US20030104495A1

US20030104495A1 - Sperm-specific AKAP protein genes and uses

Info

Publication number: US20030104495A1
Application number: US10/244,488
Authority: US
Inventors: Daniel Carr; Srinivasan Vijayaraghavan
Original assignee: Individual
Current assignee: Individual
Priority date: 1999-03-16
Filing date: 2002-09-16
Publication date: 2003-06-05
Also published as: US6451528B1

Abstract

This invention provides isolated nucleic acid encoding sperm-specific protein kinase A anchoring proteins from mammalian species, recombinant expression constructs encoding said mammalian sperm-specific protein kinase A anchoring proteins, cells transformed with said constructs, and homogenous preparations of these proteins prepared using recombinant genetic techniques. The invention also provides analytic tools such as polyclonal antisera and monoclonal antibodies specific for said sperm-specific protein kinase A anchoring proteins of the invention. The invention also provides methods for isolating nucleic acid encoding mammalian sperm-specific protein kinase A anchoring proteins and recombinant genetic methods for producing said mammalian sperm-specific protein kinase A anchoring proteins of the invention. The invention particularly provides high throughput screening methods for screening compounds that can disrupt binding between protein kinase A and the sperm-specific protein kinase A anchoring protein and methods for determining and producing compounds, particularly antisense oligonucleotides, for inhibiting or preventing expression of sperm-specific protein kinase A anchoring protein in sperm, spermatids, or progenitor cells thereof. These methods are useful for development of effective, male-specific contraceptives, which are also provided by the invention.

Description

[0001] This invention was made with government support under grant number R29HD32508 from the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to sperm-specific protein kinase A anchoring proteins from mammalian species and the genes encoding such proteins. Specifically, the invention relates to the isolation of nucleic acid encoding sperm-specific protein kinase A anchoring proteins from mammalian species, and the production of homogenous preparations of such proteins using recombinant genetic techniques. The invention also relates to the preparation of analytic tools such as polyclonal antisera and monoclonal antibodies specific for said sperm-specific protein kinase A anchoring proteins. Importantly, the invention is related to high throughput screening of compounds that can disrupt binding between protein kinase A and the sperm-specific protein kinase A anchoring proteins of the invention, or that inhibit or prevent expression of the sperm-specific protein kinase A anchoring proteins in sperm, spermatids or progenitor cells thereof, for development of effective contraceptives, and provides antisense oligonucleotides, agents and compounds detected using the screening methods of the invention as reversible, male contraceptive agents.

2. Background of the Invention

Signal transduction enzymes such as protein kinases and phosphatases play pivotal roles in mediating cellular responses to a wide variety of stimuli. Cyclic AMP (cAMP) mediates the motility of sperm and a variety of other ciliated cells (Satir, 1985, Molec. Cell Biol. 4: 1-46; Tash, 1989, Cell Motil. Cytoskel. 14: 332-339; Bedford and Hoskins 1990, in Marchall's Physiology of Reproduction, (Lamming, ed.), Churchill Livingstone: New York, p. 379). Increases in the level of this nucleotide are associated with development of sperm motility in the epididymis (Bedford and Hoskins, ibid.; Hoskins et al., 1974, J. Reprod. Fertil. 37: 131-133). Cell-permeant cAMP analogs, cAMP phosphodiesterase inhibitors, and adenylyl cyclase activators all stimulate motility of sperm from several species (Garbers et al., 1972, Biol. Reprod. 7: 132; Garbers et al., 1978, Adv. Cyclic Nucleotide Res. 9: 583-595). Flagellar activity in sperm has been shown to be regulated by cAMP (Tash, ibid.; Garbers and Kopf, 1980, in Advances in Cyclic Nucleotide Research, (Greengard and Robison, eds.), Vol. 13, Raven Press: New York, pp. 252-306; Lindemann and Kanous, 1989, Arch. Androl. 23: 1-22; Tash & Means, 1988, Prog. Clin. Biol. Res. 267: 335-355).

The cAMP-dependent protein kinase (PKA) is a ubiquitous, multifunctional enzyme involved in the regulation of a diverse array of cellular events. PKA holoenzyme consists of four subunits, two catalytic (C) and two regulatory (R). In the absence of cAMP, the regulatory subunits keep the catalytic subunits inactive. Cyclic AMP binding to the regulatory subunit promotes dissociation from and activation of the catalytic subunits. There are two R subunit classes, RI and RII, which form the type I and type II holoenzymes, respectively. Type II PKA is present in all cells, whereas the tissue distribution of type I PKA is more restricted. Subcellular localization of PKA is directed through the regulatory (R) subunit. Recent studies have shown that PKA is anchored at specific subcellular sites through the interaction of its regulatory subunit with A-kinase anchoring proteins (AKAPs) (Carr et al., 1991, J. Biol. Chem. 266: 14188-14192; Rubin, 1994, Biochim. Biophys. Acta 1224: 467-479; Dell'Aqua & Scott, 1997, J. Biol. Chem. 272: 12881-4; Faux et al., 1996, Cell 85: 9-12; Pawson et al., 1997, Science 278: 2075-2080).

A number of AKAPs have been cloned and biochemically characterized (Dell'Aqua & Scott, ibid.). All AKAPs contain a common structural motif that binds with nanomolar (nM) affinity to the regulatory subunit of PKA (Carr et al., 1991, ibid.; Carr et al., 1992, J. Biol. Chem. 267: 13376-13382). Certain of the anchoring proteins that are capable of binding both RI Huang et al., 1997, Proc. Natl. Acad. Sci. USA 94: 11184-11189). Since PKA has broad substrate specificity, presumably one of the primary functions of PKA anchoring is to spatially restrict its action, thus providing specificity of function.

Because cAMP analogs and agents that increase intracellular cAMP are potent stimulators of sperm motility, the role of PKA and AKAP subcellular localization of PKA was expected by the inventors to be important for understanding and modulating sperm motility. The highly polarized sperm cell is particularly well suited for investigating the structure and function of PKA anchoring proteins. Differentiated sperm have a number of district subcellular structures. The microtubular apparatus in mammalian sperm is surrounded by district cellular organelles such as the outer dense fibers and fibrous sheath. The cytoplasmic volume of the sperm cell is considerably lower than that of most somatic cells. Free diffusion of plasma membrane proteins is apparently restricted to three distinct domains, defined by the sperm head, midpiece and tail regions (Friend, 1982, J. Cell Biol. 93: 243-249). This unique compartmentalization of the sperm cell made it an excellent model system to study the role of targeting and anchoring of PKA and other enzymes in regulating cell function.

Regulation of sperm motility also provides an important avenue for developing a new generation of contraceptive agents. In the past, contraception has relied on barrier methods (such as condoms) and in regulating a female's capacity to be impregnated or the fertilized zygote to attach to the uterus (examples include the intrauterine device and “the Pill”). Regulation of cAMP-stimulated sperm motility by targeting the PKA/AKAP interaction provides for the first time a male-specific route of contraceptive intervention.

There thus remains a need in this art for specific, effective, reversible, reliable male contraceptive with minimal physiological or systemic side effects. As disclosed herein, one particular PKA anchoring protein, termed AKAP110, is uniquely expressed in sperm, making it possible that therapies which interfere with or prevent expression of this protein could be used as a contraceptive. One example of such an approach is to use antisense oligonucleotides to inhibit or prevent AKAP110 gene expression in developing sperm or spermatids, which have been shown to express this protein. Antisense-based therapeutics are already being used in humans, and it is likely that systemic administration of antisense reagents will be achieved in the near future. Antisense approaches to male contraception have clear advantages over other methods, such as hormonal or immunological methods, such as being completely reversible and in having a likelihood of fewer and less physiologically significant side effects. Other examples of novel contraceptive approaches is the use of peptides or small molecules that directly interfere with the AKAP110:PKA binding interaction in the absence of threshold levels of cAMP.

SUMMARY OF THE INVENTION

The present invention relates to the cloning, expression and functional characterization of mammalian sperm-specific protein kinase A anchoring protein having an apparent molecular weight of about 110 kildaltons, termed “AKAP110” herein.

This invention specifically provides isolated nucleic acid encoding sperm-specific protein kinase A anchoring protein from human, bovine and murine male germ cells, recombinant expression constructs comprising said nucleic acid, and cells transformed with such recombinant expression constructs and producing the sperm-specific protein kinase A anchoring protein thereby. The invention also provides homogenous preparations of sperm-specific protein kinase A anchoring protein, and antibodies raised against and that immunologically recognize said protein. The invention also provides methods for producing said homogeneous preparation of the sperm-specific protein kinase A anchoring proteins of the invention using said cells transformed with the recombinant expression constructs of the invention. The invention advantageously provides methods for screening compounds for the capacity to disrupt binding between the sperm-specific protein kinase A anchoring protein of the invention and protein kinase A regulatory subunits, and methods for disrupting, inhibiting or preventing expression of this protein in developing sperm cells, spermatids or progenitor cells thereof, in order to provide male-specific contraception agents. Methods of male contraception, most preferably and advantageously readily reversible methods of male contraception, and methods for the prevention of pregnancy are provided by the use of compounds that disrupt, inhibit or prevent expression of the AKAP110 protein of the invention, whereby such compounds are detected or characterized using the methods of the invention. Advantageously, the invention also provides agents, preferably pharmaceutical agents, for effecting male contraception, most preferably comprising antisense oligonucleotides for inhibiting or preventing expression of AKAP110 in sperm cells, spermatids and progenitors thereof, and peptides and small molecules for disrupting AKAP110/PKA RII binding in said cells.

Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate one- (FIG. 1A) and two-dimensional (FIG. 1B) gel electrophoretic analyses of extracted bovine sperm proteins. Numbering in the left-hand side of each Figure are molecular weight markers (in kilodaltons). Electrophoresis in each of the Figures in the vertical direction is SDS/PAGE; electrophoresis in the horizontal direction in FIG. 1B is isoelectric focusing, performed between [0014] pH 10 and pH 3 (arrayed left-to-right in the Figure). Paired lanes in FIG. 1A are labeled “S” for supernatant and “P” for pellet, as described in Example 1. The arrow in FIG. 1B denotes the position of the protein band excised for microsequencing analysis as described in Example 1.
FIG. 2 is a sequence alignment of human, bovine and murine AKAP110 amino acid sequences. Shaded areas indicate sequence identity between at least two of the three species. [0015]
FIGS. 3A, 3B and [0016] 3C depict RII overlay (FIG. 3A) and Western blot assays (FIGS. 3B and 3C). Numbering in the left-hand side of FIG. 3A represents molecular weight markers (in kilodaltons).
FIGS. 4A and 4B depict RII overlay (FIG. 4A) and Western blot assays (FIG. 4B) of identical blots containing full-length (1-864) and truncated fragments (1-470; 471-864; 1-350; 1-164; and 1-117) of murine AKAP110 protein expressed and isolated from [0017] E. coli.
FIG. 4C is a schematic diagram of RII binding to AKAP110 and fragments thereof. [0018]
FIG. 4D depicts Western blot analysis performed in the absence (left-hand lane) or presence (right-hand lane) of the peptide used to prepare the antibody with which the Western blots were probed. Numbering in the left-hand side of the Figure represents molecular weight markers (in kilodaltons). [0019]
FIG. 5A is a amino acid sequence alignment of RII binding domains of a variety of AKAP proteins: AKAP110 (Seq. ID No. 19); AKAP82 (Seq. ID No. 20); S-AKAP84 (Seq. ID No. 21); TAKAP80 (Seq. ID No. 22); MAP2 (Seq. ID No. 23); Ht31 (Seq. ID No. 24); AKAP150 (Seq. ID No. 25); AKAP75 (Seq. ID No. 26); AKAP92 (Seq. ID No. 27); AKAP120 (Seq. ID No. 28); AKAP220 (Seq. ID No. 29); Gravin (Seq. ID No. 30); AKAP-KL (Seq. ID No. 31); AKAP18 (Seq. ID No. 32) and Ezrin (Seq. ID No. 33). Shaded residues are conserved in a majority of the 15 aligned sequences. [0020]
FIGS. 5B and 5C are schematic diagrams of helical wheel representations of the AKAP consensus sequence of the RII binding domain (FIG. 5B) and the AKAP110 RII binding domain (FIG. 5C). [0021]
FIG. 6 is a schematic alignment of AKAP110 and AKAP82 amino acid sequences. The boxed regions represent portions of the amino acid sequence of at least 20 amino acids having greater than 50% sequence homology. [0022]
FIG. 7 is an autoradiogram of Northern blot hybridization of mRNAs from various mouse tissues probed with an AKAP110 cDNA. Numbering in the left-hand side of the Figure represents molecular weight markers (in kilobases). [0023]
FIGS. 8A through 8C show the results of in situ hybridization analyses performed as described in Example 6. FIG. 8A shows hybridization to frozen cryosection of mouse testis. “L” indicates the seminiferous tubule lumen; “RS” represents round spermatids of the seminiferous epithelium. Dotted lines represent boundaries between adjacent seminiferous tubules. FIG. 8B represents a higher-magnification view of a single seminiferous tubule hybridized with antisense AKAP110 probe. The bracketed areas represent the basal region of the seminiferous epithelium, containing spermatogonia, spermatocytes and Sertoli cells. “LS” represents late spermatids adjacent to the tubule lumen. Hybridization is detected only in the RS region of this section. FIG. 8C is in situ hybridization performed using a sense AKAP110 probe; no hybridization was detected. [0024]
FIGS. 9A and 9B are matched phase-contrast (FIG. 9A) and immunofluorescence (FIG. 9B) photomicrographs of caudal epididymal spermatozoa. The bar at the upper left-hand corner labeled “A” equals 15 μM. “F” indicates sperm flagella, “H” indicates sperm heads, “AC” indicates acrosomal segments, and “AC*” represents acrosomal caps released from the sperm heads. Inserts labeled “B” and “B′” are phase-contrast and immunofluorescence photomicrographs incubated with preimmune serum.[0025]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The terms “mammalian sperm-specific protein kinase A anchoring protein having an apparent molecular weight of about 110 kildaltons” and “AKAP110” as used herein are intended to encompass proteins having the biological activity of any of the sperm-specific protein kinase A anchoring proteins disclosed herein or PKA-RII subunit-binding fragments thereof. This definition is also intended to encompass natural allelic variations in the sperm-specific protein kinase A anchoring protein-encoding sequences of the invention. Cloned nucleic acid provided by the present invention encodes sperm-specific protein kinase A anchoring proteins of any species of origin, including, for example, mouse, cow, horse, pig, rat, rabbit, dog, cat, and human, but preferably the nucleic acid provided by the invention encodes sperm-specific protein kinase A anchoring protein including the murine (SEQ ID Nos.: 11 and 12), bovine (SEQ ID Nos.: 13 and 14), and human (SEQ ID Nos.: 15 and 16) homologues. In addition, sequence alignment of the nucleic acid sequences of the murine (SEQ ID No. 11), bovine (SEQ ID No. 13) and human (SEQ ID No. 15) sequences provides one of ordinary skill in the art with sufficient information to be able to identify a nucleic acid probe wherein the sequence (or the amino acid sequence encoded therein) between the three mammalian species are identical. Such a sequence provides the skilled artisan with a probe that is useful for isolating a nucleic acid encoding AKAP110 from any mammalian species. Thus, this invention provides a plurality of consensus sequences for probe production, wherein said probes are useful for isolation or detection of AKAP110 genes or gene expression from any mammalian species. [0026]
The production of proteins such as the sperm-specific protein kinase A anchoring protein of the invention from cloned genes by genetic engineering means is well known in this art. The discussion which follows is accordingly intended as an overview of this field, and is not intended to reflect the full state of the art. [0027]
Nucleic acid encoding the sperm-specific protein kinase A anchoring proteins of this invention can be obtained, in view of the instant disclosure, by chemical synthesis, by screening reverse transcripts of mRNA from testis cells or cultured cell lines derived therefrom, by screening genomic libraries from appropriate cells, or by combinations of these procedures, as illustrated below. Screening of cDNA prepared from cellular mRNA or genomic DNA may be carried out with full-length, partial or oligonucleotide probes generated from the sperm-specific protein kinase A anchoring protein nucleotide sequence information provided herein, as discussed above. Probes may be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with known procedures and used in conventional hybridization assays, as described in greater detail in the Examples below. In the alternative, sperm-specific protein kinase A anchoring protein encoding nucleic acid can be obtained by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers being produced from the sperm-specific protein kinase A anchoring protein nucleotide sequences provided herein. See U.S. Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis. In addition, consensus proteins comprising artificially produced and/or genetically engineered proteins encoding at each sequence position an amino acid comprising the “consensus” between the nucleic acid sequences identified as Seq. ID Nos. 11, 13 and 15 are also provided by the invention. [0028]
Nucleic acids and oligonucleotides of the present invention are useful as probes for screening DNA libraries to isolate and characterize additional mammalian homologues of the sperm-specific protein kinase A anchoring protein-encoding nucleic acids of the invention. For the purposes of this invention, these probes are used under high stringency hybridization conditions, defined herein as follows. The probes are labeled with 150 μCi [0029] ³²P-ATP and hybridized overnight at 42° C. in a hybridization buffer comprising 6×SSC (0.9M NaCl, 0.09M sodium citrate dihydrate (pH 7.0)), 2×Denhardt's solution (0.04% Ficoll, 0.04% polyvinylpyrrolidone, 0.04% bovine serum albumin), and 0.25% sodium dodecyl sulfate (SDS). After hybridization, filters are washed four or five times for ten minutes apiece in an excess volume of 6×SSC (0.9 M NaCl, 0.09M sodium citrate dihydride, pH 7.0) and 0.05% SDS at room temperature. Thereafter, three ten-minute washes are performed one after another in this buffer at temperatures of 55° C., 60° C. and 63° C.
Sperm-specific protein kinase A anchoring proteins may be synthesized in cells, most preferably cells comprising exogenously-added, recombinant DNA, most preferably cDNA, encoding said proteins. In particular, cells transformed with a recombinant expression construct comprising a nucleic acid encoding a sperm-specific protein kinase A anchoring protein of the invention can be used to provide a homogeneous culture of sperm-specific protein kinase A anchoring protein expressing cells. Recombinant expression constructs comprising the sperm-specific protein kinase A anchoring protein coding sequences as disclosed herein can also be comprised of a vector that is a replicable DNA construct. Vectors are used herein either to amplify DNA encoding a sperm-specific protein kinase A anchoring protein and/or to express DNA which encodes a sperm-specific protein kinase A anchoring protein. For the purposes of this invention, a recombinant expression construct is a replicable DNA construct in which a DNA sequence encoding a sperm-specific protein kinase A anchoring protein is operably linked to suitable control sequences capable of effecting the expression of the receptor in a suitable host cell. The need for such control sequence will vary depending upon the host selected and the transformation method chosen. Generally, control sequences include a transcriptional promoter, an optional operator or enhancer sequence to control or regulate transcription, a sequence encoding suitable mRNA ribosomal binding sites, and sequences which control the termination of transcription and translation, and in mammalian cells, sequences that direct 5′ terminal capping and 3′ terminal polyadenylation of the primary transcript. Amplification vectors, on the other hand, do not require expression control domains. All that is needed is the ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants. See, Sambrook et al., 1990, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press: New York). [0030]
Vectors useful for practicing the present invention include plasmids, viruses (including phage), retroviruses, and particularly integratable DNA fragments (i.e., fragments integratable into the host genome by recombination). The vector may replicate and function independently of the host genome, or more preferably, may, in some instances, integrate into the genome itself. Suitable vectors will contain replicon and control sequences which are derived from species compatible with the intended expression host. Transformed host cells are preferably prokaryotic or eukaryotic cells which have been transformed or transfected with recombinant expression constructs made using recombinant DNA techniques and comprising mammalian sperm-specific protein kinase A anchoring protein-encoding sequences. Preferred host cells are bacterial cells, most preferably [0031] Escherichia coli cells. Transformed host cells are chosen that are capable of expressing functional sperm-specific protein kinase A anchoring protein introduced using the recombinant expression construct. See, Sambrook et at., ibid.
Homogenous compositions of the mammalian sperm-specific protein kinase A anchoring proteins are provided by the invention. As used herein, the term “homogenous” is intended to encompass preparations wherein the mammalian sperm-specific protein kinase A anchoring protein of the invention is the predominant protein species in the preparation, preferably wherein the mammalian sperm-specific protein kinase A anchoring protein comprises at least 70%, more preferably wherein the mammalian sperm-specific protein kinase A anchoring protein comprises from 80-90%, and most preferably where the mammalian sperm-specific protein kinase A anchoring protein comprises greater than 90% of the protein in the preparation. In preferred embodiments, said preparations have one or a multiplicity of additional protein components wherein no one component comprises more than about 5% and more preferably about 1% of the protein in the preparation. [0032]
The invention also provides antibodies that are immunologically reactive to a mammalian sperm-specific protein kinase A anchoring protein, most preferably the bovine, murine or human homologues explicitly disclosed herein. The antibodies provided by the invention are raised in animals by inoculation with homogenous preparations of a mammalian sperm-specific protein kinase A anchoring protein of the invention or epitopes thereof, using methods well known in the art. Alternatively, peptide fragments of the mammalian sperm-specific protein kinase A anchoring protein of the invention can be chemically synthesized or isolated from the full-length sequence, covalently attached to a carrier molecule such as keyhole limpet hemocyanin, and injected into an appropriate experimental animal. Animals that are used for such inoculations include individuals from species comprising cows, sheep, pigs, mice, rats, rabbits, hamsters, goats and primates. Preferred animals for inoculation are rodents (including mice, rats, and hamsters) and rabbits. For preparing polyclonal antisera, the most preferred animal is rabbit. For preparing monoclonal antibodies, the most preferred animal is the mouse. [0033]
Cells that can be used for such inoculations, or for any of the other means used in the invention, include any cell, clonal isolate or cell line that expresses a mammalian sperm-specific protein kinase A anchoring protein or any epitope thereof, most preferably as a result of molecular or genetic engineering. Preferred cells for preparing said recombinant protein is [0034] E. coli cells, most preferably having been transformed with a recombinant expression construct comprising a nucleic acid encoding a human, bovine or murine sperm-specific protein kinase A anchoring protein or epitopes thereof, and that express the mammalian gene product, wherein it is understood that such preparations are sufficiently purified from pyrogenic or other bacterial toxins or contaminants as to be suitable for injection into an animal.
The present invention provides polyclonal antisera and monoclonal antibodies that are immunologically reactive with an epitope that is a mammalian sperm-specific protein kinase A anchoring protein or fragment thereof. These antibodies are made using methods and techniques well known to those of skill in the art. [0035]
Monoclonal antibodies provided by the present invention are produced by hybridoma cell lines, that are also provided by the invention and that are made by methods well known in the art. Hybridoma cell lines are made by fusing individual cells of a myeloma cell line with spleen cells derived from animals immunized with a mammalian sperm-specific protein kinase A anchoring protein, or cells expressing such a mammalian sperm-specific protein kinase A anchoring protein, preferably the murine, bovine or human sperm-specific protein kinase A anchoring protein, as described above. The myeloma cell lines used in the invention include lines derived from myelomas of mice, rats, hamsters, primates and humans. Preferred myeloma cell lines are from mouse, and the most preferred mouse myeloma cell line is P3X63-Ag8.653. The animals from whom spleens are obtained after immunization are rats, mice and hamsters, preferably mice, most preferably Balb/c mice. Spleen cells and myeloma cells are fused using a number of methods well known in the art, including but not limited to incubation with inactivated Sendai virus and incubation in the presence of polyethylene glycol (PEG). The most preferred method for cell fusion is incubation in the presence of a solution of 45% (w/v) PEG-1450. Monoclonal antibodies produced by hybridoma cell lines can be harvested from cell culture supernatant fluids from in vitro cell growth; alternatively, hybridoma cells can be injected subcutaneously and/or into the peritoneal cavity of an animal, most preferably a mouse, and the monoclonal antibodies obtained from blood and/or ascites fluid. [0036]
Monoclonal antibodies provided by the present invention are also produced by recombinant genetic methods well known to those of skill in the art, and the present invention encompasses antibodies made by such methods that are immunologically reactive with an epitope of a mammalian sperm-specific protein kinase A anchoring protein. [0037]
The present invention encompasses fragments of the antibody that are immunologically reactive with an epitope of a mammalian sperm-specific protein kinase A anchoring protein. Such fragments are produced by any number of methods, including but not limited to proteolytic cleavage, chemical synthesis or preparation of such fragments by means of genetic engineering technology, and include Fab fragments, Fab′ fragments, F(ab)[0038] ₂fragments, and F_vfragments. The present invention also encompasses single-chain antibodies that are immunologically reactive with an epitope of a mammalian sperm-specific protein kinase A anchoring protein made by methods known to those of skill in the art. In preferred embodiments, the mammalian sperm-specific protein kinase A anchoring protein is a bovine, murine or human homologue as provided by the invention.
The present invention also encompasses an epitope of a mammalian sperm-specific protein kinase A anchoring protein that is comprised of sequences and/or a conformation of sequences present in the mammalian sperm-specific protein kinase A anchoring protein molecule. This epitope may be naturally occurring, or may be the result of proteolytic cleavage of the mammalian sperm-specific protein kinase A anchoring protein molecule and isolation of an epitope-containing peptide or may be obtained by chemical and more preferably automated chemical synthesis of an epitope-containing peptide using methods well known to those skilled in the art. The epitope also comprises a “consensus” epitope, wherein the identity of the amino acid residue at each position in the epitope sequence represents the “consensus” residue obtained by comparison of the amino acid sequences identified as Seq. ID Nos. 12, 14 and 16. The present invention also encompasses epitope peptides produced as a result of genetic engineering technology and synthesized by genetically engineered cells, preferably prokaryotic cells and most preferably [0039] E. coli cells.
The invention also provides methods for screening compounds for the capacity to disrupt binding between the mammalian sperm-specific protein kinase A anchoring proteins of the invention and protein kinase A, most preferably the RII subunit of protein kinase A. In these methods, homogenous preparations of both the mammalian sperm-specific protein kinase A anchoring protein of the invention and protein kinase A, preferably recombinant embodiments thereof and most preferably recombinant RII subunits thereof, are used to assay the effects of the screened compounds on AKAP-PKA binding. Preferably, the mammalian sperm-specific protein kinase A anchoring protein of the invention or protein kinase A, most preferably said RII subunit thereof, are detectably labeled, which for the purposes of this aspect is intended to encompass but not be limited to radioactive, fluorescent and antigenic labels. In other preferred embodiments, the mammalian sperm-specific protein kinase A anchoring protein of the invention or protein kinase A, most preferably said RII subunit thereof, are immobilized to a substrate. For the purposes of this disclosure, the term “substrate” is intended to encompass solid platforms such as are used as understood by those with skill in the art in enzyme-linked immunosorbent assay (“ELISA”) and radioimmune assay (“RIA”) wells and the wells of plastic 96-well plates, and in addition beads and chromatographic substrates. In certain embodiments, the methods are performed as variants of the RII overlay method described herein below. In alternative embodiments, particularly suited for high throughput screening and automation, the methods are performed to resemble ELISA and RIA assays, whereby at least one of either the mammalian sperm-specific protein kinase A anchoring protein of the invention or protein kinase A are immobilized to a solid support. In preferred embodiments of each of these aspects of the methods of the invention, either the mammalian sperm-specific protein kinase A anchoring protein of the invention or protein kinase A is detectably labeled. The invention also provides the agents and compounds detecting using these methods that are capable of disrupting AKAP:PKA RII binding. [0040]
The invention also provides methods for inhibiting or preventing expression of the mammalian sperm-specific protein kinase A anchoring protein of the invention in sperm, spermatids or progenitor cells thereof, said methods comprising introducing an antisense oligonucleotide into said cell. Said methods further comprise screening candidate oligonucleotides in recombinant cells, most preferably mammalian cells, transformed with a recombinant expression construct of the invention encoding a mammalian sperm-specific protein kinase A anchoring protein as disclosed herein, and assaying said cell in the presence and absence of the candidate oligonucleotide for recombinant gene expression. The invention also provides the oligonucleotides detected using these methods that are capable of inhibiting or preventing recombinant AKAP expression. [0041]
The Examples which follow are illustrative of specific embodiments of the invention, and various uses thereof. They set forth for explanatory purposes only, and are not to be taken as limiting the invention. [0042]

EXAMPLE 1

Isolation and Characterization of Bovine AKAP110 Protein from Bovine Sperm

In order to derive a probe for isolating cDNA encoding an anchoring kinase A protein having an apparent molecular weight of 110 kD (AKAP110) in sperm, AKAP110 protein was isolated from bovine sperm as follows. [0043]
Testes from mature bulls with intact tunica were obtained from a local slaughterhouse and sperm from caput or caudal epididymis were isolated and washed as described in Vijayaraghavan et al. (1985, [0044] Biol. Reproduc. 32: 489-500). The washed sperm were fractionated into heads, tails and midpieces as described in Mohapatra et al. (1998, Biochem. Biophys. Res. Commun. 244: 540-545). Tails were pelleted at 16,000×g for 15min and resuspended in homogenization buffer (HB) comprising 50 mM Tris-HCl (pH 7.0), 2 mM EGTA, 2 mM EDTA, 0.5M NaCl, 1% Nonidet P-40 (NP-40), 0.1% β-mercaptoethanol, 10 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 10 μg/mL trypsin inhibitor, 10 μg/mL leupeptin, 1 μg/mL aprotinin and 1 μg/mL Nα-p-tosyl-L-lysine chloromethyl ketone (TLCK; all chemicals obtained from Sigma Chemical Co., St. Louis, Mo.). The resuspended tails were sonicated in HB for 20 sec at a setting of “4” on a Branson Sonifer 450. The sonicated suspension was inverted for 30 min at 4° C. and then centrifuged at 16,000×g for 15 min at 4° C. The pellet was then resuspended in HB, inverted for 30 min at 4° C., and then again centrifuged at 16,000 rpm for 15 min at 4° C. Finally, the pellet was resuspended in Sonication Buffer (SB) comprising 9M deionized urea, 4% NP-40, 2% , β-mercaptoethanol, and 2% ampholytes pH 3-10 (obtained from Pharmacia LKB, Upsala, Sweden) and rocked at room temperature for 2 h. This mixture was then used for two-dimensional electrophoresis as described below.
The identity of AKAP110 in the extracts prepared as described above was determine using an RII overlay assay. This assay was based on previous experiments which had shown that PKA anchoring proteins could be specifically detected after electrophoretic separation by overlay with labeled RII subunit of mammalian PKA (see Vijayaraghavan et al., 1997, [0045] J. Biol. Chem. 272; 47474-4752). These assays were performed as follows. Polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS/PAGE) was performed as described in Johnson et al. (1997, Devel. Biol. 192: 340-350) and Visconti et al. (1997, Devel. Biol. 192: 351-363). Approximately 50 μg protein per lane was loaded on a 10% gel and electrophoresed overnight at a constant current of 8 mA. After electrophoresis, protein bands were transferred to Immobilon P (polyvinylidene difluoride, obtained from Millipore, Bedford, Mass.) as described in the Johnson and Visconti references. After about 1 h, blot transfer was complete, and the blots were pre-treated in a solution containing 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% non-fat milk, and 0.1% BSA (termed BLOTTO buffer) to prevent non-specific binding to the membrane. The blot was then treated with ³²P labeled RIIα protein at a concentration of 500,000 cpm/mL in BLOTTO buffer. Recombinant RIIα (prepared by expression in E. coli as described above; see Carr et al., 1991, J. Biol. Chem. 266: 14188-14193) was radiolabeled by incubation with the catalytic subunit of PKA in the presence of γ-³²P-labeled ATP. Approximately 1 μg of catalytic subunit was added to 5 μg of RII plus 50 μCi of ³²P-ATP in a buffer consisting of 0.05M MOPS (pH 7.0), 0.01M MgCl₂, 0.25 mg/mL bovine serum albumin (BSA) and 0.01M dithiothreitol (DTT) and incubated for one hour at room temperature. The radiolabeled RII was then separated from the radiolabeled ATP using a KWIK-Sep™ exocellulose desalting column (Pierce Chemical, Rockford, Ill.) following the manufacturer's instructions. After incubation of the blot for four hours at room temperature, the blot was washed four times for fifteen minutes each with an excess volume of TTBS (0.1% Tween-20, 10 mM Tris-HCl, pH 7.5, 150 mM NaCl). Protein bands on the blot radiolabeled by binding to the ³²P labeled RIIα were then visualized by autoradiography by exposure to X-ray film (Kodak XAR, Eastman Kodak, Rochester, N.Y.) overnight at −80° C.
The results of one such experiment are shown in FIGS. 1A and 1B. FIG. 1A shows SDS/PAGE analysis of tail fractions extracted in HB containing 0.1, 0.2 or 0.3M NaCl ([0046] lanes 1, 2 and 3 of the Figure), 0.3M NaCl plus 0.1 or 1% SDS (lane 4 and 5), or 0.3M NaCl, 1% SDS and 7M urea (lane 6), followed by blotting and radioactive RII overlay analysis as described. (In these experiments, the HB lacked 0.5M NaCl and 1% NP-40.) These experiments illustrate the insolubility of AKAP110 protein in bovine sperm under the extraction conditions employed in these assays; the majority of the protein is seen to remain in the pellet even after extraction in high salt and/or detergent concentrations.
FIG. 1B shows the results of two-dimensional assay comprising isoelectric focusing (IEF) in the first (horizontal) dimension and SDS/PAGE in the second (vertical) dimension. These assays were performed only on sperm that had been subjected to the final extraction step using SB buffer described above. IEF was performed using standard protocols as described in Bollag & Edelstein (1991, Protein Methods, Wiley-Liss, N.Y.) using ampholytes spanning a pH range of 3-10, and SDS/PAGE was performed as described above. The results of these assays showed that AKAP110 was solubilized only by treatment with high concentrations of urea (9M). [0047]

