U.S. patent application number 11/858835 was filed with the patent office on 2008-03-13 for methods and reagents for diagnosis and treatment of diabetes.
This patent application is currently assigned to Metabolex, Inc.. Invention is credited to John E. Blume, Jeffrey D. JOHNSON, John F. Palma, Yun-Ping Zhou.
Application Number | 20080064857 11/858835 |
Document ID | / |
Family ID | 39361032 |
Filed Date | 2008-03-13 |
United States Patent
Application |
20080064857 |
Kind Code |
A1 |
JOHNSON; Jeffrey D. ; et
al. |
March 13, 2008 |
Methods and Reagents for Diagnosis and Treatment of Diabetes
Abstract
The invention relates to methods and reagents for diagnosing and
treating diabetes.
Inventors: |
JOHNSON; Jeffrey D.;
(Moraga, CA) ; Blume; John E.; (Danville, CA)
; Palma; John F.; (San Ramon, CA) ; Zhou;
Yun-Ping; (San Ramon, CA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Assignee: |
Metabolex, Inc.
Hayward
CA
|
Family ID: |
39361032 |
Appl. No.: |
11/858835 |
Filed: |
September 20, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11591922 |
Nov 2, 2006 |
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11858835 |
Sep 20, 2007 |
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10308393 |
Dec 2, 2002 |
7144985 |
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11591922 |
Nov 2, 2006 |
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60336633 |
Dec 3, 2001 |
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Current U.S.
Class: |
530/387.9 ;
530/391.1; 530/391.3 |
Current CPC
Class: |
H04L 63/062 20130101;
H04W 4/80 20180201; H04W 12/04 20130101; H04W 60/00 20130101 |
Class at
Publication: |
530/387.9 ;
530/391.1; 530/391.3 |
International
Class: |
C07K 16/00 20060101
C07K016/00 |
Claims
1. An isolated antibody that binds to SEQ ID NO:2.
2. The isolated antibody of claim 1, wherein the antibody binds to
SEQ ID NO:9.
3. The isolated antibody of claim 1, wherein the antibody binds to
SEQ ID NO:10.
4. The isolated antibody of claim 1, wherein the antibody binds to
SEQ ID NO:11.
5. The isolated antibody of claim 1, wherein the antibody binds to
SEQ ID NO:12.
6. The isolated antibody of claim 1, wherein the antibody binds to
SEQ ID NO:13.
7. The isolated antibody of claim 1, wherein the antibody binds to
SEQ ID NO:14.
8. The isolated antibody of claim 1, wherein the antibody is
monoclonal.
9. The isolated antibody of claim 1, wherein the antibody is
polyclonal.
10. The isolated antibody of claim 1, wherein the antibody is bound
to a solid substrate.
11. The isolated antibody of claim 1, wherein the antibody is
detectably labeled.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/591,992, filed Nov. 1, 2006, which is a divisional of
U.S. patent application Ser. No. 10/308,393, filed Dec. 2, 2002,
now U.S. Pat. No. 7,144,985, which claims priority to U.S.
Provisional Patent Application No. 60/336,633, filed on Dec. 3,
2001, each of which is incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0002] This invention relates to methods and reagents useful for
treatment and diagnosis of diabetes.
BACKGROUND OF THE INVENTION
[0003] Diabetes mellitus can be divided into two clinical
syndromes, Type 1 and Type 2 diabetes mellitus. Type 1, or
insulin-dependent diabetes mellitus (IDDM), is a chronic autoimmune
disease characterized by the extensive loss of beta cells in the
pancreatic Islets of Langerhans (hereinafter referred to as
"pancreatic islet cells" or "islet cells"), which produce insulin.
As these cells are progressively destroyed, the amount of secreted
insulin decreases, eventually leading to hyperglycemia (abnormally
high level of glucose in the blood) when the amount secreted drops
below the level required for euglycemia (normal blood glucose
level). Although the exact trigger for this immune response is not
known, patients with IDDM have high levels of antibodies against
pancreatic beta cells. However, not all patients with high levels
of these antibodies develop IDDM.
[0004] Type 2 diabetes develops when muscle, fat and liver cells
fail to respond normally to insulin. This failure to respond
(called insulin resistance) may be due to reduced numbers of
insulin receptors on these cells, or a dysfunction of signaling
pathways within the cells, or both. The beta cells initially
compensate for this insulin resistance by increasing their insulin
output. Over time, these cells become unable to produce enough
insulin to maintain normal glucose levels, indicating progression
to type 2 diabetes.
[0005] Type 2 diabetes is brought on by a combination of poorly
understood genetic and acquired risk factors--including a high-fat
diet, lack of exercise, and aging. Worldwide, type 2 diabetes has
become an epidemic, driven by increases in obesity and a sedentary
lifestyle, widespread adoption of western dietary habits, and the
general aging of the populations in many countries. In 1985, an
estimated 30 million people worldwide had diabetes--by 2000, this
figure had increased 5-fold, to an estimated 154 million people.
The number of people with diabetes is expected double between now
and 2025, to about 300 million.
[0006] There is no cure for diabetes. Conventional treatments for
diabetes are very limited, and focus on attempting to control blood
glucose levels in order to minimize or delay complications. The
present invention addresses these and other problems.
BRIEF SUMMARY OF THE INVENTION
[0007] This invention provides isolated nucleic acids encoding an
Archipelin polypeptide. In some embodiments, the Archipelin
polypeptide is at least 60% identical to SEQ ID NO:2. In some
embodiments, the nucleic acid encodes SEQ ID NO:2. In some
embodiments, wherein the nucleic acid comprises SEQ ID NO:1. In
some embodiments, the nucleic acid encodes SEQ ID NO:7. In some
embodiments, the nucleic acid comprises SEQ ID NO:8. In some
embodiments, the nucleic acid encodes SEQ ID NO:6. In some
embodiments, the nucleic acid comprises SEQ ID NO:5. In some
embodiments, the polypeptide comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some
embodiments, the nucleic acid is amplified by a primer set selected
from the group consisting of GCGATGTTGAAGAAGAAGTTC (SEQ ID NO:15)
and ATCGCCAAGGCCAAGA (SEQ ID NO:16).
[0008] The present invention also provides expression cassettes
comprising a promoter operably linked to a nucleic acid encoding an
polypeptide at least 605 identical to SEQ ID NO:9.
[0009] The present invention also provides isolated nucleic acids
that specifically hybridizes following at least one wash in
0.2.times.SSC at 55.degree. C. for 20 minutes to a probe comprising
SEQ ID NO:1.
[0010] The present invention also provides isolated polypeptides
comprising an amino acid sequence at least 60% identical to SEQ ID
NO:9. In some embodiments, the polypeptide comprises an amino acid
sequence selected from the group consisting of SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.
In some embodiments, the polypeptide comprises SEQ ID NO:2. In some
embodiments, the polypeptide comprises SEQ ID NO:6. In some
embodiments, the polypeptide specifically binds to antibodies
generated against SEQ ID NO:9.
[0011] The present invention also provides antibodies that
specifically hybridizes to Archipelin polypeptides. In some
embodiments, polypeptides comprising an amino acid sequence at
least 60% identical to SEQ ID NO:9.
[0012] The present invention also provides host cells transfected
with a nucleic acid encoding an polypeptide at least 60% identical
to SEQ ID NO:9. In some embodiments, the cell is a pancreatic islet
cell.
[0013] The present invention also provides pharmaceutical
compositions comprising insulin and a polypeptide at least 60%
identical to SEQ ID NO:9. In some embodiments, the polypeptide
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID
NO:13, and SEQ ID NO:14. In some embodiments, the polypeptide
comprises SEQ ID NO:2. In some embodiments, the pharmaceutical
composition is suitable for injection.
[0014] The present invention also provides methods of diagnosing
type 1 or type 2 diabetes or a predisposition for type 1 or type 2
diabetes in a patient. In some embodiments, the methods comprise
detecting the level of a polypeptide at least 60% identical to SEQ
ID NO:9 in a sample from the patient, wherein a modulated level of
the polypeptide in the sample compared to a level of the
polypeptide in a non-diabetic individual indicates that the patient
is diabetic or is predisposed for at least some pathological
aspects of diabetes. In some embodiments, the modulated level of
the polypeptide in the sample is lower than a level of the
polypeptide in a non-diabetic individual. In some embodiments, the
modulated level of the polypeptide in the sample is higher than a
level of the polypeptide in a non-diabetic individual. In some
embodiments, the polypeptide comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some
embodiments, the polypeptide comprises SEQ ID NO:2. In some
embodiments, the polypeptide is detected by an antibody. In some
embodiments, the level of the polypeptide in the patient is less
than 50% of the level from the non-diabetic individual. In some
embodiments, the level of the polypeptide in the patient is at
least 150% of the level from the non-diabetic individual.
[0015] The present invention also provides methods of treating a
patient diagnosed with type 1 or type 2 diabetes. In some
embodiments, the method comprise administering to the patient a
pharmaceutical composition comprising a therapeutically effective
amount of a compound selected from the group consisting of an
agonist of Archipelin and an agent that increases expression of
Archipelin. In some embodiments, the compound is an agonist of
Archipelin. In some embodiments, the compound is an agent that
increases expression of Archipelin. In some embodiments, the
agonist is a polypeptide at least 60% identical to SEQ ID NO:9. In
some embodiments, the agonist comprises a polypeptide comprising
SEQ ID NO:2. In some embodiments, the polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and
SEQ ID NO:14. In some embodiments, the pharmaceutical composition
comprises insulin. In some embodiments, the pharmaceutical
composition is administered parenterally. In some embodiments, the
pharmaceutical composition is administered by injection. In some
embodiments, the pharmaceutical composition is administered by a
pump device.
[0016] The present invention also provides methods of modulating
Archipelin activity in a cell. In some embodiments, the methods
comprise introducing into a pancreatic islet cell an expression
cassette comprising a promoter operably linked to a polynucleotide
encoding a polypeptide at least 60% identical to SEQ ID NO:9. In
some embodiments, the polypeptide comprises SEQ ID NO:2. In some
embodiments, the polypeptide comprises an amino acid sequence
selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10,
SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some
embodiments, the cell is introduced into a patient. In some
embodiments, the cell is from the patient. In some embodiments, the
expression cassette is contained in a viral vector.
[0017] The present invention also provides methods of identifying
an agent useful for the treatment of diabetes. In some embodiments,
the methods comprise contacting a cell with an agent; and selecting
an agent that modulates the expression in the cell of a polypeptide
at least 60% identical to SEQ ID NO:9. In some embodiments, the
polypeptide comprises an amino acid sequence selected from the
group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some embodiments, the
cell is from a diabetic animal. In some embodiments, the diabetic
animal is a human. In some embodiments, the cell is a pancreatic
islet cell. In some embodiments, the polypeptide comprises SEQ ID
NO:2. In some embodiments, the expression of the polypeptide is
increased following the contacting step.
[0018] The present invention also provides methods of treating a
patient diagnosed with type 1 or type 2 diabetes. In some
embodiments, the methods comprise administering a therapeutically
effective amount of an agent which was identified by contacting a
cell with an agent; and selecting an agent that modulates the
expression in the cell of a polypeptide at least 60% identical to
SEQ ID NO:9. In some embodiments, the agent increases the
expression of the polypeptide in the patient.
DEFINITIONS
[0019] "Antibody" refers to a polypeptide substantially encoded by
an immunoglobulin gene or immunoglobulin genes, or fragments
thereof which specifically bind and recognize an analyte (antigen).
The recognized imnmunoglobulin genes include the kappa, lambda,
alpha, gamma, delta, epsilon and mu constant region genes, as well
as the myriad immunoglobulin variable region genes. Light chains
are classified as either kappa or lambda. Heavy chains are
classified as gamma, mu, alpha, delta, or epsilon, which in turn
define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE,
respectively.
[0020] An exemplary immunoglobulin (antibody) structural unit
comprises a tetramer. Each tetramer is composed of two identical
pairs of polypeptide chains, each pair having one "light" (about 25
kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each
chain defines a variable region of about 100 to 110 or more amino
acids primarily responsible for antigen recognition. The terms
variable light chain (V.sub.L) and variable heavy chain (V.sub.H)
refer to these light and heavy chains respectively.
[0021] Antibodies exist, e.g., as intact immunoglobulins or as a
number of well characterized fragments produced by digestion with
various peptidases. Thus, for example, pepsin digests an antibody
below the disulfide linkages in the hinge region to produce
F(ab)'.sub.2, a dimer of Fab which itself is a light chain joined
to V.sub.H-C.sub.H1 by a disulfide bond. The F(ab)'.sub.2 may be
reduced under mild conditions to break the disulfide linkage in the
hinge region, thereby converting the F(ab)'.sub.2 dimer into an
Fab' monomer. The Fab' monomer is essentially an Fab with part of
the hinge region (see, Paul (Ed.) Fundamental Immunology, Third
Edition, Raven Press, NY (1993)). While various antibody fragments
are defined in terms of the digestion of an intact antibody, one of
skill will appreciate that such fragments may be synthesized de
novo either chemically or by utilizing recombinant DNA methodology.
Thus, the term antibody, as used herein, also includes antibody
fragments either produced by the modification of whole antibodies
or those synthesized de novo using recombinant DNA methodologies
(e.g., single chain Fv).
[0022] The terms "peptidomimetic" and "mimetic" refer to a
synthetic chemical compound that has substantially the same
structural and functional characteristics of the Archipelin
polypeptides of the invention. Peptide analogs are commonly used in
the pharmaceutical industry as non-peptide drugs with properties
analogous to those of the template peptide. These types of
non-peptide compound are termed "peptide mimetics" or
"peptidomimetics" (Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber
and Freidinger TINS p. 392 (1985); and Evans et al. J. Med. Chem.
30:1229 (1987), which are incorporated herein by reference).
Peptide mimetics that are structurally similar to therapeutically
useful peptides may be used to produce an equivalent or enhanced
therapeutic or prophylactic effect. Generally, peptidomimetics are
structurally similar to a paradigm polypeptide (i.e., a polypeptide
that has a biological or pharmacological activity), such as found
in Archipelin, but have one or more peptide linkages optionally
replaced by a linkage selected from the group consisting of, e.g.,
--CH2NH--, --CH2S--, --CH2-CH2-, --CH.dbd.CH-- (cis and trans),
--COCH2-, --CH(OH)CH2-, and --CH2SO--. The mimetic can be either
entirely composed of synthetic, non-natural analogues of amino
acids, or, is a chimeric molecule of partly natural peptide amino
acids and partly non-natural analogs of amino acids. The mimetic
can also incorporate any amount of natural amino acid conservative
substitutions as long as such substitutions also do not
substantially alter the mimetic's structure and/or activity. For
example, a mimetic composition is within the scope of the invention
if it is capable of carrying out the binding or enzymatic
activities of Archipelin.
[0023] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0024] The term "isolated," when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is essentially
free of other cellular components with which it is associated in
the natural state. It is preferably in a homogeneous state although
it can be in either a dry or aqueous solution. Purity and
homogeneity are typically determined using analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high
performance liquid chromatography. A protein which is the
predominant species present in a preparation is substantially
purified. In particular, an isolated gene is separated from open
reading frames which flank the gene and encode a protein other than
the gene of interest. The term "purified" denotes that a nucleic
acid or protein gives rise to essentially one band in an
electrophoretic gel. Particularly, it means that the nucleic acid
or protein is at least 85% pure, more preferably at least 95% pure,
and most preferably at least 99% pure.
[0025] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g., degenerate codon substitutions)
and complementary sequences as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608
(1985); and Cassol et al. (1992); Rossolini et al., Mol. Cell.
Probes 8:91-98 (1994)). The term nucleic acid is used
interchangeably with gene, cDNA, and mRNA encoded by a gene.
[0026] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymers. As used herein, the terms encompass amino acid
chains of any length, including full length proteins (i.e.,
antigens), wherein the amino acid residues are linked by covalent
peptide bonds.
[0027] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an a carbon that is bound to a hydrogen, a carboxyl
group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. "Amino acid mimetics" refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0028] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0029] An "Archipelin nucleic acid" or "Archipelin polynucleotide
sequence" of the invention is a subsequence or full-length
polynucleotide sequence of a gene that encodes a polypeptide
expressed in pancreatic islet cells. Exemplary Archipelin nucleic
acids of the invention include sequences substantially identical to
SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:8. Similarly,
"Archipelin polypeptide" or "Archipelin" refers to a polypeptide,
or fragment thereof, that is substantially identical to a
polypeptide encoded by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and
SEQ ID NO:8 (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:7, and SEQ ID NOs:9-14) or peptidomimetic compositions with
substantially the same activity as SEQ ID NO:2, SEQ ID NO:4, SEQ ID
NO:6, SEQ ID NO:7, and SEQ ID NOs:9-14.
[0030] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, "conservatively modified variants" refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence.
[0031] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0032] The following eight groups each contain amino acids that are
conservative substitutions for one another: [0033] 1) Alanine (A),
Glycine (G); [0034] 2) Aspartic acid (D), Glutamic acid (E); [0035]
3) Asparagine (N), Glutamine (Q); [0036] 4) Arginine (R), Lysine
(K); [0037] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine
(V); [0038] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
[0039] 7) Serine (S), Threonine (T); and [0040] 8) Cysteine (C),
Methionine (M) [0041] (see, e.g., Creighton, Proteins (1984)).
[0042] Macromolecular structures such as polypeptide structures can
be described in terms of various levels of organization. For a
general discussion of this organization, see, e.g., Alberts et al.,
Molecular Biology of the Cell (3.sup.rd ed., 1994) and Cantor and
Schimmel, Biophysical Chemistry Part I. The Conformation of
Biological Macromolecules (1980). "Primary structure" refers to the
amino acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains.
Domains are portions of a polypeptide that form a compact unit of
the polypeptide and are typically 50 to 350 amino acids long.
Typical domains are made up of sections of lesser organization such
as stretches of .beta.-sheet and .alpha.-helices. "Tertiary
structure" refers to the complete three dimensional structure of a
polypeptide monomer. "Quaternary structure" refers to the three
dimensional structure formed by the noncovalent association of
independent tertiary units. Anisotropic terms are also known as
energy terms.
[0043] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide sequence in the comparison window
may comprise additions or deletions (i.e., gaps) as compared to the
reference sequence (which does not comprise additions or deletions)
for optimal alignment of the two sequences. The percentage is
calculated by determining the number of positions at which the
identical nucleic acid base or amino acid residue occurs in both
sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
window of comparison and multiplying the result by 100 to yield the
percentage of sequence identity.
[0044] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%,
90%, or 95% identity over a specified region), when compared and
aligned for maximum correspondence over a comparison window, or
designated region as measured using one of the following sequence
comparison algorithms or by manual alignment and visual inspection.
Such sequences are then said to be "substantially identical." This
definition also refers to the complement of a test sequence.
Optionally, the identity exists over a region that is at least
about 50 amino acids or nucleotides in length, or more preferably
over a region that is 75-100 amino acids or nucleotides in
length.
[0045] The term "similarity," or percent "similarity," in the
context of two or more polypeptide sequences, refer to two or more
sequences or subsequences that have a specified percentage of amino
acid residues that are either the same or similar as defined in the
8 conservative amino acid substitutions defined above (i.e., 60%,
optionally 65%, 70%, 75%, 80%, 85%, 90%, or 95% similar over a
specified region), when compared and aligned for maximum
correspondence over a comparison window, or designated region as
measured using one of the following sequence comparison algorithms
or by manual alignment and visual inspection. Such sequences are
then said to be "substantially similar." Optionally, this identity
exists over a region that is at least about 50 amino acids in
length, or more preferably over a region that is at least about
75-100 amino acids in length.
[0046] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
[0047] A "comparison window", as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment
algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by
the search for similarity method of Pearson and Lipman (1988) Proc.
Nat'l. Acad. Sci. USA 85:2444, by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by manual alignment and visual inspection
(see, e.g., Ausubel et al., Current Protocols in Molecular Biology
(1995 supplement)).
[0048] One example of a useful algorithm is PILEUP. PILEUP creates
a multiple sequence alignment from a group of related sequences
using progressive, pairwise alignments to show relationship and
percent sequence identity. It also plots a tree or dendogram
showing the clustering relationships used to create the alignment.
