45): 26746-9, 1995. Over or under secretion
of these moieties, impacted in part by over or under formation of
adipose tissue, can lead to pathological conditions associated
directly or indirectly with obesity or anorexia.
[0005] Acrp30 is a 247 amino acid polypeptide that is expressed
exclusively by adipocytes. The Acrp30 polypeptide is composed of a
amino-terminal signal sequence, a 27 amino acid stretch of no known
homology, 22 perfect Gly-Xaa-Pro or imperfect Gly-Xaa-Xaa collagen
repeats and a carboxy terminal globular domain. See, Scherer et al.
as described above and International Patent Application No. WO
96/39429. Acrp30, an abundant human serum protein regulated by
insulin, shares structural similarity, particularly in the
carboxy-terminal globular domain, to complement factor C1q and to a
summer serum protein of hibernating Siberian chipmunks (Hib27).
Expression of Acrp30 is induced over 100-fold during adipocyte
differentiation. Acrp30 is suggested for use in modulating energy
balance and in identifying adipocytes in test samples.
[0006] Another secreted protein that appears to be exclusively
produced in adipocytes is apM1, described, for example, in Maeda et
al., Biochem. Biophys. Res. Comm. 221: 286-9, 1996. A 4517 bp clone
had a 244 amino acid open reading frame and a long 3' untranslated
region. The protein included a signal sequence, an amino-terminal
non-collagenous sequence, 22 collagen repeats (Gly-XAA-Pro or
Gly-Xaa-Xaa), and a carboxy-terminal region with homology to
collagen X, collagen VIII and complement protein C1q.
[0007] Complement factor C1q consists of six copies of three
related polypeptides (A, B and C chains), with each polypeptide
being about 225 amino acids long with a near amino-terminal
collagen domain and a carboxy-terminal globular region. Six triple
helical regions are formed by the collagen domains of the six A,
six B and six C chains, forming a central region and six stalks. A
globular head portion is formed by association of the globular
carboxy terminal domain of an A, a B and a C chain. C1q is
therefore composed of six globular heads linked via six
collagen-like stalks to a central fibril region. Sellar et al.,
Biochem. J. 274: 481-90, 1991. This configuration is often referred
to as a bouquet of flowers. Acrp30 has a similar bouquet structure
formed from a single type of polypeptide chain.
[0008] C1q has been found to stimulate defense mechanisms as well
as trigger the generation of toxic oxygen species that can cause
tissue damage (Tenner, Behring Inst. Mitt. 93:241-53, 1993). C1q
binding sites are found on platelets. Additionally complement and
C1q play a role in inflammation. The complement activation is
initiated by binding of C1q to immunoglobulins
[0009] Inhibitors of C1q and the complement pathway would be useful
for anti-inflammatory applications, inhibition of complement
activation and thrombotic activity.
[0010] The present invention provides such polypeptides for these
and other uses that should be apparent to those skilled in the art
from the teachings herein.
SUMMARY OF THE INVENTION
[0011] Within one aspect, the invention provides an isolated
polypeptide comprising a sequence of amino acid residues that is at
least 75% identical in amino acid sequence to residues 51-246 of
SEQ ID NO:2, wherein the sequence comprises: Gly-Xaa-Xaa or
Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any
amino acid; and a carboxyl-terminal C1q domain comprising 10 beta
strands. Within one embodiment the polypeptide that is at least 90%
identical in amino acid sequence to residues 23-246 of SEQ ID NO:2.
Within another embodiment the collagen domain consists of 15
Gly-Xaa-Xaa repeats and 6 Gly-Xaa-Pro repeats. Within another
embodiment the carboxyl-terminal C1q domain comprises the sequence
of SEQ ID NO:10. Within another embodiment the carboxy-terminal C1q
domain comprises amino acid residues 119-123, 140-142, 148-151,
155-158, 161-173, 175-182, 190-197, 200-212, 217-222 and 236-241 of
SEQ ID NO:2. Within another embodiment any differences between the
polypeptide and SEQ ID NO:2 are due to conservative amino acid
substitutions. Within another embodiment the polypeptide
specifically binds with an antibody that specifically binds with a
polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
Within still another embodiment the polypeptide comprises residues
23-246 of SEQ ID NO:2. Within another embodiment the collagen
domain consists of amino acid residues 51-113 of SEQ ID NO:2.
Within yet another embodiment the C1q domain consists of amino acid
residues 114-246 of SEQ ID NO:2. Within another embodiment the
polypeptide is covalently linked at the amino or carboxyl terminus
to a moiety selected from the group consisting of affinity tags,
toxins, radionucleotides, enzymes and fluorophores.
[0012] Also provided is an isolated polypeptide selected from the
group consisting of: a) a polypeptide consisting of a sequence of
amino acid residues that is 80% identical in amino acid sequence to
amino acid residue 51 to amino acid residue 113 of SEQ ID NO:2, the
polypeptide consisting of Gly-Xaa-Xaa and Gly-Xaa-Pro repeats
forming a collagen domain; b) a polypeptide consisting of a
sequence of amino acid residues that is 80% identical in amino acid
sequence to amino acid residue 114 to amino acid residue 246 of SEQ
ID NO:2 comprising the sequence of SEQ ID NO:5; and c) a
polypeptide consisting of a sequence of amino acid residues that is
80% identical in amino acid sequence to amino acid residue 51 to
246 of SEQ ID NO:2, the polypeptide consisting of Gly-Xaa-Xaa and
Gly-Xaa-Pro repeats forming a collagen domain and comprising the
sequence of SEQ ID NO:5.
[0013] Within another aspect is provided a fusion protein
comprising a first portion and a second portion joined by a peptide
bond, the first portion consisting of a polypeptide selected from
the group consisting of: a) a polypeptide comprising a sequence of
amino acid residues that is at least 75% identical in amino acid
sequence to amino acid residue 51 to amino acid residue 246 of SEQ
ID NO:2; b) a polypeptide comprising a sequence of amino acid
residues as shown in SEQ ID NO:2 from amino acid residue 1 to amino
acid residue 246; c) a portion of the zacrp3 polypeptide of SEQ ID
NO:2, comprising the collagen-like domain or a portion of the
collagen-like domain capable of dimerization or oligomerization; d)
a portion of the zacrp3 polypeptide of SEQ ID NO:2, comprising the
C1q domain or an active portion of the C1q domain; or e) a portion
of the zacrp3 polypeptide of SEQ ID NO:2 comprising of the
collagen-like domain and the C1q domain; and the second portion
comprising another polypeptide. Within one embodiment the first
portion is selected from the group consisting of: a) a polypeptide
consisting of the sequence of amino acid residue 51 to amino acid
residue 113 of SEQ ID NO:2; b) a polypeptide consisting of the
sequence of amino acid residue 114 to amino acid residue 246 of SEQ
ID NO:2; c) a polypeptide consisting of the sequence of amino acid
residue 51 to 246 of SEQ ID NO:2.
[0014] The invention also provides a polypeptide as described
above; in combination with a pharmaceutically acceptable
vehicle.
[0015] Within another aspect, the invention provides an antibody or
antibody fragment that specifically binds to a polypeptide as
described above. Within one embodiment the antibody is selected
from the group consisting of: a) polyclonal antibody; b) murine
monoclonal antibody; c) humanized antibody derived from b); and d)
human monoclonal antibody. Within another embodiment the antibody
fragment is selected from the group consisting of F(ab'), F(ab),
Fab', Fab, Fv, scFv, and minimal recognition unit. Within another
embodiment is provided an anti-idiotype antibody that specifically
binds to the antibody described above.
[0016] Within another aspect, the invention provides an isolated
polynucleotide encoding a polypeptide comprising a sequence of
amino acid residues that is at least 75% identical in amino acid
sequence to residues 51-246 of SEQ ID NO:2, wherein the sequence
comprises: Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen
domain, wherein Xaa is any amino acid; and a carboxyl-terminal C1q
domain consisting of 10 beta strands. Within one embodiment the
polypeptide is at least 90% identical in amino acid sequence to
residues 23-246 of SEQ ID NO:2. Within another embodiment the
collagen domain consists of 15 Gly-Xaa-Xaa repeats and 6
Gly-Xaa-Pro repeats. Within another embodiment the
carboxyl-terminal C1q domain comprises the sequence of SEQ ID NO:5.
Within another embodiment the carboxy-terminal C1q domain consists
of amino acid residues 119-123, 140-142, 148-151, 155-158, 161-173,
175-182, 190-197, 200-212, 217-222 and 236-241 of SEQ ID NO:2.
Within another embodiment any differences between the polypeptide
and SEQ ID NO:2 are due to conservative amino acid substitutions.
Within yet another embodiment the polypeptide specifically binds
with an antibody that specifically binds with a polypeptide
consisting of the amino acid sequence of SEQ ID NO:2. Within
another embodiment the polypeptide comprises residues 23-246 of SEQ
ID NO:2. Within another embodiment the collagen domain consists of
amino acid residues 51-113 of SEQ ID NO:2. Within yet another
embodiment the C1q domain consists of amino acid residues 114-246
of SEQ ID NO:2.
[0017] Also provided is an isolated polynucleotide selected from
the group consisting of: a) a sequence of nucleotides from
nucleotide 1 to nucleotide 1696 of SEQ ID NO:1; b) a sequence of
nucleotides from nucleotide 69 to nucleotide 806 of SEQ ID NO:1; c)
a sequence of nucleotides from nucleotide 135 to nucleotide 806 of
SEQ ID NO:1; d) a sequence of nucleotides from nucleotide 219 to
nucleotide 806 of SEQ ID NO:1; e) a sequence of nucleotides from
nucleotide 408 to nucleotide 806 of SEQ ID NO:1; f) a sequence of
nucleotides from nucleotide 69 to nucleotide 407 of SEQ ID NO:1; g)
a sequence of nucleotides from nucleotide 135 to nucleotide 407 of
SEQ ID NO:1; h) a sequence of nucleotides from nucleotide 219 to
nucleotide 407 of SEQ ID NO:1; i) a polynucleotide encoding a
polypeptide, the polypeptide consisting of a sequence of amino acid
residues that is at least 75% identical to a polypeptide consisting
of the amino acid sequence of residues 51 to 113 of SEQ ID NO:2; j)
a polynucleotide encoding a polypeptide, the polypeptide consisting
of a sequence of amino acid residues that is at least 75% identical
to a polypeptide consisting of the amino acid sequence of residues
114 to 246 of SEQ ID NO:2; k) a polynucleotide encoding a
polypeptide, the polypeptide consisting of a sequence of amino acid
residues that is at least 75% identical to a polypeptide consisting
of the amino acid sequence of residues 51 to 246 of SEQ ID NO:2; 1)
a polynucleotide encoding a polypeptide consisting of a sequence of
amino acid residues that is at least 75% identical to a polypeptide
consisting of the amino acid sequence of residues 23 to 113 of SEQ
ID NO:2; m) a polynucleotide that remains hybridized following
stringent wash conditions to a polynucleotide consisting of the
nucleotide sequence of SEQ ID NO:1, or the complement of SEQ ID
NO:1; n) nucleotide sequences complementary to a), b), c), d), e),
f), g), h), i), j), k), l) or m) and o) degenerate nucleotide
sequences of i), j), k) or l).
[0018] Also provided is an isolated polynucleotide encoding a
fusion protein comprises a first portion and a second portion
joined by a peptide bond, the first portion is selected from the
group consisting of: a) a polypeptide comprising a sequence of
amino acid residues that is at least 75% identical in amino acid
sequence to amino acid residues 51 to 246 of SEQ ID NO:2; b) a
polypeptide comprising the sequence of amino acid residues 1 to 246
of SEQ ID NO:2; c) a polypeptide comprising the sequence of amino
acid residues 23 to 246 of SEQ ID NO:2; d) a polypeptide comprising
the sequence of amino acid residues 23 to 113 of SEQ ID NO:2; e) a
polypeptide comprising the sequence of amino acid residues 1 to 113
of SEQ ID NO:2; f) a portion of a polypeptide of SEQ ID NO:2
comprising the collagen-like domain or a portion of the
collagen-like domain capable of dimerization or oligomerization; g)
a portion of the polypeptide of SEQ ID NO:2 containing the C1q
domain; or h) a portion of the polypeptide of SEQ ID NO:2 including
the collagen-like domain and the C1q domain; and the second portion
comprising another polypeptide.
[0019] Also provided is an isolated polynucleotide consisting of
the sequence of nucleotide 1 to nucleotide 738 of SEQ ID NO:10.
[0020] Within another aspect, the invention provides an expression
vector comprising the following operably linked elements: a
transcription promoter; a DNA segment encoding a polypeptide as
described above; and a transcription terminator. Within one
embodiment the DNA segment encodes a polypeptide that is at least
90% identical in amino acid sequence to residues 23-246 of SEQ ID
NO:2. Within another embodiment the collagen domain consists of 15
Gly-Xaa-Xaa repeats and 6 Gly-Xaa-Pro repeats. Within another
embodiment the carboxyl-terminal C1q domain comprises the sequence
of SEQ ID NO:10. Within another embodiment the carboxy-terminal C1q
domain consists of amino acid residues 119-123, 140-142, 148-151,
155-158, 161-173, 175-182, 190-197, 200-212, 217-222 and 236-241 of
SEQ ID NO:2. Within another embodiment differences between the
polypeptide and SEQ ID NO:2 are due to conservative amino acid
substitutions. Within yet another embodiment the polypeptide
specifically binds with an antibody that specifically binds with a
polypeptide consisting of the amino acid sequence of SEQ ID NO:2.
Within a further embodiment the DNA segment encodes a polypeptide
comprising residues 23-246 of SEQ ID NO:2. Within another
embodiment the collagen domain consists of amino acid residues
51-113 of SEQ ID NO:2. Within yet another embodiment the C1q domain
consists of amino acid residues 114-246 of SEQ ID NO:2. Within yet
another embodiment the DNA segment encodes a polypeptide covalently
linked at the amino or carboxyl terminus to an affinity tag. Within
another embodiment the DNA segment further encodes a secretory
signal sequence operably linked to the polypeptide. Within a
related embodiment the secretory signal sequence comprises residues
1-22 of SEQ ID NO:2.
[0021] Within another aspect, the invention provides a cultured
cell into which has been introduced an expression vector as
described above, wherein the cell expresses the polypeptide encoded
by the DNA segment.
[0022] Within another aspect, the invention provides a method of
producing a polypeptide comprising: culturing a cell into which has
been introduced an expression vector as describe above; whereby the
cell expresses the polypeptide encoded by the DNA segment; and
recovering the expressed polypeptide.
BRIEF DESCRIPTION OF THE DRAWING
[0023] The Figure illustrates a multiple alignment of and zacrp3
polypeptide of the present invention and human ACRP30 (ACR3) (SEQ
ID NO:3, Maeda et al., Biochem. Biophys. Res. Commun. 221:286-9,
1996) and human C1q C (SEQ ID NO:4, Sellar et al., Biochem J.
274:481-90, 1991 and Reid, Biochem J. 179:361-71, 1979). The
multiple alignment performed using a Clustalx multiple alignment
tool with the default settings: Blosum Series Weight Matricies, Gap
Opening penalty:10.0, Gap Extension penalty:0.05. Multiple
alignments were further hand tuned before computing percent
identity.
DETAILED DESCRIPTION OF THE INVENTION
[0024] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms.
[0025] The term "affinity tag" is used herein to denote a peptide
segment that can be attached to a polypeptide to provide for
purification or detection of the polypeptide or provide sites for
attachment of the 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),
substance P, Flag.TM. peptide (Hopp et al., Biotechnology
6:1204-10, 1988; available from Eastman Kodak Co., New Haven,
Conn.), 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.).
[0026] The term "allelic variant" denotes 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.
[0027] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides and proteins. Where
the context allows, these terms are used with reference to a
particular sequence or portion of a polypeptide or protein to
denote proximity or relative position. For example, a certain
sequence positioned carboxyl-terminal to a reference sequence
within a protein is located proximal to the carboxyl terminus of
the reference sequence, but is not necessarily at the carboxyl
terminus of the complete protein.
[0028] 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-complement pair preferably has a binding affinity
of <10.sup.9 M.sup.-1.
[0029] 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'
CCCGTGCAT 3'.
[0030] 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'-ATGGCTTAGCTT-3' are 5'-TAGCTTgagtct-3' and
3'-gtcgacTACCGA-5'.