A band on a gel processed in parallel and corresponding to the position of AKAP110 detected in the two-dimensional assay as described was excised and the AKAP110 protein eluted therefrom. This protein was subjected to tryptic digestion, high pressure liquid chromatographic separation, and amino acid microsequencing. As a result, seven peptide fragments were sequenced; these sequences are as follows:


	SCVETLGEHIIK	(Seq. ID No.:1)

	KGFCVDYYNTT	(Seq. ID No.:2)

	EGISACLQGSPFVTP	(Seq. ID No.:3)

	SPAVSHESSLR	(Seq. ID No.:4)

	NLLSETIFK	(Seq. ID No.:5)

	GTGTAETFLQNAYQAIHNEL; and	(Seq. ID No.:6)

	LLQLSAAAVEK	(Seq. ID No.:7)

These sequences were compared with sequences in GenBank and were determined to be unique. However, certain of these peptides (SEQ ID Nos. 6 and 7) showed approximately 90% sequence similarity to expressed sequence tags (ESTs) previously isolated from mouse and human testis-specific cDNA libraries. These included ESTs designated MTEST21 (C. Hoog, Department of Molecular Genetics, Karolinska Institute, Stockholm, Sweden), AA424242 (available royalty-free from Lawrence Livermore National Laboratory), AA92960 (available royalty-free from Lawrence Livermore National Laboratory) and AA061683 (Genbank, National Center for Biotechnology Information, Bethesda, Md). These similarities, and the uniqueness of the peptide sequences specific for bovine testis AKAP110, were used to develop probes for the library screening experiments described in Example 2. [0049]

EXAMPLE 2

Isolation and Characterization of cDNA Encoding Murine AKAP110 Protein

The results obtained in Example 1 were used to develop AKAP110-specific probes for screening a murine cDNA library for sequences encoding AKAP110. [0050]

Two of the EST sequences identified as being similar to two of the seven peptide sequences derived from bovine AKAP110 protein were used. These ESTs, AA92960 and AA061683, had the following nucleotide sequence:


AA92960:	(Seq ID No.:8)

ATGAGTGCAAACTTAAGTCTCCTCCCCACAAGATATGTGACCAAGAACAA

ACAGAAAAGAAAGATCTGATGAGTGTCATCTTCAATTTTATCCGGAACTT

ACTCAGCGAGACCATATTCAAGAGTAGCCGTAACTGTGAATCCAATGTGC

ATGAGCAGAACACTCAGGAAGAAGAGATACACCCGTGTGAAAGGCCTAAG

ACTCCATGTGAAAGGCCTATTACCCCGCCTGCCCCGAAATTCTGTGAGGA

TGAGGAGGCCACTGGTGGTGCCTTATCTGGGCTAACCAAGATGGTTGCCA

ACCAGCTAGACAACTGTATGAATGGGCAGATGGTGGAGCACCTGATGGAC

TCGGTGATGAAGTTATGCCTCATTATTGCCAAGTCCTGTGACTCTCCCCT

GTCGGAGCTGGGAGAGGAAAAGTGTGGGGATG; and

AA061683:

(Seq ID No.:9)

GTGAATCCAATGTGCATGAGCAGAACACTCAGGAAGAAGAGATACACCCG

TGTGAAAGGCCTAAGACTCCATGTGAAAGGCCTATTACCCCGCCTGCCCC

GAAATTCTGTGAGGATGAGGAGGCCACTGGTGGTGCCTTATCTGGGCTAA

CCAAGATGGTTGCCAACCAGCTAGACAACTGTATGAATGGGCAGATGGTG

GAGCACCTGATGGACTCGGTGATGAAGTTATGCCTCATTATTGCCAAGTC

CTGTGACTCTCCCCTGTCGGAGCTGGGAGAGGAAAAGTGTGGGGATGCCA

GCCGGCCAAATTCTGCCTTCCCAGATAACTTATATGAGTGCCTACCAGTC

AAGGGCACAGGGACAGCTGAAGCCCTCCTGCAGAACG.

These oligonucleotides were obtained from Research Genetics Inc. (Huntsville, Ala.). and radiolabeled by end-labeling with [0052] ³²P using terminal transferase (obtained from New England Biolabs, Beverly, Mass.) in the presence of γ³²P-ATP under conditions recommended by the supplier. Radiolabeled probes were used to screen a mixed germ cell mouse testis cDNA library prepared in the cloning vector Lambda ZAP, obtained from Stratagene (LaJolla, Calif.). Approximately 500,000 phage plaques were screened using conventional techniques (Sambrook et al., ibid.). The probes were labeled with 150 μCi ³²P-ATP and hybridized overnight at 42° C. in a hybridization buffer comprising 6×SSC (0.9M NaCl, 0.09M sodium citrate dihydrate (pH 7.0)), 2×Denhardt's solution (0.04% Ficoll, 0.04% polyvinylpyrrolidone, 0.04% bovine serum albumin), and 0.25% SDS. After hybridization, the filters were washed four or five times for ten minutes apiece in an excess volume of 6×SSC (0.9 M NaCl, 0.09M sodium citrate dihydride, pH 7.0) and 0.05% SDS at room temperature. Thereafter, three ten minute washes were performed one after another in this buffer at temperatures of 55° C., 60° C. and 63° C. Three positively-hybridizing clones were identified and plaque-purified. Insert were excised from the phage vector in vitro to provide pBluescript SK plasmids containing the inserts using an excision technique according to the manufacturer's instructions (Stratagene).
Inserts were amplified by the polymerase chain reaction for 25 cycles consisting of 30 sec at 94° C., 30 sec at 55° C. and 2 min at 72° C. using primers complementary to the pBluescript SK vector on either side of the insert (Stratagene). Amplification using the three plasmids derived from the three positively-hybridizing phage resulted in amplification products of 2.5 kb, 2.5 kb and 3.1 kb. Nucleotide sequence determination was performed using Taq polymerase (in a kit supplied by Applied Biosystems, Foster City, Calif.) by the dideoxynucleotide chain termination method (Sanger et al., 1977, [0053] Proc. Natl. Acad. Sci. USA 74: 5463-5467). The sequences of these inserts were overlapping, with the 3.1 kb insert containing an additional 600 bp of sequence information 5′ to the sequences contained in the other insert fragments. In the largest clone, an initiation Met codon is located at nucleotide residue 292, and the context of this initiation codon (residues 289-294: AGGATGG) indicates that it is a translation start site (Kozak, 1987, Nucleic Acids Res. 15: 8125-8148). The 288 residues 5′ to this putative translation start site contain 10 inframe translation termination codons, precluding the use of another contiguous ATG codon upstream. In addition, the sequence between nucleotide residue 292 and residue 2887 comprised an open reading frame encoding a protein having 864 amino acids; residues 2884-2886 encoded a translation termination codon followed by a polyA tail at residue 3079. This 3.1 kb insert was cloned into plasmid pET30A (obtained from Novagen, Madison, Wis.) after digestion with EcoRI and XhoI using conventional techniques.
The sequence of the 3.1 kb fragment is shown in FIG. 2 and comprises Seq ID Nos. 10 and 11. These sequences have been deposited with GenBank under accession number AF093406. [0054]

EXAMPLE 3

Isolation and Characterization of cDNA Encoding Bovine and Human AKAP110 Protein

The bovine and human homologues of the mouse AKAP110 protein-encoding cDNA described in Example 2 above were obtained as follows. [0055]
The mouse AKAP cDNA was used as a probe to screen a bovine oligo dT-primed cDNA library prepared in the Lambda ZAPII vector and obtained from Stratagene. 700,000 clones were screened at high stringency as described above using the 3.1 kb insert [0056] ³²P-labeled by the random primer method (Sambrook et al., ibid.). This screening resulted in 12 positively-hybridizing plaques that were plaque-purified and converted to pBluescript plasmids as described above. Nucleotide sequencing of these plasmids revealed them to be a collection of overlapping fragments, the longest of which was 2.9 kb in length and contained all but the amino-terminal 4 amino acids of the complete bovine AKAP110-encoding sequence (when compared with the putative amino acid sequence of the murine clone). The bovine sequence is shown in FIG. 2 and Seq ID Nos. 12 and 13. All seven of the peptides produced by microsequencing the bovine AKAP110 protein as described in Example 1 were found in the predicted bovine amino acid sequence. These sequences have been deposited with GenBank under accession number AF093407.
The human AKAP110 cDNA was isolated from a human testis cDNA library obtained from Clontech (Palo Alto, Calif.) and was screened using the murine 3.1 kb fragment at high stringency as described for the screening of the bovine library. Several positively-hybridizing phage plaques were obtained and plaque purified, and the size of the hybridizing insert fragments determined by restriction enzyme digestion and Southern blot analysis (Sambrook et al., ibid.). The longest hybridizing insert was 1.3 kb, which was sequenced and recognized to be too short to encode the complete 110 kD protein. The polymerase chain reaction-based RACE technique was used to obtain the missing portions of the sequence from testis cDNA (obtained from Clontech), the method being performed using a kit supplied by Clontech according to the instructions provided by the supplier. [0057]
The human sequence is shown in FIG. 2 and Seq ID Nos. 14 and 15. An initiation Met codon is located at nucleotide residue 230, and the 229 [0058] residues 5′ to this putative translation start site contain 3 inframe translation termination codons. In addition, the sequence between nucleotide residue 230 and residue 2788 comprised an open reading frame encoding a protein having 853 amino acids; residues 2789-2791 encoded a translation termination codon. These sequences have been deposited with GenBank under accession number AF0934098.

EXAMPLE 4

Sequence Characterization and Comparison of cDNA Encoding Murine, Bovine and Human AKAP110 Protein

The amino acid sequences encoded by the three mammalian AKAP110 cDNA clones of the invention are aligned in FIG. 2; regions of sequence identity are represented as shaded in the Figure. This alignment was produced using the ClustalW Alignment program (MacVector Software, Oxford Molecular Group). The nucleotide sequence of the bovine clone predicted an amino acid sequence of 859 amino acids having a calculated molecular weight of 95,578 and a calculated isoelectric point of 6.02. These results were consistent with the experimentally-observed values obtained according to Example 1. There was 65% amino acid sequence identity between the three species (554 out of 864 identical residues). There were also large domains of greater than 95% sequence homology interspersed by regions where some amino acids were deleted in one sequence compared with the other two (for example, beginning at amino acid 45), as well as a region of significant sequence diversity (e.g., the 50 amino acids between residues 475 and 525). No matches to AKAP110 were found in the Prosite database of patterns (Bairoch et al., 1997, [0059] Nucleic Acids Res. 25: 217-221), except for patterns otherwise having a high probability of occurrence, such as phosphorylation sites. Finally, the SOSUI structural analysis computer program (Hirokawa et al., 1998, Bioinformatics 14: 378-379) predicts AKAP110 to be a soluble protein.
The conserved regions of sequence are expected to correspond to functional domains. The portion of the sequence corresponding to the RII binding domain was determined using an RII overlay assay as follows. The mouse cDNA was fragmented into the following species containing the indicated portions of the full-length AKAP110 amino acid sequence: residues 1-470; 471-864; 1-350; 1-164; and 1-117. These fragments were subcloned into the pET30A vector (Novagen) and expressed in [0060] E. coli using a protocol provided by the supplier of the vector. The protein fragments were isolated from individual E. coli clones, separated by SDS/PAGE and transferred to an Immobilon P membrane as described above. This blot was then hybridized with ³²P-labeled RIIα (results shown in FIG. 4A) or with horse radish peroxidase-conjugated S-protein that detects an S-tag expressed as a fusion protein by the pET30A vector (results shown in FIG. 4B). The results shown in FIG. 4B demonstrate the presence of all six fragments on the blot, while the results shown in FIG. 4A indicate that only certain of these fragments are capable of binding the radiolabeled RIIα protein. The pattern of RIIα binding of these fragments is summarized in FIG. 4C. Deletion of the carboxyl-terminal half of the protein (residues 471-864) had no effect on RII binding, while deletion of the amino terminal half (residues 1-470) abolished RII binding. Progressive continuation of this deletion analysis limited the region of the sequence to amino acids 117 and 164.
A comparison of the portion of the sequence delineated by this analysis and several prior art AKAP sequences implicated the sequence corresponding to amino acids 124-141 in the mouse sequence as being the RII binding site. The identity of this portion of the sequence as an RIIα binding site was confirmed by preparing a synthetic peptide having amino acid sequence: [0061]
SFYANRLTNLVI (Seq ID No.: 16) [0062]
with an automated peptide synthesizer using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and base-mediated coupling (Applied Biosystems). This peptide was shown to prevent radiolabeled RIIα binding when it was included in the binding assay at a concentration of 1 μM. The results of these experiments are shown in FIG. 4D. [0063]
As a result of this alignment, a comparison was made between the RII binding domain of AKAP110 and the RII binding domains of 14 other known AKAP proteins; this alignment is shown in FIG. 5A. This alignment permitted the development of a model of the RII binding motif in AKAP proteins. Optimal alignment of the RII binding sequence from these fifteen proteins was performed using the ClustalW Alignment program (MacVector). The comparison was performed iteratively, wherein the sequences were compared and modified until a consensus sequence was obtained. This analysis revealed that 8 out of 18 positions were strongly conserved in this sequence. All of these conserved positions encoded predominantly hydrophobic amino acids. A common motif can be represented as follows: [0064]

(Seq ID No.:17)

X-{L/I/V}-X-X-X-{A/S}-X-X-{L/I/V}-{L/I/V}-X-X-

{L/I/V}-{L/I/V}-X-X-{A/S}-L/I/V}
where “X” is any amino acid and the residues in brackets are alternative, conserved amino acids at that position in the consensus sequence. Eleven of the fifteen AKAPs aligned match at least 7 of the 8 conserved positions; if additional alternative residues are allowed (such as V for A/S and T, A or M for L/I/V, then nine of the fifteen AKAPs are conserved in all eight positions and 13 out of 15 are conserved at 7 of 8 positions. Six of the eight conserved positions consist of branched aliphatic residues that are most likely to be involved in binding hydrophobic amino acids at the amino terminus of the RII subunit (see Dell'Aqua & Scott, 1997, [0065] J. Biol. Chem. 272: 12881-12884). The other two positions (position 5 and 7) are occupied by either Ala or Ser, residues having low steric bulk, amphipathic or hydrophobically neutral and ambivalent in terms of placement either on the internal or external face of a protein. Positions 2 and 18 contain the most variability in the conserved residues, suggesting that these residues flank a more sequence-restricted central core.
Arrangement of the residues in the consensus sequence into an alpha helical structure is shown in FIGS. 5B and 5C. When drawn in this way, all of the conserved positions are on one face of the helix (between [0066] residues 9 and 18 as shown). The residues between the conserved residues are predominantly hydrophilic neutral residues. These results are consistent with a model of AKAP:RII binding containing an amphipathic helix having a highly conserved, hydrophobic face and a less well conserved, hydrophilic face. The entire binding domains appears to encompass five turn of the alpha helix. It is expected that the affinity between any particular AKAP and RII pair will be related to how closely the AKAP binding domain matches the conserved motif, especially at positions (such as 6, 9, 10, 13, 14 and 17) in the middle of the domain. AKAP110 is conserved at all 6 of these central positions (as shown in FIG. 5C).
These results suggest methods for screening compounds for the ability to inhibit, modulate or enhance binding between AKAP110 and RIIα, as described in further detail herein. [0067]
The murine, bovine and human AKAP110 sequences were used to search a number of databases for related amino acid sequences. No matches were found in the Prosite database (Bairoch et al., 1997, [0068] Nucleic Acids Res. 25: 217-221). Using the Gapped BLAST program (Altschult et al., 1990, J. Molec. Biol. 215: 403-410) from the National Library of Medicine at the National Institutes of Health, AKAP110 was found to have significant amino acid sequence homology to a previously-described sperm major fibrous sheet protein, variously termed AKAP82, p82, FSC-1 and HI (see Carrera et al., 1994, Develop. Biol. 165: 272-284; Fulcher et al., 1995, Biol. Reprod. 52: 41-49; and Mohapatra et al., 1998, ibid.). Alignment using up to 20 gaps revealed 35% sequence identity between AKAP110 and AKAP82. This sequence similarity exists throughout the entire length of the protein molecule, although the longest continuous stretch of identity is 11 amino acids in length. The topography of the sequence similarities between AKAP110 and AKAP82 is illustrated in FIG. 6, where the boxes define conserved regions of sequence of at least 20 amino acids having greater than 50% homology. This comparison suggests that these proteins may share several conserved domains that may perform similar functions. Conversely over half the amino acid sequence is dissimilar, suggesting that each protein may have diverged to perform different functions. It is also interesting to note that two regions at the amino terminus are conserved, even though the mature AKAP82 protein is processed to eliminate this portion of the protein (at a cleavage site depicted in the Figure). The third domain depicted in the Figure contains the putative RII binding site for both proteins; FIG. 5A provides a comparison, showing that 12 of the 18 amino acids of the binding site are conserved.
Despite the significant sequence homology between these two proteins, there is no cross-hybridization between cDNA encoding these proteins, and no cross-reactivity between either of these proteins and antibodies raised against the other. As described below, Northern blot analysis of germ cell-specific mRNA (or, as described above, screening a cDNA library prepared therefrom) using AKAP110 cDNA as a probe detected only AKAP mRNA or cDNA. Similarly, antibodies specific for AKAP110 detected only the single 110 kD AKAP110 protein band in Western blot experiments (described below). In addition, although AKAP82 has been reported to be an RII binding protein (see Carrera et al., ibid. and Visconti et al., ibid.), and the sequence comparison disclosed herein revealed significant sequence homology between the RII binding site of AKAP110 and the putative RII binding site of AKAP82, only AKAP110 was detected in RII overlay analyses of sperm protein extracts (as described in Example 1 and shown in FIG. 1). One explanation for these results is that AKAP110 has a higher affinity for RII that AKAP82, an explanation that is supported by the differences noted in the RII binding site. Importantly, the differences in RII binding suggests that the diversity at certain conserved positions in the RII binding site may be important: for example, AKAP82 has a valine at [0069] position 6 rather than an alanine or serine, and a serine at position 10 rather than a leucine, isoleucine, valine, alanine or threonine. These results suggest that these sites are particularly important for AKAP:RII interaction, which further suggests that these sites may be important targets for disrupting the AKAP:RII interaction in vivo, as described in greater detail below.