PILEUP uses a simplification of the progressive alignment method of
Feng and Doolittle (1987) J. Mol. Evol. 35:351-360. The method used
is similar to the method described by Higgins and Sharp (1989)
CABIOS 5:151-153. The program can align up to 300 sequences, each
of a maximum length of 5,000 nucleotides or amino acids. The
multiple alignment procedure begins with the pairwise alignment of
the two most similar sequences, producing a cluster of two aligned
sequences. This cluster is then aligned to the next most related
sequence or cluster of aligned sequences. Two clusters of sequences
are aligned by a simple extension of the pairwise alignment of two
individual sequences. The final alignment is achieved by a series
of progressive, pairwise alignments. The program is run by
designating specific sequences and their amino acid or nucleotide
coordinates for regions of sequence comparison and by designating
the program parameters. Using PILEUP, a reference sequence is
compared to other test sequences to determine the percent sequence
identity relationship using the following parameters: default gap
weight (3.00), default gap length weight (0.10), and weighted end
gaps. PILEUP can be obtained from the GCG sequence analysis
software package, e.g., version 7.0 (Devereaux et al. (1984) Nuc.
Acids Res. 12:387-395).
[0049] Another example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al. (1977) Nuc. Acids Res. 25:3389-3402, and Altschul et al.
(1990) J. Mol. Biol. 215:403-410, respectively. Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) or 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad.
Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0050] The BLAST algorithm also performs a statistical analysis of
the similarity between two sequences (see, e.g., Karlin and
Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One
measure of similarity provided by the BLAST algorithm is the
smallest sum probability (P(N)), which provides an indication of
the probability by which a match between two nucleotide or amino
acid sequences would occur by chance. For example, a nucleic acid
is considered similar to a reference sequence if the smallest sum
probability in a comparison of the test nucleic acid to the
reference nucleic acid is less than about 0.2, more preferably less
than about 0.01, and most preferably less than about 0.001.
[0051] An indication that two nucleic acid sequences or
polypeptides are substantially identical is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with the antibodies raised against the polypeptide encoded by the
second nucleic acid, as described below. Thus, a polypeptide is
typically substantially identical to a second polypeptide, for
example, where the two peptides differ only by conservative
substitutions. Another indication that two nucleic acid sequences
are substantially identical is that the two molecules or their
complements hybridize to each other under stringent conditions, as
described below. Yet another indication that two nucleic acid
sequences are substantially identical is that the same primers can
be used to amplify the sequence.
[0052] The phrase "selectively (or specifically) hybridizes to"
refers to the binding, duplexing, or hybridizing of a molecule only
to a particular nucleotide sequence under stringent hybridization
conditions when that sequence is present in a complex mixture
(e.g., total cellular or library DNA or RNA).
[0053] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acid, but to
no other sequences. Stringent conditions are sequence-dependent and
will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions will be those in which the salt concentration
is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M
sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the
temperature is at least about 30.degree. C. for short probes (e.g.,
10 to 50 nucleotides) and at least about 60.degree. C. for long
probes (e.g., greater than 50 nucleotides). Stringent conditions
may also be achieved with the addition of destabilizing agents such
as formamide. For selective or specific hybridization, a positive
signal is at least two times background, optionally 10 times
background hybridization. Exemplary stringent hybridization
conditions can be as following: 50% formamide, 5.times.SSC, and 1%
SDS, incubating at 42.degree. C., or 5.times.SSC, 1% SDS,
incubating at 65.degree. C., with wash in 0.2.times.SSC, and 0.1%
SDS at 65.degree. C. Such washes can be performed for 5, 15, 30,
60, 120, or more minutes.
[0054] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. Such washes can be performed for 5,
15, 30, 60, 120, or more minutes. A positive hybridization is at
least twice background. Those of ordinary skill will readily
recognize that alternative hybridization and wash conditions can be
utilized to provide conditions of similar stringency.
[0055] The phrase "a nucleic acid sequence encoding" refers to a
nucleic acid which contains sequence information for a structural
RNA such as rRNA, a tRNA, or the primary amino acid sequence of a
specific protein or peptide, or a binding site for a trans-acting
regulatory agent. This phrase specifically encompasses degenerate
codons (i.e., different codons which encode a single amino acid) of
the native sequence or sequences which may be introduced to conform
with codon preference in a specific host cell.
[0056] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(nonrecombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under-expressed or not expressed at
all.
[0057] The term "heterologous" when used with reference to portions
of a nucleic acid indicates that the nucleic acid comprises two or
more subsequences that are not found in the same relationship to
each other in nature. For instance, the nucleic acid is typically
recombinantly produced, having two or more sequences from unrelated
genes arranged to make a new functional nucleic acid, e.g., a
promoter from one source and a coding region from another source.
Similarly, a heterologous protein indicates that the protein
comprises two or more subsequences that are not found in the same
relationship to each other in nature (e.g., a fusion protein).
[0058] A "promoter" is defined as an array of nucleic acid control
sequences that direct transcription of a nucleic acid. As used
herein, a promoter includes necessary nucleic acid sequences near
the start site of transcription, such as, in the case of a
polymerase II type promoter, a TATA element. A promoter also
optionally includes distal enhancer or repressor elements, which
can be located as much as several thousand base pairs from the
start site of transcription. A "constitutive" promoter is a
promoter that is active under most environmental and developmental
conditions. An "inducible" promoter is a promoter that is active
under environmental or developmental regulation. The term "operably
linked" refers to a functional linkage between a nucleic acid
expression control sequence (such as a promoter, or array of
transcription factor binding sites) and a second nucleic acid
sequence, wherein the expression control sequence directs
transcription of the nucleic acid corresponding to the second
sequence.
[0059] An "expression vector" is a nucleic acid construct,
generated recombinantly or synthetically, with a series of
specified nucleic acid elements that permit transcription of a
particular nucleic acid in a host cell. The expression vector can
be part of a plasmid, virus, or nucleic acid fragment. Typically,
the expression vector includes a nucleic acid to be transcribed
operably linked to a promoter.
[0060] The phrase "specifically (or selectively) binds to an
antibody" or "specifically (or selectively) immunoreactive with",
when referring to a protein or peptide, refers to a binding
reaction which is determinative of the presence of the protein in
the presence of a heterogeneous population of proteins and other
biologics. Thus, under designated immunoassay conditions, the
specified antibodies bind to a particular protein and do not bind
in a significant amount to other proteins present in the sample.
Specific binding to an antibody under such conditions may require
an antibody that is selected for its specificity for a particular
protein. For example, antibodies raised against a protein having an
amino acid sequence encoded by any of the polynucleotides of the
invention can be selected to obtain antibodies specifically
immunoreactive with that protein and not with other proteins,
except for polymorphic variants. A variety of immunoassay formats
may be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays,
Western blots, or immunohistochemistry are routinely used to select
monoclonal antibodies specifically immunoreactive with a protein.
See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, NY (1988) for a description of immunoassay
formats and conditions that can be used to determine specific
immunoreactivity. Typically, a specific or selective reaction will
be at least twice the background signal or noise and more typically
more than 10 to 100 times background.
[0061] "Inhibitors," "activators," and "modulators" of Archipelin
expression or of Archipelin activity are used to refer to
inhibitory, activating, or modulating molecules, respectively,
identified using in vitro and in vivo assays for Archipelin
expression or Archipelin signaling, e.g., ligands, agonists,
antagonists, and their homologs and mimetics. Modulators include
inhibitors and activators. Inhibitors are compounds that, e.g.,
inhibit expression of Archipelin or bind to, partially or totally
block stimulation, decrease, prevent, delay activation, inactivate,
desensitize, or down regulate the activity of Archipelin or that
bind or down regulate a receptor to which Archipelin binds. e.g.,
antagonists. Activators are compounds that, e.g., induce or
activate the expression of a Archipelin or bind to, stimulate,
increase, open, activate, facilitate, enhance activation, sensitize
or up regulate the activity of Archipelin or that bind or up
regulate a receptor to which Archipelin binds, e.g., agonists.
Modulators include compounds that, e.g., alter the interaction of
Archipelin with: extracellular proteins that bind activators or
inhibitors, receptors, including G-proteins coupled-receptors
(GPCRs), kinases, etc. Modulators include genetically modified
versions of Archipelin, e.g., with altered activity, as well as
naturally occurring and synthetic ligands, antagonists, agonists,
small chemical molecules and the like. Such assays for inhibitors
and activators include, e.g., applying putative modulator compounds
to pancreatic islet cells, in the presence or absence of Archipelin
and then determining the functional effects on Archipelin
signaling, as described above. Samples or assays comprising
Archipelin that are treated with a potential activator, inhibitor,
or modulator are compared to control samples without the inhibitor,
activator, or modulator to examine the extent of inhibition.
Control samples (untreated with inhibitors) are assigned a relative
Archipelin activity value of 100%. Inhibition of a Archipelin is
achieved when the Archipelin activity value relative to the control
is less than about 80%, optionally less than about 50% or less than
about 25-0%. Activation of a Archipelin is achieved when the
Archipelin activity value relative to the control is at least about
110%, optionally at least about 150%, optionally at least about
200-500%, or at least about 1000-3000% or higher.
BRIEF DESCRIPTION OF THE DRAWINGS
[0062] FIG. 1 illustrates the expression pattern of probe set
MBXRATISL12276 in various tissues of rat.
[0063] FIG. 2 illustrates the expression pattern of probe set
MBXRATISL12276 in various tissues of mouse.
[0064] FIG. 3 illustrates an amino acid alignment of Archipelin
sequences (SEQ ID NOS:33 and 34) compared to peptides from the CRF
peptide family (SEQ ID NOS:17-32).
[0065] FIG. 4 illustrates potential amino-terminal and
carboxyl-terminal cleavage sites of the human Archipelin proprotein
(SEQ ID NO:2). Potential amino terminal cleavage sites are marked
with a "/". Potential carboxyl terminal cleavage sites are marked
with a "!" Mature human Archipelin peptides=SEQ ID NOS:35-40.
[0066] FIG. 5 illustrates potential amino-terminal and
carboxyl-terminal cleavage sites of the mouse Archipelin proprotein
(SEQ ID NO:4). Potential amino terminal cleavage sites are marked
with a "/". Potential carboxyl terminal cleavage sites are marked
with a "!". Mature mouse Archipelin peptides=SEQ ID NOS:41-45.
[0067] FIG. 6 illustrates potential amino-terminal and
carboxyl-terminal cleavage sites of the rat Archipelin proprotein
(SEQ ID NO:6). Potential amino terminal cleavage sites are marked
with a "/". Potential carboxyl terminal cleavage sites are marked
with a "!". Mature rat Archipelin peptides=SEQ ID NOS:41, 42, 46,
47, 45 and 48, respectively.
[0068] FIG. 7 illustrates detection of Archipelin in human serum
using surface enhanced laser desorption ionization (SELDI) mass
spectroscopy.
[0069] FIG. 8 illustrates detection of Archipelin (SEQ ID
NOS:49-52) in rat serum using surface enhanced laser desorption
ionization (SELDI) mass spectroscopy.
DETAILED DESCRIPTION
I. Introduction
[0070] This invention is directed to new polypeptide and
polynucleotide sequences, designated Archipelin sequences, as well
as methods of using the sequences to diagnose and treat diabetes.
The present method also provides methods of identifying modulators
of Archipelin expression and activity. Such modulators are useful
for treating type 1 and type 2 diabetes as well as the pathological
aspects of such diseases.
II. General Recombinant Nucleic Acids Methods for Use with the
Invention
[0071] In numerous embodiments of the present invention, nucleic
acids encoding a Archipelin of interest will be isolated and cloned
using recombinant methods. Such embodiments are used, e.g., to
isolate Archipelin polynucleotides (e.g., SEQ ID NO:1, SEQ ID NO:3,
SEQ ID NO:5, and SEQ ID NO:8) for protein expression or during the
generation of variants, derivatives, expression cassettes, or other
sequences derived from an Archipelin polypeptide (e.g., SEQ ID
NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NOs:9-14),
to monitor Archipelin gene expression, for the isolation or
detection of Archipelin sequences in different species, for
diagnostic purposes in a patient, e.g., to detect mutations in
Archipelin or to detect expression levels of Archipelin nucleic
acids or Archipelin polypeptides. In some embodiments, the
sequences encoding the Archipelin of the invention are operably
linked to a heterologous promoter. In one embodiment, the nucleic
acids of the invention are from any mammal, including, in
particular, e.g., a human, a mouse, a rat, etc.
[0072] A. General Recombinant Nucleic Acids Methods
[0073] This invention relies on routine techniques in the field of
recombinant genetics. Basic texts disclosing the general methods of
use in this invention include Sambrook et al., Molecular Cloning, A
Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and
Expression: A Laboratory Manual (1990); and Current Protocols in
Molecular Biology (Ausubel et al., eds., 1994)).
[0074] For nucleic acids, sizes are given in either kilobases (kb)
or base pairs (bp). These are estimates derived from agarose or
acrylamide gel electrophoresis, from sequenced nucleic acids, or
from published DNA sequences. For proteins, sizes are given in
kilodaltons (kDa) or amino acid residue numbers. Proteins sizes are
estimated from gel electrophoresis, from sequenced proteins, from
derived amino acid sequences, or from published protein
sequences.
[0075] Oligonucleotides that are not commercially available can be
chemically synthesized according to the solid phase phosphoramidite
triester method first described by Beaucage & Caruthers,
Tetrahedron Letts. 22:1859-1862 (1981), using an automated
synthesizer, as described in Van Devanter et. al., Nucleic Acids
Res. 12:6159-6168 (1984). Purification of oligonucleotides is by
either native acrylamide gel electrophoresis or by anion-exchange
HPLC as described in Pearson & Reanier, J. Chrom. 255:137-149
(1983).
[0076] The sequence of the cloned genes and synthetic
oligonucleotides can be verified after cloning using, e.g., the
chain termination method for sequencing double-stranded templates
of Wallace et al., Gene 16:21-26 (1981).
[0077] B. Cloning Methods for the Isolation of Nucleotide Sequences
Encoding the Desired Proteins
[0078] In general, the nucleic acids encoding the subject proteins
are cloned from DNA sequence libraries that are made to encode copy
DNA (cDNA) or genomic DNA. The particular sequences can be located
by hybridizing with an oligonucleotide probe, the sequence of which
can be derived from the sequences provided herein (e.g., SEQ ID
NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:8), which provides a
reference for PCR primers and defines suitable regions for
isolating Archipelin-specific probes. Alternatively, where the
sequence is cloned into an expression library, the expressed
recombinant protein can be detected immunologically with antisera
or purified antibodies made against the Archipelin of interest.
[0079] Methods for making and screening genomic and cDNA libraries
are well known to those of skill in the art (see, e.g., Gubler and
Hoffman Gene 25:263-269 (1983); Benton and Davis Science,
196:180-182 (1977); and Sambrook, supra). A islet cells are an
example of suitable cells to isolate Archipelin RNA and cDNA.
[0080] Briefly, to make the cDNA library, one should choose a
source that is rich in mRNA. The mRNA can then be made into cDNA,
ligated into a recombinant vector, and transfected into a
recombinant host for propagation, screening and cloning. For a
genomic library, the DNA is extracted from a suitable tissue and
either mechanically sheared or enzymatically digested to yield
fragments of preferably about 5-100 kb. The fragments are then
separated by gradient centrifugation from undesired sizes and are
constructed in bacteriophage lambda vectors. These vectors and
phage are packaged in vitro, and the recombinant phages are
analyzed by plaque hybridization. Colony hybridization is carried
out as generally described in Grunstein et al., Proc. Natl. Acad.
Sci. USA., 72:3961-3965 (1975).
[0081] An alternative method combines the use of synthetic
oligonucleotide primers with polymerase extension on an mRNA or DNA
template. Suitable primers can be designed from specific Archipelin
sequences, e.g., the sequences described in SEQ ID NO:1, SEQ ID
NO:3, SEQ ID NO:5, and SEQ ID NO:8. This polymerase chain reaction
(PCR) method amplifies the nucleic acids encoding the protein of
interest directly from mRNA, cDNA, genomic libraries or cDNA
libraries. Restriction endonuclease sites can be incorporated into
the primers. Polymerase chain reaction or other in vitro
amplification methods may also be useful, for example, to clone
nucleic acids encoding specific proteins and express said proteins,
to synthesize nucleic acids that will be used as probes for
detecting the presence of mRNA encoding an Archipelin polypeptide
of the invention in physiological samples, for nucleic acid
sequencing, or for other purposes (see, U.S. Pat. Nos. 4,683,195
and 4,683,202). Genes amplified by a PCR reaction can be purified
from agarose gels and cloned into an appropriate vector.
[0082] Appropriate primers and probes for identifying the genes
encoding an Archipelin polypeptide of the invention from mammalian
tissues can be derived from the sequences provided herein, in
particular SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:8.
For a general overview of PCR, see, Innis et al. PCR Protocols: A
Guide to Methods and Applications, Academic Press, San Diego
(1990).
[0083] Synthetic oligonucleotides can be used to construct genes.
This is done using a series of overlapping oligonucleotides,
usually 40-120 bp in length, representing both the sense and
anti-sense strands of the gene. These DNA fragments are then
annealed, ligated and cloned.
[0084] A gene encoding an Archipelin polypeptide of the invention
can be cloned using intermediate vectors before transformation into
mammalian cells for expression. These intermediate vectors are
typically prokaryote vectors or shuttle vectors. The proteins can
be expressed in either prokaryotes, using standard methods well
known to those of skill in the art, or eukaryotes as described
infra.
[0085] C. Expression in Prokaryotes and Eukaryotes
[0086] To obtain high level expression of a cloned gene, such as
cDNAs encoding Archipelin, one typically subclones polynucleotides
encoding Archipelin into an expression vector that contains a
strong promoter to direct transcription, a
transcription/translation terminator, and if for a nucleic acid
encoding a protein, a ribosome binding site for translational
initiation. Suitable bacterial promoters are well known in the art
and described, e.g., in Sambrook et al. and Ausubel et al.
Bacterial expression systems for expressing the Archipelin protein
are available in, e.g., E. coli, Bacillus sp., and Salmonella
(Palva et al., Gene 22:229-235 (1983); Mosbach et al., Nature
302:543-545 (1983). Kits for such expression systems are
commercially available. Eukaryotic expression systems for mammalian
cells, yeast, and insect cells are well known in the art and are
also commercially available.
[0087] Selection of the promoter used to direct expression of a
heterologous nucleic acid depends on the particular application.
The promoter is preferably positioned about the same distance from
the heterologous transcription start site as it is from the
transcription start site in its natural setting. As is known in the
art, however, some variation in this distance can be accommodated
without loss of promoter function.
[0088] In addition to the promoter, the expression vector typically
contains a transcription unit or expression cassette that contains
all the additional elements required for the expression of
Archipelin-encoding nucleic acid in host cells. A typical
expression cassette thus contains a promoter operably linked to the
nucleic acid sequence encoding Archipelin and signals required for
efficient polyadenylation of the transcript, ribosome binding
sites, and translation termination. Additional elements of the
cassette may include enhancers and, if genomic DNA is used as the
structural gene, introns with functional splice donor and acceptor
sites.
[0089] In addition to a promoter sequence, the expression cassette
should also contain a transcription termination region downstream
of the structural gene to provide for efficient termination. The
termination region may be obtained from the same gene as the
promoter sequence or may be obtained from different genes.
[0090] The particular expression vector used to transport the
genetic information into the cell is not particularly critical. Any
of the conventional vectors used for expression in eukaryotic or
prokaryotic cells may be used. Standard bacterial expression
vectors include plasmids such as pBR322 based plasmids, pSKF,
pET23D, and fusion expression systems such as GST and LacZ. Epitope
tags can also be added to recombinant proteins to provide
convenient methods of isolation, e.g., c-myc.
[0091] Expression vectors containing regulatory elements from
eukaryotic viruses are typically used in eukaryotic expression
vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors
derived from Epstein-Barr virus. Other exemplary eukaryotic vectors
include pMSG, pAV009/A.sup.+, pMTO10/A.sup.+, pMAMneo-5,
baculovirus pDSVE, and any other vector allowing expression of
proteins under the direction of the CMV promoter, SV40 early
promoter, SV40 later promoter, metallothionein promoter, murine
mammary tumor virus promoter, Rous sarcoma virus promoter,
polyhedrin promoter, or other promoters shown effective for
expression in eukaryotic cells.