[0031] 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).
[0032] The term "expression vector" denotes 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 may include promoter
and terminator sequences, and may optionally include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, and the like. Expression
vectors are generally derived from plasmid or viral DNA, or may
contain elements of both.
[0033] 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).
[0034] 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.
[0035] The term "operably linked", when referring to DNA segments,
denotes 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.
[0036] 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.
[0037] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0038] The term "polynucleotide" denotes 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.
[0039] 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".
[0040] "Probes and/or primers" as used herein can be RNA or DNA.
DNA can be either cDNA or genomic DNA. Polynucleotide probes and
primers are single or double-stranded DNA or RNA, generally
synthetic oligonucleotides, but may be generated from cloned cDNA
or genomic sequences or its complements. Analytical probes will
generally be at least 20 nucleotides in length, although somewhat
shorter probes (14-17 nucleotides) can be used. PCR primers are at
least 5 nucleotides in length, preferably 15 or more nt, more
preferably 20-30 nt. Short polynucleotides can be used when a small
region of the gene is targeted for analysis. For gross analysis of
genes, a polynucleotide probe may comprise an entire exon or more.
Probes can be labeled to provide a detectable signal, such as with
an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer,
paramagnetic particle and the like, which are commercially
available from many sources, such as Molecular Probes, Inc.,
Eugene, Oreg., and Amersham Corp., Arlington Heights, Ill., using
techniques that are well known in the art.
[0041] The term "promoter" denotes 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.
[0042] 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 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. Most nuclear receptors also exhibit a
multi-domain structure, including an amino-terminal,
transactivating domain, a DNA binding domain and a ligand binding
domain. 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).
[0043] 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 peptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0044] 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.
[0045] 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.
[0046] 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%.
[0047] All references cited herein are incorporated by reference in
their entirety.
[0048] The present invention is based in part upon the discovery of
a novel DNA sequence that encodes a polypeptide having homology to
an adipocyte complement related protein (Acrp30). The novel DNA
sequence encodes a polypeptide having an amino-terminal signal
sequence, an adjacent N-terminal region of non-homology, a collagen
domain composed of 21 Gly-Xaa-Xaa or Gly-Xaa-Pro repeats and a
carboxy-terminal globular-like C1q domain followed by a long 3'
untranslated region. The general polypeptide structure set forth
above is shared by Acrp30 and C1q C. Other regions of homology,
found in the carboxy-terminal globular C1q domain in the aligned
proteins, are identified herein as useful primers for searching for
other family members. Acrp30 and C1q C, for example, would be
identified in a search using the primers. Intra-chain disulfide
bonding may involve the cysteines at residues 39, 42 and 43 of SEQ
ID NO:2.
[0049] The novel zacrp3 polypeptides of the present invention were
initially identified by querying an EST database for homologs of
ACRP30, characterized by a signal sequence, a collagen-like domain
and a C1q domain. Polypeptides corresponding to ESTs meeting those
search criteria were compared to known sequences to identify
proteins having homology to ACRP30. An assembled EST cluster was
discovered and predicted to be a secreted protein. To identify the
corresponding cDNA, a clone considered likely to contain the entire
coding sequence was used for sequencing. The resulting 1696 bp
sequence is disclosed in SEQ ID NO:1. Comparison of the originally
derived EST sequence with the sequence represented in SEQ ID NO:1
showed that there were two frame shifts and an unspliced intron.
The novel polypeptide encoded by the full length cDNA enabled the
identification of a homolog relationship with adipocyte complement
related protein Acrp30 (SEQ ID NO:3) and complement component C1q C
(SEQ ID NO:4) as is shown in the Figure. Zacrp3 shares 27.5 and
25.7% identity at the amino acid level with human ACRP30 and C1q C
respectfully. C1q C and ACRP30 share 32.4% identity. Within the C1q
domain, zacrp3 shares 26.3 and 26% identity at the amino acid level
when compared to human ACRP30 and C1q C respectfully. C1q C and
ACRP30 share 38.2% identity over this region.
[0050] The full sequence of the zacrp3 polypeptide was obtained
from a single clone believed to contain it, wherein the clone was
obtained from a chest wall soft tissue library. This message was
also found using electronic searches, in libraries of connective
tissues, digestive, skeletal, respiratory and nervous system
tissues as well as urinary tract tissues.
[0051] The nucleotide sequence of zacrp3 is described in SEQ ID
NO:1, and its deduced amino acid sequence is described in SEQ ID
NO:2. As described generally above, the zacrp3 polypeptide includes
a signal sequence, ranging from amino acid 1 (Met) to amino acid
residue 22 (Cys). The mature polypeptide therefore ranges from
amino acid 23 (Gln) to amino acid 246 (Lys). Within the mature
polypeptide, an N-terminal region of no known homology is found,
ranging between amino acid residue 23 (Gln) and 50 (Arg) of SEQ ID
NO:2. In addition, a collagen-like domain is found between amino
acid 51 (Gly) and 113 (Pro). In the collagen-like domain, 6 perfect
Gly-Xaa-Pro and 15 imperfect Gly-Xaa-Xaa repeats are observed.
Acrp30 contains 22 perfect or imperfect repeats. Proline residues
found in this domain at amino acid residue 56, 59, 62 and 113 of
SEQ ID NO:2 may be hydroxylated. The zacrp3 polypeptide also
includes a carboxy-terminal C1q domain, ranging from about amino
acid 114 (Pro) to 246 (Lys). There is a fair amount of conserved
structure within the C1q domain to enable proper folding. An
aromatic motif seen in all C1q domain containing proteins
(F-X(5)-[ND]-X(4)-[FYWL]-X(6)-F-X(5)-G-X-Y-X-F-X-[FY] (SEQ ID NO:5)
is found between residues 169 and 199 of SEQ ID NO:2. X represents
any amino acid residue and the number in parentheses ( ) indicates
the amino acid number of residues. The amino acid residues
contained within the square parentheses [ ] restrict the choice of
amino acid residues at that particular position. Zacrp3
polypeptide, human C1q C and Acrp30 appear to be homologous within
the collagen domain and in the C1q domain, but not in the
N-terminal portion of the mature polypeptide.
[0052] Another aspect of the present invention includes zacrp3
polypeptide fragments. Preferred fragments include those containing
the collagen-like domain of zacrp3 polypeptides, ranging from amino
acid 1 (Met), 23 (Gln) or 51 (Gly) to amino acid 1113 (Pro) of SEQ
ID NO:2, a portion of the zacrp3 polypeptide containing the
collagen-like domain or a portion of the collagen-like domain
capable of dimerization or oligomerization. As used herein the term
"collagen" or "collagen-like domain" refers to a series of
repeating triplet amino acid sequences, "repeats" or "collagen
repeats" represented by the motifs Gly-Xaa-Pro or Gly-Xaa-Xaa,
where Xaa is any amino acid reside. Such domains may contain as
many as 21 collagen repeats or more. Fragments or proteins
containing such collagen-like domains may form homomeric constructs
(dimers or oligomers of the same fragment or protein). Moreover,
such fragments or proteins containing such collagen-like domains
may form heteromeric constructs, usually trimers.
[0053] These fragments are particularly useful in the study of
collagen dimerization or oligomerization or in formation of fusion
proteins as described more fully below. Polynucleotides encoding
such fragments are also encompassed by the present invention,
including the group consisting of (a) polynucleotide molecule
comprising a sequence of nucleotides as shown in SEQ ID NO:1 from
nucleotide 1, 69, 135 or 219 to nucleotide 407; (b) polynucleotide
molecules that encode a zacrp3 polypeptide fragment that is at
least 80% identical to the amino acid sequence of SEQ ID NO:2 from
amino acid residue 51 (Gly) to amino acid residue 113 (Pro); (c)
molecules complementary to (a) or (b); and (d) degenerate
nucleotide sequences encoding a zacrp3 polypeptide collagen-like
domain fragment.
[0054] Other preferred fragments include the globular C1q domain of
zacrp3 polypeptides, ranging from amino acid 114 (Pro) to 246 (Lys)
of SEQ ID NO:2, a portion of the zacrp3 polypeptide containing the
C1q domain or an active portion of the C1q domain. Other C1q domain
containing proteins include C1q A, B and C (Sellar et al., ibid.,
Reid, ibid., and Reid et al., Biochem. J. 203: 559-69, 1982),
chipmunk hibernation-associated plasma proteins HP-20, HP-25 and
HP-27 (Takamatsu et al., Mol. Cell. Biol. 13: 1516-21, 1993 and
Kondo & Kondo, J. Biol. Chem. 267: 473-8, 1992), human
precerebellin (Urade et al., Proc. Natl. Acad. Sci. USA 88:1069-73,
1991), human endothelial cell multimerin (Hayward et al., J. Biol.
Chem. 270:18246-51, 1995) and vertebrate collagens type VIII and X
(Muragaki et al., Eur. J. Biochem. 197:615-22, 1991). The globular
C1q domain of ACRP30 has been determined to have a 10 beta strand
"jelly roll" topology (Shapiro and Scherer, Curr. Biol. 8:335-8,
1998) that shows significant homology to the TNF family and the
zacrp3 sequence as represented by SEQ ID NO:2 contains all 10
beta-strands of this structure (amino acid residues 119-123,
140-142, 148-151, 155-158, 161-173, 175-182, 190-197, 200-212,
217-222 and 236-241 of SEQ ID NO:2). These strands have been
designated "A", "A'", "B", "B'", "C", "D", "E", "F", "G" and "H"
respectively.
[0055] Zacrp3 has two receptor binding loops, at amino acid
residues 111-139 and 170-182, does zacrp3 have anything similar.
The core receptor binding region is predicted to include amino acid
residues 124-150 and 181-197 of SEQ ID NO:2. Amino acid residues
161 (Gly), 163 (Tyr), 212 (Leu) and 237 (Phe) appear to be
conserved across the superfamily including CD40, TNF.alpha., ACRP30
and zacrp3.
[0056] These fragments are particularly useful in the study or
modulation of energy balance or neurotransmission, particularly
diet- or stress-related neurotransmission. Anti-microbial activity
may also be present in such fragments. The homology to TNF proteins
suggests such fragments would be useful in obesity-related insulin
resistance, immune regulation, inflammatory response, apoptosis and
osteoclast maturation. Polynucleotides encoding such fragments are
also encompassed by the present invention, including the group
consisting of (a) polynucleotide molecules comprising a sequence of
nucleotides as shown in SEQ ID NO:1 from nucleotide 408 to
nucleotide 806; (b) polynucleotide molecules that encode a zacrp3
polypeptide fragment that is at least 80% identical to the amino
acid sequence of SEQ ID NO:2 from amino acid residue 114 (Pro) to
amino acid residue 246 (Lys); (c) molecules complementary to (a) or
(b); and (d) degenerate nucleotide sequences encoding a zacrp3
polypeptide C1q domain fragment.
[0057] Other zacrp3 polypeptide fragments of the present invention
include both the collagen-like domain and the C1q domain ranging
from amino acid residue 51 (Gly) to 806 (Lys) of SEQ ID NO:2.
Polynucleotides encoding such fragments are also encompassed by the
present invention, including the group consisting of (a)
polynucleotide molecules comprising a sequence of nucleotides as
shown in SEQ ID NO:1 from nucleotide 291 to nucleotide 806; (b)
polynucleotide molecules that encode a zacrp3 polypeptide fragment
that is at least 80% identical to the amino acid sequence of SEQ ID
NO:2 from amino acid residue 41 (Gly) to amino acid residue 246
(Lys); (c) molecules complementary to (a) or (b); and (d)
degenerate nucleotide sequences encoding a zacrp3 polypeptide
collagen-like domain-C1q domain fragment.
[0058] The highly conserved amino acids, particularly those in the
carboxy-terminal C1q domain of the zacrp3 polypeptide, 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 motifs from RNA obtained
from a variety of tissue sources. In particular, highly degenerate
primers and their complements designed from conserved sequences are
useful for this purpose. In particular, the following primers are
useful for this purpose:
[0059] Amino acid residues 215-221 of SEQ ID NO:2 TABLE-US-00001
GGN GAN SAR GTN TGG YT (SEQ ID NO:6)
[0060] Amino acid residues 160-165 of SEQ ID NO:2 TABLE-US-00002 SN
GNN NTN TAY TWY TTY R (SEQ ID NO:7)
[0061] Amino acid residues 237-242 of SEQ ID NO:2 TABLE-US-00003
TTY DSN GGN TTY YTN HT (SEQ ID NO:8)
[0062] Amino acid residues 147-153 of SEQ ID NO:2 TABLE-US-00004 Y
TWY RAY RBN WBN WSN GG (SEQ ID NO:9)
Probes corresponding to complements of the polynucleotides set
forth above are also encompassed.
[0063] The present invention also provides polynucleotide
molecules, including DNA and RNA molecules, that encode the zacrp3
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:10 is a degenerate DNA sequence
that encompasses all DNAs that encode the zacrp3 polypeptide of SEQ
ID NO:2. Those skilled in the art will recognize that the
degenerate sequence of SEQ ID NO:10 also provides all RNA sequences
encoding SEQ ID NO:2 by substituting U for T. Thus, zacrp3
polypeptide-encoding polynucleotides comprising nucleotide 1 to
nucleotide 738 of SEQ ID NO:10 and their RNA equivalents are
contemplated by the present invention. Table 1 sets forth the
one-letter codes used within SEQ ID NO:10 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. TABLE-US-00005 TABLE 1
Nucleotide Resolution Complement Resolution A A T T C C G G G G C C
T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G
W A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T
H A|C|T N A|C|G|T N A|C|G|T
[0064] The degenerate codons used in SEQ ID NO:10, encompassing all
possible codons for a given amino acid, are set forth in Table 2.
TABLE-US-00006 TABLE 2 One Amino Letter Degenerate 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 .cndot. TAA TAG
TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN
[0065] 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.
[0066] 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:10 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.
[0067] The present invention further provides variant polypeptides
and nucleic acid molecules that represent counterparts 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
zacrp3 polypeptides from other mammalian species, including murine,
porcine, ovine, bovine, canine, feline, equine, and other primate
polypeptides. A murine zacrp2 homolog (SEQ ID NO:12) has been
identified. The polynucleotide sequence encoding this murine zacrp2
polypeptide disclosed in SEQ ID NO:11. Orthologs of human zacrp3
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 zacrp3 as disclosed
herein. 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.
[0068] An zacrp3-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 with primers designed from the representative human
zacrp3 sequences 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 zacrp3 polypeptide. Similar techniques can also be applied to
the isolation of genomic clones.
[0069] Those skilled in the art will recognize that the sequence
disclosed in SEQ ID NO:1 represents a single allele of human
zacrp3, 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
nucleotide 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. cDNA molecules generated from alternatively spliced mRNAs,
which retain the properties of the zacrp3 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.
[0070] Within preferred embodiments of the invention, the isolated
nucleic acid molecules can hybridize under stringent conditions to
nucleic acid molecules having the nucleotide sequence of SEQ ID
NO:1 or to nucleic acid molecules having a nucleotide sequence
complementary to SEQ ID NO:1. 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.
[0071] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA
and DNA-RNA, can hybridize if the nucleotide sequences have some
degree of complementarity. Hybrids can tolerate mismatched base
pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases. Stringent hybridization conditions encompass
temperatures of about 5-25.degree. C. below the T.sub.m of the
hybrid and a hybridization buffer having up to 1 M Na.sup.+. Higher
degrees of stringency at lower temperatures can be achieved with
the addition of formamide which reduces the T.sub.m of the hybrid
about 1.degree. C. for each 1% formamide in the buffer solution.
Generally, such stringent conditions include temperatures of
20-70.degree. C. and a hybridization buffer containing up to
6.times.SSC and 0-50% formamide. A higher degree of stringency can
be achieved at temperatures of from 40-70.degree. C. with a
hybridization buffer having up to 4.times.SSC and from 0-50%
formamide. Highly stringent conditions typically encompass
temperatures of 42-70.degree. C. with a hybridization buffer having
up to 1.times.SSC and O-50% formamide. Different degrees of
stringency can be used during hybridization and washing to achieve
maximum specific binding to the target sequence. Typically, the
washes following hybridization are performed at increasing degrees
of stringency to remove non-hybridized polynucleotide probes from
hybridized complexes.