EXAMPLE 5

Expression of AKAP mRNA and Protein in Adult Tissues and in Sperm

AKAP110 expression in adult tissues was investigated at both the mRNA and protein levels. [0070]
In order to detect AKAP110 protein expression, polyclonal antisera were produced in rabbits. A peptide corresponding to amino acid residues 448-459 of bovine AKAP110 was synthesized using an automated peptide synthesizer and Fmoc chemistry (Applied Biosystems), having the following sequence: [0071]
SCVETLGEHIIK (Seq. ID No.: 1). [0072]
This peptide was conjugated to keyhole limpet hemocyanin according to conventional protocols (Harlow & Lane, 1988, Antibodies, Cold Spring Harbor Press: New York) and injected into two New Zealand white rabbits. The animals were bled from the ear after six weeks, the serum separated and then pooled. Ammonium sulfate was then added to the pooled serum to a final concentration of 40% to precipitate the immunoglobulin fraction. The precipitated antibodies were resuspended in phosphate buffered saline (PBS) and subjected to affinity chromatography using the original peptide (Seq ID No.: 1) conjugated to Amino Link matrix (obtained from Pierce Chemical Co.) according to the instructions from the supplier. [0073]
The plasmids prepared as described above in Example 2 were used to produce full-length AKAP110 in [0074] E. coli using ITPG induction as detailed in the instructions provided with the vector by the supplier (Novagen). The AKAP110 protein was then separated from other proteins by SDS/PAGE, and transferred to Immobilon P as described above. This Western blot was treated with BLOTTO solution (10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% non-fat milk, 0.1% BSA) and then incubated with the primary antibody for two hours at room temperature using antibody dilutions of about 1:5000. Antibody binding was detected using antirabbit secondary antibody conjugated to horse radish peroxidase and developed using an enhanced chemiluminescence kit (Renaissance from NEN, Boston, Mass.) according to instructions from the supplier.
Results of these experiments are shown in FIGS. 3A through 3C. FIGS. 3A and 3B are identical blots loaded with bacterial lysates from either uninduced (Control) or induced [0075] E. coli, and separated into pellet and supernatant fractions. FIG. 3A shows an RII overlay assay performed as described above, and confirms that intact, functional AKAP110 protein was produced and isolated from the E. coli. FIG. 3B is the result of probing this blot with the anti-AKAP110 antisera. These results show that the antisera specifically recognized the same protein band in the blot as the RII overlay assay. FIG. 3C shows a similar Western blot probed with the anti-AKAP110 antisera and containing 50 μg of bovine caudal sperm lysate (prepared as described in Example 1). These results demonstrate that the antisera were also capable of detecting native bovine AKAP110 in sperm protein lysates, and support the conclusion that the synthetic peptide raise antisera against native AKAP110.
Northern blots prepared using mouse polyA[0076] ⁺ mRNA from heart, brain, spleen, lung, liver, skeletal muscle, kidney and testis were obtained from Clontech; 2 μg of each tissue-specific mRNA were contained in each lane. These blots were hybridized according to the manufacturer's instructions with either full-length mouse AKAP110 cDNA or nucleotides 838-1055, labeled with a random hexamer primer kit (Amersham) to 1.5 million cpm/mL. The blot was thereafter washed sequentially using the following protocol: twice for 5 minutes apiece at room temperature in an excess volume of 2×SSC (0.03M sodium citrate dihydride (pH 7.0), 0.3M NaCl), and 1% SDS; twice for 30 minutes apiece at room temperature in an excess volume of 0.2×SSC and 1% SDS; eight times at 46° C. in 0.1×SSC and 0.5% SDS. The blot was visualized by autoradiography by exposure to X-ray film (Kodak XAR, Eastman Kodak) overnight at −80° C.
The results of these experiments are shown in FIG. 7. A single mouse AKAP110 mRNA of about 3.6 kb in size was detected specifically and only in mRNA from testis. These results indicate that AKAP110 expression may be sperm-specific. This conclusion is supported by the finding that all ESTs having similar sequences have been isolated only from testis cDNA libraries. The blots were probed with AKAP110 nucleotides 838-1055 to ensure that the detected mRNA was not the result of cross-hybridization with AKAP82 mRNA; this portion of the AKAP110 cDNA has no significant homology to the AKAP82 sequence. These results support the conclusion that, although the proteins are related, AKAP110 and AKAP82 cDNAs do not cross-hybridize. [0077]
In situ hybridization was performed on cryosections of fresh-frozen mouse testis tissue. Said cryosections were fixed in 4% formaldehyde in 0.1M Na-phosphate (pH 7.4), and then acetylated for 15 min in 0.25% acetic anhydride in 0.1M triethanolamine (pH 8). Sense and—antisense riboprobes were synthesized in the presence of digoxigenin-labeled nucleotides (Boehringer Mannheim, Indianapolis, Ind.) by run-off transcription of linearized pCRII plasmid (Invitrogen, San Diego, Calif.) containing a 217 bp fragment of AKAP110 (residues 838-1055). Riboprobes were denatured for 5 min at 80° C., then diluted in 50% formamide, 10% dextran sulfate, 4×SSC, 1×Denhardt's reagent, 0.5 mg/mL denatured salmon sperm DNA and 0.25 mg/mL yeast tRNA. Frozen sections were hybridized at 50° C. overnight. After hybridization, slides were rinsed at room temperature with 2×SSC, rinsed again with STE (500 mM NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA), and then incubated for 30 min at 37° C. in STE containing 40 μg/mL RNase A. The slides were then washed twice for 5 min at room temperature, first in 1×SSC and then in 0.5×SSC. Hybridization was detected using sheep anti-digoxigenin Fab fragments conjugated with alkaline phosphatase (Boehringer Mannheim). Staining was achieved by incubation of the hybridized, antibody-bound sections with BCIP/NBT (bromo-4-chloro-3-indolphosphate/nitroblue tetrazolium, a substrate for alkaline phosphatase) in a solution of 100 mnM Tris-HCl (pH 9.5), 50 mM magnesium chloride, and 1 mM levamisole according to manufacturer's instructions. [0078]
The results of these assays are shown in FIGS. 8A and 8B. Antisense AKAP110 riboprobe specifically hybridized to the seminiferous epithelium, and no hybridization was detected in either the interstitial tissue or in the peritubular and Sertoli cells of the seminiferous tubules, as shown in FIG. 8A produced by hybridization with the antisense probe. AKAP110 exhibited a stage-specific expression pattern in germ cells. No hybridization signal was detected in spermatogonia or spermatocytes, but expression was evident in round spermatids (FIG. 8B). FIG. 8C, which was hybridized with a sense-oriented probe showed no hybridization, as expected. [0079]
Intracellular localization of AKAP110 in sperm was determined by immunostaining. Ejaculated and epididymal caudal bovine sperm were collected by centrifugation at 1000×g for 10 min, then washed twice by resuspension in Hanks' saline or phosphate buffered saline (PBS) followed by centrifugation as above. Sperm were permeabilized either before or after fixation; no difference was found in the experimental results obtained. For permeabilizing sperm before fixation, the sperm were incubated at 4° C. for 30 min in a solution of 0.25% Triton X-100, 10 mm DTT, 150 mM NaCl, 25 mM Tris-HCl (pH 7.5), 2 mM benzamidine, 1 μg/mL leupeptin, and 1 μg/mL pepstatin, and then fixed in 4% formaldehyde in 0.1M sodium phosphate (pH 7.4). For permeabilizing sperm after fixation, sperm were fixed in 4% formaldehyde in 0.1M sodium phosphate (pH 7.4) at 4° C., then permeabilized for 30 min by the addition of Triton X-100 to a final concentration of 0.25%. Sperm were then attached to a poly-lysine coated coverglass which was then incubated in absolute acetone at 20° C. for 10 min and air-dried; alternatively, the coverglass was used directly for immunostaining [0080]
Immunostaining was performed as follows. Sperm prepared as described above were rinsed in a solution of 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.1% Tween 20 (termed “TNT solution”), then incubated for 1 h in a blocking solution of TNT containing 5% normal donkey serum and 2.5% BSA. In parallel, coverslips were incubated at least 1 h in equal dilutions of preimmune serum or anti-AKAP110 antisera diluted in blocking solution. After incubation, slides were rinsed three times in TNT containing 1% donkey serum; the slides were then incubated 1 h with cy3-conjugated, affinity-purified, donkey anti-rabbit IgG (obtained from Jackson ImmunoResearch Labs, West Grove, Pa.). The slide was then rinsed three times in TNT and observed by fluorescence and phase contrast microscopy; these results are shown in FIG. 9. [0081]
Ejaculated and caudal epididymal sperm exhibited identical specific staining patterns with anti-AKAP110 antisera, and no differences in staining were detected if the sperm were permeabilized before or after formaldehyde fixation. The flagellum and acrosomal regions were specifically stained with anti-AKAP110 antibody. Intense fluorescence was detected in the anterior acrosomal segment (shown in FIG. 9 and designated AC), but no staining was detected over the equatorial or post acrosomal segments of the head. Detached acrosomal caps also exhibited intense fluorescence (shown in FIG. 9 and designated AC*), and no fluorescence was detected on sperm heads after release of the acrosomal cap (shown in FIG. 9 and designated H). Specific fluorescence was detected over the full extent of the flagellum. No fluorescence staining was detected in matched sample incubated with preimmune serum (shown in FIG. 9 and designated B). [0082]
This pattern of AKAP110 localization is consistent with this protein being a dual AKAP, that is, being capable of binding to both the RI (found predominantly in sperm head) and RII (found predominantly in sperm flagellum) isoforms. The localization of AKAP in the acrosomal region of the sperm head suggests that disruption of this interaction may be a useful way of preventing fertilization. [0083]