[0092] Expression of proteins from eukaryotic vectors can be also
be regulated using inducible promoters. With inducible promoters,
expression levels are tied to the concentration of inducing agents,
such as tetracycline or ecdysone, by the incorporation of response
elements for these agents into the promoter. Generally, high level
expression is obtained from inducible promoters only in the
presence of the inducing agent; basal expression levels are
minimal. Inducible expression vectors are often chosen if
expression of the protein of interest is detrimental to eukaryotic
cells.
[0093] Some expression systems have markers that provide gene
amplification such as thymidine kinase and dihydrofolate reductase.
Alternatively, high yield expression systems not involving gene
amplification are also suitable, such as using a baculovirus vector
in insect cells, with an Archipelin-encoding sequence under the
direction of the polyhedrin promoter or other strong baculovirus
promoters.
[0094] The elements that are typically included in expression
vectors also include a replicon that functions in E. coli, a gene
encoding antibiotic resistance to permit selection of bacteria that
harbor recombinant plasmids, and unique restriction sites in
nonessential regions of the plasmid to allow insertion of
eukaryotic sequences. The particular antibiotic resistance gene
chosen is not critical, any of the many resistance genes known in
the art are suitable. The prokaryotic sequences are preferably
chosen such that they do not interfere with the replication of the
DNA in eukaryotic cells, if necessary.
[0095] Standard transfection methods are used to produce bacterial,
mammalian, yeast or insect cell lines that express large quantities
of Archipelin protein, which are then purified using standard
techniques (see, e.g., Colley et al., J. Biol. Chem.
264:17619-17622 (1989); Guide to Protein Purification, in Methods
in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of
eukaryotic and prokaryotic cells are performed according to
standard techniques (see, e.g., Morrison, J. Bact. 132:349-351
(1977); Clark-Curtiss & Curtiss, Methods in Enzymology
101:347-362 (Wu et al., eds, 1983).
[0096] Any of the well-known procedures for introducing foreign
nucleotide sequences into host cells may be used. These include the
use of calcium phosphate transfection, polybrene, protoplast
fusion, electroporation, liposomes, microinjection, plasma vectors,
viral vectors and any of the other well known methods for
introducing cloned genomic DNA, cDNA, synthetic DNA or other
foreign genetic material into a host cell (see, e.g., Sambrook et
al., supra). It is only necessary that the particular genetic
engineering procedure used be capable of successfully introducing
at least one gene into the host cell capable of expressing
Archipelin.
[0097] After the expression vector is introduced into the cells,
the transfected cells are cultured under conditions favoring
expression of Archipelin, which is recovered from the culture using
standard techniques identified below.
III. Purification of Proteins of the Invention
[0098] Either naturally occurring or recombinant Archipelin can be
purified for use in functional assays. Naturally occurring
Archipelin can be purified, e.g., from mouse or human tissue such
as islet cells or any other source of an Archipelin ortholog.
Recombinant Archipelin can be purified from any suitable expression
system.
[0099] The Archipelin may be purified to substantial purity by
standard techniques, including selective precipitation with such
substances as ammonium sulfate; column chromatography,
immunopurification methods, and others (see, e.g., Scopes, Protein
Purification: Principles and Practice (1982); U.S. Pat. No.
4,673,641; Ausubel et al., supra; and Sambrook et al., supra).
[0100] A number of procedures can be employed when recombinant
Archipelin are being purified. For example, proteins having
established molecular adhesion properties can be reversible fused
to Archipelin. With the appropriate ligand, Archipelin can be
selectively adsorbed to a purification column and then freed from
the column in a relatively pure form. The fused protein is then
removed by enzymatic activity. Finally Archipelin can be purified
using immunoaffinity columns.
[0101] A. Purification of Proteins from Recombinant Bacteria
[0102] When recombinant proteins are expressed by the transformed
bacteria in large amounts, typically after promoter induction,
although expression can be constitutive, the proteins may form
insoluble aggregates. There are several protocols that are suitable
for purification of protein inclusion bodies. For example,
purification of aggregate proteins (hereinafter referred to as
inclusion bodies) typically involves the extraction, separation
and/or purification of inclusion bodies by disruption of bacterial
cells typically, but not limited to, by incubation in a buffer of
about 100-150 .mu.g/ml lysozyme and 0.1% Nonidet P40, a non-ionic
detergent. The cell suspension can be ground using a Polytron
grinder (Brinkman Instruments, Westbury, N.Y.). Alternatively, the
cells can be sonicated on ice. Alternate methods of lysing bacteria
are described in Ausubel et al. and Sambrook et al., both supra,
and will be apparent to those of skill in the art.
[0103] The cell suspension is generally centrifuged and the pellet
containing the inclusion bodies resuspended in buffer which does
not dissolve but washes the inclusion bodies, e.g., 20 mM Tris-HCl
(pH 7.2), 1 mM EDTA, 150 mM NaCl and 2% Triton-X 100, a non-ionic
detergent. It may be necessary to repeat the wash step to remove as
much cellular debris as possible. The remaining pellet of inclusion
bodies may be resuspended in an appropriate buffer (e.g., 20 mM
sodium phosphate, pH 6.8, 150 mM NaCl). Other appropriate buffers
will be apparent to those of skill in the art.
[0104] Following the washing step, the inclusion bodies are
solubilized by the addition of a solvent that is both a strong
hydrogen acceptor and a strong hydrogen donor (or a combination of
solvents each having one of these properties). The proteins that
formed the inclusion bodies may then be renatured by dilution or
dialysis with a compatible buffer. Suitable solvents include, but
are not limited to, urea (from about 4 M to about 8 M), formamide
(at least about 80%, volume/volume basis), and guanidine
hydrochloride (from about 4 M to about 8 M). Some solvents that are
capable of solubilizing aggregate-forming proteins, such as SDS
(sodium dodecyl sulfate) and 70% formic acid, are inappropriate for
use in this procedure due to the possibility of irreversible
denaturation of the proteins, accompanied by a lack of
immunogenicity and/or activity. Although guanidine hydrochloride
and similar agents are denaturants, this denaturation is not
irreversible and renaturation may occur upon removal (by dialysis,
for example) or dilution of the denaturant, allowing re-formation
of the immunologically and/or biologically active protein of
interest. After solubilization, the protein can be separated from
other bacterial proteins by standard separation techniques.
[0105] Alternatively, it is possible to purify proteins from
bacteria periplasm. Where the protein is exported into the
periplasm of the bacteria, the periplasmic fraction of the bacteria
can be isolated by cold osmotic shock in addition to other methods
known to those of skill in the art (see, Ausubel et al., supra). To
isolate recombinant proteins from the periplasm, the bacterial
cells are centrifuged to form a pellet. The pellet is resuspended
in a buffer containing 20% sucrose. To lyse the cells, the bacteria
are centrifuged and the pellet is resuspended in ice-cold 5 mM
MgSO.sub.4 and kept in an ice bath for approximately 10 minutes.
The cell suspension is centrifuged and the supernatant decanted and
saved. The recombinant proteins present in the supernatant can be
separated from the host proteins by standard separation techniques
well known to those of skill in the art.
[0106] B. Purification of Proteins from Insect Cells
[0107] Proteins can also be purified from eukaryotic gene
expression systems as described in, e.g., Fernandez and Hoeffler,
Gene Expression Systems (1999). In some embodiments, baculovirus
expression systems are used to isolate Archipelin proteins or other
proteins of the invention. Recombinant cabulaoviruses are generally
generated by replacing the polyhedrin coding sequence of a
baculovirus with a gene to be expressed (e.g., an Archipelin
polynucleotide). Viruses lacking the polyhedrin gene have a unique
plaque morphology making them easy to recognize. In some
embodiments, a recombinant baculovirus is generated by first
cloning a polynucleotide of interest into a transfer vector (e.g.,
a pUC based vector) such that the polynucleotide is operably linked
to a polyhedrin promoter. The transfer vector is transfected with
wildtype DNA into an insect cell (e.g., Sf9, Sf21 or BT1-TN-5B1-4
cells), resulting in homologous recombination and replacement of
the polyhedrin gene in the wildtype viral DNA with the
polynucleotide of interest. Virus can then be generated and plaque
purified. Protein expression results upon viral infection of insect
cells. Expressed proteins can be harvested from cell supernatant if
secreted, or from cell lysates if intracellular. See, e.g., Ausubel
et al. and Fernandez and Hoeffler, supra.
[0108] C. Standard Protein Separation Techniques For Purifying
Proteins
[0109] 1. Solubility Fractionation
[0110] Often as an initial step, and if the protein mixture is
complex, an initial salt fractionation can separate many of the
unwanted host cell proteins (or proteins derived from the cell
culture media) from the recombinant protein of interest. The salt
used for fractionation can be, e.g., ammonium sulfate. Ammonium
sulfate precipitates proteins by effectively reducing the amount of
water in the protein mixture. Proteins then precipitate on the
basis of their solubility. The more hydrophobic a protein is, the
more likely it is to precipitate at lower ammonium sulfate
concentrations. A typical protocol is to add saturated ammonium
sulfate to a protein solution so that the resultant ammonium
sulfate concentration is between 20-30%. This will precipitate the
most hydrophobic proteins. The precipitate is discarded (unless the
protein of interest is hydrophobic) and ammonium sulfate is added
to the supernatant to a concentration known to precipitate the
protein of interest. The precipitate is then solubilized in buffer
and the excess salt removed if necessary, through either dialysis
or diafiltration. Other methods that rely on solubility of
proteins, such as cold ethanol precipitation, are well known to
those of skill in the art and can be used to fractionate complex
protein mixtures.
[0111] 2. Size Differential Filtration
[0112] Based on a calculated molecular weight, a protein of greater
and lesser size can be isolated using ultrafiltration through
membranes of different pore sizes (for example, Amicon or Millipore
membranes). As a first step, the protein mixture is ultrafiltered
through a membrane with a pore size that has a lower molecular
weight cut-off than the molecular weight of the protein of
interest. The retentate of the ultrafiltration is then
ultrafiltered against a membrane with a molecular cut off greater
than the molecular weight of the protein of interest. The
recombinant protein will pass through the membrane into the
filtrate. The filtrate can then be chromatographed as described
below.
[0113] 3. Column Chromatography
[0114] The proteins of interest can also be separated from other
proteins on the basis of their size, net surface charge,
hydrophobicity and affinity for ligands. In addition, antibodies
raised against proteins can be conjugated to column matrices and
the proteins immunopurified. All of these methods are well known in
the art.
[0115] It will be apparent to one of skill that chromatographic
techniques can be performed at any scale and using equipment from
many different manufacturers (e.g., Pharmacia Biotech).
IV. Detection of Gene Expression
[0116] Those of skill in the art will recognize that detection of
expression of Archipelin polynucleotides has many uses. For
example, as discussed herein, detection of Archipelin levels in a
patient is useful for diagnosing diabetes or a predisposition for
at least some of the pathological effects of diabetes.
[0117] A variety of methods of specific DNA and RNA measurement
using nucleic acid hybridization techniques are known to those of
skill in the art (see, Sambrook, supra). Some methods involve an
electrophoretic separation (e.g., Southern blot for detecting DNA,
and Northern blot for detecting RNA), but measurement of DNA and
RNA can also be carried out in the absence of electrophoretic
separation (e.g., by dot blot). Southern blot of genomic DNA (e.g.,
from a human) can be used for screening for restriction fragment
length polymorphism (RFLP) to detect the presence of a genetic
disorder affecting an Archipelin polypeptide of the invention.
[0118] The selection of a nucleic acid hybridization format is not
critical. A variety of nucleic acid hybridization formats are known
to those skilled in the art. For example, common formats include
sandwich assays and competition or displacement assays.
Hybridization techniques are generally described in Hames and
Higgins Nucleic Acid Hybridization, A Practical Approach, IRL Press
(1985); Gall and Pardue, Proc. Natl. Acad. Sci. U.S.A., 63:378-383
(1969); and John et al. Nature, 223:582-587 (1969).
[0119] Detection of a hybridization complex may require the binding
of a signal generating complex to a duplex of target and probe
polynucleotides or nucleic acids. Typically, such binding occurs
through ligand and anti-ligand interactions as between a
ligand-conjugated probe and an anti-ligand conjugated with a
signal. The binding of the signal generation complex is also
readily amenable to accelerations by exposure to ultrasonic
energy.
[0120] The label may also allow indirect detection of the
hybridization complex. For example, where the label is a hapten or
antigen, the sample can be detected by using antibodies. In these
systems, a signal is generated by attaching fluorescent or enzyme
molecules to the antibodies or in some cases, by attachment to a
radioactive label (see, e.g., Tijssen, "Practice and Theory of
Enzyme Immunoassays," Laboratory Techniques in Biochemistry and
Molecular Biology, Burdon and van Knippenberg Eds., Elsevier
(1985), pp. 9-20).
[0121] The probes are typically labeled either directly, as with
isotopes, chromophores, lumiphores, chromogens, or indirectly, such
as with biotin, to which a streptavidin complex may later bind.
Thus, the detectable labels used in the assays of the present
invention can be primary labels (where the label comprises an
element that is detected directly or that produces a directly
detectable element) or secondary labels (where the detected label
binds to a primary label, e.g., as is common in immunological
labeling). Typically, labeled signal nucleic acids are used to
detect hybridization. Complementary nucleic acids or signal nucleic
acids may be labeled by any one of several methods typically used
to detect the presence of hybridized polynucleotides. The most
common method of detection is the use of autoradiography with
.sup.3H, .sup.125I, .sup.35S, .sup.14C, or .sup.32P-labeled probes
or the like.
[0122] Other labels include, e.g., ligands which bind to labeled
antibodies, fluorophores, chemiluminescent agents, enzymes, and
antibodies which can serve as specific binding pair members for a
labeled ligand. An introduction to labels, labeling procedures and
detection of labels is found in Polak and Van Noorden Introduction
to Immunocytochemistry, 2nd ed., Springer Verlag, NY (1997); and in
Haugland Handbook of Fluorescent Probes and Research Chemicals, a
combined handbook and catalogue Published by Molecular Probes, Inc.
(1996).
[0123] In general, a detector which monitors a particular probe or
probe combination is used to detect the detection reagent label.
Typical detectors include spectrophotometers, phototubes and
photodiodes, microscopes, scintillation counters, cameras, film and
the like, as well as combinations thereof. Examples of suitable
detectors are widely available from a variety of commercial sources
known to persons of skill in the art. Commonly, an optical image of
a substrate comprising bound labeling moieties is digitized for
subsequent computer analysis.
[0124] Most typically, the amount of, for example, an Archipelin
RNA is measured by quantitating the amount of label fixed to the
solid support by binding of the detection reagent. Typically, the
presence of a modulator during incubation will increase or decrease
the amount of label fixed to the solid support relative to a
control incubation which does not comprise the modulator, or as
compared to a baseline established for a particular reaction type.
Means of detecting and quantitating labels are well known to those
of skill in the art.
[0125] In some embodiments, the target nucleic acid or the probe is
immobilized on a solid support. Solid supports suitable for use in
the assays of the invention are known to those of skill in the art.
As used herein, a solid support is a matrix of material in a
substantially fixed arrangement.
[0126] A variety of automated solid-phase assay techniques are also
appropriate. For instance, very large scale immobilized polymer
arrays (VLSIPS.TM.), available from Affymetrix, Inc. in Santa
Clara, Calif. can be used to detect changes in expression levels of
a plurality of genes involved in the same regulatory pathways
simultaneously. See, Tijssen, supra., Fodor et al. (1991) Science,
251: 767- 777; Sheldon et al. (1993) Clinical Chemistry 39(4):
718-719, and Kozal et al. (1996) Nature Medicine 2(7): 753-759.
[0127] Detection can be accomplished, for example, by using a
labeled detection moiety that binds specifically to duplex nucleic
acids (e.g., an antibody that is specific for RNA-DNA duplexes).
One example uses an antibody that recognizes DNA-RNA heteroduplexes
in which the antibody is linked to an enzyme (typically by
recombinant or covalent chemical bonding). The antibody is detected
when the enzyme reacts with its substrate, producing a detectable
product. Coutlee et al. (1989) Analytical Biochemistry 181:153-162;
Bogulavski (1986) et al. J. Immunol. Methods 89:123-130;
Prooijen-Knegt (1982) Exp. Cell Res. 141:397-407; Rudkin (1976)
Nature 265:472-473, Stollar (1970) PNAS 65:993-1000; Ballard (1982)
Mol. Immunol. 19:793-799; Pisetsky and Caster (1982) Mol. Immunol.
19:645-650; Viscidi et al. (1988) J. Clin. Microbial. 41:199-209;
and Kiney et al. (1989) J. Clin. Microbiol. 27:6-12 describe
antibodies to RNA duplexes, including homo and heteroduplexes. Kits
comprising antibodies specific for DNA:RNA hybrids are available,
e.g., from Digene Diagnostics, Inc. (Beltsville, Md.).
[0128] In addition to available antibodies, one of skill in the art
can easily make antibodies specific for nucleic acid duplexes using
existing techniques, or modify those antibodies which are
commercially or publicly available. In addition to the art
referenced above, general methods for producing polyclonal and
monoclonal antibodies are known to those of skill in the art (see,
e.g., Paul (ed) Fundamental Immunology, Third Edition Raven Press,
Ltd., NY (1993); Coligan Current Protocols in Immunology
Wiley/Greene, NY (1991); Harlow and Lane Antibodies: A Laboratory
Manual Cold Spring Harbor Press, NY (1989); Stites et al. (eds.)
Basic and Clinical Immunology (4th ed.) Lange Medical Publications,
Los Altos, Calif., and references cited therein; Goding Monoclonal
Antibodies: Principles and Practice (2d ed.) Academic Press, New
York, N.Y., (1986); and Kohler and Milstein Nature 256: 495-497
(1975)). Other suitable techniques for antibody preparation include
selection of libraries of recombinant antibodies in phage or
similar vectors (see, Huse et al. Science 246:1275-1281 (1989); and
Ward et al. Nature 341:544-546 (1989)). Specific monoclonal and
polyclonal antibodies and antisera will usually bind with a K.sub.D
of at least about 0.1 .mu.M, preferably at least about 0.01 .mu.M
or better, and most typically and preferably, 0.001 .mu.M or
better.
[0129] The nucleic acids used in this invention can be either
positive or negative probes. Positive probes bind to their targets
and the presence of duplex formation is evidence of the presence of
the target. Negative probes fail to bind to the suspect target and
the absence of duplex formation is evidence of the presence of the
target. For example, the use of a wild type specific nucleic acid
probe or PCR primers may serve as a negative probe in an assay
sample where only the nucleotide sequence of interest is
present.
[0130] The sensitivity of the hybridization assays may be enhanced
through use of a nucleic acid amplification system that multiplies
the target nucleic acid being detected. Examples of such systems
include the polymerase chain reaction (PCR) system and the ligase
chain reaction (LCR) system. Other methods described in the art are
the nucleic acid sequence based amplification (NASBA, Cangene,
Mississauga, Ontario) and Q Beta Replicase systems. These systems
can be used to directly identify mutants where the PCR or LCR
primers are designed to be extended or ligated only when a selected
sequence is present. Alternatively, the selected sequences can be
generally amplified using, for example, nonspecific PCR primers and
the amplified target region later probed for a specific sequence
indicative of a mutation.
[0131] An alternative means for determining the level of expression
of the nucleic acids of the present invention is in situ
hybridization. In situ hybridization assays are well known and are
generally described in Angerer et al., Methods Enzymol. 152:649-660
(1987). In an in situ hybridization assay, cells, preferentially
human pancreatic cells such as islet cells, are fixed to a solid
support, typically a glass slide. If DNA is to be probed, the cells
are denatured with heat or alkali. The cells are then contacted
with a hybridization solution at a moderate temperature to permit
annealing of specific probes that are labeled. The probes are
preferably labeled with radioisotopes or fluorescent reporters.
V. Immunological Detection of Archipelin
[0132] In addition to the detection of Archipelin genes and gene
expression using nucleic acid hybridization technology, one can
also use immunoassays to detect Archipelin polypeptides.
Immunoassays can be used to qualitatively or quantitatively analyze
Archipelin. A general overview of the applicable technology can be
found in Harlow & Lane, Antibodies. A Laboratory Manual
(1988).
[0133] A. Antibodies to Target Proteins
[0134] Methods for producing polyclonal and monoclonal antibodies
that react specifically with a protein of interest are known to
those of skill in the art (see, e.g., Coligan, supra; and Harlow
and Lane, supra; Stites et al., supra and references cited therein;
Goding, supra; and Kohler and Milstein Nature, 256:495-497 (1975)).