[0072] The above conditions are meant to serve as a guide and it is
well within the abilities of one skilled in the art to adapt these
conditions for use with a particular polypeptide hybrid. The
T.sub.m for a specific target sequence is the temperature (under
defined conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those conditions
which influence the T.sub.m include, the size and base pair content
of the polynucleotide probe, the ionic strength of the
hybridization solution, and the presence of destabilizing agents in
the hybridization solution. Numerous equations for calculating
T.sub.m are known in the art, and are specific for DNA, RNA and
DNA-RNA hybrids and polynucleotide probe sequences of varying
length (see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989);
Ausubel et al., (eds.), Current Protocols in Molecular Biology
(John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide
to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and
Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence
analysis software, such as OLIGO 6.0 (LSR; Long Lake, Minn.) and
Primer Premier 4.0 (Premier Biosoft International; Palo Alto,
Calif.), as well as sites on the Internet, are available tools for
analyzing a given sequence and calculating T.sub.m based on user
defined criteria. Such programs can also analyze a given sequence
under defined conditions and identify suitable probe sequences.
Typically, hybridization of longer polynucleotide sequences, >50
base pairs, is performed at temperatures of about 20-25.degree. C.
below the calculated T.sub.m. For smaller probes, <50 base
pairs, hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below. This allows for the maximum rate of
hybridization for DNA-DNA and DNA-RNA hybrids.
[0073] The length of the polynucleotide sequence influences the
rate and stability of hybrid formation. Smaller probe sequences,
<50 base pairs, reach equilibrium with complementary sequences
rapidly, but may form less stable hybrids. Incubation times of
anywhere from minutes to hours can be used to achieve hybrid
formation. Longer probe sequences come to equilibrium more slowly,
but form more stable complexes even at lower temperatures.
Incubations are allowed to proceed overnight or longer. Generally,
incubations are carried out for a period equal to three times the
calculated Cot time. Cot time, the time it takes for the
polynucleotide sequences to reassociate, can be calculated for a
particular sequence by methods known in the art.
[0074] The base pair composition of polynucleotide sequence will
effect the thermal stability of the hybrid complex, thereby
influencing the choice of hybridization temperature and the ionic
strength of the hybridization buffer. A-T pairs are less stable
than G-C pairs in aqueous solutions containing sodium chloride.
Therefore, the higher the G-C content, the more stable the hybrid.
Even distribution of G and C residues within the sequence also
contribute positively to hybrid stability. In addition, the base
pair composition can be manipulated to alter the T.sub.m of a given
sequence. For example, 5-methyldeoxycytidine can be substituted for
deoxycytidine and 5-bromodeoxuridine can be substituted for
thymidine to increase the T.sub.m, whereas
7-deazz-2'-deoxyguanosine can be substituted for guanosine to
reduce dependence on T.sub.m.
[0075] The ionic concentration of the hybridization buffer also
affects the stability of the hybrid. Hybridization buffers
generally contain blocking agents such as Denhardt's solution
(Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA,
tRNA, milk powders (BLOTTO), heparin or SDS, and a Na.sup.+ source,
such as SSC (1.times.SSC: 0.15 M sodium chloride, 15 mM sodium
citrate) or SSPE (1.times.SSPE: 1.8 M NaCl, 10 mM
NaH.sub.2PO.sub.4, 1 mM EDTA, pH 7.7). By decreasing the ionic
concentration of the buffer, the stability of the hybrid is
increased. Typically, hybridization buffers contain from between 10
mM-1 M Na.sup.+. The addition of destabilizing or denaturing agents
such as formamide, tetralkylammonium salts, guanidinium cations or
thiocyanate cations to the hybridization solution will alter the
T.sub.m of a hybrid. Typically, formamide is used at a
concentration of up to 50% to allow incubations to be carried out
at more convenient and lower temperatures. Formamide also acts to
reduce non-specific background when using RNA probes.
[0076] As an illustration, a nucleic acid molecule encoding a
variant zacrp3 polypeptide can be hybridized with a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1 (or its
complement) at 42.degree. C. overnight in a solution comprising 50%
formamide, 5.times.SSC (1.times.SSC: 0.15 M sodium chloride and 15
mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution (100.times. Denhardt's solution: 2% (w/v)
Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine
serum albumin), 10% dextran sulfate, and 20 .mu.g/ml denatured,
sheared salmon sperm DNA. One of skill in the art can devise
variations of these hybridization conditions. For example, the
hybridization mixture can be incubated at a higher or lower
temperature, such as about 65.degree. C., in a solution that does
not contain formamide. Moreover, premixed hybridization solutions
are available (e.g., EXPRESSHYB. Hybridization Solution from
CLONTECH Laboratories, Inc.), and hybridization can be performed
according to the manufacturer's instructions.
[0077] Following hybridization, the nucleic acid molecules can be
washed to remove non-hybridized nucleic acid molecules under
stringent conditions, or under highly stringent conditions. Typical
stringent washing conditions include washing in a solution of
0.5.times.-2.times.SSC with 0.1% sodium dodecyl sulfate (SDS) at
55-65.degree. C. That is, nucleic acid molecules encoding a variant
zacrp3 polypeptide hybridize with a nucleic acid molecule having
the nucleotide sequence of SEQ ID NO:1 (or its complement) under
stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 50-65.degree.
C., including 0.5.times.SSC with 0.1% SDS at 55.degree. C., or
2.times.SSC with 0.1% SDS at 65.degree. C. One of skill in the art
can readily devise equivalent conditions, for example, by
substituting SSPE for SSC in the wash solution.
[0078] Typical highly stringent washing conditions include washing
in a solution of 0.1.times.-0.2.times.SSC with 0.1% sodium dodecyl
sulfate (SDS) at 50-65.degree. C. In other words, nucleic acid
molecules encoding a variant zacrp3 polypeptide hybridize with a
nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1
(or its complement) under highly stringent washing conditions, in
which the wash stringency is equivalent to 0.1.times.-0.2.times.SSC
with 0.1% SDS at 50-65.degree. C., including 0.1.times.SSC with
0.1% SDS at 50.degree. C., or 0.2.times.SSC with 0.1% SDS at
65.degree. C.
[0079] The present invention also provides isolated zacrp3
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO:2, or their orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides having at least 70%, at least 80%, at least 90%, at
least 95% or greater than 95% sequence identity to the sequences
shown in SEQ ID NO:2, or their orthologs. The present invention
also includes polypeptides that comprise an amino acid sequence
having at least 70%, at least 80%, at least 90%, at least 95% or
greater than 95% sequence identity to the sequence of amino acid
residues 51 to 246 of SEQ ID NO:2. The present invention further
includes nucleic acid molecules that encode such polypeptides.
Methods for determining percent identity are described below.
[0080] The present invention also contemplates zacrp3 variant
nucleic acid molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence of SEQ ID NO:2, and a hybridization
assay, as described above. Such zacrp3 variants include nucleic
acid molecules (1) that hybridize with a nucleic acid molecule
having the nucleotide sequence of SEQ ID NO:1 (or its complement)
under stringent washing conditions, in which the wash stringency is
equivalent to 0.5.times.-2.times.SSC with 0.1% SDS at 50-65.degree.
C., and (2) that encode a polypeptide having at least 70%, at least
80%, at least 90%, at least 95% or greater than 95% sequence
identity to the amino acid sequence of SEQ ID NO: 2. Alternatively,
zacrp3 variants can be characterized as nucleic acid molecules (1)
that hybridize with a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:1 (or its complement) under highly stringent
washing conditions, in which the wash stringency is equivalent to
0.1.times.-0.2.times.SSC with 0.1% SDS at 50-65.degree. C., and (2)
that encode a polypeptide having at least 70%, at least 80%, at
least 90%, at least 95% or greater than 95% sequence identity to
the amino acid sequence of SEQ ID NO:2.
[0081] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio.
48:603, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915, 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 "BLOSUM62" 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: ([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])(100).
TABLE-US-00007 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
[0082] 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 zacrp3. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat. Acad. Sci.
USA 85:2444, 1988, and by Pearson, Meth. Enzymol. 183:63, 1990.
[0083] 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 re-scored 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. Illustrative
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.
[0084] 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 four to six.
[0085] The present invention includes nucleic acid molecules that
encode a polypeptide having one or more "conservative amino acid
substitutions," compared with the amino acid sequence of SEQ ID
NO:2. Conservative amino acid substitutions can be based upon the
chemical properties of the amino acids. That is, variants can be
obtained that contain one or more amino acid substitutions of SEQ
ID NO:2, in which an alkyl amino acid is substituted for an alkyl
amino acid in a zacrp3 amino acid sequence, an aromatic amino acid
is substituted for an aromatic amino acid in a zacrp3 amino acid
sequence, a sulfur-containing amino acid is substituted for a
sulfur-containing amino acid in a zacrp3 amino acid sequence, a
hydroxy-containing amino acid is substituted for a
hydroxy-containing amino acid in a zacrp3 amino acid sequence, an
acidic amino acid is substituted for an acidic amino acid in a
zacrp3 amino acid sequence, a basic amino acid is substituted for a
basic amino acid in a zacrp3 amino acid sequence, or a dibasic
monocarboxylic amino acid is substituted for a dibasic
monocarboxylic amino acid in a zacrp3 amino acid sequence.
[0086] Among the common amino acids, for example, a "conservative
amino acid substitution" is illustrated by a substitution among
amino acids within each of the following groups: (1) glycine,
alanine, valine, leucine, and isoleucine, (2) phenylalanine,
tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate
and glutamate, (5) glutamine and asparagine, and (6) lysine,
arginine and histidine.
[0087] 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.
Natl. 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. Although it is possible to
design amino acid substitutions based solely upon chemical
properties (as discussed above), the language "conservative amino
acid substitution" preferably 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. According to this system,
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).
[0088] Conservative amino acid changes in a zacrp3 gene can be
introduced by substituting nucleotides for the nucleotides recited
in SEQ ID NO:1. Such "conservative amino acid" variants can be
obtained, for example, by oligonucleotide-directed mutagenesis,
linker-scanning mutagenesis, mutagenesis using the polymerase chain
reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22;
and McPherson (ed.), Directed Mutagenesis: A Practical Approach
(IRL Press 1991)). The ability of such variants to promote the
energy balance modulating or other properties of the wild-type
protein can be determined using a standard methods, such as the
assays described herein. Alternatively, a variant zacrp3
polypeptide can be identified by the ability to specifically bind
anti-zacrp3 antibodies.
[0089] 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-methyl-glycine, 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 typically carried out in a
cell-free system comprising an E. coli 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 Enzmmol. 202:301, 1991,
Chung et al., Science 259:806, 1993, and Chung et al., Proc. Nat.
Acad. Sci. USA 90:10145, 1993.
[0090] 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, 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, 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, 1993).
[0091] 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 zacrp3 amino acid residues.
[0092] 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, 1988) or
Bowie and Sauer (Proc. Nat. Acad. Sci. USA 86:2152, 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, 1991, Ladner et al., U.S. Pat. No.
5,223,409, Huse, international publication No. WO 92/06204, and
region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986,
and Ner et al., DNA 7:127, 1988).
[0093] Variants of the disclosed zacrp3 nucleotide and polypeptide
sequences can also be generated through DNA shuffling as disclosed
by Stemmer, Nature 370:389, 1994, Stemmer, Proc. Nat. Acad. Sci.
USA 91:10747, 1994, and international publication No. WO 97/20078.
Briefly, variant DNA molecules 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 DNA molecules, such as allelic variants or DNA molecules
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.
[0094] 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 biologically active polypeptides, or
polypeptides that bind with anti-zacrp3 antibodies, 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.
[0095] 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, 1989, Bass et
al., Proc. Nat. Acad. Sci. USA 88:4498, 1991, Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). 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, 1996. The identities of essential amino acids can
also be inferred from analysis of homologies with zacrp3.
[0096] The location of zacrp3 receptor binding domains can be
identified 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, 1992, Smith et al., J. Mol. Biol. 224:899,
1992, and Wlodaver et al., FEBS Lett. 309:59, 1992. Moreover,
zacrp3 labeled with biotin or FITC can be used for expression
cloning of zacrp3 receptors.
[0097] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a zacrp3
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).
[0098] 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.
[0099] 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 zacrp3 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).
[0100] Regardless of the particular nucleotide sequence of a
variant zacrp3 gene, the gene encodes a polypeptide that is
characterized by its energy balance modulating activity or other
activities of the wild-type protein, or by the ability to bind
specifically to an anti-zacrp3 antibody. More specifically, variant
zacrp3 genes encode polypeptides which exhibit at least 50%, and
preferably, greater than 70, 80, or 90%, of the activity of
polypeptide encoded by the human zacrp3 gene described herein.
[0101] For any zacrp3 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. Moreover,
those of skill in the art can use standard software to devise
zacrp3 variants based upon the nucleotide and amino acid sequences
described herein. Accordingly, the present invention includes a
computer-readable medium encoded with a data structure that
provides at least one of the following sequences: SEQ ID NO:1, SEQ
ID NO:2, and SEQ ID NO:10. Suitable forms of computer-readable
media include magnetic media and optically-readable media. Examples
of magnetic media include a hard or fixed drive, a random access
memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk
cache, and a ZIP disk. Optically readable media are exemplified by
compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW),
and CD-recordable), and digital versatile/video discs (DVD) (e.g.,
DVD-ROM, DVD-RAM, and DVD+RW).
[0102] The present invention also provides zacrp3 fusion proteins.
For example, fusion proteins of the present invention encompass (1)
a polypeptide selected from the group consisting of: (a)
polypeptide molecules comprising a sequence of amino acid residues
as shown in SEQ ID NO:2 from amino acid residue 1 (Met), 23 (Gln)
or 51 (Gly) to amino acid residue 246 (Lys); (b) polypeptide
molecules ranging from amino acid 51 (Gly) to amino acid 113 (Pro)
of SEQ ID NO:2, a portion of the zacrp3 polypeptide containing the
collagen-like domain or a portion of the collagen-like domain
capable of dimerization or oligomerization; (c) polypeptide
molecules ranging from amino acid 114 (Pro) to 246 (Lys) of SEQ ID
NO:2, a portion of the zacrp3 polypeptide containing the C1q domain
or an active portion of the C1q domain; or (d) polypeptide
molecules ranging from amino acid 51 (Gly) to 246 (Lys), a portion
of the zacrp3 polypeptide including the collagen-like domain and
the C1q domain; and (2) another polypeptide. The other polypeptide
may be alternative or additional C1q domain, an alternative or
additional collagen-like domain, a signal peptide to facilitate
secretion of the fusion protein or the like. The globular domain of
complement binds IgG, thus, the globular domain of zacrp3
polypeptide, fragment or fusion may have a similar role.
[0103] Zacrp3 polypeptides, ranging from amino acid 1 (Met) to
amino acid 246 (Lys); the mature zacrp3 polypeptides, ranging from
amino acid 23 (Gln) to amino acid 246 (Lys); or the secretion
leader fragments thereof, which fragments range from amino acid 1
(Met) to amino acid 22 (Cys) may be used in the study of secretion
of proteins from cells. In preferred embodiments of this aspect of
the present invention, the mature polypeptides are formed as fusion
proteins with putative secretory signal sequences; plasmids bearing
regulatory regions capable of directing the expression of the
fusion protein is introduced into test cells; and secretion of
mature protein is monitored. The monitoring may be done by
techniques known in the art, such as HPLC and the like.
[0104] The polypeptides of the present invention, including
full-length proteins, fragments thereof and fusion proteins, 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., ibid., and Ausubel
et al. ibid.
[0105] In general, a DNA sequence encoding a zacrp3 polypeptide of
the present invention 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.
[0106] To direct a zacrp3 polypeptide into the secretory pathway of
a host cell, a secretory signal sequence (also known as a leader
sequence, signal sequence, prepro sequence or pre-sequence) is
provided in the expression vector. The secretory signal sequence
may be that of the zacrp3 polypeptide, or may be derived from
another secreted protein (e.g., t-PA) or synthesized de novo. The
secretory signal sequence is joined to the zacrp3 polypeptide DNA
sequence in the correct reading frame. Secretory signal sequences
are commonly positioned 5' to the DNA sequence encoding the
polypeptide of interest, although certain 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). Conversely, the signal sequence portion of the
zacrp3 polypeptide (amino acid residues 1-22 of SEQ ID NO:2) may be
employed to direct the secretion of an alternative protein by
analogous methods.