EXAMPLE 6

Disruption of AKAP110/PKA Binding to Achieve Contraception in Adults

Isolation of mammalian AKAP110 cDNA as disclosed above enabled production of AKAP110 by recombinant genetic means. As described above, the cDNA inserts were cloned into an expression vector, pET30A, and introduced into [0084] E. coli cells. pET30A contains a Histidine tag sequence that was used to purify the fusion protein. Recombinant AKAP110 protein was recovered from said E. coli cells transformed with recombinant expression constructs comprising AKAP110-encoding cDNA.
The observation that disruption of AKAP:PKA binding inhibits sperm motility suggested that a high throughput in vitro screening system for detecting compounds that inhibit the AKAP:PKA interaction is useful for developing compounds useful as contraceptives. For example, a pharmaceutical compound directed at preventing or inhibiting sperm motility would provide for the first time a “male” contraceptive. This approach is particularly attractive, since Northern blot analysis of AKAP110 mRNA expression in a variety of tissues indicated that AKAP110 was specifically and exclusively expressed in male germ cells such as spermatogonia. Thus, any compound directed at specifically disrupting AKAP110:PKA binding has the potential for being used to develop a male contraceptive having minimal side effects on AKAP:PKA interactions in cell types other than sperm. [0085]
One embodiment of a high throughput analytical system comprises using a variant of the RII overlay assay described above to screen compounds for the capacity to prevent binding of a detectably-labeled RII protein. Recombinant RIIα is commercially available (Promega, Madison, Wis.), and can be detectably labeled with a radioactive, fluorescent or antigenic label. Alternatively, RII binding can be detected using RII-specific antibodies, which are also commercially available (Transduction Labs, Lexington, Ky.). Such assays are performed whereby the anti-RII antibodies are labeled directly, or in so-called “sandwich” assays using a labeled antibody specific for the anti-RII antibody (such as a using a labeled goat anti-rabbit antibody to detect rabbit polyclonal antisera as described above in Example 5). RII binding, and the disruption of RII binding in the presence of a test compound, can thereby be detected using either detectably-labeled recombinant RII or antibodies therefore, in binding assays using the recombinantly-produced AKAP110 proteins of the invention. [0086]
In alternative embodiments of these types of assays, recombinantly available AKAP110 or PKA is attached to a solid substrate, covalently or otherwise, and RII binding of said substrate is assayed analogous to either ELISA or radioimmune assay. In other embodiments, said solid substrate comprises materials such as chromatographic beads and other supports to which binding can be detected. These embodiments are particularly adapted for developing automated assays for screening a large number of compounds simultaneously. Also provided are competitive assays used to characterize binding affinities and inhibition constants of various binding inhibitors. [0087]
Alternatively, antisense oligonucleotides are used to inhibit, disrupt or prevent expression of AKAP110 in testis cells. An example of such an antisense oligonucleotide is one complementary to the sequence surrounding the initiation codon of AKAP110: [0088]
GCTTTGTAACCAGTCAACCTTTTCTGACAT (SEQ ID No. 18). [0089]
It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and that all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims. [0090]
1 33 1 12 PRT Bos sp. Description of Unknown Organism cow 1 Ser Cys Val Glu Thr Leu Gly Glu His Ile Ile Lys 1 5 10 2 11 PRT Bos sp. 2 Lys Gly Phe Cys Val Asp Tyr Tyr Asn Thr Thr 1 5 10 3 15 PRT Bos sp. 3 Glu Gly Ile Ser Ala Cys Leu Gln Gly Ser Pro Phe Val Thr Pro 1 5 10 15 4 11 PRT Bos sp. 4 Ser Pro Ala Val Ser His Glu Ser Ser Leu Arg 1 5 10 5 9 PRT Bos sp. 5 Asn Leu Leu Ser Glu Thr Ile Phe Lys 1 5 6 20 PRT Bos sp. 6 Gly Thr Gly Thr Ala Glu Thr Phe Leu Gln Asn Ala Tyr Gln Ala Ile 1 5 10 15 His Asn Glu Leu 20 7 11 PRT Bos sp. 7 Leu Leu Gln Leu Ser Ala Ala Ala Val Glu Lys 1 5 10 8 432 DNA Mus musculus 8 atgagtgcaa acttaagtct cctccccaca agatatgtga ccaagaacaa acagaaaaga 60 aagatctgat gagtgtcatc ttcaatttta tccggaactt actcagcgag accatattca 120 agagtagccg taactgtgaa tccaatgtgc atgagcagaa cactcaggaa gaagagatac 180 acccgtgtga aaggcctaag actccatgtg aaaggcctat taccccgcct gccccgaaat 240 tctgtgagga tgaggaggcc actggtggtg ccttatctgg gctaaccaag atggttgcca 300 accagctaga caactgtatg aatgggcaga tggtggagca cctgatggac tcggtgatga 360 agttatgcct cattattgcc aagtcctgtg actctcccct gtcggagctg ggagaggaaa 420 agtgtgggga tg 432 9 387 DNA Homo sapiens 9 gtgaatccaa tgtgcatgag cagaacactc aggaagaaga gatacacccg tgtgaaaggc 60 ctaagactcc atgtgaaagg cctattaccc cgcctgcccc gaaattctgt gaggatgagg 120 aggccactgg tggtgcctta tctgggctaa ccaagatggt tgccaaccag ctagacaact 180 gtatgaatgg gcagatggtg gagcacctga tggactcggt gatgaagtta tgcctcatta 240 ttgccaagtc ctgtgactct cccctgtcgg agctgggaga ggaaaagtgt ggggatgcca 300 gccggccaaa ttctgccttc ccagataact tatatgagtg cctaccagtc aagggcacag 360 ggacagctga agccctcctg cagaacg 387 10 3103 DNA Mus musculus 5′UTR (1)..(291) CDS (292)..(2883) 3′UTR (2884)..(3078) 10 agaacatccc agcatcttca ttgactttaa aagtatattc tggagtcttc tgtggttccc 60 tataacagag cttcagaggt tctccgattt taagaaagat gagctgcctg gaattacaaa 120 tgggcaggac ttttctattc actctttcaa caggaaaatt actaagcacc aacgttaaag 180 agtccttata tattacatgg caggaggggt gggtagactt agggatagtg aagacaccaa 240 taaggctcat tcggagtctt ccttgtatct caaaacccat cctcaataag g atg gcg 297 Met Ala 1 gat agg gtt gac tgg tta caa agt caa agt ggc gtt tgc aaa gtt ggt 345 Asp Arg Val Asp Trp Leu Gln Ser Gln Ser Gly Val Cys Lys Val Gly 5 10 15 gtc tat tca cct gga gac aac caa cac caa gac tgg aaa atg gac aca 393 Val Tyr Ser Pro Gly Asp Asn Gln His Gln Asp Trp Lys Met Asp Thr 20 25 30 tca aca gat cct gtc cga gtg ctc agc tgg ctc cgc aaa gac ctg gag 441 Ser Thr Asp Pro Val Arg Val Leu Ser Trp Leu Arg Lys Asp Leu Glu 35 40 45 50 aaa agt aca gcg ggc ttc cag gac tcg agg ttc aag cct gga gag tca 489 Lys Ser Thr Ala Gly Phe Gln Asp Ser Arg Phe Lys Pro Gly Glu Ser 55 60 65 tcg ttt gtg gag gaa gtg gct tac cca gtg gac caa cga aaa ggt ttc 537 Ser Phe Val Glu Glu Val Ala Tyr Pro Val Asp Gln Arg Lys Gly Phe 70 75 80 tgt gtt gat tat tac aat acc acc aac aag ggc agt cca gga aga ttg 585 Cys Val Asp Tyr Tyr Asn Thr Thr Asn Lys Gly Ser Pro Gly Arg Leu 85 90 95 cat ttt gag atg tct cac aag gag aac cct tcc cag ggc ctc att tcc 633 His Phe Glu Met Ser His Lys Glu Asn Pro Ser Gln Gly Leu Ile Ser 100 105 110 cat gtt ggt aat ggg ggt tcc ata gac gaa gtt tcc ttc tat gcc aac 681 His Val Gly Asn Gly Gly Ser Ile Asp Glu Val Ser Phe Tyr Ala Asn 115 120 125 130 cgc ctc aca aac cta gtg atc gcc atg gcc cga aag gag atc aat gag 729 Arg Leu Thr Asn Leu Val Ile Ala Met Ala Arg Lys Glu Ile Asn Glu 135 140 145 aag atc cac ggc gct gaa aac aaa tgt gtc cat cag tca ttg tat atg 777 Lys Ile His Gly Ala Glu Asn Lys Cys Val His Gln Ser Leu Tyr Met 150 155 160 ggg gat gag ccc aca ccc cac aaa agc ttg agt aca gtg gcc tct gag 825 Gly Asp Glu Pro Thr Pro His Lys Ser Leu Ser Thr Val Ala Ser Glu 165 170 175 ctc gtg aac gag aca gtc acc gca tgt tcc aag aac att tcc agt gac 873 Leu Val Asn Glu Thr Val Thr Ala Cys Ser Lys Asn Ile Ser Ser Asp 180 185 190 aaa gct ccc ggc tct gga gac agg gcc tcg ggg tcg tca cag gcc cct 921 Lys Ala Pro Gly Ser Gly Asp Arg Ala Ser Gly Ser Ser Gln Ala Pro 195 200 205 210 ggt cta aga tac atg agc act ctg aag atc aag gag agt aca aag gaa 969 Gly Leu Arg Tyr Met Ser Thr Leu Lys Ile Lys Glu Ser Thr Lys Glu 215 220 225 ggc aag tgt cca gac gac aag ccc ggc act aag aag tct ttc ttc tat 1017 Gly Lys Cys Pro Asp Asp Lys Pro Gly Thr Lys Lys Ser Phe Phe Tyr 230 235 240 aag gaa gtg ttt gag tcc cgg aat gca gga gat gcc aag gag ggc ggg 1065 Lys Glu Val Phe Glu Ser Arg Asn Ala Gly Asp Ala Lys Glu Gly Gly 245 250 255 agg tcc tta ccc gga gat caa aaa ctg ttc agg acc agc ccc gac aac 1113 Arg Ser Leu Pro Gly Asp Gln Lys Leu Phe Arg Thr Ser Pro Asp Asn 260 265 270 agg cct gat gac ttt tca aac tct atc agt caa ggg atc atg acc tac 1161 Arg Pro Asp Asp Phe Ser Asn Ser Ile Ser Gln Gly Ile Met Thr Tyr 275 280 285 290 gcc aac agc gtg gtg tct gac atg atg gtc tcc atc atg aag acg ctg 1209 Ala Asn Ser Val Val Ser Asp Met Met Val Ser Ile Met Lys Thr Leu 295 300 305 aag atc cag gtg aag gac aca acc atc gcc acg att ctg ctg aag aag 1257 Lys Ile Gln Val Lys Asp Thr Thr Ile Ala Thr Ile Leu Leu Lys Lys 310 315 320 gtg ctg atg aag cat gca aag gag gtt gtc tcc gac ctc atc gac tcc 1305 Val Leu Met Lys His Ala Lys Glu Val Val Ser Asp Leu Ile Asp Ser 325 330 335 ttc atg aag aac ctc cac ggc gtc acg gga agc ctc atg act gac aca 1353 Phe Met Lys Asn Leu His Gly Val Thr Gly Ser Leu Met Thr Asp Thr 340 345 350 gac ttt gtc tcg gcc gtg aaa cga agt ttt ttt tct cat gga agc caa 1401 Asp Phe Val Ser Ala Val Lys Arg Ser Phe Phe Ser His Gly Ser Gln 355 360 365 370 aag gcc aca gat atc atg gat gcc atg ctg ggc aag cta tac aat gtg 1449 Lys Ala Thr Asp Ile Met Asp Ala Met Leu Gly Lys Leu Tyr Asn Val 375 380 385 atg ttt gcc aag aaa ttc cct gag aac atc cgg aga gcc agg gac aag 1497 Met Phe Ala Lys Lys Phe Pro Glu Asn Ile Arg Arg Ala Arg Asp Lys 390 395 400 tcg gag agt tac tcc ctt atc tcc acg aaa tca cgg gct ggt gac cca 1545 Ser Glu Ser Tyr Ser Leu Ile Ser Thr Lys Ser Arg Ala Gly Asp Pro 405 410 415 aag ctc tca aat ttg aac ttt gcg atg aag tca gaa tca aag ctg aaa 1593 Lys Leu Ser Asn Leu Asn Phe Ala Met Lys Ser Glu Ser Lys Leu Lys 420 425 430 gaa aat ttg ttt tct aca tgc aaa cta gag aaa gag aag acg tgt gcc 1641 Glu Asn Leu Phe Ser Thr Cys Lys Leu Glu Lys Glu Lys Thr Cys Ala 435 440 445 450 gaa act ctg ggt gag cat att att aag gag gga ctg cac atg tgg cac 1689 Glu Thr Leu Gly Glu His Ile Ile Lys Glu Gly Leu His Met Trp His 455 460 465 aag agt cag caa aaa tct cct ggc ttg gag cgt gcc gca aaa ctg ggt 1737 Lys Ser Gln Gln Lys Ser Pro Gly Leu Glu Arg Ala Ala Lys Leu Gly 470 475 480 aac gct cca cag gag gtc tcc ttt gag tgc cca gat cct tgt gag gca 1785 Asn Ala Pro Gln Glu Val Ser Phe Glu Cys Pro Asp Pro Cys Glu Ala 485 490 495 aac cct cct cac caa cct cag cca cca gag aat ttt gca aat ttt atg 1833 Asn Pro Pro His Gln Pro Gln Pro Pro Glu Asn Phe Ala Asn Phe Met 500 505 510 tgt gac tca gac tcc tgg gcc aag gac ctg att gta tct gcc ctg ctt 1881 Cys Asp Ser Asp Ser Trp Ala Lys Asp Leu Ile Val Ser Ala Leu Leu 515 520 525 530 ctg att cag tat cac ctg gca cag gga gga aag atg gat gct cag agc 1929 Leu Ile Gln Tyr His Leu Ala Gln Gly Gly Lys Met Asp Ala Gln Ser 535 540 545 ttc ctg gaa gct gct gcc agc acc aat ttt ccc acc aac aag cca cct 1977 Phe Leu Glu Ala Ala Ala Ser Thr Asn Phe Pro Thr Asn Lys Pro Pro 550 555 560 cct cct tct cct gta gtt cag gat gag tgc aaa ctt aag tct cct ccc 2025 Pro Pro Ser Pro Val Val Gln Asp Glu Cys Lys Leu Lys Ser Pro Pro 565 570 575 cac aag ata tgt gac caa gaa caa aca gaa aag aaa gat ctg atg agt 2073 His Lys Ile Cys Asp Gln Glu Gln Thr Glu Lys Lys Asp Leu Met Ser 580 585 590 gtc atc ttc aat ttt atc cgg aac tta ctc agc gag acc ata ttc aag 2121 Val Ile Phe Asn Phe Ile Arg Asn Leu Leu Ser Glu Thr Ile Phe Lys 595 600 605 610 agt agc cgt aac tgt gaa tcc aat gtg cat gag cag aac act cag gaa 2169 Ser Ser Arg Asn Cys Glu Ser Asn Val His Glu Gln Asn Thr Gln Glu 615 620 625 gaa gag ata cac ccg tgt gaa agg cct aag act cca tgt gaa agg cct 2217 Glu Glu Ile His Pro Cys Glu Arg Pro Lys Thr Pro Cys Glu Arg Pro 630 635 640 att acc ccg cct gcc ccg aaa ttc tgt gag gat gag gag gcc act ggt 2265 Ile Thr Pro Pro Ala Pro Lys Phe Cys Glu Asp Glu Glu Ala Thr Gly 645 650 655 ggt gcc tta tct ggg cta acc aag atg gtt gcc aac cag cta gac aac 2313 Gly Ala Leu Ser Gly Leu Thr Lys Met Val Ala Asn Gln Leu Asp Asn 660 665 670 tgt atg aat ggg cag atg gtg gag cac ctg atg gac tcg gtg atg aag 2361 Cys Met Asn Gly Gln Met Val Glu His Leu Met Asp Ser Val Met Lys 675 680 685 690 tta tgc ctc att att gcc aag tcc tgt gac tct ccc ctg tcg gag ctg 2409 Leu Cys Leu Ile Ile Ala Lys Ser Cys Asp Ser Pro Leu Ser Glu Leu 695 700 705 gga gag gaa aag tgt ggg gat gcc agc cgg cca aat tct gcc ttc cca 2457 Gly Glu Glu Lys Cys Gly Asp Ala Ser Arg Pro Asn Ser Ala Phe Pro 710 715 720 gat aac tta tat gag tgc cta cca gtc aag ggc aca ggg aca gct gaa 2505 Asp Asn Leu Tyr Glu Cys Leu Pro Val Lys Gly Thr Gly Thr Ala Glu 725 730 735 gcc ctc ctg cag aac gcc tac ctc acc atc cat aat gaa ctg aga ggt 2553 Ala Leu Leu Gln Asn Ala Tyr Leu Thr Ile His Asn Glu Leu Arg Gly 740 745 750 ttg tca gga cag ccc ccc gag ggc tgt gaa atc ccc aag gtg atc gtc 2601 Leu Ser Gly Gln Pro Pro Glu Gly Cys Glu Ile Pro Lys Val Ile Val 755 760 765 770 agc aac cac aat ctg gct gac acc gtt cag aac aag caa ctg caa gct 2649 Ser Asn His Asn Leu Ala Asp Thr Val Gln Asn Lys Gln Leu Gln Ala 775 780 785 gtc ctt cag tgg gtg gct gcc tca gag ctc aat gtc cct att ttg tac 2697 Val Leu Gln Trp Val Ala Ala Ser Glu Leu Asn Val Pro Ile Leu Tyr 790 795 800 ttt gct ggt gac gat gaa gga atc cag gag aag ctg ctt cag ctc tca 2745 Phe Ala Gly Asp Asp Glu Gly Ile Gln Glu Lys Leu Leu Gln Leu Ser 805 810 815 gcc act gcc gtg gag aaa ggc cgc agc gtt ggg gag gtt ctg cag tcg 2793 Ala Thr Ala Val Glu Lys Gly Arg Ser Val Gly Glu Val Leu Gln Ser 820 825 830 gtg ctg agg tac gag aag gag cga cag ctg gat gaa gca gtg gga aat 2841 Val Leu Arg Tyr Glu Lys Glu Arg Gln Leu Asp Glu Ala Val Gly Asn 835 840 845 850 gtc acg cgg ctg cag ctg ctg gac tgg ctg atg gca aac ctg 2883 Val Thr Arg Leu Gln Leu Leu Asp Trp Leu Met Ala Asn Leu 855 860 tgattggggc ctaccctgag ttccctcagc gggccgagtc cccgccccct cagccccctc 2943 catgccccac agagccctaa agtcccctcc atgccaccca cactaaacac gccatctaac 3003 gctactcact ggattttgca gattttcttg tccatgcgag caaggacata aatgaaaaga 3063 ttacagttaa agggcaaaaa aaaaaaaaaa aaaaaaaaaa 3103 11 864 PRT Mus musculus 11 Met Ala Asp Arg Val Asp Trp Leu Gln Ser Gln Ser Gly Val Cys Lys 1 5 10 15 Val Gly Val Tyr Ser Pro Gly Asp Asn Gln His Gln Asp Trp Lys Met 20 25 30 Asp Thr Ser Thr Asp Pro Val Arg Val Leu Ser Trp Leu Arg Lys Asp 35 40 45 Leu Glu Lys Ser Thr Ala Gly Phe Gln Asp Ser Arg Phe Lys Pro Gly 50 55 60 Glu Ser Ser Phe Val Glu Glu Val Ala Tyr Pro Val Asp Gln Arg Lys 65 70 75 80 Gly Phe Cys Val Asp Tyr Tyr Asn Thr Thr Asn Lys Gly Ser Pro Gly 85 90 95 Arg Leu His Phe Glu Met Ser His Lys Glu Asn Pro Ser Gln Gly Leu 100 105 110 Ile Ser His Val Gly Asn Gly Gly Ser Ile Asp Glu Val Ser Phe Tyr 115 120 125 Ala Asn Arg Leu Thr Asn Leu Val Ile Ala Met Ala Arg Lys Glu Ile 130 135 140 Asn Glu Lys Ile His Gly Ala Glu Asn Lys Cys Val His Gln Ser Leu 145 150 155 160 Tyr Met Gly Asp Glu Pro Thr Pro His Lys Ser Leu Ser Thr Val Ala 165 170 175 Ser Glu Leu Val Asn Glu Thr Val Thr Ala Cys Ser Lys Asn Ile Ser 180 185 190 Ser Asp Lys Ala Pro Gly Ser Gly Asp Arg Ala Ser Gly Ser Ser Gln 195 200 205 Ala Pro Gly Leu Arg Tyr Met