Such techniques include antibody preparation by selection of
antibodies from libraries of recombinant antibodies in phage or
similar vectors (see, Huse et al., supra; and Ward et al., supra).
For example, in order to produce antisera for use in an
immunoassay, the protein of interest or an antigenic fragment
thereof, is isolated as described herein. For example, a
recombinant protein is produced in a transformed cell line. An
inbred strain of mice or rabbits is immunized with the protein
using a standard adjuvant, such as Freund's adjuvant, and a
standard immunization protocol. Alternatively, a synthetic peptide
derived from the sequences disclosed herein and conjugated to a
carrier protein can be used as an immunogen.
[0135] Polyclonal sera are collected and titered against the
immunogen protein in an immunoassay, for example, a solid phase
immunoassay with the immunogen immobilized on a solid support.
Polyclonal antisera with a titer of 10.sup.4 or greater are
selected and tested for their crossreactivity against
non-Archipelin proteins or even other homologous proteins from
other organisms, using a competitive binding immunoassay. Specific
monoclonal and polyclonal antibodies and antisera will usually bind
with a K.sub.D of at least about 0.1 mM, more usually at least
about 1 .mu.M, preferably at least about 0.1 .mu.M or better, and
most preferably, 0.01 .mu.M or better.
[0136] For preparation of antibodies, e.g., recombinant,
monoclonal, or polyclonal antibodies, many technique known in the
art can be used (see, e.g., Kohler & Milstein, Nature
256:495-497 (1975); Kozbor et al., Immunology Today 4: 72 (1983);
Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy,
Alan R. Liss, Inc. (1985); Coligan, Current Protocols in Immunology
(1991); Harlow & Lane, Antibodies, A Laboratory Manual (1988);
and Goding, Monoclonal Antibodies: Principles and Practice (2d ed.
1986)). The genes encoding the heavy and light chains of an
antibody of interest can be cloned from a cell, e.g., the genes
encoding a monoclonal antibody can be cloned from a hybridoma and
used to produce a recombinant monoclonal antibody. Gene libraries
encoding heavy and light chains of monoclonal antibodies can also
be made from hybridoma or plasma cells. Random combinations of the
heavy and light chain gene products generate a large pool of
antibodies with different antigenic specificity (see, e.g., Kuby,
Immunology (3rd ed. 1997)). Techniques for the production of single
chain antibodies or recombinant antibodies (U.S. Pat. No.
4,946,778, U.S. Pat. No. 4,816,567) can be adapted to produce
antibodies to polypeptides of this invention. Also, transgenic
mice, or other organisms such as other mammals, may be used to
express humanized or human antibodies (see, e.g., U.S. Pat. Nos.
5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016,
Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al.,
Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994);
Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger,
Nature Biotechnology 14:826 (1996); and Lonberg & Huszar,
Intern. Rev. Immunol. 13:65-93 (1995)). Alternatively, phage
display technology can be used to identify antibodies and
heteromeric Fab fragments that specifically bind to selected
antigens (see, e.g., McCafferty et al., Nature 348:552-554 (1990);
Marks et al., Biotechnology 10:779-783 (1992)). Antibodies can also
be made bispecific, i.e., able to recognize two different antigens
(see, e.g., WO 93/08829, Traunecker et al., EMBO J. 10:3655-3659
(1991); and Suresh et al., Methods in Enzymology 121:210 (1986)).
Antibodies can also be heteroconjugates, e.g., two covalently
joined antibodies, or immunotoxins (see, e.g., U.S. Pat. No.
4,676,980, WO 91/00360; WO 92/200373; and EP 03089).
[0137] Methods for humanizing or primatizing non-human antibodies
are well known in the art. Generally, a humanized antibody has one
or more amino acid residues introduced into it from a source which
is non-human. These non-human amino acid residues are often
referred to as import residues, which are typically taken from an
import variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (see, e.g., Jones et
al., Nature 321:522-525 (1986); Riechmann et al., Nature
332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such humanized
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0138] A number of proteins of the invention comprising immunogens
may be used to produce antibodies specifically or selectively
reactive with the proteins of interest. Recombinant protein is an
exemplary immunogen for the production of monoclonal or polyclonal
antibodies. Naturally occurring protein may also be used either in
pure or impure form. Synthetic peptides made using the protein
sequences described herein may also be used as an immunogen for the
production of antibodies to the protein. Recombinant protein can be
expressed in eukaryotic or prokaryotic cells and purified as
generally described supra. The product is then injected into an
animal capable of producing antibodies. Either monoclonal or
polyclonal antibodies may be generated for subsequent use in
immunoassays to measure the protein.
[0139] Methods of production of polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, preferably a
purified protein, is mixed with an adjuvant and animals are
immunized. The animal's immune response to the immunogen
preparation is monitored by taking test bleeds and determining the
titer of reactivity to the Archipelin of interest. When
appropriately high titers of antibody to the immunogen are
obtained, blood is collected from the animal and antisera are
prepared. Further fractionation of the antisera to enrich for
antibodies reactive to the protein can be done if desired (see,
Harlow and Lane, supra).
[0140] Monoclonal antibodies may be obtained using various
techniques familiar to those of skill in the art. Typically, spleen
cells from an animal immunized with a desired antigen are
immortalized, commonly by fusion with a myeloma cell (see, Kohler
and Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative
methods of immortalization include, e.g., transformation with
Epstein Barr Virus, oncogenes, or retroviruses, or other methods
well known in the art. Colonies arising from single immortalized
cells are screened for production of antibodies of the desired
specificity and affinity for the antigen, and yield of the
monoclonal antibodies produced by such cells may be enhanced by
various techniques, including injection into the peritoneal cavity
of a vertebrate host. Alternatively, one may isolate DNA sequences
which encode a monoclonal antibody or a binding fragment thereof by
screening a DNA library from human B cells according to the general
protocol outlined by Huse et al., supra.
[0141] Once target protein specific antibodies are available, the
protein can be measured by a variety of immunoassay methods with
qualitative and quantitative results available to the clinician.
For a review of immunological and immunoassay procedures in general
see, Stites, supra. Moreover, the immunoassays of the present
invention can be performed in any of several configurations, which
are reviewed extensively in Maggio Enzyme Immunoassay, CRC Press,
Boca Raton, Fla. (1980); Tijssen, supra; and Harlow and Lane,
supra.
[0142] Immunoassays to measure target proteins in a human sample
may use a polyclonal antiserum which was raised to the protein
(e.g., SEQ ID NO:2, SEQ ID NO:7 and SEQ ID NOs:9-14) at least
partially encoded by a sequence described herein (e.g., SEQ ID NO:1
and SEQ ID NO:8) or a fragment thereof. This antiserum is selected
to have low cross-reactivity against non-Archipelin proteins and
any such cross-reactivity is removed by immunoabsorption prior to
use in the immunoassay.
[0143] Polyclonal antibodies that specifically bind to an
Archipelin of interest from a particular species can be made by
subtracting out cross-reactive antibodies using Archipelin
homologs. In an analogous fashion, antibodies specific to a
particular Archipelin (e.g., the human Archipelin polypeptide) can
be obtained in an organism with multiple Archipelin genes by
subtracting out cross-reactive antibodies using other
Archipelin.
[0144] B. Immunological Binding Assays
[0145] In some embodiments, a protein of interest is detected
and/or quantified using any of a number of well known immunological
binding assays (see, e.g., U.S. Pat. 4,366,241; 4,376,110;
4,517,288; and 4,837,168). For a review of the general
immunoassays, see also Asai Methods in Cell Biology Volume 37:
Antibodies in Cell Biology, Academic Press, Inc. NY (1993); Stites,
supra. Immunological binding assays (or immunoassays) typically
utilize a "capture agent" to specifically bind to and often
immobilize the analyte (in this case an Archipelin of the present
invention or antigenic subsequences thereof). The capture agent is
a moiety that specifically binds to the analyte. In some
embodiments, the capture agent is an antibody that specifically
binds, for example, an Archipelin polypeptide of the invention. The
antibody (e.g., anti-Archipelin antibody) may be produced by any of
a number of means well known to those of skill in the art and as
described above.
[0146] Immunoassays also often utilize a labeling agent to
specifically bind to and label the binding complex formed by the
capture agent and the analyte. The labeling agent may itself be one
of the moieties comprising the antibody/analyte complex. Thus, the
labeling agent may be a labeled Archipelin polypeptide or a labeled
anti-Archipelin receptor antibody. Alternatively, the labeling
agent may be a third moiety, such as another antibody, that
specifically binds to the antibody/protein complex.
[0147] In some embodiments, the labeling agent is a second antibody
bearing a label. Alternatively, the second antibody may lack a
label, but it may, in turn, be bound by a labeled third antibody
specific to antibodies of the species from which the second
antibody is derived. The second antibody can be modified with a
detectable moiety, such as biotin, to which a third labeled
molecule can specifically bind, such as enzyme-labeled
streptavidin.
[0148] Other proteins capable of specifically binding
immunoglobulin constant regions, such as protein A or protein G,
can also be used as the label agents. These proteins are normal
constituents of the cell walls of streptococcal bacteria. They
exhibit a strong non-immunogenic reactivity with immunoglobulin
constant regions from a variety of species (see, generally,
Kronval, et al. J. Immunol., 111: 1401-1406 (1973); and Akerstrom,
et al. J. Immunol., 135:2589-2542 (1985)).
[0149] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. The incubation time will depend
upon the assay format, analyte, volume of solution, concentrations,
and the like. Usually, the assays will be carried out at ambient
temperature, although they can be conducted over a range of
temperatures, such as 10.degree. C. to 40.degree. C.
[0150] 1. Non-Competitive Assay Formats
[0151] Immunoassays for detecting proteins of interest from tissue
samples may be either competitive or noncompetitive. Noncompetitive
immunoassays are assays in which the amount of captured analyte (in
this case the protein) is directly measured. In one "sandwich"
assay, for example, the capture agent (e.g., anti-Archipelin
antibodies) can be bound directly to a solid substrate where it is
immobilized. These immobilized antibodies then capture the
Archipelin present in the test sample. The Archipelin thus
immobilized is then bound by a labeling agent, such as a second
anti-Archipelin receptor antibody bearing a label. Alternatively,
the second antibody may lack a label, but it may, in turn, be bound
by a labeled third antibody specific to antibodies of the species
from which the second antibody is derived. The second can be
modified with a detectable moiety, such as biotin, to which a third
labeled molecule can specifically bind, such as enzyme-labeled
streptavidin.
[0152] 2. Competitive Assay Formats
[0153] In competitive assays, the amount of target protein
(analyte) present in the sample is measured indirectly by measuring
the amount of an added (exogenous) analyte (i.e., the Archipelin of
interest) displaced (or competed away) from a capture agent (i.e.,
anti antibody) by the analyte present in the sample. In one
competitive assay, a known amount of, in this case, the protein of
interest is added to the sample and the sample is then contacted
with a capture agent, in this case an antibody that specifically
binds to the Archipelin of interest. The amount of Archipelin bound
to the antibody is inversely proportional to the concentration of
Archipelin present in the sample. In some embodiments, the antibody
is immobilized on a solid substrate. The amount of the Archipelin
bound to the antibody may be determined either by measuring the
amount of subject protein present in a Archipelin protein/antibody
complex or, alternatively, by measuring the amount of remaining
uncomplexed protein. The amount of Archipelin protein may be
detected by providing a labeled Archipelin protein molecule.
[0154] A hapten inhibition assay is another exemplary competitive
assay. In this assay, a known analyte, in this case the target
protein, is immobilized on a solid substrate. A known amount of
anti-Archipelin antibody is added to the sample, and the sample is
then contacted with the immobilized target. In this case, the
amount of anti-Archipelin antibody bound to the immobilized
Archipelin is inversely proportional to the amount of Archipelin
protein present in the sample. Again, the amount of immobilized
antibody may be detected by detecting either the immobilized
fraction of antibody or the fraction of the antibody that remains
in solution. Detection may be direct where the antibody is labeled
or indirect by the subsequent addition of a labeled moiety that
specifically binds to the antibody as described above.
[0155] Immunoassays in the competitive binding format can be used
for cross-reactivity determinations. For example, the protein
encoded by the sequences described herein can be immobilized on a
solid support. Proteins are added to the assay which compete with
the binding of the antisera to the immobilized antigen. The ability
of the above proteins to compete with the binding of the antisera
to the immobilized protein is compared to that of the protein
encoded by any of the sequences described herein. The percent
cross-reactivity for the above proteins is calculated, using
standard calculations. Those antisera with less than 10%
cross-reactivity with each of the proteins listed above are
selected and pooled. The cross-reacting antibodies are optionally
removed from the pooled antisera by immunoabsorption with the
considered proteins, e.g., distantly related homologs.
[0156] The immunoabsorbed and pooled antisera are then used in a
competitive binding immunoassay as described above to compare a
second protein, thought to be perhaps a protein of the present
invention, to the immunogen protein. In order to make this
comparison, the two proteins are each assayed at a wide range of
concentrations and the amount of each protein required to inhibit
50% of the binding of the antisera to the immobilized protein is
determined. If the amount of the second protein required is less
than 10 times the amount of the protein partially encoded by a
sequence herein that is required, then the second protein is said
to specifically bind to an antibody generated to an immunogen
consisting of the target protein.
[0157] 3. Other Assay Formats
[0158] In some embodiments, western blot (immunoblot) analysis is
used to detect and quantify the presence of an Archipelin of the
invention in the sample. The technique generally comprises
separating sample proteins by gel electrophoresis on the basis of
molecular weight, transferring the separated proteins to a suitable
solid support (such as, e.g., a nitrocellulose filter, a nylon
filter, or a derivatized nylon filter) and incubating the sample
with the antibodies that specifically bind the protein of interest.
For example, the anti-Archipelin antibodies specifically bind to
the Archipelin on the solid support. These antibodies may be
directly labeled or alternatively may be subsequently detected
using labeled antibodies (e.g., labeled sheep anti-mouse
antibodies) that specifically bind to the antibodies against the
protein of interest.
[0159] Other assay formats include liposome immunoassays (LIA),
which use liposomes designed to bind specific molecules (e.g.,
antibodies) and release encapsulated reagents or markers. The
released chemicals are then detected according to standard
techniques (see, Monroe et al. (1986) Amer. Clin. Prod. Rev.
5:34-41).
[0160] 4. Labels
[0161] The particular label or detectable group used in the assay
is not a critical aspect of the invention, as long as it does not
significantly interfere with the specific binding of the antibody
used in the assay. The detectable group can be any material having
a detectable physical or chemical property. Such detectable labels
have been well-developed in the field of immunoassays and, in
general, most labels useful in such methods can be applied to the
present invention. Thus, a label is any composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels in the present
invention include magnetic beads (e.g., Dynabeads.TM.), fluorescent
dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and
the like), radiolabels (e.g., .sup.3H, .sup.125I, .sup.35S,
.sup.14C, or .sup.32P), enzymes (e.g., horse radish peroxidase,
alkaline phosphatase and others commonly used in an ELISA), and
colorimetric labels such as colloidal gold or colored glass or
plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
[0162] The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. As indicated above, a wide variety of labels may be used,
with the choice of label depending on the sensitivity required, the
ease of conjugation with the compound, stability requirements,
available instrumentation, and disposal provisions.
[0163] Non-radioactive labels are often attached by indirect means.
The molecules can also be conjugated directly to signal generating
compounds, e.g., by conjugation with an enzyme or fluorescent
compound. A variety of enzymes and fluorescent compounds can be
used with the methods of the present invention and are well-known
to those of skill in the art (for a review of various labeling or
signal producing systems which may be used, see, e.g., U.S. Pat.
No. 4,391,904).
[0164] Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter or
photographic film as in autoradiography. Where the label is a
fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the
resulting fluorescence. The fluorescence may be detected visually,
by means of photographic film, by the use of electronic detectors
such as charge coupled devices (CCDs) or photomultipliers and the
like. Similarly, enzymatic labels may be detected by providing the
appropriate substrates for the enzyme and detecting the resulting
reaction product. Finally simple colorimetric labels may be
detected directly by observing the color associated with the label.
Thus, in various dipstick assays, conjugated gold often appears
pink, while various conjugated beads appear the color of the
bead.
[0165] Some assay formats do not require the use of labeled
components. For instance, agglutination assays can be used to
detect the presence of the target antibodies. In this case,
antigen-coated particles are agglutinated by samples comprising the
target antibodies. In this format, none of the components need to
be labeled and the presence of the target antibody is detected by
simple visual inspection.
VI. Screening for Modulators of Archipelin
[0166] Modulators of Archipelin, i.e. agonists or antagonists or
agents of Archipelin activity or modulators of Archipelin
polypeptide or polynucleotide expression, are useful for treating a
number of human diseases, including diabetes. Administration of
Archipelin agonists or agents that increase expression of
Archipelin can be used to treat diabetic patients. For example,
insufficient Archipelin due to functional impairment of islets may
contribute to some of the pathologies associated with diabetes.
Thus, restoration of Archipelin ameliorates some of these
pathologies.
[0167] Conversely, under conditions of islet hyperactivity, such as
occurs in an insulin resistant states, islet expansion may lead to
overproduction of Archipelin. Overproduction leads to a different
set of deleterious physiological effects that can be relieved by
Archipelin antagonists. Archipelin agonists or antagonists may have
beneficial physiological effects in diabetes whether or not the
endogenous level of the peptide is abnormal.
[0168] A. Methods for identifying Modulators of Archipelin
[0169] A number of different screening protocols can be utilized to
identify agents that modulate the level of expression or activity
of Archipelin in cells, particularly mammalian cells, and
especially human cells. In general terms, the screening methods
involve screening a plurality of agents to identify an agent that
modulates the activity of Archipelin by binding to Archipelin,
preventing an inhibitor from binding to Archipelin or activating
expression of Archipelin, for example.
[0170] 1. Archipelin Binding Assays
[0171] Preliminary screens can be conducted by screening for
compounds capable of binding to Archipelin, as at least some of the
compounds so identified are likely Archipelin activators. The
binding assays usually involve contacting an Archipelin protein
with one or more test compounds and allowing sufficient time for
the protein and test compounds to form a binding complex. Any
binding complexes formed can be detected using any of a number of
established analytical techniques. Protein binding assays include,
but are not limited to, methods that measure co-precipitation,
co-migration on non-denaturing SDS-polyacrylamide gels, and
co-migration on Western blots (see, e.g., Bennet, J. P. and
Yamamura, H. I. (1985) "Neurotransmitter, Hormone or Drug Receptor
Binding Methods," in Neurotransmitter Receptor Binding (Yamamura,
H. I., et al., eds.), pp. 61-89) as well as phage display and other
binding assays known to those of skill in the art. The Archipelin
protein utilized in such assays can be naturally expressed, cloned
or synthesized Archipelin. In some embodiments, two hybrid assays,
or other expression-based in vivo binding assays can be used. See,
e.g., Fields, et al., Nature 340(6230):245-6 (1989).
[0172] Binding assays are also useful, e.g., for identifying
endogenous proteins that interact with Archipelin. For example,
receptors that bind Archipelin can be identified in binding
assays.
[0173] 2. Expression Assays
[0174] Certain screening methods involve screening for a compound
that up-regulates the expression of Archipelin. Such methods
generally involve conducting cell-based assays in which test
compounds are contacted with one or more cells expressing
Archipelin and then detecting an increase or decrease in Archipelin
expression (either transcript or translation product). Some assays
are performed with pancreatic islet cells, or other cells, that
express endogenous Archipelin.
[0175] Archipelin expression can be detected in a number of
different ways. As described herein, the expression level of
Archipelin in a cell can be determined by probing the mRNA
expressed in a cell with a probe that specifically hybridizes with
a transcript (or complementary nucleic acid derived therefrom) of
Archipelin. Probing can be conducted by lysing the cells and
conducting Northern blots or without lysing the cells using in
situ-hybridization techniques (see above). Alternatively,
Archipelin protein can be detected using immunological methods in
which a cell lysate is probe with antibodies that specifically bind
to Archipelin.
[0176] Other cell-based assays are reporter assays conducted with
cells that do not express Archipelin. Certain of these assays are
conducted with a heterologous nucleic acid construct that includes
a Archipelin promoter that is operably linked to a reporter gene
that encodes a detectable product. A number of different reporter
genes can be utilized. Some reporters are inherently detectable. An
example of such a reporter is green fluorescent protein that emits
fluorescence that can be detected with a fluorescence detector.