[0107] The secretory signal sequence contained in the polypeptides
of the present invention can be used to direct other polypeptides
into the secretory pathway. The present invention provides for such
fusion polypeptides. A signal fusion polypeptide can be made
wherein a secretory signal sequence derived from amino acid
residues 1-22 of SEQ ID NO:2 is operably linked to another
polypeptide using methods known in the art and disclosed herein.
The secretory signal sequence contained in the fusion polypeptides
of the present invention is preferably fused amino-terminally to an
additional peptide to direct the additional peptide into the
secretory pathway. Such constructs have numerous applications known
in the art. For example, these novel secretory signal sequence
fusion constructs can direct the secretion of an active component
of a normally non-secreted protein, such as a receptor. Such
fusions may be used in vivo or in vitro to direct peptides through
the secretory pathway.
[0108] 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. Pat. 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-KL1; 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, Manassas, Va. 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.
[0109] 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 may 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, placental alkaline phosphatase may be used to
sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0110] 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 and Possee, The Baculovirus Expression System: A Laboratory
Guide, London, Chapman & Hall; O'Reilly 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 zacrp3
baculovirus utilizes a transposon-based system described by Luckow
(Luckow 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 zacrp3 polypeptide into a
baculovirus genome maintained in E. coli as a large plasmid called
a "bacmid." The pFastBac1.TM. transfer vector utilizes the AcNPV
polyhedrin promoter to drive the expression of the gene of
interest, in this case zacrp3. However, pFastBac1.TM. can be
modified to a considerable degree. The polyhedrin promoter can be
removed and substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is expressed
earlier in the baculovirus infection, and has been shown to be
advantageous for expressing secreted proteins. See, Hill-Perkins
and Possee, J. Gen. Virol. 71:971-6, 1990; Bonning et al., J. Gen.
Virol. 75:1551-6, 1994; and, Chazenbalk, and Rapoport, J. Biol.
Chem. 270:1543-9, 1995. In such transfer vector constructs, a short
or long version of the basic protein promoter can be used.
Moreover, transfer vectors can be constructed which replace the
native zacrp3 secretory signal sequences with secretory signal
sequences derived from insect proteins. For example, a secretory
signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey
bee Melittin (Invitrogen, Carlsbad, Calif.), or baculovirus gp67
(PharMingen, San Diego, Calif.) can be used in constructs to
replace the native zacrp3 secretory signal sequence. 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 zacrp3
polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et
al., Proc. Natl. Acad. Sci. 82:7952-4, 1985). Using a technique
known in the art, a transfer vector containing zacrp3 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
zacrp3 is subsequently produced. Recombinant viral stocks are made
by methods commonly used the art.
[0111] 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 ESF921.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
and Possee, ibid.; O'Reilly et al., ibid.; Richardson, ibid.).
Subsequent purification of the zacrp3 polypeptide from the
supernatant can be achieved using methods described herein.
[0112] 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.
[0113] 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.
[0114] 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 zacrp3 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.
[0115] 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.
[0116] Expressed recombinant zacrp3 polypeptides (or chimeric
zacrp3 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.
[0117] The polypeptides of the present invention can be isolated by
exploitation of their structural or binding properties. For
example, immobilized metal ion adsorption (IMAC) chromatography can
be used to purify histidine-rich proteins or proteins having a His
tag. 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", Deutscher,
(ed.), Acad. Press, San Diego, 1990, pp. 529-39). Within an
additional preferred embodiments of the invention, a fusion of the
polypeptide of interest and an affinity tag (e.g., maltose-binding
protein, FLAG, Glu-Glu, an immunoglobulin domain) may be
constructed to facilitate purification as is discussed in greater
detail in the Example sections below.
[0118] Protein refolding (and optionally, reoxidation) procedures
may be advantageously used. It is preferred to purify the protein
to >80% purity, more preferably to >90% purity, even more
preferably >95%, 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 protein is substantially free of
other proteins, particularly other proteins of animal origin.
[0119] Zacrp3 polypeptides or fragments thereof may also be
prepared through chemical synthesis by methods well known in the
art. Such zacrp3 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.
[0120] A ligand-binding polypeptide, such as a zacrp3-binding
polypeptide, can also be used for purification of ligand. 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 ligand are passed through the column one or more times
to allow ligand to bind to the ligand-binding polypeptide. The
ligand is then eluted using changes in salt concentration,
chaotropic agents (guanidine HCl), or pH to disrupt ligand-receptor
binding.
[0121] An assay system that uses a ligand-binding receptor (or an
antibody, one member of a complement/anti-complement pair) or a
binding fragment thereof, and a commercially available biosensor
instrument (BIAcore.TM., 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
complement/anti-complement 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.
[0122] Ligand-binding polypeptides 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).
[0123] The invention also provides anti-zacrp3 antibodies.
Antibodies to zacrp3 can be obtained, for example, using as an
antigen the product of a zacrp3 expression vector, or zacrp3
isolated from a natural source. Particularly useful anti-zacrp3
antibodies "bind specifically" with zacrp3. Antibodies are
considered to be specifically binding if the antibodies bind to a
zacrp3 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, Ann. NY Acad. Sci. 51:660, 1949). Suitable antibodies
include antibodies that bind with zacrp3 in particular domains.
[0124] Anti-zacrp3 antibodies can be produced using antigenic
zacrp3 epitope-bearing peptides and polypeptides. Antigenic
epitope-bearing peptides and polypeptides of the present invention
contain a sequence of at least nine, preferably between 15 to about
30 amino acids contained within SEQ ID NO:2. However, peptides or
polypeptides comprising a larger portion of an amino acid sequence
of the invention, containing from 30 to 50 amino acids, or any
length up to and including the entire amino acid sequence of a
polypeptide of the invention, also are useful for inducing
antibodies that bind with zacrp3. It is desirable that the amino
acid sequence of the epitope-bearing peptide is selected to provide
substantial solubility in aqueous solvents (i.e., the sequence
includes relatively hydrophilic residues, while hydrophobic
residues are preferably avoided). Hydrophilic peptides can be
predicted by one of skill in the art from a hydrophobicity plot,
see for example, Hopp and Woods (Proc. Nat. Acad. Sci. USA
78:3824-8, 1981) and Kyte and Doolittle (J. Mol. Biol. 157:
105-142, 1982). Moreover, amino acid sequences containing proline
residues may be also be desirable for antibody production.
[0125] Polyclonal antibodies to recombinant zacrp3 protein or to
zacrp3 isolated from natural sources can be prepared using methods
well-known to those of skill in the art. See, for example, Green et
al., "Production of Polyclonal Antisera," in Immunochemical
Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and
Williams et al., "Expression of foreign proteins in E. coli using
plasmid vectors and purification of specific polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995). The
immunogenicity of a zacrp3 polypeptide can 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
zacrp3 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.
[0126] Although polyclonal antibodies are typically raised in
animals such as horses, cows, dogs, chicken, rats, mice, rabbits,
hamsters, guinea pigs, goats, or sheep, an anti-zacrp3 antibody of
the present invention may also be derived from a subhuman primate
antibody. General techniques for raising diagnostically and
therapeutically useful antibodies in baboons may be found, for
example, in Goldenberg et al., international patent publication No.
WO 91/11465, and in Losman et al., Int. J. Cancer 46:310, 1990.
Antibodies can also be raised in transgenic animals such as
transgenic sheep, cows, goats or pigs, and can also be expressed in
yeast and fungi in modified forms as will as in mammalian and
insect cells.
[0127] Alternatively, monoclonal anti-zacrp3 antibodies can be
generated. Rodent monoclonal antibodies to specific antigens may be
obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991), Picksley et al., "Production of
monoclonal antibodies against proteins expressed in E. coli," in
DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.
(eds.), page 93 (Oxford University Press 1995)).
[0128] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a zacrp3 gene product, verifying
the presence of antibody production by removing a serum sample,
removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0129] In addition, an anti-zacrp3 antibody of the present
invention may be derived from a human monoclonal antibody. Human
monoclonal antibodies are obtained from transgenic mice that have
been engineered to produce specific human antibodies in response to
antigenic challenge. In this technique, elements of the human heavy
and light chain locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy chain and light chain loci. The transgenic
mice can synthesize human antibodies specific for human antigens,
and the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described, for example, by Green et al., Nature Genet.
7:13, 1994, Lonberg et al., Nature 368:856, 1994, and Taylor et
al., Int. Immun. 6:579, 1994.
[0130] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0131] For particular uses, it may be desirable to prepare
fragments of anti-zacrp3 antibodies. Such antibody fragments can be
obtained, for example, by proteolytic hydrolysis of the antibody.
Antibody fragments can be obtained by pepsin or papain digestion of
whole antibodies by conventional methods. As an illustration,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent to produce 3.5S Fab' monovalent fragments.
Optionally, the cleavage reaction can be performed using a blocking
group for the sulfhydryl groups that result from cleavage of
disulfide linkages. As an alternative, an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys.
89:230, 1960, Porter, Biochem. J. 73:119, 1959, Edelman et al., in
Methods in Enzymology Vol. 1, page 422 (Academic Press 1967), and
by Coligan, ibid.
[0132] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0133] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described by Inbar et al., Proc. Natl. Acad. Sci. USA 69:2659,
1972. Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
gluteraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437, 1992).
[0134] The Fv fragments may comprise V.sub.H and V.sub.L chains
which are connected by a peptide linker. These single-chain antigen
binding proteins (scFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains which are connected by an oligonucleotide. The structural
gene is inserted into an expression vector which is subsequently
introduced into a host cell, such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing scFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology 2:97, 1991, also see, Bird et al., Science
242:423, 1988, Ladner et al., U.S. Pat. No. 4,946,778, Pack et al.,
Bio/Technology 11:1271, 1993, and Sandhu, ibid.
[0135] As an illustration, a scFV can be obtained by exposing
lymphocytes to zacrp3 polypeptide in vitro, and selecting antibody
display libraries in phage or similar vectors (for instance,
through use of immobilized or labeled zacrp3 protein or peptide).
Genes encoding polypeptides having potential zacrp3 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, Ladner
et al., U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of
Peptides and Proteins (Academic Press, Inc. 1996)) 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 zacrp3 sequences disclosed herein to identify
proteins which bind to zacrp3.
[0136] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106, 1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press, 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0137] Alternatively, an anti-zacrp3 antibody may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain. Typical residues of
human antibodies are then substituted in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat.
Acad. Sci. USA 86:3833, 1989. Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522, 1986, Carter et al., Proc. Nat. Acad. Sci. USA
89:4285, 1992, Sandhu, Crit. Rev. Biotech. 12:437, 1992, Singer et
al., J. Immun. 150:2844, 1993, Sudhir (ed.), Antibody Engineering
Protocols (Humana Press, Inc. 1995), Kelley, "Engineering
Therapeutic Antibodies," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 399-434 (John Wiley &
Sons, Inc. 1996), and by Queen et al., U.S. Pat. No. 5,693,762
(1997).
[0138] Polyclonal anti-idiotype antibodies can be prepared by
immunizing animals with anti-zacrp3 antibodies or antibody
fragments, using standard techniques. See, for example, Green et
al., "Production of Polyclonal Antisera," in Methods In Molecular
Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana
Press 1992). Also, see Coligan, ibid. at pages 2.4.1-2.4.7.
Alternatively, monoclonal anti-idiotype antibodies can be prepared
using anti-zacrp3 antibodies or antibody fragments as immunogens
with the techniques, described above. As another alternative,
humanized anti-idiotype antibodies or subhuman primate
anti-idiotype antibodies can be prepared using the above-described
techniques. Methods for producing anti-idiotype antibodies are
described, for example, by Irie, U.S. Pat. No. 5,208,146, Greene,
et. al., U.S. Pat. No. 5,637,677, and Varthakavi and Minocha, J.
Gen. Virol. 77:1875, 1996.
[0139] Genes encoding polypeptides having potential zacrp3
polypeptide binding domains, "binding proteins", can be obtained by
screening random or directed 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. Alternatively, constrained phage display libraries can
also be produced. These 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 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 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.). Peptide display libraries
can be screened using the zacrp3 sequences disclosed herein to
identify proteins which bind to zacrp3. These "binding proteins"
which interact with zacrp3 polypeptides can be used essentially
like an antibody.
[0140] A variety of assays known to those skilled in the art can be
utilized to detect antibodies and/or binding proteins which
specifically bind to zacrp3 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 zacrp3 protein or polypeptide.
[0141] Antibodies and binding proteins to zacrp3 may be used for
tagging cells that express zacrp3; for isolating zacrp3 by affinity
purification; for diagnostic assays for determining circulating
levels of zacrp3 polypeptides; for detecting or quantitating
soluble zacrp3 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 zacrp3
polypeptide modulation of spermatogenesis or like activity 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. Moreover,
antibodies to zacrp3 or fragments thereof may be used in vitro to
detect denatured zacrp3 or fragments thereof in assays, for
example, Western Blots or other assays known in the art.
[0142] 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
(receptor or antigen, respectively, for instance). More
specifically, zacrp3 polypeptides or anti-zacrp3 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.
[0143] An additional aspect of the present invention provides
methods for identifying agonists or antagonists of the zacrp3
polypeptides disclosed above, which agonists or antagonists may
have valuable properties as discussed further herein. Within one
embodiment, there is provided a method of identifying zacrp3
polypeptide agonists, comprising providing cells responsive
thereto, culturing the cells in the presence of a test compound and
comparing the cellular response with the cell cultured in the
presence of the zacrp3 polypeptide, and selecting the test
compounds for which the cellular response is of the same type.
[0144] Within another embodiment, there is provided a method of
identifying antagonists of zacrp3 polypeptide, comprising providing
cells responsive to a zacrp3 polypeptide, culturing a first portion
of the cells in the presence of zacrp3 polypeptide, culturing a
second portion of the cells in the presence of the zacrp3
polypeptide and a test compound, and detecting a decrease in a
cellular response of the second portion of the cells as compared to
the first portion of the cells. In addition to those assays
disclosed herein, samples can be tested for inhibition of zacrp3
activity within a variety of assays designed to measure receptor
binding or the stimulation/inhibition of zacrp3-dependent cellular
responses. For example, zacrp3-responsive cell lines can be
transfected with a reporter gene construct that is responsive to a
zacrp3-stimulated cellular pathway. Reporter gene constructs of
this type are known in the art, and will generally comprise a
zacrp3-DNA response element operably linked to a gene encoding an
assayable protein, such as luciferase. 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. Candidate compounds,
solutions, mixtures or extracts are tested for the ability to
inhibit the activity of zacrp3 on the target cells as evidenced by
a decrease in zacrp3 stimulation of reporter gene expression.
Assays of this type will detect compounds that directly block
zacrp3 binding to cell-surface receptors, as well as compounds that
block processes in the cellular pathway subsequent to
receptor-ligand binding. In the alternative, compounds or other
samples can be tested for direct blocking of zacrp3 binding to
receptor using zacrp3 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 zacrp3 to the receptor is indicative of
inhibitory activity, which can be confirmed through secondary
assays. Receptors used within binding assays may be cellular
receptors or isolated, immobilized receptors.
[0145] Based on homology to other adipocyte complement related
proteins, zacrp3 polypeptides, fragments, fusions, agonists or
antagonists can be used to modulate energy balance in mammals or to
protect endothelial cells from injury. With regard to modulating
energy balance, zacrp3 polypeptides modulate cellular metabolic
reactions. Such metabolic reactions include adipogenesis,
gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake,
protein synthesis, thermogenesis, oxygen utilization and the like.
Zacrp3 polypeptides may also find use as neurotransmitters or as
modulators of neurotransmission, as indicated by expression of the
polypeptide in tissues associated with the sympathetic or
parasympathetic nervous system. In this regard, zacrp3 polypeptides
may find utility in modulating nutrient uptake, as demonstrated,
for example, by 2-deoxy-glucose uptake in the brain or the
like.
[0146] Among other methods known in the art or described herein,
mammalian energy balance may be evaluated by monitoring one or more
of the following metabolic functions: adipogenesis,
gluconeogenesis, glycogenolysis, lipogenesis, glucose uptake,
protein synthesis, thermogenesis, oxygen utilization or the like.