Ser Thr Leu Lys Ile Lys Glu Ser Thr 210 215 220 Lys Glu Gly Lys Cys Pro Asp Asp Lys Pro Gly Thr Lys Lys Ser Phe 225 230 235 240 Phe Tyr Lys Glu Val Phe Glu Ser Arg Asn Ala Gly Asp Ala Lys Glu 245 250 255 Gly Gly Arg Ser Leu Pro Gly Asp Gln Lys Leu Phe Arg Thr Ser Pro 260 265 270 Asp Asn Arg Pro Asp Asp Phe Ser Asn Ser Ile Ser Gln Gly Ile Met 275 280 285 Thr Tyr Ala Asn Ser Val Val Ser Asp Met Met Val Ser Ile Met Lys 290 295 300 Thr Leu Lys Ile Gln Val Lys Asp Thr Thr Ile Ala Thr Ile Leu Leu 305 310 315 320 Lys Lys Val Leu Met Lys His Ala Lys Glu Val Val Ser Asp Leu Ile 325 330 335 Asp Ser Phe Met Lys Asn Leu His Gly Val Thr Gly Ser Leu Met Thr 340 345 350 Asp Thr Asp Phe Val Ser Ala Val Lys Arg Ser Phe Phe Ser His Gly 355 360 365 Ser Gln Lys Ala Thr Asp Ile Met Asp Ala Met Leu Gly Lys Leu Tyr 370 375 380 Asn Val Met Phe Ala Lys Lys Phe Pro Glu Asn Ile Arg Arg Ala Arg 385 390 395 400 Asp Lys Ser Glu Ser Tyr Ser Leu Ile Ser Thr Lys Ser Arg Ala Gly 405 410 415 Asp Pro Lys Leu Ser Asn Leu Asn Phe Ala Met Lys Ser Glu Ser Lys 420 425 430 Leu Lys Glu Asn Leu Phe Ser Thr Cys Lys Leu Glu Lys Glu Lys Thr 435 440 445 Cys Ala Glu Thr Leu Gly Glu His Ile Ile Lys Glu Gly Leu His Met 450 455 460 Trp His Lys Ser Gln Gln Lys Ser Pro Gly Leu Glu Arg Ala Ala Lys 465 470 475 480 Leu Gly Asn Ala Pro Gln Glu Val Ser Phe Glu Cys Pro Asp Pro Cys 485 490 495 Glu Ala Asn Pro Pro His Gln Pro Gln Pro Pro Glu Asn Phe Ala Asn 500 505 510 Phe Met Cys Asp Ser Asp Ser Trp Ala Lys Asp Leu Ile Val Ser Ala 515 520 525 Leu Leu Leu Ile Gln Tyr His Leu Ala Gln Gly Gly Lys Met Asp Ala 530 535 540 Gln Ser Phe Leu Glu Ala Ala Ala Ser Thr Asn Phe Pro Thr Asn Lys 545 550 555 560 Pro Pro Pro Pro Ser Pro Val Val Gln Asp Glu Cys Lys Leu Lys Ser 565 570 575 Pro Pro His Lys Ile Cys Asp Gln Glu Gln Thr Glu Lys Lys Asp Leu 580 585 590 Met Ser Val Ile Phe Asn Phe Ile Arg Asn Leu Leu Ser Glu Thr Ile 595 600 605 Phe Lys Ser Ser Arg Asn Cys Glu Ser Asn Val His Glu Gln Asn Thr 610 615 620 Gln Glu Glu Glu Ile His Pro Cys Glu Arg Pro Lys Thr Pro Cys Glu 625 630 635 640 Arg Pro Ile Thr Pro Pro Ala Pro Lys Phe Cys Glu Asp Glu Glu Ala 645 650 655 Thr Gly Gly Ala Leu Ser Gly Leu Thr Lys Met Val Ala Asn Gln Leu 660 665 670 Asp Asn Cys Met Asn Gly Gln Met Val Glu His Leu Met Asp Ser Val 675 680 685 Met Lys Leu Cys Leu Ile Ile Ala Lys Ser Cys Asp Ser Pro Leu Ser 690 695 700 Glu Leu Gly Glu Glu Lys Cys Gly Asp Ala Ser Arg Pro Asn Ser Ala 705 710 715 720 Phe Pro Asp Asn Leu Tyr Glu Cys Leu Pro Val Lys Gly Thr Gly Thr 725 730 735 Ala Glu Ala Leu Leu Gln Asn Ala Tyr Leu Thr Ile His Asn Glu Leu 740 745 750 Arg Gly Leu Ser Gly Gln Pro Pro Glu Gly Cys Glu Ile Pro Lys Val 755 760 765 Ile Val Ser Asn His Asn Leu Ala Asp Thr Val Gln Asn Lys Gln Leu 770 775 780 Gln Ala Val Leu Gln Trp Val Ala Ala Ser Glu Leu Asn Val Pro Ile 785 790 795 800 Leu Tyr Phe Ala Gly Asp Asp Glu Gly Ile Gln Glu Lys Leu Leu Gln 805 810 815 Leu Ser Ala Thr Ala Val Glu Lys Gly Arg Ser Val Gly Glu Val Leu 820 825 830 Gln Ser Val Leu Arg Tyr Glu Lys Glu Arg Gln Leu Asp Glu Ala Val 835 840 845 Gly Asn Val Thr Arg Leu Gln Leu Leu Asp Trp Leu Met Ala Asn Leu 850 855 860 12 2562 DNA Bos sp. CDS (1)..(2562) 12 gtt gac tgg tta caa agc caa aat gga gta tgc aaa gtt gac gtc tat 48 Val Asp Trp Leu Gln Ser Gln Asn Gly Val Cys Lys Val Asp Val Tyr 1 5 10 15 tcc ccc gga gac agc caa ccc caa gac tgg aaa atg tcg gat gaa tcc 96 Ser Pro Gly Asp Ser Gln Pro Gln Asp Trp Lys Met Ser Asp Glu Ser 20 25 30 ttg tct att ttt aag gaa gcc tca cac gat ccc atc aga gtg ctc agc 144 Leu Ser Ile Phe Lys Glu Ala Ser His Asp Pro Ile Arg Val Leu Ser 35 40 45 tgg ctc cgc aga gac ctg gaa aaa agc aca gca gga ttc caa gac att 192 Trp Leu Arg Arg Asp Leu Glu Lys Ser Thr Ala Gly Phe Gln Asp Ile 50 55 60 agg ttc aag ccc gga gaa tca tcg ctc ggc agg gaa atg gtc agc tca 240 Arg Phe Lys Pro Gly Glu Ser Ser Leu Gly Arg Glu Met Val Ser Ser 65 70 75 80 gga gac cca cgc aaa ggt ttc tgc gtg gac tat tac aac acc acc tcc 288 Gly Asp Pro Arg Lys Gly Phe Cys Val Asp Tyr Tyr Asn Thr Thr Ser 85 90 95 agg gga agt cca ggg agg ctg cat ttt gag atg agt cac aga gag aac 336 Arg Gly Ser Pro Gly Arg Leu His Phe Glu Met Ser His Arg Glu Asn 100 105 110 cca cac cag ggc ccc acc tgc aat ggg agc tcc gta gat gaa gtc tcc 384 Pro His Gln Gly Pro Thr Cys Asn Gly Ser Ser Val Asp Glu Val Ser 115 120 125 ttc tac gcc aac cgc ctc aca aat ctt gtc atc gcc atg gcc cgc aag 432 Phe Tyr Ala Asn Arg Leu Thr Asn Leu Val Ile Ala Met Ala Arg Lys 130 135 140 gag atc aac gag aag att gac ggc tct gaa aac agg tgt gtc cac caa 480 Glu Ile Asn Glu Lys Ile Asp Gly Ser Glu Asn Arg Cys Val His Gln 145 150 155 160 tca gtg tac atg ggg gat gag cct cca ccc aac aag agc ctg agc aag 528 Ser Val Tyr Met Gly Asp Glu Pro Pro Pro Asn Lys Ser Leu Ser Lys 165 170 175 gtg gcg tcg gag ctg gtg aat gag acc gtc act gcc tgc tcc aaa aac 576 Val Ala Ser Glu Leu Val Asn Glu Thr Val Thr Ala Cys Ser Lys Asn 180 185 190 acc cca tca gat aag gct ccg ggc tct ggt gac aga gcc tcg ggg aca 624 Thr Pro Ser Asp Lys Ala Pro Gly Ser Gly Asp Arg Ala Ser Gly Thr 195 200 205 tta cag agc ccc cca aat tta aaa tac aaa agc act ttg aag atc aag 672 Leu Gln Ser Pro Pro Asn Leu Lys Tyr Lys Ser Thr Leu Lys Ile Lys 210 215 220 gaa agc aac aag gga ggc agg ggt cca gac gac agg cct agc tct aag 720 Glu Ser Asn Lys Gly Gly Arg Gly Pro Asp Asp Arg Pro Ser Ser Lys 225 230 235 240 aag tct ctc ttc tac aag gaa gta ttc gaa tct cgt aac gcg ggt gat 768 Lys Ser Leu Phe Tyr Lys Glu Val Phe Glu Ser Arg Asn Ala Gly Asp 245 250 255 gcc aaa gag gga gga agg acc tta ccg gcg gag aga aag atg ttc aga 816 Ala Lys Glu Gly Gly Arg Thr Leu Pro Ala Glu Arg Lys Met Phe Arg 260 265 270 ggg tat gaa agg ccc gat gac ttc aca gct tcc atc agt caa ggg atc 864 Gly Tyr Glu Arg Pro Asp Asp Phe Thr Ala Ser Ile Ser Gln Gly Ile 275 280 285 atg acc tat gcc aac agt gtg gtg tcc gac atg atg gtc tcc atc atg 912 Met Thr Tyr Ala Asn Ser Val Val Ser Asp Met Met Val Ser Ile Met 290 295 300 aag acc ctg aag atc cag gtg aag gac acg acc att gcc acc atc gtg 960 Lys Thr Leu Lys Ile Gln Val Lys Asp Thr Thr Ile Ala Thr Ile Val 305 310 315 320 ctg aag aag gtc ctg atc aag cac gcc aaa gag gtg gtc tcc gac ctc 1008 Leu Lys Lys Val Leu Ile Lys His Ala Lys Glu Val Val Ser Asp Leu 325 330 335 atc gac tcc ttc atg aag aac ctc cac aac gtc acg ggg acc ctc atg 1056 Ile Asp Ser Phe Met Lys Asn Leu His Asn Val Thr Gly Thr Leu Met 340 345 350 acg gac acg gac ttt gtc tcg gcc gtg aaa aga agc ttc ttc tct cat 1104 Thr Asp Thr Asp Phe Val Ser Ala Val Lys Arg Ser Phe Phe Ser His 355 360 365 gga agc cag aag gcc acg gac atc atg gac gcc atg ctg ggg aaa ctc 1152 Gly Ser Gln Lys Ala Thr Asp Ile Met Asp Ala Met Leu Gly Lys Leu 370 375 380 tat tcg gtg ata ttt gcc aag aaa ccc cct gag aca gtc agg aaa acc 1200 Tyr Ser Val Ile Phe Ala Lys Lys Pro Pro Glu Thr Val Arg Lys Thr 385 390 395 400 aag gat aag tct gaa agt tac tcc ctc gtc tct atg aaa ggg atg ggt 1248 Lys Asp Lys Ser Glu Ser Tyr Ser Leu Val Ser Met Lys Gly Met Gly 405 410 415 gac cct aaa cac cga aac gtc aac ttc gcc tcc atg aaa tca gaa ggt 1296 Asp Pro Lys His Arg Asn Val Asn Phe Ala Ser Met Lys Ser Glu Gly 420 425 430 aaa gtg agg gag aga gta tgc acc ccc acg ccc aaa ccc gag aag tcc 1344 Lys Val Arg Glu Arg Val Cys Thr Pro Thr Pro Lys Pro Glu Lys Ser 435 440 445 tgt gtg gaa act ctg ggg gaa cac att atc aaa gag ggg ctg acc ctg 1392 Cys Val Glu Thr Leu Gly Glu His Ile Ile Lys Glu Gly Leu Thr Leu 450 455 460 tgg cac aac agc cag cag aag gaa ggc ata tct gca tgc ctc cag ggc 1440 Trp His Asn Ser Gln Gln Lys Glu Gly Ile Ser Ala Cys Leu Gln Gly 465 470 475 480 tca ccc ttc gtc act ccc aaa aga cag tat aaa cct gtg ccg gac ttt 1488 Ser Pro Phe Val Thr Pro Lys Arg Gln Tyr Lys Pro Val Pro Asp Phe 485 490 495 ccc ctg ggg ttc cct ttt gac ccc tgc aac ttc agc ctc cct atg cag 1536 Pro Leu Gly Phe Pro Phe Asp Pro Cys Asn Phe Ser Leu Pro Met Gln 500 505 510 tgc cca gaa aaa cct gag aat ttt atg tgc gac tca gac tcc tgg gcc 1584 Cys Pro Glu Lys Pro Glu Asn Phe Met Cys Asp Ser Asp Ser Trp Ala 515 520 525 aaa gac ctg ctt gtg tct gct tta ctt ttg atc cag tac cac ctg gcc 1632 Lys Asp Leu Leu Val Ser Ala Leu Leu Leu Ile Gln Tyr His Leu Ala 530 535 540 cag gga gga aac atg gat gca cag agc ttt ctc gaa gct gct ggc acc 1680 Gln Gly Gly Asn Met Asp Ala Gln Ser Phe Leu Glu Ala Ala Gly Thr 545 550 555 560 tcc aac ctg tat ccc act aag tct cca gcc gtt tcc cat gag tcc agc 1728 Ser Asn Leu Tyr Pro Thr Lys Ser Pro Ala Val Ser His Glu Ser Ser 565 570 575 ctc cgg tct ccc caa gtg gga gct gac cca gaa gaa gtg gag aag aag 1776 Leu Arg Ser Pro Gln Val Gly Ala Asp Pro Glu Glu Val Glu Lys Lys 580 585 590 gac cta atg agc gtt ttc ttc aac ttt atc cgg aac ttg ctc agt gag 1824 Asp Leu Met Ser Val Phe Phe Asn Phe Ile Arg Asn Leu Leu Ser Glu 595 600 605 acc att ttc aag agc gac cat agc tgt gat ccc aag gca acc aag gaa 1872 Thr Ile Phe Lys Ser Asp His Ser Cys Asp Pro Lys Ala Thr Lys Glu 610 615 620 gac aac agc ccc cag tgt gaa aga ccc gtc acc cct tct ccc gcc aaa 1920 Asp Asn Ser Pro Gln Cys Glu Arg Pro Val Thr Pro Ser Pro Ala Lys 625 630 635 640 tta aat gaa tgt gat gag act ggg ggt gct ttt gcg ggg ctc acc aag 1968 Leu Asn Glu Cys Asp Glu Thr Gly Gly Ala Phe Ala Gly Leu Thr Lys 645 650 655 atg gtg gct aac aaa ctt gat ggc cac atg aac ggg cag atg gta gac 2016 Met Val Ala Asn Lys Leu Asp Gly His Met Asn Gly Gln Met Val Asp 660 665 670 cat ctg atg gac tca gtg atg aag ttg tgt ctc atc att gcc aag tcc 2064 His Leu Met Asp Ser Val Met Lys Leu Cys Leu Ile Ile Ala Lys Ser 675 680 685 tgc gac tct cca ttg gca gaa ctg gga gac gac aag tct ggg gat gcc 2112 Cys Asp Ser Pro Leu Ala Glu Leu Gly Asp Asp Lys Ser Gly Asp Ala 690 695 700 agc agg cca aca tca gcc ttc cca gag agt tta tat gag tgt ttg tcc 2160 Ser Arg Pro Thr Ser Ala Phe Pro Glu Ser Leu Tyr Glu Cys Leu Ser 705 710 715 720 acc aag ggc aca ggg act gca gag aca ttc cta cag aat gcc tat cag 2208 Thr Lys Gly Thr Gly Thr Ala Glu Thr Phe Leu Gln Asn Ala Tyr Gln 725 730 735 gct atc cac aac gaa ctg agg agt cta tca gca cag ccc cca gaa ggg 2256 Ala Ile His Asn Glu Leu Arg Ser Leu Ser Ala Gln Pro Pro Glu Gly 740 745 750 tgc aca gca ccc aaa gtg atc gtc agc aac cac aac ctg act gac acc 2304 Cys Thr Ala Pro Lys Val Ile Val Ser Asn His Asn Leu Thr Asp Thr 755 760 765 gtg cag aac aag cag ctc caa gcc gtt ctt cag tgg gtg gcc gcc tct 2352 Val Gln Asn Lys Gln Leu Gln Ala Val Leu Gln Trp Val Ala Ala Ser 770 775 780 gag ctc aac gtc ccc att ctg tac ttt gcg ggt gac gac gaa gga atc 2400 Glu Leu Asn Val Pro Ile Leu Tyr Phe Ala Gly Asp Asp Glu Gly Ile 785 790 795 800 cag gag aag cta ctt cag ctc tcg gca gct gcc gtg gaa aag ggg cgc 2448 Gln Glu Lys Leu Leu Gln Leu Ser Ala Ala Ala Val Glu Lys Gly Arg 805 810 815 agc gta ggt gag gtt cta cag tcg gtg ctg cgg tac gag aag gag cga 2496 Ser Val Gly Glu Val Leu Gln Ser Val Leu Arg Tyr Glu Lys Glu Arg 820 825 830 cag ctg gac gag gcg gtg ggc aac gtc aca cgg ctg cag ctg ctg gac 2544 Gln Leu Asp Glu Ala Val Gly Asn Val Thr Arg Leu Gln Leu Leu Asp 835 840 845 tgg ctg atg gtg aac ctg 2562 Trp Leu Met Val Asn Leu 850 13 854 PRT Bos sp. 13 Val Asp Trp Leu Gln Ser Gln Asn Gly Val Cys Lys Val Asp Val Tyr 1 5 10 15 Ser Pro Gly Asp Ser Gln Pro Gln Asp Trp Lys Met Ser Asp Glu Ser 20 25 30 Leu Ser Ile Phe Lys Glu Ala Ser His Asp Pro Ile Arg Val Leu Ser 35 40 45 Trp Leu Arg Arg Asp Leu Glu Lys Ser Thr Ala Gly Phe Gln Asp Ile 50 55 60 Arg Phe Lys Pro Gly Glu Ser Ser Leu Gly Arg Glu Met Val Ser Ser 65 70 75 80 Gly Asp Pro Arg Lys Gly Phe Cys Val Asp Tyr Tyr Asn Thr Thr Ser 85 90 95 Arg Gly Ser Pro Gly Arg Leu His Phe Glu Met Ser His Arg Glu Asn 100 105 110 Pro His Gln Gly Pro Thr Cys Asn Gly Ser Ser Val Asp Glu Val Ser 115 120 125 Phe Tyr Ala Asn Arg Leu Thr Asn Leu Val Ile Ala Met Ala Arg Lys 130 135 140 Glu Ile Asn Glu Lys Ile Asp Gly Ser Glu Asn Arg Cys Val His Gln 145 150 155 160 Ser Val Tyr Met Gly Asp Glu Pro Pro Pro Asn Lys Ser Leu Ser Lys 165 170 175 Val Ala Ser Glu Leu Val Asn Glu Thr Val Thr Ala Cys Ser Lys Asn 180 185 190 Thr Pro Ser Asp Lys Ala Pro Gly Ser Gly Asp Arg Ala Ser Gly Thr 195 200 205 Leu Gln Ser Pro Pro Asn Leu Lys Tyr Lys Ser Thr Leu Lys Ile Lys 210 215 220 Glu Ser Asn Lys Gly Gly Arg Gly Pro Asp Asp Arg Pro Ser Ser Lys 225 230 235 240 Lys Ser Leu Phe Tyr Lys Glu Val Phe Glu Ser Arg Asn Ala Gly Asp 245 250 255 Ala Lys Glu Gly Gly Arg Thr Leu Pro Ala Glu Arg Lys Met Phe Arg 260 265 270 Gly Tyr Glu Arg Pro Asp Asp Phe Thr Ala Ser Ile Ser Gln Gly Ile 275 280 285 Met Thr Tyr Ala Asn Ser Val Val Ser Asp Met Met Val Ser Ile Met 290 295 300 Lys Thr Leu Lys Ile Gln Val Lys Asp Thr Thr Ile Ala Thr Ile Val 305 310 315 320 Leu Lys Lys Val Leu Ile Lys His Ala Lys Glu Val Val Ser Asp Leu 325 330 335 Ile Asp Ser Phe Met Lys Asn Leu His Asn Val Thr Gly Thr Leu Met 340 345 350 Thr Asp Thr Asp Phe Val Ser Ala Val Lys Arg Ser Phe Phe Ser His 355 360 365 Gly Ser Gln Lys Ala Thr Asp Ile Met Asp Ala Met Leu Gly Lys Leu 370 375 380 Tyr Ser Val Ile Phe Ala Lys Lys Pro Pro Glu Thr Val Arg Lys Thr 385 390 395 400 Lys Asp Lys Ser Glu Ser Tyr Ser Leu Val Ser Met Lys Gly Met Gly 405 410 415 Asp Pro Lys His Arg Asn Val Asn Phe Ala Ser Met Lys Ser Glu Gly 420 425 430 Lys Val Arg Glu Arg Val Cys Thr Pro Thr Pro Lys Pro Glu Lys Ser 435 440 445 Cys Val Glu Thr Leu Gly Glu His Ile Ile Lys Glu Gly Leu Thr Leu 450 455 460 Trp His Asn Ser Gln Gln Lys Glu Gly Ile Ser Ala Cys Leu Gln Gly 465 470 475 480 Ser Pro Phe Val Thr Pro Lys Arg Gln Tyr Lys Pro Val Pro Asp Phe 485 490 495 Pro Leu Gly Phe Pro Phe Asp Pro Cys Asn Phe Ser Leu Pro Met Gln 500 505 510 Cys Pro Glu Lys Pro Glu Asn Phe Met Cys Asp Ser Asp Ser Trp Ala 515 520 525 Lys Asp Leu Leu Val Ser Ala Leu Leu Leu Ile Gln Tyr His Leu Ala 530 535 540 Gln Gly Gly Asn Met Asp Ala Gln Ser Phe Leu Glu Ala Ala Gly Thr 545 550 555 560 Ser Asn Leu Tyr Pro Thr Lys Ser Pro Ala Val Ser His Glu Ser Ser 565 570 575 Leu Arg Ser Pro Gln Val Gly Ala Asp Pro Glu Glu Val Glu Lys Lys 580 585 590 Asp Leu Met Ser Val Phe Phe Asn Phe Ile Arg Asn Leu Leu Ser Glu 595 600 605 Thr Ile Phe Lys Ser Asp His Ser Cys Asp Pro Lys Ala Thr Lys Glu 610 615 620 Asp Asn Ser Pro Gln Cys Glu Arg Pro Val Thr Pro Ser Pro Ala Lys 625 630 635 640 Leu Asn Glu Cys Asp Glu Thr Gly Gly Ala Phe Ala Gly Leu Thr Lys 645 650 655 Met Val Ala Asn Lys Leu Asp Gly His Met Asn Gly Gln Met Val Asp 660 665 670 His Leu Met Asp Ser Val Met Lys Leu Cys Leu Ile Ile Ala Lys Ser 675 680 685 Cys Asp Ser Pro Leu Ala Glu Leu Gly Asp Asp Lys Ser Gly Asp Ala 690 695 700 Ser Arg Pro Thr Ser Ala Phe Pro Glu Ser Leu Tyr Glu Cys Leu Ser 705 710 715 720 Thr Lys Gly Thr Gly Thr Ala Glu Thr Phe Leu Gln Asn Ala Tyr Gln 725 730 735 Ala Ile His Asn Glu Leu Arg Ser Leu Ser Ala Gln Pro Pro Glu Gly 740 745 750 Cys Thr Ala Pro Lys Val Ile Val Ser Asn His Asn Leu Thr Asp Thr 755 760 765 Val Gln Asn Lys Gln Leu Gln Ala Val Leu Gln Trp Val Ala Ala Ser 770 775 780 Glu Leu Asn Val Pro Ile Leu Tyr Phe Ala Gly Asp Asp Glu Gly Ile 785 790 795 800 Gln Glu Lys Leu Leu Gln Leu Ser Ala Ala Ala Val Glu Lys Gly Arg 805 810 815 Ser Val Gly Glu Val Leu Gln Ser Val Leu Arg Tyr Glu Lys Glu Arg 820 825 830 Gln Leu Asp Glu Ala Val Gly Asn Val Thr Arg Leu Gln Leu Leu Asp 835 840 845 Trp Leu Met Val Asn Leu 850 14 3014 DNA Homo sapiens CDS (230)..