Other reporters generate a detectable product. Often such reporters
are enzymes. Exemplary enzyme reporters include, but are not
limited to, .beta.-glucuronidase, CAT (chloramphenicol acetyl
transferase; Alton and Vapnek (1979) Nature 282:864-869),
luciferase, .beta.-galactosidase and alkaline phosphatase (Toh, et
al. (1980) Eur. J. Biochem. 182:231-238; and Hall et al. (1983) J.
Mol. Appl. Gen. 2: 101).
[0177] In these assays, cells harboring the reporter construct are
contacted with a test compound. A test compound that either
modulates the activity of the promoter by binding to it or triggers
a cascade that produces a molecule that modulates the promoter
causes expression of the detectable reporter. Certain other
reporter assays are conducted with cells that harbor a heterologous
construct that includes a transcriptional control element that
activates expression of Archipelin and a reporter operably linked
thereto. Here, too, an agent that binds to the transcriptional
control element to activate expression of the reporter or that
triggers the formation of an agent that binds to the
transcriptional control element to activate reporter expression,
can be identified by the generation of signal associated with
reporter expression.
[0178] The level of expression or activity can be compared to a
baseline value. As indicated above, the baseline value can be a
value for a control sample or a statistical value that is
representative of Archipelin expression levels for a control
population (e.g., healthy individuals not having or at risk for
type 1 or type 2 diabetes). Expression levels can also be
determined for cells that do not express Archipelin as a negative
control. Such cells generally are otherwise substantially
genetically the same as the test cells.
[0179] A variety of different types of cells can be utilized in the
reporter assays. As stated above, certain cells are nerve cells
that express an endogenous Archipelin. Cells not expressing
Archipelin can be prokaryotic, but preferably are eukaryotic. The
eukaryotic cells can be any of the cells typically utilized in
generating cells that harbor recombinant nucleic acid constructs.
Exemplary eukaryotic cells include, but are not limited to, yeast,
and various higher eukaryotic cells such as the COS, CHO and HeLa
cell lines.
[0180] Various controls can be conducted to ensure that an observed
activity is authentic including running parallel reactions with
cells that lack the reporter construct or by not contacting a cell
harboring the reporter construct with test compound. Compounds can
also be further validated as described below.
[0181] 3. Validation
[0182] Compounds that are initially identified by any of the
foregoing screening methods can be further tested to validate the
apparent activity. Preferably such studies are conducted with
suitable animal models. The basic format of such methods involves
administering a lead compound identified during an initial screen
to an animal that serves as a model for humans and then determining
if Archipelin is in fact modulated. The animal models utilized in
validation studies generally are mammals of any kind. Specific
examples of suitable animals include, but are not limited to,
primates, mice and rats.
[0183] B. Modulators of Archipelin
[0184] The compounds tested as modulators of Archipelin can be any
small chemical compound, or a biological entity, such as a protein,
sugar, nucleic acid or lipid. Alternatively, modulators can be
genetically altered versions of an Archipelin gene or gene product.
Typically, test compounds will be small chemical molecules and
peptides. Essentially any chemical compound can be used as a
potential modulator or ligand in the assays of the invention,
although most often compounds that can be dissolved in aqueous or
organic (especially DMSO-based) solutions are used. The assays are
designed to screen large chemical libraries by automating the assay
steps and providing compounds from any convenient source to assays,
which are typically run in parallel (e.g., in microtiter formats on
microtiter plates in robotic assays). It will be appreciated that
there are many suppliers of chemical compounds, including Sigma
(St. Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St.
Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs,
Switzerland) and the like.
[0185] In some embodiments, high throughput screening methods
involve providing a combinatorial chemical or peptide library
containing a large number of potential therapeutic compounds
(potential modulator or ligand compounds). Such "combinatorial
chemical libraries" or "ligand libraries" are then screened in one
or more assays, as described herein, to identify those library
members (particular chemical species or subclasses) that display a
desired characteristic activity. The compounds thus identified can
serve as conventional "lead compounds" or can themselves be used as
potential or actual therapeutics.
[0186] A combinatorial chemical library is a collection of diverse
chemical compounds generated by either chemical synthesis or
biological synthesis, by combining a number of chemical "building
blocks" such as reagents. For example, a linear combinatorial
chemical library such as a polypeptide library is formed by
combining a set of chemical building blocks (amino acids) in every
possible way for a given compound length (i.e., the number of amino
acids in a polypeptide compound). Millions of chemical compounds
can be synthesized through such combinatorial mixing of chemical
building blocks.
[0187] Preparation and screening of combinatorial chemical
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al., Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used. Such chemistries include, but
are not limited to: peptoids (e.g., PCT Publication No. WO
91/19735), encoded peptides (e.g., PCT Publication WO 93/20242),
random bio-oligomers (e.g., PCT Publication No. WO 92/00091),
benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such
as hydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc.
Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides
(Hagihara et al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidal
peptidomimetics with glucose scaffolding (Hirschmann et al., J.
Amer. Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses
of small compound libraries (Chen et al., J. Amer. Chem. Soc.
116:2661 (1994)), oligocarbamates (Cho et al., Science 261:1303
(1993)), and/or peptidyl phosphonates (Campbell et al., J. Org.
Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger
and Sambrook, all supra), peptide nucleic acid libraries (see,
e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g.,
Vaughn et al., Nature Biotechnology, 14(3):309-314 (1996) and
PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,
Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), small
organic molecule libraries (see, e.g., benzodiazepines, Baum
C&EN, January 18, page 33 (1993); isoprenoids, U.S. Pat. No.
5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No.
5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134;
morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines,
U.S. Pat. No. 5,288,514, and the like).
[0188] Devices for the preparation of combinatorial libraries are
commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem
Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied
Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford,
Mass.). In addition, numerous combinatorial libraries are
themselves commercially available (see, e.g., ComGenex, Princeton,
N.J., Tripos, Inc., St. Louis, Mo., 3D Pharmaceuticals, Exton, Pa.,
Martek Biosciences, Columbia, Md., etc.).
[0189] C. Solid Phase and Soluble High Throughput Assays
[0190] In the high throughput assays of the invention, it is
possible to screen up to several thousand different modulators or
ligands in a single day. In particular, each well of a microtiter
plate can be used to run a separate assay against a selected
potential modulator, or, if concentration or incubation time
effects are to be observed, every 5-10 wells can test a single
modulator. Thus, a single standard microtiter plate can assay about
100 (e.g., 96) modulators. If 1536 well plates are used, then a
single plate can easily assay from about 100 to about 1500
different compounds. It is possible to assay several different
plates per day; assay screens for up to about 6,000-20,000
different compounds are possible using the integrated systems of
the invention. More recently, microfluidic approaches to reagent
manipulation have been developed.
[0191] The molecule of interest can be bound to the solid state
component, directly or indirectly, via covalent or non covalent
linkage, e.g., via a tag. The tag can be any of a variety of
components. In general, a molecule that binds the tag (a tag
binder) is fixed to a solid support, and the tagged molecule of
interest (e.g., Archipelin) is attached to the solid support by
interaction of the tag and the tag binder.
[0192] A number of tags and tag binders can be used, based upon
known molecular interactions well described in the literature. For
example, where a tag has a natural binder, for example, biotin,
protein A, or protein G, it can be used in conjunction with
appropriate tag binders (avidin, streptavidin, neutravidin, the Fc
region of an immunoglobulin, etc.) Antibodies to molecules with
natural binders such as biotin are also widely available and
appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue
SIGMA, St. Louis Mo.).
[0193] Similarly, any haptenic or antigenic compound can be used in
combination with an appropriate antibody to form a tag/tag binder
pair. Thousands of specific antibodies are commercially available
and many additional antibodies are described in the literature. For
example, in one common configuration, the tag is a first antibody
and the tag binder is a second antibody which recognizes the first
antibody. In addition to antibody-antigen interactions,
receptor-ligand interactions are also appropriate as tag and
tag-binder pairs, such as agonists and antagonists of cell membrane
receptors (e.g., cell receptor-ligand interactions such as
transferrin, c-kit, viral receptor ligands, cytokine receptors,
chemokine receptors, interleukin receptors, immunoglobulin
receptors and antibodies, the cadherin family, the integrin family,
the selectin family, and the like; see, e.g., Pigott & Power,
The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and
venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.),
intracellular receptors (e.g., which mediate the effects of various
small ligands, including steroids, thyroid hormone, retinoids and
vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both
linear and cyclic polymer configurations), oligosaccharides,
proteins, phospholipids and antibodies can all interact with
various cell receptors.
[0194] Synthetic polymers, such as polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyimides, and polyacetates
can also form an appropriate tag or tag binder. Many other tag/tag
binder pairs are also useful in assay systems described herein, as
would be apparent to one of skill upon review of this
disclosure.
[0195] Common linkers such as peptides, polyethers, and the like
can also serve as tags, and include polypeptide sequences, such as
poly Gly sequences of between about 5 and 200 amino acids (SEQ ID
NO:53). Such flexible linkers are known to those of skill in the
art. For example, poly(ethylene glycol) linkers are available from
Shearwater Polymers, Inc., Huntsville, Ala. These linkers
optionally have amide linkages, sulfhydryl linkages, or
heterofunctional linkages.
[0196] Tag binders are fixed to solid substrates using any of a
variety of methods currently available. Solid substrates are
commonly derivatized or functionalized by exposing all or a portion
of the substrate to a chemical reagent which fixes a chemical group
to the surface which is reactive with a portion of the tag binder.
For example, groups which are suitable for attachment to a longer
chain portion would include amines, hydroxyl, thiol, and carboxyl
groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to
functionalize a variety of surfaces, such as glass surfaces. The
construction of such solid phase biopolymer arrays is well
described in the literature (see, e.g., Merrifield, J. Am. Chem.
Soc. 85:2149-2154 (1963) (describing solid phase synthesis of,
e.g., peptides); Geysen et al., J. Immun. Meth. 102:259-274 (1987)
(describing synthesis of solid phase components on pins); Frank and
Doring, Tetrahedron 44:60316040 (1988) (describing synthesis of
various peptide sequences on cellulose disks); Fodor et al.,
Science, 251:767-777 (1991); Sheldon et al., Clinical Chemistry
39(4):718-719 (1993); and Kozal et al., Nature Medicine 2(7):753759
(1996) (all describing arrays of biopolymers fixed to solid
substrates). Non-chemical approaches for fixing tag binders to
substrates include other common methods, such as heat,
cross-linking by UV radiation, and the like.
[0197] The invention provides in vitro assays for identifying, in a
high throughput format, compounds that can modulate the expression
or activity of Archipelin. Control reactions that measure
Archipelin activity of the cell in a reaction that does not include
a potential modulator are optional, as the assays are highly
uniform. Such optional control reactions are appropriate and
increase the reliability of the assay. Accordingly, in some
embodiments, the methods of the invention include such a control
reaction. For each of the assay formats described, "no modulator"
control reactions which do not include a modulator provide a
background level of binding activity.
[0198] In some assays it will be desirable to have positive
controls to ensure that the components of the assays are working
properly. At least two types of positive controls are appropriate.
First, a known activator of Archipelin of the invention can be
incubated with one sample of the assay, and the resulting increase
in signal resulting from an increased expression level or activity
of Archipelin determined according to the methods herein. Second, a
known inhibitor of Archipelin can be added, and the resulting
decrease in signal for the expression or activity of Archipelin can
be similarly detected. It will be appreciated that modulators can
also be combined with activators or inhibitors to find modulators
which inhibit the increase or decrease that is otherwise caused by
the presence of the known modulator of Archipelin.
[0199] D. Computer-Based Assays
[0200] Yet another assay for compounds that modulate the activity
of Archipelin involves computer assisted drug design, in which a
computer system is used to generate a three-dimensional structure
of Archipelin based on the structural information encoded by its
amino acid sequence. The input amino acid sequence interacts
directly and actively with a pre-established algorithm in a
computer program to yield secondary, tertiary, and quaternary
structural models of the protein. Similar analyses can be performed
on potential receptors of Archipelin. The models of the protein
structure are then examined to identify regions of the structure
that have the ability to bind, e.g., Archipelin. These regions are
then used to identify polypeptides that bind to Archipelin.
[0201] The three-dimensional structural model of the protein is
generated by entering protein amino acid sequences of at least 10
amino acid residues or corresponding nucleic acid sequences
encoding a potential Archipelin receptor into the computer system.
The amino acid sequences encoded by the nucleic acid sequences
provided herein represent the primary sequences or subsequences of
the proteins, which encode the structural information of the
proteins. At least 10 residues of an amino acid sequence (or a
nucleotide sequence encoding 10 amino acids) are entered into the
computer system from computer keyboards, computer readable
substrates that include, but are not limited to, electronic storage
media (e.g., magnetic diskettes, tapes, cartridges, and chips),
optical media (e.g., CD ROM), information distributed by internet
sites, and by RAM. The three-dimensional structural model of the
protein is then generated by the interaction of the amino acid
sequence and the computer system, using software known to those of
skill in the art.
[0202] The amino acid sequence represents a primary structure that
encodes the information necessary to form the secondary, tertiary
and quaternary structure of the protein of interest. The software
looks at certain parameters encoded by the primary sequence to
generate the structural model. These parameters are referred to as
"energy terms," and primarily include electrostatic potentials,
hydrophobic potentials, solvent accessible surfaces, and hydrogen
bonding. Secondary energy terms include van der Waals potentials.
Biological molecules form the structures that minimize the energy
terms in a cumulative fashion. The computer program is therefore
using these terms encoded by the primary structure or amino acid
sequence to create the secondary structural model.
[0203] The tertiary structure of the protein encoded by the
secondary structure is then formed on the basis of the energy terms
of the secondary structure. The user at this point can enter
additional variables such as whether the protein is membrane bound
or soluble, its location in the body, and its cellular location,
e.g., cytoplasmic, surface, or nuclear. These variables along with
the energy terms of the secondary structure are used to form the
model of the tertiary structure. In modeling the tertiary
structure, the computer program matches hydrophobic faces of
secondary structure with like, and hydrophilic faces of secondary
structure with like.
[0204] Once the structure has been generated, potential ligand
binding regions are identified by the computer system.
Three-dimensional structures for potential ligands are generated by
entering amino acid or nucleotide sequences or chemical formulas of
compounds, as described above. The three-dimensional structure of
the potential ligand is then compared to that of Archipelin to
identify binding sites of Archipelin. Binding affinity between the
protein and ligands is determined using energy terms to determine
which ligands have an enhanced probability of binding to the
protein.
[0205] Computer systems are also used to screen for mutations,
polymorphic variants, alleles and interspecies homologs of genes
encoding an Archipelin polypeptide of the invention. Such mutations
can be associated with disease states or genetic traits. As
described above, GeneChip.TM. and related technology can also be
used to screen for mutations, polymorphic variants, alleles and
interspecies homologs. Once the variants are identified, diagnostic
assays can be used to identify patients having such mutated genes.
Identification of the mutated Archipelin genes involves receiving
input of a first amino acid sequence of a Archipelin (or of a first
nucleic acid sequence encoding a Archipelin of the invention),
e.g., any amino acid sequence having at least 60%, optionally at
least 85%, identity with the amino acid sequence of the polypeptide
encoded by the nucleic acid sequence set forth in SEQ ID NO:1, SEQ
ID NO:3, SEQ ID NO:5, and SEQ ID NO:8, or conservatively modified
versions thereof. The sequence is entered into the computer system
as described above. The first nucleic acid or amino acid sequence
is then compared to a second nucleic acid or amino acid sequence
that has substantial identity to the first sequence. The second
sequence is entered into the computer system in the manner
described above. Once the first and second sequences are compared,
nucleotide or amino acid differences between the sequences are
identified. Such sequences can represent allelic differences in
various Archipelin genes, and mutations associated with disease
states and genetic traits.
VII. Compositions, Kits and Integrated Systems
[0206] The invention provides compositions, kits and integrated
systems for practicing the assays described herein using nucleic
acids encoding the Archipelin polypeptides of the invention, or
Archipelin proteins, anti-Archipelin antibodies, etc.
[0207] The invention provides assay compositions for use in solid
phase assays; such compositions can include, for example, one or
more nucleic acids encoding an Archipelin immobilized on a solid
support, and a labeling reagent. In each case, the assay
compositions can also include additional reagents that are
desirable for hybridization. Modulators of expression or activity
of an Archipelin of the invention can also be included in the assay
compositions. Solid supports include, e.g., petri plates,
microtiter dishes and microarrays.
[0208] The invention also provides kits for carrying out the assays
of the invention. The kits typically include a probe which
comprises an antibody that specifically binds to Archipelin or a
polynucleotide sequence encoding an Archipelin polypeptide, and a
label for detecting the presence of the probe. The kits may include
several polynucleotide sequences encoding Archipelin polypeptides
of the invention. Kits can include any of the compositions noted
above, and optionally further include additional components such as
instructions to practice a high-throughput method of assaying for
an effect on expression of the genes encoding the Archipelin
polypeptides of the invention, or on activity of the Archipelin
polypeptides of the invention, one or more containers or
compartments (e.g., to hold the probe, labels, or the like), a
control modulator of the expression or activity of Archipelin
polypeptides, a robotic armature for mixing kit components or the
like.
[0209] The invention also provides integrated systems for
high-throughput screening of potential modulators for an effect on
the expression or activity of the Archipelin polypeptides of the
invention. The systems typically include a robotic armature which
transfers fluid from a source to a destination, a controller which
controls the robotic armature, a label detector, a data storage
unit which records label detection, and an assay component such as
a microtiter dish comprising a well having a reaction mixture or a
substrate comprising a fixed nucleic acid or immobilization
moiety.
[0210] A number of robotic fluid transfer systems are available, or
can easily be made from existing components. For example, a Zymate
XP (Zymark Corporation; Hopkinton, Mass.) automated robot using a
Microlab 2200 (Hamilton; Reno, Nev.) pipetting station can be used
to transfer parallel samples to 96 well microtiter plates to set up
several parallel simultaneous binding assays.
[0211] Optical images viewed (and, optionally, recorded) by a
camera or other recording device (e.g., a photodiode and data
storage device) are optionally further processed in any of the
embodiments herein, e.g., by digitizing the image and storing and
analyzing the image on a computer. A variety of commercially
available peripheral equipment and software is available for
digitizing, storing and analyzing a digitized video or digitized
optical image, e.g., using PC (Intel x86 or Pentium chip-compatible
DOS.RTM., OS2.RTM. WINDOWS.RTM., WINDOWS NT.RTM. or WINDOWS95.RTM.
based computers), MACINTOSH.RTM., or UNIX.RTM. based (e.g.,
SUN.RTM. work station) computers.
[0212] One conventional system carries light from the specimen
field to a cooled charge-coupled device (CCD) camera, in common use
in the art. A CCD camera includes an array of picture elements
(pixels). The light from the specimen is imaged on the CCD.
Particular pixels corresponding to regions of the specimen (e.g.,
individual hybridization sites on an array of biological polymers)
are sampled to obtain light intensity readings for each position.
Multiple pixels are processed in parallel to increase speed. The
apparatus and methods of the invention are easily used for viewing
any sample, e.g., by fluorescent or dark field microscopic
techniques.
VIII. Gene Therapy Applications
[0213] A variety of human diseases can be treated by therapeutic
approaches that involve stably introducing a gene into a human cell
such that the gene is transcribed and the gene product is produced
in the cell. Diseases amenable to treatment by this approach
include inherited diseases, including those in which the defect is
in a single gene. Gene therapy is also useful for treatment of
acquired diseases and other conditions. For discussions on the
application of gene therapy towards the treatment of genetic as
well as acquired diseases, see, Miller Nature 357:455-460 (1992);
and Mulligan Science 260:926-932 (1993).
[0214] In the context of the present invention, gene therapy can be
used for treating a variety of disorders and/or diseases in which
Archipelin has been implicated. For example, introduction by gene
therapy of polynucleotides encoding an Archipelin polypeptide of
the invention can be used to treat, e.g., diabetes.
[0215] A. Vectors for Gene Delivery
[0216] For delivery to a cell or organism, the nucleic acids of the
invention can be incorporated into a vector. Examples of vectors
used for such purposes include expression plasmids capable of
directing the expression of the nucleic acids in the target cell.
In other instances, the vector is a viral vector system wherein the
nucleic acids are incorporated into a viral genome that is capable
of transfecting the target cell. In embodiments, the nucleic acids
can be operably linked to expression and control sequences that can
direct expression of the gene in the desired target host cells.