These metabolic functions are monitored by techniques (assays or
animal models) known to one of ordinary skill in the art, as is
more fully set forth below. For example, the glucoregulatory
effects of insulin are predominantly exerted in the liver, skeletal
muscle and adipose tissue. Insulin binds to its cellular receptor
in these three tissues and initiates tissue-specific actions that
result in, for example, the inhibition of glucose production and
the stimulation of glucose utilization. In the liver, insulin
stimulates glucose uptake and inhibits gluconeogenesis and
glycogenolysis. In skeletal muscle and adipose tissue, insulin acts
to stimulate the uptake, storage and utilization of glucose.
[0147] Art-recognized methods exist for monitoring all of the
metabolic functions recited above. Thus, one of ordinary skill in
the art is able to evaluate zacrp3 polypeptides, fragments, fusion
proteins, antibodies, agonists and antagonists for metabolic
modulating functions. Exemplary modulating techniques are set forth
below.
[0148] Adipogenesis, gluconeogenesis and glycogenolysis are
interrelated components of mammalian energy balance, which may be
evaluated by known techniques using, for example, ob/ob mice or
db/db mice. The ob/ob mice are inbred mice that are homozygous for
an inactivating mutation at the ob (obese) locus. Such ob/ob mice
are hyperphagic and hypometabolic, and are believed to be deficient
in production of circulating OB protein. The db/db mice are inbred
mice that are homozygous for an inactivating mutation at the db
(diabetes) locus. The db/db mice display a phenotype similar to
that of ob/ob mice, except db/db mice also display a diabetic
phenotype. Such db/db mice are believed to be resistant to the
effects of circulating OB protein. Also, various in vitro methods
of assessing these parameters are known in the art.
[0149] Insulin-stimulated lipogenesis, for example, may be
monitored by measuring the incorporation of .sup.14C-acetate into
triglyceride (Mackall et al. J. Biol. Chem. 251:6462-4, 1976) or
triglyceride accumulation (Kletzien et al., Mol. Pharmacol.
41:393-8, 1992).
[0150] Glucose uptake may be evaluated, for example, in an assay
for insulin-stimulated glucose transport. Non-transfected,
differentiated L6 myotubes (maintained in the absence of G418) are
placed in DMEM containing 1 g/l glucose, 0.5 or 1.0% BSA, 20 mM
Hepes, and 2 mM glutamine. After two to five hours of culture, the
medium is replaced with fresh, glucose-free DMEM containing 0.5 or
1.0% BSA, 20 mM Hepes, 1 mM pyruvate, and 2 mM glutamine.
Appropriate concentrations of insulin or IGF-1, or a dilution
series of the test substance, are added, and the cells are
incubated for 20-30 minutes. .sup.3H or .sup.14C-labeled
deoxyglucose is added to .apprxeq.50 1M final concentration, and
the cells are incubated for approximately 10-30 minutes. The cells
are then quickly rinsed with cold buffer (e.g. PBS), then lysed
with a suitable lysing agent (e.g. 1% SDS or 1 N NaOH). The cell
lysate is then evaluated by counting in a scintillation counter.
Cell-associated radioactivity is taken as a measure of glucose
transport after subtracting non-specific binding as determined by
incubating cells in the presence of cytocholasin b, an inhibitor of
glucose transport. Other methods include those described by, for
example, Manchester et al., Am. J. Physiol. 266 (Endocrinol. Metab.
29):E326-E333, 1994 (insulin-stimulated glucose transport).
[0151] Protein synthesis may be evaluated, for example, by
comparing precipitation of .sup.35S-methionine-labeled proteins
following incubation of the test cells with .sup.35S-methionine and
.sup.35S-methionine and a putative modulator of protein
synthesis.
[0152] Thermogenesis may be evaluated as described by B. Stanley in
The Biology of Neuropeptide Y and Related Peptides, W. Colmers and
C. Wahlestedt (eds.), Humana Press, Ottawa, 1993, pp. 457-509; C.
Billington et al., Am. J. Physiol. 260:R321, 1991; N. Zarjevski et
al., Endocrinology 133:1753, 1993; C. Billington et al., Am. J.
Physiol. 266:R1765, 1994; Heller et al., Am. J. Physiol. 252(4 Pt
2): R661-7, 1987; and Heller et al., Am. J. Physiol. 245: R321-8,
1983. Also, metabolic rate, which may be measured by a variety of
techniques, is an indirect measurement of thermogenesis.
[0153] Oxygen utilization may be evaluated as described by Heller
et al., Pflugers Arch 369: 55-9, 1977. This method also involved an
analysis of hypothalmic temperature and metabolic heat production.
Oxygen utilization and thermoregulation have also been evaluated in
humans as described by Haskell et al., J. Appl. Physiol. 51:
948-54, 1981.
[0154] Neurotransmission functions may be evaluated by monitoring
2-deoxy-glucose uptake in the brain. This parameter is monitored by
techniques (assays or animal models) known to one of ordinary skill
in the art, for example, autoradiography. Useful monitoring
techniques are described, for example, by Kilduff et al., J.
Neurosci. 10 2463-75, 1990, with related techniques used to
evaluate the "hibernating heart" as described in Gerber et al.
Circulation 94: 651-8, 1996, and Fallavollita et al., Circulation
95: 1900-9, 1997.
[0155] In addition, zacrp3 polypeptides, fragments, fusions
agonists or antagonists thereof may be therapeutically useful for
anti-microbial applications. For example, complement component C1q
plays a role in host defense against infectious agents, such as
bacteria and viruses. C1q is known to exhibit several specialized
functions. For example, C1q triggers the complement cascade via
interaction with bound antibody or C-reactive protein (CRP). Also,
C1q interacts directly with certain bacteria, RNA viruses,
mycoplasma, uric acid crystals, the lipid A component of bacterial
endotoxin and membranes of certain intracellular organelles. C1q
binding to the C1q receptor is believed to promote phagocytosis.
C1q also appears to enhance the antibody formation aspect of the
host defense system. See, for example, Johnston, Pediatr. Infect.
Dis. J. 12(11): 933-41, 1993. Thus, soluble C1q-like molecules may
be useful as anti-microbial agents, promoting lysis or phagocytosis
of infectious agents.
[0156] Zacrp3 fragments as well as zacrp3 polypeptides, fusion
proteins, agonists, antagonists or antibodies may be evaluated with
respect to their anti-microbial properties according to procedures
known in the art. See, for example, Barsum et al., Eur. Respir. J.
8(5): 709-14, 1995; Sandovsky-Losica et al., J. Med. Vet. Mycol
(England) 28(4): 279-87, 1990; Mehentee et al., J. Gen. Microbiol.
(England) 135 (Pt. 8): 2181-8, 1989; Segal and Savage, J. Med. Vet.
Mycol. 24: 477-9, 1986 and the like. If desired, the performance of
zacrp3 in this regard can be compared to proteins known to be
functional in this regard, such as proline-rich proteins, lysozyme,
histatins, lactoperoxidase or the like. In addition, zacrp3
fragments, polypeptides, fusion proteins, agonists, antagonists or
antibodies may be evaluated in combination with one or more
anti-microbial agents to identify synergistic effects. One of
ordinary skill in the art will recognize that the anti-microbial
properties of zacrp3 polypeptides, fragments, fusion proteins,
agonists, antagonists and antibodies may be similarly
evaluated.
[0157] As neurotransmitters or neurotransmission modulators, zacrp3
polypeptide fragments as well as zacrp3 polypeptides, fusion
proteins, agonists, antagonists or antibodies of the present
invention may also modulate calcium ion concentration, muscle
contraction, hormone secretion, DNA synthesis or cell growth,
inositol phosphate turnover, arachidonate release, phospholipase-C
activation, gastric emptying, human neutrophil activation or ADCC
capability, superoxide anion production and the like. Evaluation of
these properties can be conducted by known methods, such as those
set forth herein.
[0158] The impact of zacrp3 polypeptide, fragment, fusion,
antibody, agonist or antagonist on intracellular calcium level may
be assessed by methods known in the art, such as those described by
Dobrzanski et al., Requlatory Peptides 45: 341-52, 1993, and the
like. The impact of zacrp3 polypeptide, fragment, fusion, agonist
or antagonist on muscle contraction may be assessed by methods
known in the art, such as those described by Smits & Lebebvre,
J. Auton. Pharmacol. 14: 383-92, 1994, Belloli et al., J. Vet.
Pharmacol. Therap. 17: 379-83, 1994, Maggi et al., Regulatory
Peptides 53: 259-74, 1994, and the like. The impact of zacrp3
polypeptide, fragment, fusion, agonist or antagonist on hormone
secretion may be assessed by methods known in the art, such as
those for prolactin release described by Henriksen et al., J.
Recep. Sig. Transd. Res. 15(1-4): 529-41, 1995, and the like. The
impact of zacrp3 polypeptide, fragment, fusion, agonist or
antagonist on DNA synthesis or cell growth may be assessed by
methods known in the art, such as those described by Dobrzanski et
al., Regulatory Peptides 45: 341-52, 1993, and the like. The impact
of zacrp3 polypeptide, fragment, fusion, agonist or antagonist on
inositol phosphate turnover may be assessed by methods known in the
art, such as those described by Dobrzanski et al., Regulatory
Peptides 45: 341-52, 1993, and the like.
[0159] Also, the impact of zacrp3 polypeptide, fragment, fusion,
agonist or antagonist on arachidonate release may be assessed by
methods known in the art, such as those described by Dobrzanski et
al., Regulatory Peptides 45: 341-52, 1993, and the like. The impact
of zacrp3 polypeptide, fragment, fusion, agonist or antagonist on
phospholipase-C activation may be assessed by methods known in the
art, such as those described by Dobrzanski et al., Regulatory
Peptides 45: 341-52, 1993, and the like. The impact of zacrp3
polypeptide, fragment, fusion, agonist or antagonist on gastric
emptying may be assessed by methods known in the art, such as those
described by Varga et al., Eur. J. Pharmacol. 286: 109-112, 1995,
and the like. The impact of zacrp3 polypeptide, fragment, fusion,
agonist or antagonist on human neutrophil activation and ADCC
capability may be assessed by methods known in the art, such as
those described by Wozniak et al., Immunology 78: 629-34, 1993, and
the like. The impact of zacrp3 polypeptide, fragment, fusion,
agonist or antagonist on superoxide anion production may be
assessed by methods known in the art, such as those described by
Wozniak et al., Immunology 78: 629-34, 1993, and the like.
[0160] Collagen is a potent inducer of platelet aggregation. This
poses risks to patients recovering from vascular injures.
Inhibitors of collagen-induced platelet aggregation would be useful
for blocking the binding of platelets to collagen-coated surfaces
and reducing associated collagen-induced platelet aggregation. C1q
is a component of the complement pathway and has been found to
stimulate defense mechanisms as well as trigger the generation of
toxic oxygen species that can cause tissue damage (Tenner, Behring
Inst. Mitt. 93:241-53, 1993). C1q binding sites are found on
platelets. C1q, independent of an immune binding partner, has been
found to inhibit platelet aggregation but not platelet adhesion or
shape change. The amino terminal region of C1q shares homology with
collagen (Peerschke and Ghebrehiwet, J. Immunol. 145:2984-88,
1990). Inhibition of C1q and the complement pathway can be
determined using methods disclosed herein or know in the art, such
as described in Suba and Csako, J. Immunol. 117:304-9, 1976.
[0161] The impact of zacrp3 polypeptide, fragments, fusions,
agonists or antagonists on collagen-mediated platelet adhesion,
activation and aggregation may be evaluated using methods described
herein or known in the art, such as the platelet aggregation assay
(Chiang et al., Thrombosis Res. 37:605-12, 1985) and platelet
adhesion assays (Peerschke and Ghebrehiwet, J. Immunol. 144:221-25,
1990). Assays for platelet adhesion to collagen and inhibition of
collagen-induced platelet aggregation can be measured using methods
described in Keller et al., J. Biol. Chem. 268:5450-6, 1993; Waxman
and Connolly, J. Biol. Chem. 268:5445-9, 1993; Noeske-Jungblut et
al., J. Biol. Chem. 269:5050-3 or 1994 Deckmyn et al., Blood
85:712-9, 1995.
[0162] The impact of zacrp3 polypeptide, fragments, fusions,
agonists or antagonists on vasodilation of aortic rings can be
measured according to the methods of Dainty et al., J. Pharmacol.
100:767, 1990 and Rhee et al., Neurotox. 16:179, 1995.
[0163] Various in vitro and in vivo models are available for
assessing the effects of zacrp3 polypeptides, fragments, fusion
proteins, antibodies, agonists and antagonists on ischemia and
reperfusion injury. See for example, Shandelya et al., Circulation
88:2812-26, 1993; Weisman et al., Science 249:146-151, 1991; Buerke
et al., Circulation 91:393-402, 1995; Horstick et al., Circulation
95:701-8, 1997 and Burke et al., J. Phar. Exp. Therp. 286:429-38,
1998. An ex vivo hamster platelet aggregation assay is described by
Deckmyn et al., ibid. Bleeding times in hamsters and baboons can be
measured following injection of zacrp3 polypeptides using the model
described by Deckmyn et al., ibid. The formation of thrombus in
response to administration of proteins of the present invention can
be measured using the hamster femoral vein thrombosis model is
provided by Deckmyn et al., ibid. Changes in platelet adhesion
under flow conditions following administration of zacrp3 can be
measured using the method described in Harsfalvi et al., Blood
85:705-11, 1995.
[0164] Complement inhibition and wound healing can be zacrp3
polypeptides, fragments, fusion proteins, antibodies, agonists or
antagonists be assayed alone or in combination with other know
inhibitors of collagen-induced platelet activation and aggregation,
such as palldipin, moubatin or calin, for example.
[0165] Zacrp3 polypeptides, fragments, fusion proteins, antibodies,
agonists or antagonists can be evaluated using methods described
herein or known in the art, such as healing of dermal layers in
pigs (Lynch et al., Proc. Natl. Acad. Sci. USA 84: 7696-700, 1987)
and full-thickness skin wounds in genetically diabetic mice
(Greenhalgh et al., Am. J. Pathol. 136: 1235-46, 1990), for
example. The polypeptides of the present invention can be assayed
alone or in combination with other known complement inhibitors as
described above.
[0166] 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 the designing
of PCR primers suitable for use with chromosomal radiation hybrid
mapping panels. Commercially available radiation hybrid mapping
panels which cover the entire human genome, such as the Stanford G3
RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc.,
Huntsville, Ala.), are available. 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 in a number of
ways including: 1) determining if a sequence is part of an existing
contig and obtaining additional surrounding genetic sequences in
various forms such as YAC-, BAC- or cDNA clones, 2) providing a
possible candidate gene for an inheritable disease which shows
linkage to the same chromosomal region, and 3) for
cross-referencing model organisms such as mouse which may be
beneficial in helping to determine what function a particular gene
might have.
[0167] The results showed linkage of Zacrp3 to the human chromosome
5 framework marker SHGC-56588 with a LOD score of 15.58 and at a
distance of 0 cR.sub.--10000 from the marker. The use of
surrounding markers positions Zacrp3 in the 5p12 region on the
integrated LDB human chromosome 5 map. The present invention also
provides reagents which will find use in diagnostic applications.
For example, the zacrp3 gene, a probe comprising zacrp3 DNA or RNA,
or a subsequence thereof can be used to determine if the zacrp3
gene is present on chromosome 5 or if a mutation has occurred.
Detectable chromosomal aberrations at the zacrp3 gene locus
include, but are not limited to, aneuploidy, gene copy number
changes, insertions, deletions, restriction site changes and
rearrangements. These aberrations can occur within the coding
sequence, within introns, or within flanking sequences, including
upstream promoter and regulatory regions, and may be manifested as
physical alterations within a coding sequence or changes in gene
expression level.