(2788) 5′UTR (1)..(229) 3′UTR (2789)..(3014) 14 ggtacatgga aggccacagg aagaaacaag atcttgagct gagcaagaac atcccagcat 60 cttcattgac tttaaaagta tattctggag tcttccgtgg ttcactattc cagtactaca 120 gagattcctt atattacatg gcaggagggg ggtaaactga gggatagtga agacaacaat 180 aaattaatca agagctttcc tcatatctca gaacctatcc tctgtaaga atg tca gaa 238 Met Ser Glu 1 aag gtt gac tgg tta caa agc caa aat gga gta tgc aaa gtt gat gtc 286 Lys Val Asp Trp Leu Gln Ser Gln Asn Gly Val Cys Lys Val Asp Val 5 10 15 tat tct cct gga gac aac caa gcc cag gac tgg aaa atg gac acc tcc 334 Tyr Ser Pro Gly Asp Asn Gln Ala Gln Asp Trp Lys Met Asp Thr Ser 20 25 30 35 acg gat cct gtc aga gtg ctc agc tgg ctc cgc aga gac ctg gag aag 382 Thr Asp Pro Val Arg Val Leu Ser Trp Leu Arg Arg Asp Leu Glu Lys 40 45 50 agt aca gca gag ttc caa gat gtt cgg ttc aaa ccc gga gaa tca ttt 430 Ser Thr Ala Glu Phe Gln Asp Val Arg Phe Lys Pro Gly Glu Ser Phe 55 60 65 ggt ggg gaa acg tcc aac tca gga gac cca cac aaa ggt ttc tct gta 478 Gly Gly Glu Thr Ser Asn Ser Gly Asp Pro His Lys Gly Phe Ser Val 70 75 80 gac tat tac aac acc acc acc aag ggc act cca gaa aga ttg cat ttt 526 Asp Tyr Tyr Asn Thr Thr Thr Lys Gly Thr Pro Glu Arg Leu His Phe 85 90 95 gag atg act cac aaa gag att cct tgc cag ggc ccc agg gcc caa ctt 574 Glu Met Thr His Lys Glu Ile Pro Cys Gln Gly Pro Arg Ala Gln Leu 100 105 110 115 ggc aac ggg agt tca gta gat gaa gtt tcc ttc tat gct aac cgc ctc 622 Gly Asn Gly Ser Ser Val Asp Glu Val Ser Phe Tyr Ala Asn Arg Leu 120 125 130 acg aat cta gtc ata gcc atg gcc cgc aaa gag atc aat gag aag atc 670 Thr Asn Leu Val Ile Ala Met Ala Arg Lys Glu Ile Asn Glu Lys Ile 135 140 145 gat ggc tct gaa aac aaa tgt gtc tat cag tca ttg tac atg ggg aat 718 Asp Gly Ser Glu Asn Lys Cys Val Tyr Gln Ser Leu Tyr Met Gly Asn 150 155 160 gaa ccc aca ccc acc aaa agc ctc agt aag ata gca tca gag ctt gtg 766 Glu Pro Thr Pro Thr Lys Ser Leu Ser Lys Ile Ala Ser Glu Leu Val 165 170 175 aat gag acc gtc tct gca tgt tcc agg aat gct gcc cca gac aag gct 814 Asn Glu Thr Val Ser Ala Cys Ser Arg Asn Ala Ala Pro Asp Lys Ala 180 185 190 195 cct ggc tct gga gac aga gtc tca gga tca tca caa agt ccc cca aat 862 Pro Gly Ser Gly Asp Arg Val Ser Gly Ser Ser Gln Ser Pro Pro Asn 200 205 210 ttg aaa tac aag tcc act ttg aag atc aag gag agc acc aaa gaa aga 910 Leu Lys Tyr Lys Ser Thr Leu Lys Ile Lys Glu Ser Thr Lys Glu Arg 215 220 225 cag ggt cca gat gac aag cct cct tct aag aag tct ttc ttc tat aag 958 Gln Gly Pro Asp Asp Lys Pro Pro Ser Lys Lys Ser Phe Phe Tyr Lys 230 235 240 gaa gtg ttt gaa tct cgt aac gga gat tat gcc aga gag ggt gga agg 1006 Glu Val Phe Glu Ser Arg Asn Gly Asp Tyr Ala Arg Glu Gly Gly Arg 245 250 255 ttc ttt cct cgg gag aga aag agg ttt cga ggg cag gaa agg cct gat 1054 Phe Phe Pro Arg Glu Arg Lys Arg Phe Arg Gly Gln Glu Arg Pro Asp 260 265 270 275 gac ttt acg gct tct gtt agt gaa ggg atc atg acc tat gct aac agt 1102 Asp Phe Thr Ala Ser Val Ser Glu Gly Ile Met Thr Tyr Ala Asn Ser 280 285 290 gtg gta tct gat atg atg gtc tcc atc atg aag aca ctg aag atc caa 1150 Val Val Ser Asp Met Met Val Ser Ile Met Lys Thr Leu Lys Ile Gln 295 300 305 gtg aaa gac aca acc att gcc acc atc cta ctg aag aag gtt ctg ctc 1198 Val Lys Asp Thr Thr Ile Ala Thr Ile Leu Leu Lys Lys Val Leu Leu 310 315 320 aag cat gca aaa gag gtg gtc tcg gat ctc atc gac tcc ttc ttg agg 1246 Lys His Ala Lys Glu Val Val Ser Asp Leu Ile Asp Ser Phe Leu Arg 325 330 335 aat ctc cac agc gtc aca ggg acc ctc atg act gac aca cag ttt gtc 1294 Asn Leu His Ser Val Thr Gly Thr Leu Met Thr Asp Thr Gln Phe Val 340 345 350 355 tcg gct gtg aaa aga act gtc ttc tct cat gga agc caa aag gcc aca 1342 Ser Ala Val Lys Arg Thr Val Phe Ser His Gly Ser Gln Lys Ala Thr 360 365 370 gat atc atg gat gcc atg cta agg aag ctg tac aat gta atg ttt gcc 1390 Asp Ile Met Asp Ala Met Leu Arg Lys Leu Tyr Asn Val Met Phe Ala 375 380 385 aag aaa gtc cct gag cat gtc agg aaa gcc caa gac aag gct gag agt 1438 Lys Lys Val Pro Glu His Val Arg Lys Ala Gln Asp Lys Ala Glu Ser 390 395 400 tat tcc ctc atc tcc atg aaa gga atg ggt gat cct aaa aac cga aat 1486 Tyr Ser Leu Ile Ser Met Lys Gly Met Gly Asp Pro Lys Asn Arg Asn 405 410 415 gtg aac ttt gcc atg aaa tct gaa act aaa ttg aga gaa aaa atg tat 1534 Val Asn Phe Ala Met Lys Ser Glu Thr Lys Leu Arg Glu Lys Met Tyr 420 425 430 435 tct gaa ccc aaa tca gag gag gag act tgt gcg aaa act ctg ggt gag 1582 Ser Glu Pro Lys Ser Glu Glu Glu Thr Cys Ala Lys Thr Leu Gly Glu 440 445 450 cac att atc aaa gag ggg ctt acc ctg tgg cat aaa agt cag cag aaa 1630 His Ile Ile Lys Glu Gly Leu Thr Leu Trp His Lys Ser Gln Gln Lys 455 460 465 gaa tgt aaa tct cta ggt ttc cag cat gca gca ttc gaa gct ccc aac 1678 Glu Cys Lys Ser Leu Gly Phe Gln His Ala Ala Phe Glu Ala Pro Asn 470 475 480 aca cag cgt aag cct gca tca gac att tcc ttt gag tac cct gaa gat 1726 Thr Gln Arg Lys Pro Ala Ser Asp Ile Ser Phe Glu Tyr Pro Glu Asp 485 490 495 att ggc aac ctc agc ctt cct cca tat cct cca gag aaa cct gag aat 1774 Ile Gly Asn Leu Ser Leu Pro Pro Tyr Pro Pro Glu Lys Pro Glu Asn 500 505 510 515 ttt atg tat gat tca gac tcc tgg gcc aag gac ctg atc gtg tct gcc 1822 Phe Met Tyr Asp Ser Asp Ser Trp Ala Lys Asp Leu Ile Val Ser Ala 520 525 530 ctg ctt ctg att caa tat cac ctg gcc cag gga gga aga agg gat gca 1870 Leu Leu Leu Ile Gln Tyr His Leu Ala Gln Gly Gly Arg Arg Asp Ala 535 540 545 cgg agc ttc gtt gaa gct gct ggc acc acc aac ttt cct gcc aat gaa 1918 Arg Ser Phe Val Glu Ala Ala Gly Thr Thr Asn Phe Pro Ala Asn Glu 550 555 560 cct cct gta gct ccc gat gaa tct tgc ctt aag tct gct ccc att gta 1966 Pro Pro Val Ala Pro Asp Glu Ser Cys Leu Lys Ser Ala Pro Ile Val 565 570 575 ggt gac caa gaa caa gca gaa aag aag gac cta agg agt gtt ttc ttt 2014 Gly Asp Gln Glu Gln Ala Glu Lys Lys Asp Leu Arg Ser Val Phe Phe 580 585 590 595 aat ttc atc cgg aac tta ctt agt gag acc att ttc aag cgt gac cag 2062 Asn Phe Ile Arg Asn Leu Leu Ser Glu Thr Ile Phe Lys Arg Asp Gln 600 605 610 agc cct gaa ccc aag gtg ccg gaa cag cca gtt aag gaa gat agg aag 2110 Ser Pro Glu Pro Lys Val Pro Glu Gln Pro Val Lys Glu Asp Arg Lys 615 620 625 ttg tgt gaa aga ccg ttg gcg tct tct ccc ccc agg cta tat gag gat 2158 Leu Cys Glu Arg Pro Leu Ala Ser Ser Pro Pro Arg Leu Tyr Glu Asp 630 635 640 gat gag acc cct ggt gcc ctt tct ggg ctg acc aag atg gct gtc agc 2206 Asp Glu Thr Pro Gly Ala Leu Ser Gly Leu Thr Lys Met Ala Val Ser 645 650 655 cag ata gat ggc cac atg agt ggg cag atg gta gaa cat ctg atg aac 2254 Gln Ile Asp Gly His Met Ser Gly Gln Met Val Glu His Leu Met Asn 660 665 670 675 tca gtg atg aag ctg tgt gtc atc att gct aag tcc tgt gat gct tcg 2302 Ser Val Met Lys Leu Cys Val Ile Ile Ala Lys Ser Cys Asp Ala Ser 680 685 690 ttg gca gag ctg gga gat gac aag tct gga gat gcc agt agg cta act 2350 Leu Ala Glu Leu Gly Asp Asp Lys Ser Gly Asp Ala Ser Arg Leu Thr 695 700 705 tcg gcc ttc cca gat agt tta tat gag tgc tta cca gcc aag ggc aca 2398 Ser Ala Phe Pro Asp Ser Leu Tyr Glu Cys Leu Pro Ala Lys Gly Thr 710 715 720 ggg tca gca gaa gct gtc ctg cag aat gcc tat caa gct atc cat aat 2446 Gly Ser Ala Glu Ala Val Leu Gln Asn Ala Tyr Gln Ala Ile His Asn 725 730 735 gaa atg aga ggc aca tca gga cag ccc cct gaa ggg tgt gca gca ccc 2494 Glu Met Arg Gly Thr Ser Gly Gln Pro Pro Glu Gly Cys Ala Ala Pro 740 745 750 755 acg gtg att gtc agc aat cac aac cta acg gac aca gtt cag aac aag 2542 Thr Val Ile Val Ser Asn His Asn Leu Thr Asp Thr Val Gln Asn Lys 760 765 770 caa ctc caa gcc gtc ctt caa tgg gta gct gcc tct gag ctc aat gtc 2590 Gln Leu Gln Ala Val Leu Gln Trp Val Ala Ala Ser Glu Leu Asn Val 775 780 785 cct att ttg tat ttt gct ggt gat gat gaa ggg atc cag gag aag cta 2638 Pro Ile Leu Tyr Phe Ala Gly Asp Asp Glu Gly Ile Gln Glu Lys Leu 790 795 800 ctt cag ctc tca gct gct gct gtg gac aaa gga tgc agt gtg ggc gag 2686 Leu Gln Leu Ser Ala Ala Ala Val Asp Lys Gly Cys Ser Val Gly Glu 805 810 815 gtt ctg cag tcg gtg ctg cgc tat gag aag gag cgc cag ctg aat gag 2734 Val Leu Gln Ser Val Leu Arg Tyr Glu Lys Glu Arg Gln Leu Asn Glu 820 825 830 835 gcg gtg ggg aat gtc aca ccg ctg cag ctg ctg gac tgg ctg atg gtg 2782 Ala Val Gly Asn Val Thr Pro Leu Gln Leu Leu Asp Trp Leu Met Val 840 845 850 aac ctg taatcggcaa ccccactgct ttcccctctt ctggcagtgg ggccggccct 2838 Asn Leu tatccccgcc cttctttctc acttccacat ctccccctct atatcctcac agagccctaa 2898 cattatcttc acaccactct catcaaagac atgtcatctt gtgctagcca ctggattttg 2958 cagattttcc tgtccatgca agcaaggacg taaaattaaa aaattacaat taaaaa 3014 15 853 PRT Homo sapiens 15 Met Ser Glu Lys Val Asp Trp Leu Gln Ser Gln Asn Gly Val Cys Lys 1 5 10 15 Val Asp Val Tyr Ser Pro Gly Asp Asn Gln Ala Gln Asp Trp Lys Met 20 25 30 Asp Thr Ser Thr Asp Pro Val Arg Val Leu Ser Trp Leu Arg Arg Asp 35 40 45 Leu Glu Lys Ser Thr Ala Glu Phe Gln Asp Val Arg Phe Lys Pro Gly 50 55 60 Glu Ser Phe Gly Gly Glu Thr Ser Asn Ser Gly Asp Pro His Lys Gly 65 70 75 80 Phe Ser Val Asp Tyr Tyr Asn Thr Thr Thr Lys Gly Thr Pro Glu Arg 85 90 95 Leu His Phe Glu Met Thr His Lys Glu Ile Pro Cys Gln Gly Pro Arg 100 105 110 Ala Gln Leu Gly Asn Gly Ser Ser Val Asp Glu Val Ser Phe Tyr Ala 115 120 125 Asn Arg Leu Thr Asn Leu Val Ile Ala Met Ala Arg Lys Glu Ile Asn 130 135 140 Glu Lys Ile Asp Gly Ser Glu Asn Lys Cys Val Tyr Gln Ser Leu Tyr 145 150 155 160 Met Gly Asn Glu Pro Thr Pro Thr Lys Ser Leu Ser Lys Ile Ala Ser 165 170 175 Glu Leu Val Asn Glu Thr Val Ser Ala Cys Ser Arg Asn Ala Ala Pro 180 185 190 Asp Lys Ala Pro Gly Ser Gly Asp Arg Val Ser Gly Ser Ser Gln Ser 195 200 205 Pro Pro Asn Leu Lys Tyr Lys Ser Thr Leu Lys Ile Lys Glu Ser Thr 210 215 220 Lys Glu Arg Gln Gly Pro Asp Asp Lys Pro Pro Ser Lys Lys Ser Phe 225 230 235 240 Phe Tyr Lys Glu Val Phe Glu Ser Arg Asn Gly Asp Tyr Ala Arg Glu 245 250 255 Gly Gly Arg Phe Phe Pro Arg Glu Arg Lys Arg Phe Arg Gly Gln Glu 260 265 270 Arg Pro Asp Asp Phe Thr Ala Ser Val Ser Glu Gly Ile Met Thr Tyr 275 280 285 Ala Asn Ser Val Val Ser Asp Met Met Val Ser Ile Met Lys Thr Leu 290 295 300 Lys Ile Gln Val Lys Asp Thr Thr Ile Ala Thr Ile Leu Leu Lys Lys 305 310 315 320 Val Leu Leu Lys His Ala Lys Glu Val Val Ser Asp Leu Ile Asp Ser 325 330 335 Phe Leu Arg Asn Leu His Ser Val Thr Gly Thr Leu Met Thr Asp Thr 340 345 350 Gln Phe Val Ser Ala Val Lys Arg Thr Val Phe Ser His Gly Ser Gln 355 360 365 Lys Ala Thr Asp Ile Met Asp Ala Met Leu Arg Lys Leu Tyr Asn Val 370 375 380 Met Phe Ala Lys Lys Val Pro Glu His Val Arg Lys Ala Gln Asp Lys 385 390 395 400 Ala Glu Ser Tyr Ser Leu Ile Ser Met Lys Gly Met Gly Asp Pro Lys 405 410 415 Asn Arg Asn Val Asn Phe Ala Met Lys Ser Glu Thr Lys Leu Arg Glu 420 425 430 Lys Met Tyr Ser Glu Pro Lys Ser Glu Glu Glu Thr Cys Ala Lys Thr 435 440 445 Leu Gly Glu His Ile Ile Lys Glu Gly Leu Thr Leu Trp His Lys Ser 450 455 460 Gln Gln Lys Glu Cys Lys Ser Leu Gly Phe Gln His Ala Ala Phe Glu 465 470 475 480 Ala Pro Asn Thr Gln Arg Lys Pro Ala Ser Asp Ile Ser Phe Glu Tyr 485 490 495 Pro Glu Asp Ile Gly Asn Leu Ser Leu Pro Pro Tyr Pro Pro Glu Lys 500 505 510 Pro Glu Asn Phe Met Tyr Asp Ser Asp Ser Trp Ala Lys Asp Leu Ile 515 520 525 Val Ser Ala Leu Leu Leu Ile Gln Tyr His Leu Ala Gln Gly Gly Arg 530 535 540 Arg Asp Ala Arg Ser Phe Val Glu Ala Ala Gly Thr Thr Asn Phe Pro 545 550 555 560 Ala Asn Glu Pro Pro Val Ala Pro Asp Glu Ser Cys Leu Lys Ser Ala 565 570 575 Pro Ile Val Gly Asp Gln Glu Gln Ala Glu Lys Lys Asp Leu Arg Ser 580 585 590 Val Phe Phe Asn Phe Ile Arg Asn Leu Leu Ser Glu Thr Ile Phe Lys 595 600 605 Arg Asp Gln Ser Pro Glu Pro Lys Val Pro Glu Gln Pro Val Lys Glu 610 615 620 Asp Arg Lys Leu Cys Glu Arg Pro Leu Ala Ser Ser Pro Pro Arg Leu 625 630 635 640 Tyr Glu Asp Asp Glu Thr Pro Gly Ala Leu Ser Gly Leu Thr Lys Met 645 650 655 Ala Val Ser Gln Ile Asp Gly His Met Ser Gly Gln Met Val Glu His 660 665 670 Leu Met Asn Ser Val Met Lys Leu Cys Val Ile Ile Ala Lys Ser Cys 675 680 685 Asp Ala Ser Leu Ala Glu Leu Gly Asp Asp Lys Ser Gly Asp Ala Ser 690 695 700 Arg Leu Thr Ser Ala Phe Pro Asp Ser Leu Tyr Glu Cys Leu Pro Ala 705 710 715 720 Lys Gly Thr Gly Ser Ala Glu Ala Val Leu Gln Asn Ala Tyr Gln Ala 725 730 735 Ile His Asn Glu Met Arg Gly Thr Ser Gly Gln Pro Pro Glu Gly Cys 740 745 750 Ala Ala Pro Thr Val Ile Val Ser Asn His Asn Leu Thr Asp Thr Val 755 760 765 Gln Asn Lys Gln Leu Gln Ala Val Leu Gln Trp Val Ala Ala Ser Glu 770 775 780 Leu Asn Val Pro Ile Leu Tyr Phe Ala Gly Asp Asp Glu Gly Ile Gln 785 790 795 800 Glu Lys Leu Leu Gln Leu Ser Ala Ala Ala Val Asp Lys Gly Cys Ser 805 810 815 Val Gly Glu Val Leu Gln Ser Val Leu Arg Tyr Glu Lys Glu Arg Gln 820 825 830 Leu Asn Glu Ala Val Gly Asn Val Thr Pro Leu Gln Leu Leu Asp Trp 835 840 845 Leu Met Val Asn Leu 850 16 12 PRT Mus musculus 16 Ser Phe Tyr Ala Asn Arg Leu Thr Asn Leu Val Ile 1 5 10 17 18 PRT Artificial Sequence Description of Artificial Sequence This is a consensus sequence of the RII subunit binding site of AKAP110 from several tissue types 17 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa 18 30 DNA Artificial Sequence Description of Artificial Sequence antisense oligonucleotide comprising sequence complementary to the translation initiation codon of human akap110 18 gctttgtaac cagtcaacct tttctgacat 30 19 18 PRT Bos sp. 19 Glu Val Ser Phe Tyr Ala Asn Arg Leu Thr Asn Leu Val Ile Ala Met 1 5 10 15 Ala Arg 20 18 PRT Bos sp. 20 Asp Val Ser Phe Tyr Val Asn Arg Leu Thr Ser Leu Val Ile Gln Met 1 5 10 15 Ala Arg 21 18 PRT Bos sp. 21 Glu Ile Lys Arg Ala Ala Phe Gln Ile Ile Ser Gln Val Ile Ser Glu 1 5 10 15 Ala Thr 22 18 PRT Bos sp. 22 Arg Met Asn Glu Ile Ala Arg Thr Val Val Glu Gly Val Leu Ala Ala 1 5 10 15 Ser Val 23 18 PRT Bos sp. 23 Thr Ala Glu Glu Val Ser Ala Arg Ile Val Gln Val Val Thr Ala Glu 1 5 10 15 Ala Val 24 18 PRT Bos sp. 24 Leu Ile Glu Glu Ala Ala Ser Arg Ile Val Asp Ala Val Ile Glu Gln 1 5 10 15 Val Lys 25 18 PRT Bos sp. 25 Leu Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn Ala Ile Glu Leu 1 5 10 15 Ser Val 26 18 PRT Bos sp. 26 Leu Leu Ile Glu Thr Ala Ser Ser Leu Val Lys Asn Ala Ile Gln Leu 1 5 10 15 Ser Ile 27 18 PRT Bos sp. 27 Thr Pro Glu Glu Val Ala Ala Glu Val Leu Ala Glu Val Ile Thr Ala 1 5 10 15 Ala Val 28 18 PRT Bos sp. 28 Leu Glu Glu Lys Val Ala Ala Ala Leu Val Ser Gln Val Gln Leu Glu 1 5 10 15 Ala Val 29 18 PRT Bos sp. 29 Thr Val Glu Gln Tyr Ala Arg Lys Val Val Gly Asp Thr Leu Glu Leu 1 5 10 15 Ser Leu 30 18 PRT Bos sp. 30 Glu Leu Glu Thr Lys Ser Ser Lys Leu Val Gln Asn Ile Ile Gln Thr 1 5 10 15 Ala Val 31 18 PRT Bos frontalis 31 Pro Leu Glu Tyr Gln Ala Gly Leu Leu Val Gln Asn Ala Ile Gln Gln 1 5 10 15 Ala Ile 32 18 PRT Bos frontalis 32 Glu Leu Val Arg Leu Ser Lys Arg Leu Val Glu Asn Ala Val Leu Lys 1 5 10 15 Ala Val 33 18 PRT Bos sp. 33 Ser Gln Glu Gln Leu Ala Ala Glu Leu Ala Gly Tyr Thr Ala Lys Ile 1 5 10 15 Ala Leu