Thus, one can achieve expression of the nucleic acid under
appropriate conditions in the target cell.
[0217] B. Gene Delivery Systems
[0218] Viral vector systems useful in the expression of the nucleic
acids include, for example, naturally occurring or recombinant
viral vector systems. Depending upon the particular application,
suitable viral vectors include replication competent, replication
deficient, and conditionally replicating viral vectors. For
example, viral vectors can be derived from the genome of human or
bovine adenoviruses, vaccinia virus, herpes virus, adeno-associated
virus, minute virus of mice (MVM), HIV, sindbis virus, and
retroviruses (including but not limited to Rous sarcoma virus), and
MoMLV. Typically, the genes of interest are inserted into such
vectors to allow packaging of the gene construct, typically with
accompanying viral DNA, followed by infection of a sensitive host
cell and expression of the gene of interest.
[0219] As used herein, "gene delivery system" refers to any means
for the delivery of a nucleic acid of the invention to a target
cell. In some embodiments of the invention, nucleic acids are
conjugated to a cell receptor ligand for facilitated uptake (e.g.,
invagination of coated pits and internalization of the endosome)
through an appropriate linking moiety, such as a DNA linking moiety
(Wu et al., J. Biol. Chem. 263:14621-14624 (1988); WO 92/06180).
For example, nucleic acids can be linked through a polylysine
moiety to asialo-oromucocid, which is a ligand for the
asialoglycoprotein receptor of hepatocytes.
[0220] Similarly, viral envelopes used for packaging gene
constructs that include the nucleic acids of the invention can be
modified by the addition of receptor ligands or antibodies specific
for a receptor to permit receptor-mediated endocytosis into
specific cells (see, e.g., WO 93/20221, WO 93/14188, and WO
94/06923). In some embodiments of the invention, the DNA constructs
of the invention are linked to viral proteins, such as adenovirus
particles, to facilitate endocytosis (Curiel et al., Proc. Natl.
Acad. Sci. U.S.A. 88:8850-8854 (1991)). In other embodiments,
molecular conjugates of the instant invention can include
microtubule inhibitors (WO/9406922), synthetic peptides mimicking
influenza virus hemagglutinin (Plank et al., J. Biol. Chem.
269:12918-12924 (1994)), and nuclear localization signals such as
SV40 T antigen (WO93/19768).
[0221] Retroviral vectors are also useful for introducing the
nucleic acids of the invention into target cells or organisms.
Retroviral vectors are produced by genetically manipulating
retroviruses. The viral genome of retroviruses is RNA. Upon
infection, this genomic RNA is reverse transcribed into a DNA copy
which is integrated into the chromosomal DNA of transduced cells
with a high degree of stability and efficiency. The integrated DNA
copy is referred to as a provirus and is inherited by daughter
cells as is any other gene. The wild type retroviral genome and the
proviral DNA have three genes: the gag, the pol and the env genes,
which are flanked by two long terminal repeat (LTR) sequences. The
gag gene encodes the internal structural (nucleocapsid) proteins;
the pol gene encodes the RNA directed DNA polymerase (reverse
transcriptase); and the env gene encodes viral envelope
glycoproteins. The 5' and 3' LTRs serve to promote transcription
and polyadenylation of virion RNAs. Adjacent to the 5' LTR are
sequences necessary for reverse transcription of the genome (the
tRNA primer binding site) and for efficient encapsulation of viral
RNA into particles (the Psi site) (see, Mulligan, In: Experimental
Manipulation of Gene Expression, Inouye (ed), 155-173 (1983); Mann
et al., Cell 33:153-159 (1983); Cone and Mulligan, Proceedings of
the National Academy of Sciences, U.S.A., 81:6349-6353 (1984)).
[0222] The design of retroviral vectors is well known to those of
ordinary skill in the art. In brief, if the sequences necessary for
encapsidation (or packaging of retroviral RNA into infectious
virions) are missing from the viral genome, the result is a cis
acting defect which prevents encapsidation of genomic RNA. However,
the resulting mutant is still capable of directing the synthesis of
all virion proteins. Retroviral genomes from which these sequences
have been deleted, as well as cell lines containing the mutant
genome stably integrated into the chromosome are well known in the
art and are used to construct retroviral vectors. Preparation of
retroviral vectors and their uses are described in many
publications including, e.g., European Patent Application EPA 0 178
220; U.S. Pat. No. 4,405,712, Gilboa Biotechniques 4:504-512
(1986); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan
Proc. Natl. Acad. Sci. USA 81:6349-6353 (1984); Eglitis et al.
Biotechniques 6:608-614 (1988); Miller et al. Biotechniques
7:981-990 (1989); Miller (1992) supra; Mulligan (1993), supra; and
WO 92/07943.
[0223] The retroviral vector particles are prepared by
recombinantly inserting the desired nucleotide sequence into a
retrovirus vector and packaging the vector with retroviral capsid
proteins by use of a packaging cell line. The resultant retroviral
vector particle is incapable of replication in the host cell but is
capable of integrating into the host cell genome as a proviral
sequence containing the desired nucleotide sequence. As a result,
the patient is capable of producing, for example, an Archipelin
polypeptide of interest and thus restore the cells to a normal
phenotype.
[0224] Packaging cell lines that are used to prepare the retroviral
vector particles are typically recombinant mammalian tissue culture
cell lines that produce the necessary viral structural proteins
required for packaging, but which are incapable of producing
infectious virions. The defective retroviral vectors that are used,
on the other hand, lack these structural genes but encode the
remaining proteins necessary for packaging. To prepare a packaging
cell line, one can construct an infectious clone of a desired
retrovirus in which the packaging site has been deleted. Cells
comprising this construct will express all structural viral
proteins, but the introduced DNA will be incapable of being
packaged. Alternatively, packaging cell lines can be produced by
transforming a cell line with one or more expression plasmids
encoding the appropriate core and envelope proteins. In these
cells, the gag, pol, and env genes can be derived from the same or
different retroviruses.
[0225] A number of packaging cell lines suitable for the present
invention are also available in the prior art. Examples of these
cell lines include Crip, GPE86, PA317 and PG13 (see Miller et al.,
J. Virol. 65:2220-2224 (1991)). Examples of other packaging cell
lines are described in Cone and Mulligan Proceedings of the
National Academy of Sciences, USA, 81:6349-6353 (1984); Danos and
Mulligan Proceedings of the National Academy of Sciences, USA,
85:6460-6464 (1988); Eglitis et al. (1988), supra; and Miller
(1990), supra.
[0226] Packaging cell lines capable of producing retroviral vector
particles with chimeric envelope proteins may be used.
Alternatively, amphotropic or xenotropic envelope proteins, such as
those produced by PA317 and GPX packaging cell lines may be used to
package the retroviral vectors.
[0227] In some embodiments of the invention, an antisense nucleic
acid is administered which hybridizes to a gene encoding an
Archipelin of the invention or to a transcript thereof. The
antisense nucleic acid can be provided as an antisense
oligonucleotide (see, e.g., Murayama et al., Antisense Nucleic Acid
Drug Dev. 7:109-114 (1997)). Genes encoding an antisense nucleic
acid can also be provided; such genes can be introduced into cells
by methods known to those of skill in the art. For example, one can
introduce a gene that encodes an antisense nucleic acid in a viral
vector, such as, for example, in hepatitis B virus (see, e.g., Ji
et al., J. Viral Hepat. 4:167-173 (1997)), in adeno-associated
virus (see, e.g., Xiao et al., Brain Res. 756:76-83 (1997)), or in
other systems including, but not limited, to an HVJ (Sendai
virus)-liposome gene delivery system (see, e.g., Kaneda et al.,
Ann. NY Acad. Sci. 811:299-308 (1997)), a "peptide vector" (see,
e.g., Vidal et al., CR Acad. Sci III 32:279-287 (1997)), as a gene
in an episomal or plasmid vector (see, e.g., Cooper et al., Proc.
Natl. Acad. Sci. U.S.A. 94:6450-6455 (1997), Yew et al. Hum Gene
Ther. 8:575-584 (1997)), as a gene in a peptide-DNA aggregate (see,
e.g., Niidome et al., J. Biol. Chem. 272:15307-15312 (1997)), as
"naked DNA" (see, e.g., U.S. Pat. Nos. 5,580,859 and 5,589,466), in
lipidic vector systems (see, e.g., Lee et al., Crit Rev Ther Drug
Carrier Syst. 14:173-206 (1997)), polymer coated liposomes (U.S.
Pat. Nos. 5,213,804 and 5,013,556), cationic liposomes (Epand et
al., U.S. Pat. Nos. 5,283,185; 5,578,475; 5,279,833; and
5,334,761), gas filled microspheres (U.S. Pat. No. 5,542,935),
ligand-targeted encapsulated macromolecules (U.S. Pat. Nos.
5,108,921; 5,521,291; 5,554,386; and 5,166,320).
[0228] C. Pharmaceutical Formulations
[0229] When used for pharmaceutical purposes, the vectors used for
gene therapy are formulated in a suitable buffer, which can be any
pharmaceutically acceptable buffer, such as phosphate buffered
saline or sodium phosphate/sodium sulfate, Tris buffer, glycine
buffer, sterile water, and other buffers known to the ordinarily
skilled artisan such as those described by Good et al. Biochemistry
5:467 (1966).
[0230] The compositions can additionally include a stabilizer,
enhancer or other pharmaceutically acceptable carriers or vehicles.
A pharmaceutically acceptable carrier can contain a physiologically
acceptable compound that acts, for example, to stabilize the
nucleic acids of the invention and any associated vector. A
physiologically acceptable compound can include, for example,
carbohydrates, such as glucose, sucrose or dextrans, antioxidants,
such as ascorbic acid or glutathione, chelating agents, low
molecular weight proteins or other stabilizers or excipients. Other
physiologically acceptable compounds include wetting agents,
emulsifying agents, dispersing agents or preservatives, which are
particularly useful for preventing the growth or action of
microorganisms. Various preservatives are well known and include,
for example, phenol and ascorbic acid. Examples of carriers,
stabilizers or adjuvants can be found in Remington's Pharmaceutical
Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed.
(1985).
[0231] D. Administration of Formulations
[0232] The formulations of the invention can be delivered to any
tissue or organ using any delivery method known to the ordinarily
skilled artisan. In some embodiments of the invention, the nucleic
acids of the invention are formulated in mucosal, topical, and/or
buccal formulations, particularly mucoadhesive gel and topical gel
formulations. Exemplary permeation enhancing compositions, polymer
matrices, and mucoadhesive gel preparations for transdermal
delivery are disclosed in U.S. Pat. No. 5,346,701.
[0233] E. Methods of Treatment
[0234] The gene therapy formulations of the invention are typically
administered to a cell. The cell can be provided as part of a
tissue, such as an epithelial membrane, or as an isolated cell,
such as in tissue culture. The cell can be provided in vivo, ex
vivo, or in vitro.
[0235] The formulations can be introduced into the tissue of
interest in vivo or ex vivo by a variety of methods. In some
embodiments of the invention, the nucleic acids of the invention
are introduced into cells by such methods as microinjection,
calcium phosphate precipitation, liposome fusion, or biolistics. In
further embodiments, the nucleic acids are taken up directly by the
tissue of interest.
[0236] In some embodiments of the invention, the nucleic acids of
the invention are administered ex vivo to cells or tissues
explanted from a patient, then returned to the patient. Examples of
ex vivo administration of therapeutic gene constructs include Nolta
et al., Proc Natl. Acad. Sci. USA 93(6):2414-9 (1996); Koc et al.,
Seminars in Oncology 23 (1):46-65 (1996); Raper et al., Annals of
Surgery 223(2):116-26 (1996); Dalesandro et al., J. Thorac. Cardi.
Surg., 11(2):416-22 (1996); and Makarov et al., Proc. Natl. Acad.
Sci. USA 93(1):402-6 (1996).
IX. Administration and Pharmaceutical Compositions
[0237] Modulators of Archipelin (e.g., agonists, including
Archipelin polypeptides, and antagonists) can be administered
directly to the mammalian subject for modulation of Archipelin
signaling in vivo. Administration is by any of the routes normally
used for introducing a modulator compound into ultimate contact
with the tissue to be treated and well known to those of skill in
the art. Although more than one route can be used to administer a
particular composition, a particular route can often provide a more
immediate and more effective reaction than another route.
[0238] The compounds of the present invention can also be used
effectively in combination with one or more additional active
agents depending on the desired target therapy (see, e.g., Turner,
N. et al. Prog. Drug Res. (1998) 51: 33-94; Haffner, S. Diabetes
Care (1998) 21: 160-178; and DeFronzo, R. et al. (eds.), Diabetes
Reviews (1997) Vol. 5 No. 4). A number of studies have investigated
the benefits of combination therapies with oral agents (see, e.g.,
Mahler, R., J. Clin. Endocrinol. Metab. (1999) 84: 1165-71; United
Kingdom Prospective Diabetes Study Group: UKPDS 28, Diabetes Care
(1998) 21: 87-92; Bardin, C. W.,(ed.), Current Therapy In
Endocrinology And Metabolism, 6th Edition (Mosby--Year Book, Inc.,
St. Louis, Mo. 1997); Chiasson, J. et al., Ann. Intern. Med. (1994)
121: 928-935; Coniff, R. et al., Clin. Ther. (1997) 19: 16-26;
Coniff, R. et al., Am. J. Med. (1995) 98: 443-451; and Iwamoto, Y.
et al., Diabet. Med. (1996) 13 365-370; Kwiterovich, P. Am. J.
Cardiol (1998) 82(12A): 3U-17U). These studies indicate that
modulation of diabetes and hyperlipidemia, among other diseases,
can be further improved by the addition of a second agent to the
therapeutic regimen. Combination therapy includes administration of
a single pharmaceutical dosage formulation which contains an
Archipelin modulator of the invention and one or more additional
active agents, as well as administration of an Archipelin modulator
and each active agent in its own separate pharmaceutical dosage
formulation. For example, an Archipelin modulator and a
thiazolidinedione can be administered to the human subject together
in a single oral dosage composition, such as a tablet or capsule,
or each agent can be administered in separate oral dosage
formulations. Where separate dosage formulations are used, an
Archipelin modulator and one or more additional active agents can
be administered at essentially the same time (i.e., concurrently),
or at separately staggered times (i.e., sequentially). Combination
therapy is understood to include all these regimens.
[0239] Still another example of combination therapy can be seen in
modulating diabetes (or treating diabetes and its related symptoms,
complications, and disorders), wherein the AKR1C modulators can be
effectively used in combination with, for example, sulfonylureas
(such as chlorpropamide, tolbutamide, acetohexamide, tolazamide,
glyburide, gliclazide, glynase, glimepiride, and glipizide),
biguanides (such as metformin), a PPAR beta delta agonist, a ligand
or agonist of PPAR alpha such as thiazolidinediones (such as
ciglitazone, pioglitazone (see, e.g., U.S. Pat. No. 6,218,409),
troglitazone, and rosiglitazone (see, e.g., U.S. Pat. No.
5,859,037)); dehydroepiandrosterone (also referred to as DHEA or
its conjugated sulphate ester, DHEA-SO4); antiglucocorticoids;
TNF.alpha. inhibitors; .alpha.-glucosidase inhibitors (such as
acarbose, miglitol, and voglibose), amylin and amylin derivatives
(such as pramlintide, (see, also, U.S. Pat. Nos. 5,902,726;
5,124,314; 5,175,145 and 6,143,718, 6,136,784)), insulin
secretogogues (such as repaglinide, gliquidone, and nateglinide
(see, also, U.S. Pat. Nos. 6,251,856; 6,251,865; 6,221,633;
6,174,856)), insulin, as well as the active agents discussed above
for treating atherosclerosis.
[0240] The pharmaceutical compositions of the invention may
comprise a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers are determined in part by the particular
composition being administered, as well as by the particular method
used to administer the composition. Accordingly, there is a wide
variety of suitable formulations of pharmaceutical compositions of
the present invention (see, e.g., Remington's Pharmaceutical
Sciences, 17.sup.th ed. 1985)).
[0241] The modulators (e.g., agonists or antagonists) of the
expression or activity of the Archipelin, alone or in combination
with other suitable components, can be prepared for injection or
for use in a pump device. Pump devices (also known as "insulin
pumps") are commonly used to administer insulin to patients and
therefore can be easily adapted to include compositions of the
present invention. Manufacturers of insulin pumps include Animas,
Disetronic and MiniMed.
[0242] The modulators (e.g., agonists or antagonists) of the
expression or activity of the Archipelin, alone or in combination
with other suitable components, can be made into aerosol
formulations (i.e., they can be "nebulized") to be administered via
inhalation. Aerosol formulations can be placed into pressurized
acceptable propellants, such as dichlorodifluoromethane, propane,
nitrogen, and the like.
[0243] Formulations suitable for administration include aqueous and
non-aqueous solutions, isotonic sterile solutions, which can
contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic, and aqueous and non-aqueous
sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. In
the practice of this invention, compositions can be administered,
for example, orally, nasally, topically, intravenously,
intraperitoneally, or intrathecally. The formulations of compounds
can be presented in unit-dose or multi-dose sealed containers, such
as ampoules and vials. Solutions and suspensions can be prepared
from sterile powders, granules, and tablets of the kind previously
described. The modulators can also be administered as part a of
prepared food or drug.
[0244] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
response in the subject over time. The optimal dose level for any
patient will depend on a variety of factors including the efficacy
of the specific modulator employed, the age, body weight, physical
activity, and diet of the patient, on a possible combination with
other drugs, and on the severity of the case of diabetes. It is
recommended that the daily dosage of the modulator be determined
for each individual patient by those skilled in the art in a
similar way as for known insulin compositions. The size of the dose
also will be determined by the existence, nature, and extent of any
adverse side-effects that accompany the administration of a
particular compound or vector in a particular subject.
[0245] In determining the effective amount of the modulator to be
administered a physician may evaluate circulating plasma levels of
the modulator, modulator toxicity, and the production of
anti-modulator antibodies. In general, the dose equivalent of a
modulator is from about 1 ng/kg to 10 mg/kg for a typical
subject.
[0246] For administration, Archipelin modulators of the present
invention can be administered at a rate determined by the LD-50 of
the modulator, and the side-effects of the inhibitor at various
concentrations, as applied to the mass and overall health of the
subject. Administration can be accomplished via single or divided
doses.
X. Diagnosis of Diabetes
[0247] The present invention also provides methods of diagnosing
diabetes or a predisposition of at least some of the pathologies of
diabetes. Diagnosis involves determining the level of Archipelin in
a patient and then comparing the level to a baseline or range.
Typically, the baseline value is representative of Archipelin in a
healthy (i.e., non-diabetic) person. As discussed above, variation
of levels (either high or low) of Archipelin from the baseline
range suggests that the patient is either diabetic or at risk of
developing at least some of the pathologies of diabetes. Variation
van be, e.g., at least 5%, 10%, 20%, 50%, 200%, 400%, 500%, or
1000% or more of a baseline value or range. In some embodiments,
the level of Archipelin are measured by taking a blood sample from
a patient and measuring the amount of Archipelin in the sample
using any number of detection methods, such as those discussed
herein. For instance, fasting and fed blood or urine levels can be
tested.
[0248] Glucose tolerance tests can also be used to detect the
effect of glucose levels on Archipelin levels. In glucose tolerance
tests, the patient's ability to tolerate a standard oral glucose
load is evaluated by assessing serum and urine specimens for
glucose levels. Blood samples are taken before the glucose is
ingested, glucose is given by mouth, and blood or urine glucose
levels are tested at set intervals after glucose ingestion.
[0249] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0250] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
EXAMPLES
[0251] The following examples are offered to illustrate, but not to
limit the claimed invention.
Example 1
[0252] This example describes the discovery of a new member of the
CRF/urocortin family of peptides that is also highly abundant in
pancreatic islets and is a hormone important for the treatment of
diabetes mellitus.
[0253] The probe set MBXRATISL12276 was identified as a gene
specifically expressed in rat islets using rat tissue map
experiments on rat islet GeneChips. Analysis of a second, more
extensive, rat tissue mapping experiment also revealed that probe
set MBXRATISL12276 was abundant in rat islet samples and absent in
10 samples from 10 other tissues. This probe set was present in 5
out of 5 rat islet samples with an average Average Difference of
>4000 and was absent in all 10 other tissues with an average
Average Difference value of 0 or less. This represents an extremely
islet specific expression pattern. See FIG. 1. A mouse tissue map
experiment also showed an extremely islet specific expression
pattern. See FIG. 2. The predicted product of the gene
corresponding to these probe sets was designated Archipelin.