[0168] In general, these diagnostic methods comprise the steps of
(a) obtaining a genetic sample from a patient; (b) incubating the
genetic sample with a polynucleotide probe or primer as disclosed
above, under conditions wherein the polynucleotide will hybridize
to complementary polynucleotide sequence, to produce a first
reaction product; and (iii) comparing the first reaction product to
a control reaction product. A difference between the first reaction
product and the control reaction product is indicative of a genetic
abnormality in the patient. Genetic samples for use within the
present invention include genomic DNA, cDNA, and RNA. The
polynucleotide probe or primer can be RNA or DNA, and will comprise
a portion of SEQ ID NO:1, the complement of SEQ ID NO:1, or an RNA
equivalent thereof. Suitable assay methods in this regard include
molecular genetic techniques known to those in the art, such as
restriction fragment length polymorphism (RFLP) analysis, short
tandem repeat (STR) analysis employing PCR techniques, ligation
chain reaction (Barany, PCR Methods and Applications 1:5-16, 1991),
ribonuclease protection assays, and other genetic linkage analysis
techniques known in the art (Sambrook et al., ibid.; Ausubel et.
al., ibid.; Marian, Chest 108:255-65, 1995). Ribonuclease
protection assays (see, e.g., Ausubel et al., ibid., ch. 4)
comprise the hybridization of an RNA probe to a patient RNA sample,
after which the reaction product (RNA-RNA hybrid) is exposed to
RNase. Hybridized regions of the RNA are protected from digestion.
Within PCR assays, a patient's genetic sample is incubated with a
pair of polynucleotide primers, and the region between the primers
is amplified and recovered. Changes in size or amount of recovered
product are indicative of mutations in the patient. Another
PCR-based technique that can be employed is single strand
conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods
and Applications 1:34-8, 1991).
[0169] Zacrp3 polypeptides may be used in the analysis of energy
efficiency of a mammal. Zacrp3 polypeptides found in serum or
tissue samples may be indicative of a mammals ability to store
food, with more highly efficient mammals tending toward obesity.
More specifically, the present invention contemplates methods for
detecting zacrp3 polypeptide comprising: [0170] exposing a sample
possibly containing zacrp3 polypeptide to an antibody attached to a
solid support, wherein said antibody binds to an epitope of a
zacrp3 polypeptide; [0171] washing said immobilized
antibody-polypeptide to remove unbound contaminants; [0172]
exposing the immobilized antibody-polypeptide to a second antibody
directed to a second epitope of a zacrp3 polypeptide, wherein the
second antibody is associated with a detectable label; and [0173]
detecting the detectable label. The concentration of zacrp3
polypeptide in the test sample appears to be indicative of the
energy efficiency of a mammal. This information can aid nutritional
analysis of a mammal. Potentially, this information may be useful
in identifying and/or targeting energy deficient tissue.
[0174] A further aspect of the invention provides a method for
studying insulin. Such methods of the present invention comprise
incubating adipocytes in a culture medium comprising zacrp3
polypeptide, monoclonal antibody, agonist or antagonist thereof
.+-. insulin and observing changes in adipocyte protein secretion
or differentiation.
[0175] Anti-microbial protective agents may be directly acting or
indirectly acting. Such agents operating via membrane association
or pore forming mechanisms of action directly attach to the
offending microbe. Anti-microbial agents can also act via an
enzymatic mechanism, breaking down microbial protective substances
or the cell wall/membrane thereof. Anti-microbial agents, capable
of inhibiting microorganism proliferation or action or of
disrupting microorganism integrity by either mechanism set forth
above, are useful in methods for preventing contamination in cell
culture by microbes susceptible to that anti-microbial activity.
Such techniques involve culturing cells in the presence of an
effective amount of said zacrp3 polypeptide or an agonist or
antagonist thereof.
[0176] Also, zacrp3 polypeptides or agonists thereof may be used as
cell culture reagents in in vitro studies of exogenous
microorganism infection, such as bacterial, viral or fungal
infection. Such moieties may also be used in in vivo animal models
of infection.
[0177] The present invention also provides methods of studying
mammalian cellular metabolism. Such methods of the present
invention comprise incubating cells to be studied, for example,
human vascular endothelial cells, .+-. zacrp3 polypeptide,
monoclonal antibody, agonist or antagonist thereof and observing
changes in adipogenesis, gluconeogenesis, glycogenolysis,
lipogenesis, glucose uptake, or the like.
[0178] An additional aspect of the invention provides a method for
studying dimerization or oligomerization. Such methods of the
present invention comprise incubating zacrp3 polypeptides or
fragments or fusion proteins thereof containing a collagen-like
domain alone or in combination with other polypeptides bearing
collagen-like domains and observing the associations formed between
the collagen like domains. Such associations are indicated by HPLC,
circular dichroism or the like.
[0179] Zacrp3 polypeptides, fragments, fusion proteins, antibodies,
agonists or antagonists of the present invention can be used in
methods for promoting blood flow within the vasculature of a mammal
by reducing the number of platelets that adhere and are activated
and the size of platelet aggregates. Used to such an end, Zacrp3
can be administered prior to, during or following an acute vascular
injury in the mammal. Vascular injury may be due to vascular
reconstruction, including but not limited to, angioplasty, coronary
artery bypass graft, microvascular repair or anastomosis of a
vascular graft. Also contemplated are vascular injuries due to
trauma, stroke or aneurysm. In other preferred methods the vascular
injury is due to plaque rupture, degradation of the vasculature,
complications associated with diabetes and atherosclerosis. Plaque
rupture in the coronary artery induces heart attack and in the
cerebral artery induces stroke. Use of zacrp3 polypeptides,
fragments, fusion proteins, antibodies, agonists or antagonists in
such methods would also be useful for ameliorating whole system
diseases of the vasculature associated with the immune system, such
as disseminated intravascular coagulation (DIC) and SIDs.
Additionally the complement inhibiting activity would be useful for
treating non-vasculature immune diseases such as
arteriolosclerosis.
[0180] A correlation has been found between the presence of C1q in
localized ischemic myocardium and the accumulation of leukocytes
following coronary occlusion and reperfusion. Release of cellular
components following tissue damage triggers complement activation
which results in toxic oxygen products that may be the primary
cause of myocardial damage (Rossen et al., Circ. Res. 62:572-84,
1998 and Tenner, ibid.). Blocking the complement pathway was found
to protect ischemic myocardium from reperfusion injury (Buerke et
al., J. Pharm. Exp. There. 286:429-38, 1998). Proteins having
complement inhibition and C1q binding activity would be useful for
such purposes.
[0181] Collagen and C1q binding capabilities of adipocyte
complement related protein homologs such as zacrp3 would be useful
to pacify damaged collagenous tissues preventing platelet adhesion,
activation or aggregation, and the activation of inflammatory
processes which lead to the release of toxic oxygen products. By
rendering the exposed tissue inert towards such processes as
complement activity, thrombotic activity and immune activation,
reduces the injurious effects of ischemia and reperfusion. In
particular, such injuries would include trauma injury ischemia,
intestinal strangulation, and injury associated with pre- and
post-establishment of blood flow. Such polypeptides would be useful
in the treatment of cardiopulmonary bypass ischemia and
recesitation, myocardial infarction and post trauma vasospasm, such
as stroke or percutanious transluminal angioplasty as well as
accidental or surgical-induced vascular trauma.
[0182] Additionally such collagen- and C1q-binding polypeptides
would be useful to pacify prosthetic biomaterials and surgical
equipment to render the surface of the materials inert towards
complement activation, thrombotic activity or immune activation.
Such materials include, but are not limited to, collagen or
collagen fragment-coated biomaterials, gelatin-coated biomaterials,
fibrin-coated biomaterials, fibronectin-coated biomaterials,
heparin-coated biomaterials, collagen and gel-coated stents,
arterial grafts, synthetic heart valves, artificial organs or any
prosthetic application exposed to blood that will bind zsig37 at
greater than 1.times.10.sup.8. Coating such materials can be done
using methods known in the art, see for example, Rubens, U.S. Pat.
No. 5,272,074.
[0183] Complement and C1q play a role in inflammation. The
complement activation is initiated by binding of C1q to
immunoglobulins (Johnston, Pediatr. Infect. Dis. J. 12:933-41,
1993; Ward and Ghetie, Therap. Immunol. 2:77-94, 1995). Inhibitors
of C1q and complement would be useful as anti-inflammatory agents.
Such application can be made to prevent infection. Additionally,
such inhibitors can be administrated to an individual suffering
from inflammation mediated by complement activation and binding of
immune complexes to C1q. Inhibitors of C1q and complement would be
useful in methods of mediating wound repair, enhancing progression
in wound healing by overcoming impaired wound healing. Progression
in wound healing would include, for example, such elements as a
reduction in inflammation, fibroblasts recruitment, wound
retraction and reduction in infection.
[0184] Ability of tumor cells to bind to collagen may contribute to
the metastasis of tumors. Inhibitors of collagen binding are also
useful for mediating the adhesive interactions and metastatic
spread of tumors (Noeske-Jungbult et al., U.S. Pat. No.
5,723,312).
[0185] In addition, zacrp3 polypeptides, fragments, fusions
agonists or antagonists thereof may be therapeutically useful for
anti-microbial applications. For example, complement component C1q
plays a role in host defense against infectious agents, such as
bacteria and viruses. C1q is known to exhibit several specialized
functions. For example, C1q triggers the complement cascade via
interaction with bound antibody or C-reactive protein (CRP). Also,
C1q interacts directly with certain bacteria, RNA viruses,
mycoplasma, uric acid crystals, the lipid A component of bacterial
endotoxin and membranes of certain intracellular organelles. C1q
binding to the C1q receptor is believed to promote phagocytosis.
C1q also appears to enhance the antibody formation aspect of the
host defense system. See, for example, Johnston, Pediatr. Infect.
Dis. J. 12(11): 933-41, 1993. Thus, soluble C1q-like molecules may
be useful as anti-microbial agents, promoting lysis or phagocytosis
of infectious agents.
[0186] The positively charged, extracellular, triple helix,
collagenous domains of C1q and macrophage scavenger receptor were
determined to play a role in ligand binding and were shown to have
a broad binding specificity for polyanions (Acton et al., J. Biol.
Chem. 268:3530-37, 1993). Lysophospholipid growth factor
(lysophosphatidic acid, LPA) and other mitogenic anions localize at
the site of damaged tissues and assist in wound repair. LPA exerts
many biological effects including activation of platelets and
up-regulation of matrix assembly. It is thought that LPA synergizes
with other blood coagulation factors and mediates wound
healing.
[0187] The collagenous domains of proteins such as C1q and
macrophage scavenger receptor are know to bind acidic phospholipids
such as LPA. The interaction of zacrp3 polypeptides, fragments,
fusions, agonists or antagonists with mitogenic anions such as LPA
can be determined using assays known in the art, see for example,
Acton et al., ibid. Inhibition of inflammatory processes by
polypeptides and antibodies of the present invention would also be
useful in preventing infection at the wound site.
[0188] For pharmaceutical use, the proteins of the present
invention can be formulated with pharmaceutically acceptable
carriers for parenteral, oral, nasal, rectal, topical, transdermal
administration or the like, according to conventional methods. In a
preferred embodiment administration is made at or near the site of
vascular injury. In general, pharmaceutical formulations will
include a zacrp3 protein 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., 19.sup.th ed., 1995.
Therapeutic doses will generally be 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.
[0189] As used herein a "pharmaceutically effective amount" of a
zsig37 polypeptide, fragment, fusion protein, agonist or antagonist
is an amount sufficient to induce a desired biological result. The
result can be alleviation of the signs, symptoms, or causes of a
disease, or any other desired alteration of a biological system.
For example, an effective amount of a zacrp3 polypeptide is that
which provides either subjective relief of symptoms or an
objectively identifiable improvement as noted by the clinician or
other qualified observer. Such an effective amount of a zacrp3
polypeptide would provide, for example, inhibition of
collagen-activated platelet activation and the complement pathway,
including C1q, increase localized blood flow within the vasculature
of a patient and/or reduction in injurious effects of ischemia and
reperfusion. Effective amounts of the zacrp3 polypeptides can vary
widely depending on the disease or symptom to be treated. The
amount of the polypeptide to be administered and its concentration
in the formulations, depends upon the vehicle selected, route of
administration, the potency of the particular polypeptide, the
clinical condition of the patient, the side effects and the
stability of the compound in the formulation. Thus, the clinician
will employ the appropriate preparation containing the appropriate
concentration in the formulation, as well as the amount of
formulation administered, depending upon clinical experience with
the patient in question or with similar patients. Such amounts will
depend, in part, on the particular condition to be treated, age,
weight, and general health of the patient, and other factors
evident to those skilled in the art. Typically a dose will be in
the range of 0.01-100 mg/kg of subject. In applications such as
balloon catheters the typical dose range would be 0.05-5 mg/kg of
subject. Doses for specific compounds may be determined from in
vitro or ex vivo studies in combination with studies on
experimental animals. Concentrations of compounds found to be
effective in vitro or ex vivo provide guidance for animal studies,
wherein doses are calculated to provide similar concentrations at
the site of action.
[0190] Polynucleotides encoding zacrp3 polypeptides are useful
within gene therapy applications where it is desired to increase or
inhibit zacrp3 activity. If a mammal has a mutated or absent zacrp3
gene, the zacrp3 gene can be introduced into the cells of the
mammal. In one embodiment, a gene encoding a zacrp3 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).
[0191] In another embodiment, a zacrp3 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; WIPO Publication WO 95/07358; 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.
[0192] 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.
[0193] Antisense methodology can be used to inhibit zacrp3 gene
transcription, such as to inhibit cell proliferation in vivo.
Polynucleotides that are complementary to a segment of a
zacrp3-encoding polynucleotide (e.g., a polynucleotide as set froth
in SEQ ID NO:1) are designed to bind to zacrp3-encoding mRNA and to
inhibit translation of such mRNA. Such antisense polynucleotides
are used to inhibit expression of zacrp3 polypeptide-encoding genes
in cell culture or in a subject.
[0194] Transgenic mice, engineered to express the zacrp3 gene, and
mice that exhibit a complete absence of zacrp3 gene function,
referred to as "knockout mice" (Snouwaert et al., Science 257:1083,
1992), may also be generated (Lowell et al., Nature 366:740-42,
1993). These mice may be employed to study the zacrp3 gene and the
protein encoded thereby in an in vivo system.
[0195] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
Chromosomal Assignment and Placement of Zacrp3
[0196] Zacrp3 was mapped to human chromosome 5 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.
[0197] For the mapping of zacrp3 with the Stanford G3 RH Panel, 20
.mu.l reactions were set up in a 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, ZC
21,913 (SEQ ID NO:13), 1 .mu.l antisense primer, ZC 21,914 (SEQ ID
NO:14), 2 .mu.l RediLoad (Research Genetics, Inc.), 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
ddH.sub.2O 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 62.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.).
[0198] The results showed linkage of Zacrp3 to the human chromosome
5 framework marker SHGC-56588 with a LOD score of 15.58 and at a
distance of 0 cR.sub.--10000 from the marker. The use of
surrounding markers positions Zacrp3 in the 5p12 region on the
integrated LDB human chromosome 5 map (The Genetic Location
Database, University of Southhampton, WWW server:
http://cedar.genetics.soton.ac. uk/public_html/).
Example 2
Baculovirus Expression of Zacrp3
[0199] An expression vector, pzacrp3cee, was prepared to express
human zacrp3 polypeptides having a carboxy-terminal Glu-Glu tag, in
insect cells.
A. Construction of pzacrp3cee
[0200] A 766 bp fragment containing sequence for zacrp3 (SEQ ID
NO:1) and a polynucleotide sequence encoding BamHI and Xba1
restriction sites on the 5' and 3' ends, respectively, was
generated by PCR amplification from a plasmid containing zacrp3
cDNA using primers ZC23377 (SEQ ID NO:15) and ZC23378 (SEQ ID
NO:16). The PCR reaction conditions were as follows: 1 cycle of
94.degree. C. for 4 minutes, followed by 25 cycles of 94.degree. C.
for 45 seconds, 50.degree. C. for 45 seconds, and 72.degree. C. for
2 minutes; 1 cycle at 72.degree. C. for 10 min; followed by a 4C
soak. The fragment was visualized by gel electrophoresis (1%
Seaplaque/1% NuSieve). The band was excised, diluted to 0.5%
agarose with 2 mM MgCl.sub.2, melted at 65.degree. C. and ligated
into an BamHI/XbaI digested baculovirus expression donor vector,
pZBV32L. The pZBV32L vector is a modification of the pFastBac1.TM.