Claims

We claim:

1. A homogeneous composition of a mammalian sperm-specific protein kinase A anchoring protein having an apparent molecular weight of about 110 kildaltons.

2. A homogeneous composition of a human sperm-specific protein kinase A anchoring protein according to claim 1, having an amino acid sequence identified by Seq. ID No. 16.

3. A homogeneous composition of a bovine sperm-specific protein kinase A anchoring protein according to claim 1, having an amino acid sequence identified by Seq. ID No. 14.

4. A homogeneous composition of a murine sperm-specific protein kinase A anchoring protein according to claim 1, having an amino acid sequence identified by Seq. ID No. 12.

5. A method of screening a compound as an inhibitor of binding between a mammalian sperm-specific protein kinase A anchoring protein having an apparent molecular weight of about 110 kilodaltons and a regulatory subunit of protein kinase A, the method comprising the following steps:

(a) providing a first preparation of an isolated mammalian sperm-specific protein kinase A anchoring protein having an apparent molecular weight of about 110 kilodaltons;

(b) providing a second preparation of an isolated regulatory subunit of protein kinase A;

(c) contacting the first and second preparations under conditions permissible for binding of the protein kinase A anchoring protein and the regulatory subunit of protein kinase A in the presence or absence of the compound; and

(d) detecting a reduction in binding between the protein kinase A anchoring protein and the regulatory subunit of protein kinase A in the presence of the compound compared with binding in the absence of the compound.

6. The method of claim 5, wherein either the protein kinase A anchoring protein or the regulatory subunit of protein kinase A is detectably-labeled.

7. The method of claim 5, wherein the protein kinase A anchoring protein has an amino acid sequence identified by Seq. ID No. 16.

8. The method of claim 5, wherein the protein kinase A anchoring protein has an amino acid sequence identified by Seq. ID No. 14.

9. The method of claim 5, wherein the protein kinase A anchoring protein has an amino acid sequence identified by Seq. ID No. 12.

10. The method of claim 5, wherein either the protein kinase A anchoring protein or the regulatory subunit of protein kinase A is covalently attached to a solid substrate.

11. An antibody or fragment thereof that is immunologically reactive to a mammalian sperm-specific protein kinase A anchoring protein having an apparent molecular weight of 110 kilodaltons.

12. The antibody or fragment thereof according to claim 11, wherein the antibody is a monoclonal antibody.

13. The antibody or fragment thereof according to claim 11, wherein the protein kinase A anchoring protein is human sperm-specific protein kinase A anchoring protein having amino acid sequence identified by Seq. ID No. 16.

14. The antibody or fragment thereof according to claim 11, wherein the protein kinase A anchoring protein is bovine sperm-specific protein kinase A anchoring protein having amino acid sequence identified by Seq. ID No. 14.

15. The antibody or fragment thereof according to claim 11, wherein the protein kinase A anchoring protein is murine sperm-specific protein kinase A anchoring protein having amino acid sequence identified by Seq. ID No. 12.

16. A cell line which produces an antibody or fragment thereof that is immunologically reactive to a mammalian sperm-specific protein kinase A anchoring protein having an apparent molecular weight of 110 kilodaltons.

17. The cell line according to claim 17, wherein the antibody is a monoclonal antibody.

18. The cell line according to claim 17, wherein the mammalian protein kinase A anchoring protein is human sperm-specific protein kinase A anchoring protein having amino acid sequence identified by Seq. ID No. 16.

19. The cell line according to claim 17, wherein the mammalian protein kinase A anchoring protein is bovine sperm-specific protein kinase A anchoring protein having amino acid sequence identified by Seq. ID No. 14.

20. The cell line according to claim 17, wherein the mammalian protein kinase A anchoring protein is murine sperm-specific protein kinase A anchoring protein having amino acid sequence identified by Seq. ID No. 12.

21. An epitope of a mammalian protein kinase A anchoring protein is murine sperm-specific protein kinase A wherein the epitope is immunologically reactive to the antibody or fragment thereof according to claim 11.

22. The epitope according to claim 21 wherein the mammalian protein kinase A anchoring protein is human sperm-specific protein kinase A.

23. The epitope according to claim 21 wherein the mammalian protein kinase A anchoring protein is murine sperm-specific protein kinase A.

24. The epitope according to claim 21 wherein the mammalian protein kinase A anchoring protein is bovine sperm-specific protein kinase A.

25. The epitope according to claim 24 wherein the epitope has an amino acid sequence identified selected from the group consisting of Seq. ID Nos.: 1, 2, 3, 4, 5, 6, 7, 16 and 17.