[0254] Expression of Archipelin in diabetic animal models was also
determined. This data indicated that probe set MBXRATISL12276
expression is 3-fold lower in Zucker Diabetic Fatty male rats
versus ZLC control rats. This probe set is also lower in diabetic
(fat fed) ZDF female rats. This data indicates that this gene is
down-regulated in the islets of these animal models of Type 2
diabetes. These findings indicate that pancreatic islets are the
major site of Archipelin production and suggests that this hormone
is inappropriately expressed in individuals with diabetes mellitus.
Type 1 diabetes is characterized by destruction of islets, and this
will very likely lead to Archipelin levels to be reduced or absent.
Islet disfunction is also a critical aspect of Type 2 diabetes and
therefore also effects Archipelin production.
[0255] The nucleotide sequence of the mouse, rat and human
Archipelin clones were determined. SEQ ID NO:1 and SEQ ID NO:8
display two variants of the human sequence, SEQ ID NO:3 shows the
mouse sequence and SEQ ID NO:5 shows the rat sequence. The
predicted amino acid sequences of unprocessed Archipelin are shown
for human (SEQ ID NOs:2 and 7), mouse (SEQ ID NO:4) and rat (SEQ ID
NO:6). Depending on the human clone sequenced, amino acid 94 was
either arginine or glycine. Multiple clones encoded each amino
acid.
[0256] Using MBXRATISL12276 to BLAST search a human islet endocrine
cell database revealed ortholog clones among the human islet ESTs.
cDNA clones for Archipelin were also abundant in the mouse and rat
islet est databases. Subsequent Northern blot hybridizations with
both rat and human cDNA clones of Archipelin confirm the islet
specific expression of an approximately 1.2 kb Archipelin
transcript.
[0257] A BLAST search of the public databases revealed significant
similarity to swissprot+database entry Q9i8e5 (fugu rubripes
(japanese pufferfish) (takifugu rubripes)), which was designated as
the fugu urocortin precursor. Further alignments suggested that the
Archipelin peptide was also related to Corticotropin Releasing
Factor (CRF) family. CRF is a key regulatory component of the
hypothalamus-pituitary-adrenal axis. See, FIG. 3 illustrating an
alignment of Archipelin amino acid sequences with members of the
CRF peptide family. In light of the sequence analysis, it was
determined that the Archipelin peptide was a processed, secreted
peptide. FIGS. 3-5 illustrate the possible processing sites of the
human, mouse and rat Archipelin peptides, respectively.
[0258] As noted above, the predicted Archipelin peptide sequence
has a CRF family signature as well as a C-terminal proteolytic
processing sequence (glycine-basic-basic-basic) that is
characteristic for this family and results in C-terminal amidation.
In the case of Archipelin from mouse, rat and human, this will
result in an isoleucyl-amide. There are also basic residues in the
region that is expected to be an N-terminal processing site (FIGS.
4-6) producing a mature peptide containing between 50 and 38 amino
acid residues. More than one of these may exist physiologically and
one or all of these may be biologically active. It is also possible
that a larger version containing some or all of the propeptide has
biological function.
[0259] A predicted mature peptide sequence for rat Archipelin
(TKFTLSLDVPTNIMNILFNIDKAKNLRAKAAANAQLMAQI-CONH2; SEQ ID NO:41) was
obtained and used in a variety of biological studies. Without
intending to limit the scope of the invention, it is predicted that
this peptide represents a likely mature, processed version of the
peptide.
[0260] In addition, the peptide sequence Cys-LFNIDKAKNLRAK (SEQ ID
NO:54), which represents residues 137 to 149 of the coding sequence
of both rat and mouse Archipelin precursors was used to develop
anti-sera. An affinity-purified version of this antibody recognizes
the in vitro translated 160 amino acid rat Archipelin precursor
molecule. This antiserum also recognizes the Archipelin precursor
expressed by transfection of the rat Archipelin cDNA in HEK 293
cells on a western blot, as well as the 40-mer form of Archipelin
(TKFTLSLDVPTNIMNILFNIDKAKNLRAKAAANAQLMAQI-CONH2; SEQ ID NO:41).
[0261] Immunohistochemical studies using these antibodies
demonstrated that Archipelin is highly expressed in islet cells,
and in particular, in .beta.-cells.
[0262] Moreover, in diabetes animal model systems, the animals
typically displayed reduced levels of Archipelin their blood before
a drop in insulin levels. These results indicate that monitoring
Archipelin levels is particularly useful as an early indicator of a
predisposition for diabetes.
Archipelin in Human Serum Samples
[0263] Human archipelin peptide was detected in normal human serum
samples using surface enhanced laser desorption/ionization (SELDI)
mass spectroscopy of peptides specifically captured on a surface
coated with affinity-purified anti-archipelin antibody (FIG. 7).
This was accomplished using a Ciphergen protein chip reader
instrument (Series PBS II). Affinity purified antibody was
covalently bound to a PS20 pre-activated chip surface for the
affinity capture of archipelin. Antibody was incubated on the chip
surface overnight at 2-8.degree. C. The 8 spot chip was then
blocked for 30 minutes using a free amine from a 1M solution of
ethanolamine. Dilutions (1:70) of human serum in PBS containing
0.05% Triton X were then applied to each of the spots and incubated
for two hours at room temperature. Non-specific proteins were
removed through post binding washes in PBS containing 0.05%
triton-X. The energy-absorbing molecule CHCA was applied to the
chip surface to form a crystal surface. Once crystal formation
occurred the captured protein was ionized and read with the protein
chip reader. The results revealed highly reproducible peaks at
masses of 4472-4476 (monomer) and 8932-8936 (dimer). This size is
consistent with the major serum form of the archipelin peptide
being 40 or 41 amino acids
(TKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI (SEQ ID NO:55) or
RTKFTLSLDVPTNIMNLLFNIAKAKNLRAQAAANAHLMAQI (SEQ ID NO:56)).
Trypsin Digestion Pattern of Rat Archipelin
[0264] After antibody capture of archipelin from rat serum using a
PS 20 pre-activated Ciphergen chip, the peptide was digested using
modified sequencing grade trypsin. Resulting peaks were identified
based on predicted sequence from peptide cutter (expasy.org) (FIG.
8). The mass of the thirty-eight amino acid peptide of rat
archipelin from rat was 4172 daltons. Capture of the native form of
archipelin from serum was approximately 4150 daltons, which is
within 0.5% of the expected mass. The major serum form of rat
archipelin was therefore the 38-mer
(FTLSLDVPTNIMNILFNIDKAKNLRAKAAANAQLMAQI; SEQ ID NO:57). Predicted
trypsin digestion products closely matched the observed archipelin
digestion.
[0265] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
57 1 1080 DNA Homo sapiens human Archipelin A full-length cDNA
modified_base (1)..(1080) n = g, a, c or t 1 cacagcccac ttaggaacaa
taccggagaa gcaggagccg agaccccgga gcagccacaa 60 gttcatgggg
acgtgcacgg ggccgccctc ctggccctga agctgcgccg gcctccctga 120
gcgtttcgct gcggagggaa gtccactctc ggggagagat gctgatgccg gtccacttcc
180 tgctgctcct gctgctgctc ctggggggcc ccaggacagg cctcccccac
aagttctaca 240 aagccaagcc catcttcagc tgcctcaaca ccgccctgtc
tgaggctgag aagggccagt 300 gggaggatgc atccctgctg agcaagagga
gcttccacta cctgcgcagc agagacgcct 360 cttcgggaga ggaggaggag
ggcaaagaga aaaagacttt ccccatctct ggggccaggg 420 gtggagccag
aggcacccgg tacagatacg tgtcccaagc acagcccagg ggaaagccac 480
gccaggacac ggccaagagt ccccaccgca ccaagttcac cctgtccctc gacgtcccca
540 ccaacatcat gaacctcctc ttcaacatcg ccaaggccaa gaacctgcgt
gcccaggcgg 600 ccgccaatgc ccacctgatg gcgcaaattg ggaggaagaa
gtagaggcgg aggctggacg 660 ggagggcagc ggggtgngga gggggagggg
agggggaggg gagggcnagg gggggagggg 720 agggggaggg tgctgtctgc
tggnttgtgt tttgtgggat cagtcagttt tacaggttgc 780 tgcactgctg
agcccctctg atctcttctg gcctttgacc ctgtctccct cctgctctgt 840
ctgtacacac agaagtgcag tattgtccaa ccttcccaga cacaaagcag ctaacnttcc
900 tccctgtact caacgtctcc ttcctccctc cccacagcaa gaggcaaagt
tcatgcattc 960 ctcctctcca gtcttctctc tgttgacccc atgcctgaga
agagagcgtt cagggctcct 1020 ctcccacaca tcaacttctg ccagggcaga
aagaggagct gcagcactcg ccttcctgac 1080 2 161 PRT Homo sapiens human
unprocessed Archipelin A 2 Met Leu Met Pro Val His Phe Leu Leu Leu
Leu Leu Leu Leu Leu Gly 1 5 10 15 Gly Pro Arg Thr Gly Leu Pro His
Lys Phe Tyr Lys Ala Lys Pro Ile 20 25 30 Phe Ser Cys Leu Asn Thr
Ala Leu Ser Glu Ala Glu Lys Gly Gln Trp 35 40 45 Glu Asp Ala Ser
Leu Leu Ser Lys Arg Ser Phe His Tyr Leu Arg Ser 50 55 60 Arg Asp
Ala Ser Ser Gly Glu Glu Glu Glu Gly Lys Glu Lys Lys Thr 65 70 75 80
Phe Pro Ile Ser Gly Ala Arg Gly Gly Ala Arg Gly Thr Arg Tyr Arg 85
90 95 Tyr Val Ser Gln Ala Gln Pro Arg Gly Lys Pro Arg Gln Asp Thr
Ala 100 105 110 Lys Ser Pro His Arg Thr Lys Phe Thr Leu Ser Leu Asp
Val Pro Thr 115 120 125 Asn Ile Met Asn Leu Leu Phe Asn Ile Ala Lys
Ala Lys Asn Leu Arg 130 135 140 Ala Gln Ala Ala Ala Asn Ala His Leu
Met Ala Gln Ile Gly Arg Lys 145 150 155 160 Lys 3 1278 DNA Mus sp.
mouse unprocessed Archipelin full-length cDNA 3 ggcacgaggc
ttgtatttta aatgaaatct catcctaatg tagacactac atctggaatt 60
acgatccacc ctgtgatctg ccacttttac atatatgcac aggggagtgg agcggtttcc
120 atagagagga acgatcacag cccactacaa tcagtaccag agaagacaaa
gctgcaaccc 180 tgaacagtca gaagttcaga ggacctgcag ggagtagcct
cctggcccgg aagctgtgcc 240 cctcgacctg agcatttcca ctccagagca
aagtccactt acagggagcg atgctgatgc 300 ccacctactt cctgctgcca
cttctgctgc tcctaggagg tccaaggaca agcctctccc 360 acaagttcta
caacactgga ccagtcttca gctgcctcaa cacagcccta tctgaggtca 420
agaagaacaa gctggaagat gtgcccttgc tgagcaagaa gagctttggc cacctgccca
480 cacaagaccc ctcaggggaa gaagatgaca accaaacgca cctccagatc
aaaagaactt 540 tctcaggtgc cgcgggtggg aatggagctg ggagcacccg
gtacagatac caatcccagg 600 cacagcacaa ggggaagctg tacccagaca
agcccaaaag cgaccggggc accaagttca 660 ccctttccct tgatgttccc
actaacatca tgaacatcct cttcaacatc gacaaggcca 720 agaatttgcg
agccaaggca gctgccaatg ctcagctcat ggcacagatt gggaagaaga 780
agtaaagcaa agcccaggca tgagggtggc acatcaagac aagggccccg gagtaaaggg
840 taaaggaaac tgaggacgtg ccctcgaatt tcaaaggaca gtctgttttc
ccaggctgct 900 ccactactgt gcccctctga tcctctcttg cttccagtcc
ggtttccctt ctcagtacat 960 acacactcaa gcgcagtatt gctcagccca
tatccacaat ggaggttaac gtctctcccc 1020 gaatcctgtg tctgctttct
ggttccctgt aacaacagac aagttcatgt ggtgcccctc 1080 cttcccaagc
tcctccctgg tgtcaccatg tctgcagaga gagctttcag gctccctcct 1140
tgtcccacat tggtctgagc ctcggcagag acacagagat gcacagctcc ccctcttgat
1200 accaaatacc tcccctactt cctcatctgg attaaagtca gtggcttctt
gaaaaaaaaa 1260 aaaaaaaaaa aaaaaaaa 1278 4 164 PRT Mus sp. mouse
unprocessed Archipelin 4 Met Leu Met Pro Thr Tyr Phe Leu Leu Pro
Leu Leu Leu Leu Leu Gly 1 5 10 15 Gly Pro Arg Thr Ser Leu Ser His
Lys Phe Tyr Asn Thr Gly Pro Val 20 25 30 Phe Ser Cys Leu Asn Thr
Ala Leu Ser Glu Val Lys Lys Asn Lys Leu 35 40 45 Glu Asp Val Pro
Leu Leu Ser Lys Lys Ser Phe Gly His Leu Pro Thr 50 55 60 Gln Asp
Pro Ser Gly Glu Glu Asp Asp Asn Gln Thr His Leu Gln Ile 65 70 75 80
Lys Arg Thr Phe Ser Gly Ala Ala Gly Gly Asn Gly Ala Gly Ser Thr 85
90 95 Arg Tyr Arg Tyr Gln Ser Gln Ala Gln His Lys Gly Lys Leu Tyr
Pro 100 105 110 Asp Lys Pro Lys Ser Asp Arg Gly Thr Lys Phe Thr Leu
Ser Leu Asp 115 120 125 Val Pro Thr Asn Ile Met Asn Ile Leu Phe Asn
Ile Asp Lys Ala Lys 130 135 140 Asn Leu Arg Ala Lys Ala Ala Ala Asn
Ala Gln Leu Met Ala Gln Ile 145 150 155 160 Gly Lys Lys Lys 5 1186
DNA Rattus sp. rat Archipelin unprocessed full-length cDNA 5
ccggcgtgca cagctggctc tagttgtatt ttaaatgaaa tctcatccta aagtagacac
60 tacgtctgga attacgatcc accctgtgat ctgtcacttt tacatacatg
cacaggggag 120 tggagcggtt tccatagaga ggaactacca cagctcacta
cgaacaatac cagagaagac 180 gaagctgcga ccccgaacaa gaggacctgc
ggggagcagc cctccgggcc cagaagctgt 240 gcccctcgcc ctgagcactt
ccaccctaga gcaaagtcct cttacagtac agggagcgat 300 gctgatgccc
acttacttcc tgctgcttct gctgctgctc ctagggggcc caaggacaag 360
cctctcccac aagttctaca acgcaggacc aatcttcagc tgcctcaaca cagccctgtc
420 tgaggtcaag aagaacaagc tggaggatgt gccggtgctg agcaagaaga
actttggcta 480 cctgcccaca caagaccctt cgggagaaga agaggatgaa
caaaaacaca tcaagaacaa 540 aagaactttc tcagacgctg tgggtgggaa
tggaggtaga agcatccggt acagatacca 600 atccccagca cagcccaaag
gaaagctgta cccggacaag gtcaaaaacg accggggcac 660 caagttcact
ctgtccctcg acgttcccac taacatcatg aacatcctct tcaacattga 720
caaggccaag aatttgcgag ccaaggcagc ggccaatgct caactcatgg cacagattgg
780 gaaaaagaaa taaagcaaag gccaggcagg agggcgcccc atcgagacaa
gagccccaga 840 ataaagggga gggaaactga ggacgtgccc tcgaattgca
aaggacggat ggtccgtttt 900 cacaggctac tccactgctg tacccctctg
accctctcct gcttccagcc tgtgcccaca 960 atggaggtta acgtctctcc
ccatatcttg tgtttgcttt ctagttccct gtggcaacag 1020 acaagttcac
acagtgcccc tcctttccaa gttcctccct gatgtcccat gtctgaagag 1080
agagctctcg ggctccctct ttgttctatg ttggtctgag cctcggcaga gacacggaga
1140 agcacagctc accctcttgc taccaagtac ctcccctact tcctca 1186 6 164
PRT Rattus sp. rat unprocessed Archipelin 6 Met Leu Met Pro Thr Tyr
Phe Leu Leu Leu Leu Leu Leu Leu Leu Gly 1 5 10 15 Gly Pro Arg Thr
Ser Leu Ser His Lys Phe Tyr Asn Ala Gly Pro Ile 20 25 30 Phe Ser
Cys Leu Asn Thr Ala Leu Ser Glu Val Lys Lys Asn Lys Leu 35 40 45
Glu Asp Val Pro Val Leu Ser Lys Lys Asn Phe Gly Tyr Leu Pro Thr 50
55 60 Gln Asp Pro Ser Gly Glu Glu Glu Asp Glu Gln Lys His Ile Lys
Asn 65 70 75 80 Lys Arg Thr Phe Ser Asp Ala Val Gly Gly Asn Gly Gly
Arg Ser Ile 85 90 95 Arg Tyr Arg Tyr Gln Ser Pro Ala Gln Pro Lys
Gly Lys Leu Tyr Pro 100 105 110 Asp Lys Val Lys Asn Asp Arg Gly Thr
Lys Phe Thr Leu Ser Leu Asp 115 120 125 Val Pro Thr Asn Ile Met Asn
Ile Leu Phe Asn Ile Asp Lys Ala Lys 130 135 140 Asn Leu Arg Ala Lys
Ala Ala Ala Asn Ala Gln Leu Met Ala Gln Ile 145 150 155 160 Gly Lys
Lys Lys 7 161 PRT Homo sapiens human unprocessed Archipelin B 7 Met
Leu Met Pro Val His Phe Leu Leu Leu Leu Leu Leu Leu Leu Gly 1 5 10
15 Gly Pro Arg Thr Gly Leu Pro His Lys Phe Tyr Lys Ala Lys Pro Ile
20 25 30 Phe Ser Cys Leu Asn Thr Ala Leu Ser Glu Ala Glu Lys Gly
Gln Trp 35 40 45 Glu Asp Ala Ser Leu Leu Ser Lys Arg Ser Phe His
Tyr Leu Arg Ser 50 55 60 Arg Asp Ala Ser Ser Gly Glu Glu Glu Glu
Gly Lys Glu Lys Lys Thr 65 70 75 80 Phe Pro Ile Ser Gly Ala Arg Gly
Gly Ala Gly Gly Thr Arg Tyr Arg 85 90 95 Tyr Val Ser Gln Ala Gln
Pro Arg Gly Lys Pro Arg Gln Asp Thr Ala 100 105 110 Lys Ser Pro His
Arg Thr Lys Phe Thr Leu Ser Leu Asp Val Pro Thr 115 120 125 Asn Ile
Met Asn Leu Leu Phe Asn Ile Ala Lys Ala Lys Asn Leu Arg 130 135 140
Ala Gln Ala Ala Ala Asn Ala His Leu Met Ala Gln Ile Gly Arg Lys 145
150 155 160 Lys 8 1080 DNA Homo sapiens human Archipelin B
full-length cDNA modified_base (1)..(1080) n = g, a, c or t 8
cacagcccac ttaggaacaa taccggagaa gcaggagccg agaccccgga gcagccacaa
60 gttcatgggg acgtgcacgg ggccgccctc ctggccctga agctgcgccg