(Life Technologies) expression vector, where the polyhedron
promoter has been removed and replaced with the late activating
Basic Protein Promoter and the coding sequence for the Glu-Glu tag
(SEQ ID NO:17) as well as a stop signal is inserted at the 3' end
of the multiple cloning region). About 11 nanograms of the
restriction digested zacrp3 insert and about 23 ng of the
corresponding vector were ligated overnight at 16.degree. C. The
ligation mix was diluted 3 fold in TE (10 mM Tris-HCl, pH 7.5 and 1
mM EDTA) and 4 fmol of the diluted ligation mix was transformed
into DH5.alpha. Library Efficiency competent cells (Life
Technologies) according to manufacturer's direction by heat shock
for 45 seconds in a 42.degree. C. waterbath. The transformed DNA
and cells were diluted in 450 .mu.l of SOC media (2% Bacto
Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl, 1.5 mM KCl, 10
mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM glucose) and plated onto
LB plates containing 100 .mu.g/ml ampicillin. Clones were analyzed
by restriction digests and 1 .mu.l of the positive clone was
transformed into 20 .mu.l DH10Bac Max Efficiency competent cells
(GIBCO-BRL, Gaithersburg, Md.) according to manufacturer's
instruction, by heat shock for 45 seconds in a 42.degree. C.
waterbath. The transformed cells were then diluted in 980 .mu.l SOC
media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl,
1.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM glucose)
out grown in shaking incubator at 37 C for four hours and plated
onto Luria Agar plates containing 50 .mu.g/ml kanamycin, 7 .mu.g/ml
gentamicin, 10 .mu.g/ml tetracycline, IPTG and Bluo Gal. The plated
cells were incubated for 48 hours at 37.degree. C. A color
selection was used to identify those cells having zacrp3cee
encoding donor insert that had incorporated into the plasmid
(referred to as a "bacmid"). Those colonies, which were white in
color, were picked for analysis. Bacmid DNA was isolated from
positive colonies using the QiaVac Miniprep8 system (Qiagen)
according the manufacturer's directions. Clones were screened for
the correct insert by amplifying DNA using primers to the
transposable element in the bacmid via PCR using primers ZC447 (SEQ
ID NO:18) and ZC976 (SEQ ID NO:19). The PCR reaction conditions
were as follows: 35 cycles of 94.degree. C. for 45 seconds,
50.degree. C. for 45 seconds, and 72.degree. C. for 5 minutes; 1
cycle at 72.degree. C. for 10 min.; followed by 4.degree. C. soak.
The PCR product was run on a 1% agarose gel to check the insert
size. Those having the correct insert were used to transfect
Spodoptera frugiperda (Sf9) cells.
B. Transfection
[0201] Sf9 cells were seeded at 5.times.10.sup.6 cells per 35 mm
plate and allowed to attach for 1 hour at 27.degree. C. Five
microliters of bacmid DNA was diluted with 100 .mu.l Sf-900 II SFM
(Life Technologies). Six .mu.l of CellFECTIN Reagent (Life
Technologies) was diluted with 100 .mu.l Sf-900 II SFM. The bacmid
DNA and lipid solutions were gently mixed and incubated 30-45
minutes at room temperature. The media from one plate of cells were
aspirated, the cells were washed 1.times. with 2 ml fresh Sf-900 II
SFM media. Eight hundred microliters of Sf-900 II SFM was added to
the lipid-DNA mixture. The wash media was aspirated and the
DNA-lipid mix added to the cells. The cells were incubated at
27.degree. C. for 4-5 hours. The DNA-lipid mix was aspirated and 2
ml of Sf-900 II media was added to each plate. The plates were
incubated at 27.degree. C., 90% humidity, for 96 hours after which
the virus was harvested.
C. Primary Amplification
[0202] Sf9 cells were grown in 50 ml Sf-900 II SFM in a 125 ml
shake flask to an approximate density of 0.41-0.52.times.10.sup.5
cells/ml. They were then infected with 150 .mu.l of the virus stock
from above and incubated at 27.degree. C. for 3 days after which
time the virus was harvested according to standard methods known in
the art.
Example 3
Purification of Baculovirus Expressed Glu-Glu-Tagged zacrp3
Polypeptides
[0203] Unless otherwise noted, all operations were carried out at
4.degree. C. A mixture of protease inhibitors were added to a 2
liter sample of conditioned media from C-terminal Glu-Glu (EE)
tagged zacrp3 baculovirus-infected Sf9 cells 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).
The sample was centrifuged at 10,000 rpm for 30 min at 4.degree. C.
in a Beckman JLA-10.5 rotor (Beckman Instruments) in a Beckman
Avanti J25I centrifuge (Beckman Instruments) to remove cell debris.
To the supernatant fraction was added a 50.0 ml sample of anti-EE
Sepharose, prepared as described below, and the mixture was gently
agitated on a Wheaton (Millville, N.J.) roller culture apparatus
for 18.0 h at 4.degree. C.
[0204] The mixture was poured into a 5.0.times.20.0 cm Econo-Column
(Bio-Rad Laboratories) and the gel was washed with 30 column
volumes of phosphate buffered saline (PBS). The unretained
flow-through fraction was discarded. Once the absorbance of the
effluent at 280 nM was less than 0.05, flow through the column was
reduced to zero and the anti-EE Sepharose gel was washed with 2.0
column volumes of PBS containing 0.2 mg/ml of EE peptide (AnaSpec,
San Jose, Calif.). The peptide used has the sequence
Glu-Tyr-Met-Pro-Val-Asp (SEQ ID NO:20). After 1.0 hour at 4.degree.
C., flow was resumed and the eluted protein was collected. This
fraction was referred to as the peptide elution. The anti-EE
Sepharose gel was washed with 2.0 column volumes of 0.1 M glycine,
pH 2.5, and the glycine wash was collected separately. The pH of
the glycine-eluted fraction was adjusted to 7.0 by the addition of
a small volume of 10.times.PBS and stored at 4.degree. C.
[0205] The peptide elution was concentrated to 5.0 ml using a 5,000
molecular weight cutoff membrane concentrator (Millipore) according
to the manufacturer's instructions. The concentrated peptide
elution was separated from free peptide by chromatography on a
1.5.times.50 cm Sephadex G-50 (Pharmacia) column equilibrated in
PBS at a flow rate of 1.0 ml/min using a BioCad Sprint HPLC
(PerSeptive BioSystems). Two ml fractions were collected and the
absorbance at 280 nM was monitored. The first peak of material
absorbing at 280 nM and eluting near the void volume of the column
was collected. This material represented purified zacrp3CEE and was
composed of two major bands of apparent molecular weights.
Preparation of Anti-EE Sepharose
[0206] A 100 ml bed volume of protein G-Sepharose (Pharmacia) was
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 was washed
with 6.0 volumes of 200 mM triethanolamine, pH 8.2 (TEA, Sigma),
and an equal volume of EE antibody solution containing 900 mg of
antibody was added. After an overnight incubation at 4.degree. C.,
unbound antibody was removed by washing the resin with 5 volumes of
200 mM TEA as described above. The resin was resuspended in 2
volumes of TEA, transferred to a suitable container, and
dimethylpimilimidate-2 HCl (Pierce), dissolved in TEA, was added to
a final concentration of 36 mg/ml of gel. The gel was rocked at
room temperature for 45 min and the liquid was removed using the
filter unit as described above. Nonspecific sites on the gel were
then blocked by incubating for 10 minutes at room temperature with
5 volumes of 20 mM ethanolamine in 200 mM TEA. The gel was then
washed with 5 volumes of PBS containing 0.02% sodium azide and
stored in this solution at 4.degree. C.
Example 4
Adhesion Molecule Assays
[0207] Upon stimulation with inflammatory cytokines such as TNF
(tumor necrosis factor), human microvascular bone marrow cells
(TRBMEC) express cell surface adhesion molecules, including
E-selectin (endothelial leukocyte adhesion molecule), V-CAM
(vascular cell adhesion molecule), and I-CAM (intercellular
adhesion molecule.
[0208] The effect of zacrp3 on expression of cell surface adhesion
molecules was determined using microvascular bone marrow cells
(TRBMEC) in a cell based ELISA according to Ouchi et al.,
(Circulation 100:2473-7, 1999). Briefly, TRBMEC cells were grown in
96 well, flat bottom plates (Costar, Pleasanton, Calif.) until
confluent. Both wild type control media and baculovirus-expressed
zacrp3 media was concentrated 10.times. before testing (Centricon
Centrifugal Filtration Unit 5,000K cutoff, Millipore Corp.,
Bedford, Mass.) according to the manufacturer's instructions. To
each well 90 .mu.l of zacrp3-containing media or control media was
added, and the plates were incubated at 37.degree. C., 5% CO.sub.2
overnight. The next day, half of the samples received 10 .mu.l of
TNF.alpha. (10 ng/ml, R&D Systems, Minneapolis, Minn.), the
other samples were untreated, measuring basal expression. The
plates were then incubated at 37.degree. C., 5% CO.sub.2 for 4
hours.
[0209] Following incubation, the media was removed from the plates
and 50 .mu.l anti-human VCAM antibody (1:1000 dilution of a 1 mg/ml
stock, R&D Systems), 50 .mu.l of anti-human ICAM-1 monoclonal
antibody (1:1000 dilution of a 1 mg/ml stock, R&D Systems), or
50 .mu.l of anti-human E-selectin antibodies (1:1000 dilution of a
1 mg/ml stock, R&D Systems) were then added to triplicate wells
and the plates were incubated at 37.degree. C., 5% CO.sub.2 for 1
hour.
[0210] The antibody solution was removed and the plates were washed
three times in warm RPMI+5% FBS. Following the last wash, 100
.mu.l/well of an 0.05% gluteraldehyde solution (1:1000 of 50%
gluteraldehyde in PBS) was added to the wells and the plates were
incubated at room temperature for 10 minutes. The plates were
washed three times with PBS and 50 .mu.l/well of secondary antibody
(1:1000 dilution of goat .alpha.-mouse IgG whole molecule HRP
conjugate, (Sigma Chemical Co., St. Louis, Mo.) was added to all
wells. The plates were incubated for one hour at 37.degree. C.
[0211] The plates were then washed five times with washing buffer
(PBS+0.05% Tween 20) and 100 .mu.l/well TMB solution (100 .mu.l of
4 mg/ml Tetra methyl benzidine (Sigma) in DMSO, in 10 ml 60 mM Na
Acetate pH 5.0 and 100 .mu.l 1.2% H.sub.2O.sub.2) was added to each
well. The plates were allowed to develop at room temperature for
15-20 minutes at which time the reaction was quenched by adding 100
.mu.l/well 1M H.sub.2SO.sub.4. Plates were read at 450 nm with
reference wavelength of 655 nm.
[0212] Zacrp3 showed no effect on ICAM-1 expression. Zacrp3 did
show an effect on VCAM-1 expression. When compared to the maximal
TNF response, zacrp3 treated cells showed about 50% inhibition.
Zacrp3 also had an effect, although less, 10% inhibition of
E-selection expression.
[0213] VCAM-1 expression was measured following direct adenovirus
infection of TRBMEC cells. Briefly, TRBMEC cells were directly
infected with an adenovirus containing zacrp3 or the parental
adenovirus strain. The virus was added at various multiplicities of
infection (moi 500, 1,000 and 5,000). Cells were incubated at
37.degree. C., 5% CO.sub.2 for 43 hours. Following infection, the
adenovirus-infected cells were challenged with TNF.alpha. (1 ng/ml)
for four hours. VCAM expression was measured as described above.
Inhibition of VCAM-1 expression was about 13% at moi 5000, about 5%
at moi 1000 and no effect was seen at moi 500.
[0214] Adenovirus conditioned media was concentrated 10.times.
(Centricon Centrifugal Filtration Unit 5,000K cutoff, Millipore
Corp., Bedford, Mass.) according to the manufacturer's instructions
followed by heat inactivation at 56.degree. C. for 30 minutes). The
concentrated heat inactivated samples were assayed as described
above. For VCAM-1 and E-selectin, inhibition was 100%. Similar
results were observed when either IL-1 or LPS was used to induce
adhesion molecule expression. For ICAM-1, inhibition was reduced to
nearly baseline. The experiment was repeated with varying
concentrations of zacrp3 heat inactivated adenovirus conditioned
media. Complete inhibition was seen at 5.times., 50% inhibition at
2.5.times. and no inhibition at 0.5.times..
[0215] A THP-1 monocyte adherence assay according to Ouchi et al.,
(ibid.) and Cybulsky and Gimbrone, (Science 251:788-91, 1991)
showed the same results as were seen for VCAM-1 above.