gcctccctga 120 gcgtttcgct gcggagggaa gtccactctc ggggagagat
gctgatgccg gtccacttcc 180 tgctgctcct gctgctgctc ctggggggcc
ccaggacagg cctcccccac aagttctaca 240 aagccaagcc catcttcagc
tgcctcaaca ccgccctgtc tgaggctgag aagggccagt 300 gggaggatgc
atccctgctg agcaagagga gcttccacta cctgcgcagc agagacgcct 360
cttcgggaga ggaggaggag ggcaaagaga aaaagacttt ccccatctct ggggccaggg
420 gtggagccgg aggcacccgg tacagatacg tgtcccaagc acagcccagg
ggaaagccac 480 gccaggacac ggccaagagt ccccaccgca ccaagttcac
cctgtccctc gacgtcccca 540 ccaacatcat gaacctcctc ttcaacatcg
ccaaggccaa gaacctgcgt gcccaggcgg 600 ccgccaatgc ccacctgatg
gcgcaaattg ggaggaagaa gtagaggcgg aggctggacg 660 ggagggcagc
ggggtgngga gggggagggg agggggaggg gagggcnagg gggggagggg 720
agggggaggg tgctgtctgc tggnttgtgt tttgtgggat cagtcagttt tacaggttgc
780 tgcactgctg agcccctctg atctcttctg gcctttgacc ctgtctccct
cctgctctgt 840 ctgtacacac agaagtgcag tattgtccaa ccttcccaga
cacaaagcag ctaacnttcc 900 tccctgtact caacgtctcc ttcctccctc
cccacagcaa gaggcaaagt tcatgcattc 960 ctcctctcca gtcttctctc
tgttgacccc atgcctgaga agagagcgtt cagggctcct 1020 ctcccacaca
tcaacttctg ccagggcaga aagaggagct gcagcactcg ccttcctgac 1080 9 38
PRT Homo sapiens human Archipelin cleavage product 9 Phe Thr Leu
Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu Leu Phe 1 5 10 15 Asn
Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala Ala Asn Ala 20 25
30 His Leu Met Ala Gln Ile 35 10 40 PRT Homo sapiens human
Archipelin cleavage product 10 Thr Lys Phe Thr Leu Ser Leu Asp Val
Pro Thr Asn Ile Met Asn Leu 1 5 10 15 Leu Phe Asn Ile Ala Lys Ala
Lys Asn Leu Arg Ala Gln Ala Ala Ala 20 25 30 Asn Ala His Leu Met
Ala Gln Ile 35 40 11 42 PRT Homo sapiens human Archipelin cleavage
product 11 Ser Pro His Arg Phe Thr Leu Ser Leu Asp Val Pro Thr Asn
Ile Met 1 5 10 15 Asn Leu Leu Phe Asn Ile Ala Lys Ala Lys Asn Leu
Arg Ala Gln Ala 20 25 30 Ala Ala Asn Ala His Leu Met Ala Gln Ile 35
40 12 49 PRT Homo sapiens human Archipelin cleavage product 12 Gln
Asp Thr Ala Lys Ser Pro His Arg Thr Lys Phe Thr Leu Ser Leu 1 5 10
15 Asp Val Pro Thr Asn Ile Met Asn Leu Leu Phe Asn Ile Ala Lys Ala
20 25 30 Lys Asn Leu Arg Ala Gln Ala Ala Ala Asn Ala His Leu Met
Ala Gln 35 40 45 Ile 13 51 PRT Homo sapiens human Archipelin
cleavage product 13 Pro Arg Gln Asp Thr Ala Lys Ser Pro His Arg Thr
Lys Phe Thr Leu 1 5 10 15 Ser Leu Asp Val Pro Thr Asn Ile Met Asn
Leu Leu Phe Asn Ile Ala 20 25 30 Lys Ala Lys Asn Leu Arg Ala Gln
Ala Ala Ala Asn Ala His Leu Met 35 40 45 Ala Gln Ile 50 14 53 PRT
Homo sapiens human Archipelin cleavage product 14 Gly Lys Pro Arg
Gln Asp Thr Ala Lys Ser Pro His Arg Thr Lys Phe 1 5 10 15 Thr Leu
Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu Leu Phe Asn 20 25 30
Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala Ala Asn Ala His 35
40 45 Leu Met Ala Gln Ile 50 15 21 DNA Artificial Sequence
Description of Artificial Sequencereverse amplification primer for
human Archipelin 15 gcgatgttga agaagaagtt c 21 16 16 DNA Artificial
Sequence Description of Artificial Sequenceforward amplification
primer for human Archipelin 16 atcgccaagg ccaaga 16 17 32 PRT
Artificial Sequence Description of Artificial Sequencetobacco
hornworm diuretic hormone 1 precursor corticotropin-releasing
factor (CRF) family peptide 17 Pro Ser Leu Ser Ile Asp Leu Pro Met
Ser Val Leu Arg Gln Lys Leu 1 5 10 15 Ser Leu Glu Lys Glu Arg Lys
Val His Ala Leu Arg Ala Ala Ala Asn 20 25 30 18 32 PRT Artificial
Sequence Description of Artificial Sequencetobacco hornworm
diuretic hormone 2 corticotropin-releasing factor (CRF) family
peptide MOD_RES (1)..(32) Xaa = any amino acid 18 Xaa Ser Phe Ser
Val Asn Pro Ala Val Asp Ile Leu Gln His Arg Tyr 1 5 10 15 Met Glu
Lys Val Ala Gln Asn Asn Arg Asn Phe Leu Asn Arg Val Xaa 20 25 30 19
32 PRT Artificial Sequence Description of Artificial Sequencehouse
cricket diuretic hormone corticotropin-releasing factor (CRF)
family peptide 19 Gln Ser Leu Ser Ile Val Ala Pro Leu Asp Val Leu
Arg Gln Arg Leu 1 5 10 15 Met Asn Glu Leu Asn Arg Arg Arg Met Arg
Glu Leu Gln Gly Ser Arg 20 25 30 20 32 PRT Artificial Sequence
Description of Artificial Sequencesauvage's leaf frog sauvagine
corticotropin-releasing factor (CRF) family peptide 20 Pro Pro Ile
Ser Ile Asp Leu Ser Leu Glu Leu Leu Arg Lys Met Ile 1 5 10 15 Glu
Ile Glu Lys Gln Glu Lys Glu Lys Gln Gln Ala Ala Asn Asn Arg 20 25
30 21 32 PRT Artificial Sequence Description of Artificial
Sequencewhite sucker corticoliberin 1 precursor, human pig and rat
corticoliberin precursor corticotropin-releasing factor (CRF)
family peptide 21 Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu
Arg Glu Val Leu 1 5 10 15 Glu Met Ala Arg Ala Glu Gln Leu Ala Gln
Gln Ala His Ser Asn Arg 20 25 30 22 32 PRT Artificial Sequence
Description of Artificial Sequencewhite sucker corticoliberin 2
precursor corticotropin-releasing factor (CRF) family peptide 22
Pro Pro Ile Ser Leu Asp Leu Thr Phe His Leu Leu Arg Glu Val Leu 1 5
10 15 Glu Met Ala Arg Ala Glu Gln Leu Val Gln Gln Ala His Ser Asn
Arg 20 25 30 23 32 PRT Artificial Sequence Description of
Artificial Sequencesheep corticoliberin precursor
corticotropin-releasing factor (CRF) family peptide 23 Pro Pro Ile
Ser Leu Asp Leu Thr Phe His Leu Leu Arg Glu Val Leu 1 5 10 15 Glu
Met Thr Lys Ala Asp Gln Leu Ala Gln Gln Ala His Ser Asn Arg 20 25
30 24 32 PRT Artificial Sequence Description of Artificial
Sequencexenopus corticoliberin precursor corticotropin-releasing
factor (CRF) family peptide 24 Pro Pro Ile Ser Leu Asp Leu Thr Phe
His Leu Leu Arg Glu Val Leu 1 5 10 15 Glu Met Ala Arg Ala Glu Gln
Ile Ala Gln Gln Ala His Ser Asn Arg 20 25 30 25 32 PRT Artificial
Sequence Description of Artificial Sequencelocust diuretic hormone
corticotropin-releasing factor (CRF) family peptide 25 Pro Ser Leu
Ser Ile Val Asn Pro Met Asp Val Leu Arg Gln Arg Leu 1 5 10 15 Leu
Leu Glu Ile Ala Arg Arg Arg Leu Arg Asp Ala Glu Glu Gln Ile 20 25
30 26 32 PRT Artificial Sequence Description of Artificial
Sequencestable fly diuretic hormone corticotropin-releasing factor
(CRF) family peptide 26 Pro Ser Leu Ser Ile Val Asn Pro Leu Asp Val
Leu Arg Gln Arg Leu 1 5 10 15 Leu Leu Glu Ile Ala Arg Arg Gln Met
Lys Glu Asn Thr Arg Gln Val 20 25 30 27 32 PRT Artificial
Sequence
Description of Artificial Sequencecockroach diuretic hormone
corticotropin-releasing factor (CRF) family peptide 27 Pro Ser Leu
Ser Ile Val Asn Pro Leu Asp Val Leu Arg Gln Arg Leu 1 5 10 15 Leu
Leu Glu Ile Ala Arg Arg Arg Met Arg Gln Ser Gln Asp Gln Ile 20 25
30 28 32 PRT Artificial Sequence Description of Artificial
Sequencewhite sucker urotensin I corticotropin-releasing factor
(CRF) family peptide 28 Pro Pro Ile Ser Ile Asp Leu Thr Phe His Leu
Leu Arg Asn Met Ile 1 5 10 15 Glu Met Ala Arg Ile Glu Asn Glu Arg
Glu Gln Ala Gly Leu Asn Arg 20 25 30 29 32 PRT Artificial Sequence
Description of Artificial Sequencecommon carp urotensin I precursor
corticotropin-releasing factor (CRF) family peptide 29 Pro Pro Ile
Ser Ile Asp Leu Thr Phe His Leu Leu Arg Asn Met Ile 1 5 10 15 Glu
Met Ala Arg Asn Glu Asn Gln Arg Glu Gln Ala Gly Leu Asn Arg 20 25
30 30 32 PRT Artificial Sequence Description of Artificial
Sequencehuman urocortin precursor corticotropin-releasing factor
(CRF) family peptide 30 Pro Ser Leu Ser Ile Asp Leu Thr Phe His Leu
Leu Arg Thr Leu Leu 1 5 10 15 Glu Leu Ala Arg Thr Gln Ser Gln Arg
Glu Arg Ala Glu Gln Asn Arg 20 25 30 31 32 PRT Artificial Sequence
Description of Artificial Sequencerat and mouse urocortin precursor
corticotropin-releasing factor (CRF) family peptide 31 Pro Pro Leu
Ser Ile Asp Leu Thr Phe His Leu Leu Arg Thr Leu Leu 1 5 10 15 Glu
Leu Ala Arg Thr Gln Ser Gln Arg Glu Arg Ala Glu Gln Asn Arg 20 25
30 32 32 PRT Artificial Sequence Description of Artificial
Sequencepufferfish urocortin precursor corticotropin-releasing
factor (CRF) family peptide 32 Leu Thr Leu Ser Leu Asp Val Pro Thr
Asn Ile Met Asn Val Leu Phe 1 5 10 15 Asp Val Ala Lys Ala Lys Asn
Leu Arg Ala Lys Ala Ala Glu Asn Ala 20 25 30 33 32 PRT Artificial
Sequence Description of Artificial Sequencehuman Archipelin peptide
33 Phe Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu Leu Phe
1 5 10 15 Asn Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala Ala
Asn Ala 20 25 30 34 32 PRT Artificial Sequence Description of
Artificial Sequencerat and mouse Archipelin peptide 34 Phe Thr Leu
Ser Leu Asp Val Pro Thr Asn Ile Met Asn Ile Leu Phe 1 5 10 15 Asn
Ile Asp Lys Ala Lys Asn Leu Arg Ala Lys Ala Ala Ala Asn Ala 20 25
30 35 38 PRT Homo sapiens MOD_RES (38) Xaa = isoleucinamide 35 Phe
Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu Leu Phe 1 5 10
15 Asn Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala Ala Asn Ala
20 25 30 His Leu Met Ala Gln Xaa 35 36 40 PRT Homo sapiens MOD_RES
(40) Xaa = isoleucinamide 36 Thr Lys Phe Thr Leu Ser Leu Asp Val
Pro Thr Asn Ile Met Asn Leu 1 5 10 15 Leu Phe Asn Ile Ala Lys Ala
Lys Asn Leu Arg Ala Gln Ala Ala Ala 20 25 30 Asn Ala His Leu Met
Ala Gln Xaa 35 40 37 44 PRT Homo sapiens MOD_RES (44) Xaa =
isoleucinamide 37 Ser Pro His Arg Thr Lys Phe Thr Leu Ser Leu Asp
Val Pro Thr Asn 1 5 10 15 Ile Met Asn Leu Leu Phe Asn Ile Ala Lys
Ala Lys Asn Leu Arg Ala 20 25 30 Gln Ala Ala Ala Asn Ala His Leu
Met Ala Gln Xaa 35 40 38 49 PRT Homo sapiens MOD_RES (49) Xaa =
isoleucinamide 38 Gln Asp Thr Ala Lys Ser Pro His Arg Thr Lys Phe
Thr Leu Ser Leu 1 5 10 15 Asp Val Pro Thr Asn Ile Met Asn Leu Leu
Phe Asn Ile Ala Lys Ala 20 25 30 Lys Asn Leu Arg Ala Gln Ala Ala
Ala Asn Ala His Leu Met Ala Gln 35 40 45 Xaa 39 51 PRT Homo sapiens
MOD_RES (51) Xaa = isoleucinamide 39 Pro Arg Gln Asp Thr Ala Lys
Ser Pro His Arg Thr Lys Phe Thr Leu 1 5 10 15 Ser Leu Asp Val Pro
Thr Asn Ile Met Asn Leu Leu Phe Asn Ile Ala 20 25 30 Lys Ala Lys
Asn Leu Arg Ala Gln Ala Ala Ala Asn Ala His Leu Met 35 40 45 Ala
Gln Xaa 50 40 53 PRT Homo sapiens MOD_RES (53) Xaa = isoleucinamide
40 Gly Lys Pro Arg Gln Asp Thr Ala Lys Ser Pro His Arg Thr Lys Phe
1 5 10 15 Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu Leu
Phe Asn 20 25 30 Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala
Ala Asn Ala His 35 40 45 Leu Met Ala Gln Xaa 50 41 40 PRT Mus sp.
and Rattus sp. MOD_RES (40) Xaa = isoleucinamide 41 Thr Lys Phe Thr
Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Ile 1 5 10 15 Leu Phe
Asn Ile Asp Lys Ala Lys Asn Leu Arg Ala Lys Ala Ala Ala 20 25 30
Asn Ala Gln Leu Met Ala Gln Xaa 35 40 42 41 PRT Mus sp. and Rattus
sp. MOD_RES (41) Xaa = isoleucinamide 42 Gly Thr Lys Phe Thr Leu
Ser Leu Asp Val Pro Thr Asn Ile Met Asn 1 5 10 15 Ile Leu Phe Asn
Ile Asp Lys Ala Lys Asn Leu Arg Ala Lys Ala Ala 20 25 30 Ala Asn
Ala Gln Leu Met Ala Gln Xaa 35 40 43 44 PRT Mus sp. MOD_RES (44)
Xaa = isoleucinamide 43 Ser Asp Arg Gly Thr Lys Phe Thr Leu Ser Leu
Asp Val Pro Thr Asn 1 5 10 15 Ile Met Asn Ile Leu Phe Asn Ile Asp
Lys Ala Lys Asn Leu Arg Ala 20 25 30 Lys Ala Ala Ala Asn Ala Gln
Leu Met Ala Gln Xaa 35 40 44 46 PRT Mus sp. MOD_RES (46) Xaa =
isoleucinamide 44 Pro Lys Ser Asp Arg Gly Thr Lys Phe Thr Leu Ser
Leu Asp Val Pro 1 5 10 15 Thr Asn Ile Met Asn Ile Leu Phe Asn Ile
Asp Lys Ala Lys Asn Leu 20 25 30 Arg Ala Lys Ala Ala Ala Asn Ala
Gln Leu Met Ala Gln Xaa 35 40 45 45 51 PRT Mus sp. and Rattus sp.
MOD_RES (51) Xaa = isoleucinamide 45 Leu Tyr Pro Asp Lys Pro Lys
Ser Asp Arg Gly Thr Lys Phe Thr Leu 1 5 10 15 Ser Leu Asp Val Pro
Thr Asn Ile Met Asn Ile Leu Phe Asn Ile Asp 20 25 30 Lys Ala Lys
Asn Leu Arg Ala Lys Ala Ala Ala Asn Ala Gln Leu Met 35 40 45 Ala
Gln Xaa 50 46 44 PRT Rattus sp. MOD_RES (44) Xaa = isoleucinamide
46 Asn Asp Arg Gly Thr Lys Phe Thr Leu Ser Leu Asp Val Pro Thr Asn
1 5 10 15 Ile Met Asn Ile Leu Phe Asn Ile Asp Lys Ala Lys Asn Leu
Arg Ala 20 25 30 Lys Ala Ala Ala Asn Ala Gln Leu Met Ala Gln Xaa 35
40 47 46 PRT Rattus sp. MOD_RES (46) Xaa = isoleucinamide 47 Val
Lys Asn Asp Arg Gly Thr Lys Phe Thr Leu Ser Leu Asp Val Pro 1 5 10
15 Thr Asn Ile Met Asn Ile Leu Phe Asn Ile Asp Lys Ala Lys Asn Leu
20 25 30 Arg Ala Lys Ala Ala Ala Asn Ala Gln Leu Met Ala Gln Xaa 35
40 45 48 52 PRT Rattus sp. MOD_RES (52) Xaa = isoleucinamide 48 Gly
Leu Tyr Pro Asp Lys Val Lys Asn Asp Arg Gly Thr Lys Phe Thr 1 5 10
15 Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Ile Leu Phe Asn Ile
20 25 30 Asp Lys Ala Lys Asn Leu Arg Ala Lys Ala Ala Ala Asn Ala
Gln Leu 35 40 45 Met Ala Gln Xaa 50 49 11 PRT Rattus sp. rat
Archipelin peptide from rat serum 49 Ala Ala Ala Asn Ala Gln Leu
Met Ala Gln Ile 1 5 10 50 13 PRT Rattus sp. rat Archipelin peptide
from rat serum 50 Ala Lys Ala Ala Ala Asn Ala Gln Leu Met Ala Gln
Ile 1 5 10 51 22 PRT Rattus sp. rat Archipelin peptide from rat
serum 51 Phe Thr Leu Ser Leu Asp Val Pro Thr Asn Ile Met Asn Ile
Leu Phe 1 5 10 15 Asn Ile Asp Lys Ala Lys 20 52 20 PRT Rattus sp.
rat Archipelin peptide from rat serum 52 Phe Thr Leu Ser Leu Asp
Val Pro Thr Asn Ile Met Asn Ile Leu Phe 1 5 10 15 Asn Ile Asp Lys
20 53 200 PRT Artificial Sequence Description of Artificial
Sequencepoly Gly flexible linker MOD_RES (6)...(200) Gly at
positions 6-200 may be present or absent 53 Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 1 5 10 15 Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 20 25 30 Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 35 40 45
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 50
55 60 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly 65 70 75 80 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly 85 90 95 Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly 100 105 110 Gly Gly Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly 115 120 125 Gly Gly Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly 130 135 140 Gly Gly Gly Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 145 150 155 160 Gly Gly
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 165 170 175
Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly 180
185 190 Gly Gly Gly Gly Gly Gly Gly Gly 195 200 54 14 PRT
Artificial Sequence Description of Artificial Sequencerat and mouse
Archipelin peptide used to develop antisera 54 Cys Leu Phe Asn Ile
Asp Lys Ala Lys Asn Leu Arg Ala Lys 1 5 10 55 40 PRT Homo sapiens
major serum form of human Archipelin peptide 55 Thr Lys Phe Thr Leu
Ser Leu Asp Val Pro Thr Asn Ile Met Asn Leu 1 5 10 15 Leu Phe Asn
Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala Ala 20 25 30 Asn
Ala His Leu Met Ala Gln Ile 35 40 56 41 PRT Homo sapiens major
serum form of human Archipelin peptide 56 Arg Thr Lys Phe Thr Leu
Ser Leu Asp Val Pro Thr Asn Ile Met Asn 1 5 10 15 Leu Leu Phe Asn
Ile Ala Lys Ala Lys Asn Leu Arg Ala Gln Ala Ala 20 25 30 Ala Asn
Ala His Leu Met Ala Gln Ile 35 40 57 38 PRT Rattus sp. major serum
form of rat Archipelin peptide 57 Phe Thr Leu Ser Leu Asp Val Pro
Thr Asn Ile Met Asn Ile Leu Phe 1 5 10 15 Asn Ile Asp Lys Ala Lys
Asn Leu Arg Ala Lys Ala Ala Ala Asn Ala 20 25 30 Gln Leu Met Ala
Gln Ile 35
* * * * *
References