Sequence CWU 1
1
20 1 1696 DNA Homo sapiens CDS (69)...(806) 1 cccgaggaga ccacgctcct
ggagctctgc tgtcttctca gggagactct gaggctctgt 60 tgagaatc atg ctt tgg
agg cag ctc atc tat tgg caa ctg ctg gct ttg 110 Met Leu Trp Arg Gln
Leu Ile Tyr Trp Gln Leu Leu Ala Leu 1 5 10 ttt ttc ctc cct ttt tgc
ctg tgt caa gat gaa tac atg gag tct cca 158 Phe Phe Leu Pro Phe Cys
Leu Cys Gln Asp Glu Tyr Met Glu Ser Pro 15 20 25 30 caa acc gga gga
cta ccc cca gac tgc agt aag tgt tgt cat gga gac 206 Gln Thr Gly Gly
Leu Pro Pro Asp Cys Ser Lys Cys Cys His Gly Asp 35 40 45 tac agc
ttt cga ggc tac caa ggc ccc cct ggg cca ccg ggc cct cct 254 Tyr Ser
Phe Arg Gly Tyr Gln Gly Pro Pro Gly Pro Pro Gly Pro Pro 50 55 60
ggc att cca gga aac cat gga aac aat ggc aac aat gga gcc act ggt 302
Gly Ile Pro Gly Asn His Gly Asn Asn Gly Asn Asn Gly Ala Thr Gly 65
70 75 cat gaa gga gcc aaa ggt gag aag ggc gac aaa ggt gac ctg ggg
cct 350 His Glu Gly Ala Lys Gly Glu Lys Gly Asp Lys Gly Asp Leu Gly
Pro 80 85 90 cga ggg gag cgg ggg cag cat ggc ccc aaa gga gag aag
ggc tac ccg 398 Arg Gly Glu Arg Gly Gln His Gly Pro Lys Gly Glu Lys
Gly Tyr Pro 95 100 105 110 ggg att cca cca gaa ctt cag att gca ttc
atg gct tct ctg gca acc 446 Gly Ile Pro Pro Glu Leu Gln Ile Ala Phe
Met Ala Ser Leu Ala Thr 115 120 125 cac ttc agc aat cag aac agt ggg
att atc ttc agc agt gtt gag acc 494 His Phe Ser Asn Gln Asn Ser Gly
Ile Ile Phe Ser Ser Val Glu Thr 130 135 140 aac att gga aac ttc ttt
gat gtc atg act ggt aga ttt ggg gcc cca 542 Asn Ile Gly Asn Phe Phe
Asp Val Met Thr Gly Arg Phe Gly Ala Pro 145 150 155 gta tca ggt gtg
tat ttc ttc acc ttc agc atg atg aag cat gag gat 590 Val Ser Gly Val
Tyr Phe Phe Thr Phe Ser Met Met Lys His Glu Asp 160 165 170 gtt gag
gaa gtg tat gtg tac ctt atg cac aat ggc aac aca gtc ttc 638 Val Glu
Glu Val Tyr Val Tyr Leu Met His Asn Gly Asn Thr Val Phe 175 180 185
190 agc atg tac agc tat gaa atg aag ggc aaa tca gat aca tcc agc aat
686 Ser Met Tyr Ser Tyr Glu Met Lys Gly Lys Ser Asp Thr Ser Ser Asn
195 200 205 cat gct gtg ctg aag cta gcc aaa ggg gat gag gtt tgg ctg
cga atg 734 His Ala Val Leu Lys Leu Ala Lys Gly Asp Glu Val Trp Leu
Arg Met 210 215 220 ggc aat ggc gct ctc cat ggg gac cac caa cgc ttc
tcc acc ttt gca 782 Gly Asn Gly Ala Leu His Gly Asp His Gln Arg Phe
Ser Thr Phe Ala 225 230 235 gga ttc ctg ctc ttt gaa act aag
taaatatatg actagaatag ctccactttg 836 Gly Phe Leu Leu Phe Glu Thr
Lys 240 245 gggaagactt gtagctgagc tgatttgtta cgatctgagg aacattaaag
ttgagggttt 896 tacattgctg tattcaaaaa attattggtt gcaatgttgt
tcacgctaca ggtacaccaa 956 taatgttgga caattcaggg gctcagaaga
atcaaccaca aaatagtctt ctcagatgac 1016 cttgactaat atactcagca
tctttatcac tctttccttg gcacctaaaa gataattctc 1076 ctctgacgca
ggttggaaat atttttttct atcacagaag tcatttgcaa agaattttga 1136
ctactctgct tttaatttaa taccagtttt caggaacccc tgaagtttta agttcattat
1196 tctttataac atttgagaga atcggatgta gtgatatgac agggctgggg
caagaacagg 1256 ggcactagct gccttattag ctaatttagt gccctccgtg
ttcagcttag cctttgaccc 1316 tttccttttg atccacaaaa tacattaaaa
ctctgaattc acatacaatg ctattttaaa 1376 gtcaatagat tttagctata
aagtgcttga ccagtaatgt ggttgtaatt ttgtgtatgt 1436 tcccccacat
cgcccccaac ttcggatgtg gggtcaggag gttgaggttc actattaaca 1496
aatgtcataa atatctcata gaggtacagt gccaatagat attcaaatgt tgcatgttga
1556 ccagagggat tttatatctg aagaacatac actattaata aataccttag
agaaagattt 1616 tgacctggct ttagataaaa ctgtggcaag aaaaatgtaa
tgagcaatat atggaaataa 1676 acacaccttt gttaaagata 1696 2 246 PRT
Homo sapiens 2 Met Leu Trp Arg Gln Leu Ile Tyr Trp Gln Leu Leu Ala
Leu Phe Phe 1 5 10 15 Leu Pro Phe Cys Leu Cys Gln Asp Glu Tyr Met
Glu Ser Pro Gln Thr 20 25 30 Gly Gly Leu Pro Pro Asp Cys Ser Lys
Cys Cys His Gly Asp Tyr Ser 35 40 45 Phe Arg Gly Tyr Gln Gly Pro
Pro Gly Pro Pro Gly Pro Pro Gly Ile 50 55 60 Pro Gly Asn His Gly
Asn Asn Gly Asn Asn Gly Ala Thr Gly His Glu 65 70 75 80 Gly Ala Lys
Gly Glu Lys Gly Asp Lys Gly Asp Leu Gly Pro Arg Gly 85 90 95 Glu
Arg Gly Gln His Gly Pro Lys Gly Glu Lys Gly Tyr Pro Gly Ile 100 105
110 Pro Pro Glu Leu Gln Ile Ala Phe Met Ala Ser Leu Ala Thr His Phe
115 120 125 Ser Asn Gln Asn Ser Gly Ile Ile Phe Ser Ser Val Glu Thr
Asn Ile 130 135 140 Gly Asn Phe Phe Asp Val Met Thr Gly Arg Phe Gly
Ala Pro Val Ser 145 150 155 160 Gly Val Tyr Phe Phe Thr Phe Ser Met
Met Lys His Glu Asp Val Glu 165 170 175 Glu Val Tyr Val Tyr Leu Met
His Asn Gly Asn Thr Val Phe Ser Met 180 185 190 Tyr Ser Tyr Glu Met
Lys Gly Lys Ser Asp Thr Ser Ser Asn His Ala 195 200 205 Val Leu Lys
Leu Ala Lys Gly Asp Glu Val Trp Leu Arg Met Gly Asn 210 215 220 Gly
Ala Leu His Gly Asp His Gln Arg Phe Ser Thr Phe Ala Gly Phe 225 230
235 240 Leu Leu Phe Glu Thr Lys 245 3 244 PRT Homo sapiens 3 Met
Leu Leu Leu Gly Ala Val Leu Leu Leu Leu Ala Leu Pro Gly His 1 5 10
15 Asp Gln Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro
20 25 30 Lys Gly Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His
Pro Gly 35 40 45 His Asn Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly
Thr Pro Gly Glu 50 55 60 Lys Gly Glu Lys Gly Asp Pro Gly Leu Ile
Gly Pro Lys Gly Asp Ile 65 70 75 80 Gly Glu Thr Gly Val Pro Gly Ala
Glu Gly Pro Arg Gly Phe Pro Gly 85 90 95 Ile Gln Gly Arg Lys Gly
Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg 100 105 110 Ser Ala Phe Ser
Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met 115 120 125 Pro Ile
Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp 130 135 140
Gly Ser Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe 145
150 155 160 Ala Tyr His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser
Leu Phe 165 170 175 Lys Lys Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln
Tyr Gln Glu Asn 180 185 190 Asn Val Asp Gln Ala Ser Gly Ser Val Leu
Leu His Leu Glu Val Gly 195 200 205 Asp Gln Val Trp Leu Gln Val Tyr
Gly Glu Gly Glu Arg Asn Gly Leu 210 215 220 Tyr Ala Asp Asn Asp Asn
Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr 225 230 235 240 His Asp Thr
Asn 4 245 PRT Homo sapiens 4 Met Asp Val Gly Pro Ser Ser Leu Pro
His Leu Gly Leu Lys Leu Leu 1 5 10 15 Leu Leu Leu Leu Leu Leu Ala
Leu Arg Gly Gln Ala Asn Thr Gly Cys 20 25 30 Tyr Gly Ile Pro Gly
Met Pro Gly Leu Pro Gly Ala Pro Gly Lys Asp 35 40 45 Gly Tyr Asp
Gly Leu Pro Gly Pro Lys Gly Glu Pro Gly Ile Pro Ala 50 55 60 Ile
Pro Gly Ile Arg Gly Pro Lys Gly Gln Lys Gly Glu Pro Gly Leu 65 70
75 80 Pro Gly His Pro Gly Lys Asn Gly Pro Met Gly Pro Pro Gly Met
Pro 85 90 95 Gly Val Pro Gly Pro Met Gly Ile Pro Gly Glu Pro Gly
Glu Glu Gly 100 105 110 Arg Tyr Lys Gln Lys Phe Gln Ser Val Phe Thr
Val Thr Arg Gln Thr 115 120 125 His Gln Pro Pro Ala Pro Asn Ser Leu
Ile Arg Phe Asn Ala Val Leu 130 135 140 Thr Asn Pro Gln Gly Asp Tyr
Asp Thr Ser Thr Gly Lys Phe Thr Cys 145 150 155 160 Lys Val Pro Gly
Leu Tyr Tyr Phe Val Tyr His Ala Ser His Thr Ala 165 170 175 Asn Leu
Cys Val Leu Leu Tyr Arg Ser Gly Val Lys Val Val Thr Phe 180 185 190
Cys Gly His Thr Ser Lys Thr Asn Gln Val Asn Ser Gly Gly Val Leu 195
200 205 Leu Arg Leu Gln Val Gly Glu Glu Val Trp Leu Ala Val Asn Asp
Tyr 210 215 220 Tyr Asp Met Val Gly Ile Gln Gly Ser Asp Ser Val Phe
Ser Gly Phe 225 230 235 240 Leu Leu Phe Pro Asp 245 5 31 PRT
Artificial Sequence Aromatic motif 5 Phe Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Phe Xaa Xaa
Xaa Xaa Xaa Gly Xaa Tyr Xaa Phe Xaa Xaa 20 25 30 6 17 DNA
Artificial Sequence Degenerate nucleotide primer 6 ggngansarg
tntggyt 17 7 18 DNA Artificial Sequence Degenerate nucleotide
primer 7 sngnnntnta ytwyttyr 18 8 17 DNA Artificial Sequence
Degenerate nucleotide primer 8 ttydsnggnt tyytnht 17 9 18 DNA
Artificial Sequence Degenerate nucleotide primer 9 ytwyrayrbn
wbnwsngg 18 10 738 DNA Artificial Sequence Degenerate nucleotide
sequence encoding the polypeptide of SEQ ID NO2 10 atgytntggm
gncarytnat htaytggcar ytnytngcny tnttyttyyt nccnttytgy 60
ytntgycarg aygartayat ggarwsnccn caracnggng gnytnccncc ngaytgywsn
120 aartgytgyc ayggngayta ywsnttymgn ggntaycarg gnccnccngg
nccnccnggn 180 ccnccnggna thccnggnaa ycayggnaay aayggnaaya
ayggngcnac nggncaygar 240 ggngcnaarg gngaraargg ngayaarggn
gayytnggnc cnmgnggnga rmgnggncar 300 cayggnccna arggngaraa
rggntayccn ggnathccnc cngarytnca rathgcntty 360 atggcnwsny
tngcnacnca yttywsnaay caraaywsng gnathathtt ywsnwsngtn 420
garacnaaya thggnaaytt yttygaygtn atgacnggnm gnttyggngc nccngtnwsn
480 ggngtntayt tyttyacntt ywsnatgatg aarcaygarg aygtngarga
rgtntaygtn 540 tayytnatgc ayaayggnaa yacngtntty wsnatgtayw
sntaygarat gaarggnaar 600 wsngayacnw snwsnaayca ygcngtnytn
aarytngcna arggngayga rgtntggytn 660 mgnatgggna ayggngcnyt
ncayggngay caycarmgnt tywsnacntt ygcnggntty 720 ytnytnttyg aracnaar
738 11 1117 DNA Mus musculus CDS (111)...(848) 11 gctgcagctc
tcatctccaa acctggcatt tgcctgaggc gaccacggta cctccagccc 60
ctgtcaagct tccctgcgag actcttgtcg atttgccgat ttgccgagcc atg ctc 116
Met Leu 1 ggg agg cag cgc atc tgg tgg cac ctg ctg cct ttg ctt ttc
ctc cca 164 Gly Arg Gln Arg Ile Trp Trp His Leu Leu Pro Leu Leu Phe
Leu Pro 5 10 15 ttt tgc ctg tgt caa gat gaa tac atg gag tct cca caa
gct gga gga 212 Phe Cys Leu Cys Gln Asp Glu Tyr Met Glu Ser Pro Gln
Ala Gly Gly 20 25 30 ctg ccc cca gac tgc agc aag tgt tgc cat gga
gat tat gga ttt cgt 260 Leu Pro Pro Asp Cys Ser Lys Cys Cys His Gly
Asp Tyr Gly Phe Arg 35 40 45 50 ggt tac caa ggg ccc cct gga cct cca
ggt cct cct ggc att cca gga 308 Gly Tyr Gln Gly Pro Pro Gly Pro Pro
Gly Pro Pro Gly Ile Pro Gly 55 60 65 aac cat gga aac aat ggg aac
aat gga gct act ggc cat gaa ggg gcc 356 Asn His Gly Asn Asn Gly Asn
Asn Gly Ala Thr Gly His Glu Gly Ala 70 75 80 aaa ggt gag aaa gga
gac aaa ggc gac cta ggc cct cga gga gaa cgg 404 Lys Gly Glu Lys Gly
Asp Lys Gly Asp Leu Gly Pro Arg Gly Glu Arg 85 90 95 ggg cag cat
ggc ccc aaa gga gag aaa ggc tac cca ggg gtg cca cca 452 Gly Gln His
Gly Pro Lys Gly Glu Lys Gly Tyr Pro Gly Val Pro Pro 100 105 110 gaa
ctg cag att gca ttc atg gct tct cta gca act cac ttc agc aat 500 Glu
Leu Gln Ile Ala Phe Met Ala Ser Leu Ala Thr His Phe Ser Asn 115 120
125 130 cag aac agt ggc att atc ttc agc agt gtt gag acc aac att gga
aac 548 Gln Asn Ser Gly Ile Ile Phe Ser Ser Val Glu Thr Asn Ile Gly
Asn 135 140 145 ttc ttc gat gtc atg act ggg aga ttt ggg gcc ccc gta
tca ggt gtg 596 Phe Phe Asp Val Met Thr Gly Arg Phe Gly Ala Pro Val
Ser Gly Val 150 155 160 tat ttc ttc acc ttc agc atg atg aag cat gag
gac gta gag gaa gtg 644 Tyr Phe Phe Thr Phe Ser Met Met Lys His Glu
Asp Val Glu Glu Val 165 170 175 tat gtg tac ctt atg cac aac ggc aac
aca gtc ttc agc atg tac agc 692 Tyr Val Tyr Leu Met His Asn Gly Asn
Thr Val Phe Ser Met Tyr Ser 180 185 190 tat gaa aca aag gga aaa tca
gat aca tcc agc aac cat gca gtg ctg 740 Tyr Glu Thr Lys Gly Lys Ser
Asp Thr Ser Ser Asn His Ala Val Leu 195 200 205 210 aag ttg gcc aaa
gga gat gaa gtc tgg cta aga atg ggc aac ggt gcc 788 Lys Leu Ala Lys
Gly Asp Glu Val Trp Leu Arg Met Gly Asn Gly Ala 215 220 225 ctc cac
ggg gac cac cag cgc ttc tcc acc ttc gca ggc ttt ctg ctc 836 Leu His
Gly Asp His Gln Arg Phe Ser Thr Phe Ala Gly Phe Leu Leu 230 235 240
ttt gaa act aag tgacaaggaa gacaggatat ctccactttg ggggcaattt 888 Phe
Glu Thr Lys 245 atagctgagc tagggttgtt aggatatgaa ggatgttgaa
gtcgggggtt ctttatggag 948 catttaagtg ttgcattggt cacactgcta
ctcattctaa tggcatacca ataatgttgg 1008 atgcttcagg ggctcactgc
tactcattct aatggcatac caataatgtt ggatgcttca 1068 ggggctcact
gctactcatt ctaatggcaa taccaataat gttggatgc 1117 12 246 PRT Mus
musculus 12 Met Leu Gly Arg Gln Arg Ile Trp Trp His Leu Leu Pro Leu
Leu Phe 1 5 10 15 Leu Pro Phe Cys Leu Cys Gln Asp Glu Tyr Met Glu
Ser Pro Gln Ala 20 25 30 Gly Gly Leu Pro Pro Asp Cys Ser Lys Cys
Cys His Gly Asp Tyr Gly 35 40 45 Phe Arg Gly Tyr Gln Gly Pro Pro
Gly Pro Pro Gly Pro Pro Gly Ile 50 55 60 Pro Gly Asn His Gly Asn
Asn Gly Asn Asn Gly Ala Thr Gly His Glu 65 70 75 80 Gly Ala Lys Gly
Glu Lys Gly Asp Lys Gly Asp Leu Gly Pro Arg Gly 85 90 95 Glu Arg
Gly Gln His Gly Pro Lys Gly Glu Lys Gly Tyr Pro Gly Val 100 105 110
Pro Pro Glu Leu Gln Ile Ala Phe Met Ala Ser Leu Ala Thr His Phe 115
120 125 Ser Asn Gln Asn Ser Gly Ile Ile Phe Ser Ser Val Glu Thr Asn
Ile 130 135 140 Gly Asn Phe Phe Asp Val Met Thr Gly Arg Phe Gly Ala
Pro Val Ser 145 150 155 160 Gly Val Tyr Phe Phe Thr Phe Ser Met Met
Lys His Glu Asp Val Glu 165 170 175 Glu Val Tyr Val Tyr Leu Met His
Asn Gly Asn Thr Val Phe Ser Met 180 185 190 Tyr Ser Tyr Glu Thr Lys
Gly Lys Ser Asp Thr Ser Ser Asn His Ala 195 200 205 Val Leu Lys Leu
Ala Lys Gly Asp Glu Val Trp Leu Arg Met Gly Asn 210 215 220 Gly Ala
Leu His Gly Asp His Gln Arg Phe Ser Thr Phe Ala Gly Phe 225 230 235
240 Leu Leu Phe Glu Thr Lys 245 13 18 DNA Artificial Sequence
Oligonucleotide ZC21913 13 tgaccagagg gattttat 18 14 18 DNA
Artificial Sequence Oligonucleotide ZC21914 14 ttgccacagt tttatcta
18 15 24 DNA Artificial Sequence Oligonucleotide ZC23377 15
ctctgttggg atccatgctt tgga 24 16 22 DNA Artificial Sequence ZC23378
16 gtcatctaga tacttagttt ca 22 17 7 PRT Artificial Sequence Glu-Glu
tag 17 Glu Glu Tyr Met Pro Met Glu 1 5 18 17 DNA Artificial
Sequence ZC447 18 taacaatttc acacagg 17 19 18 DNA Artificial
Sequence Oligonucleotide ZC976 19 cgttgtaaaa cgacggcc 18 20 6 PRT
Artificial Sequence purification peptide 20 Glu Tyr Met Pro Val Asp
1 5
* * * * *
References