JP2003520589A - Sperm-specific protein CBP86 - Google Patents

Abstract

SUMMARY The present invention is directed to an acidic (pI 4.0), 86 kDa isoform of a novel polymorphic testis-specific protein, termed calcium binding protein 86 (CBP86). This protein is tyrosine phosphorylated during in vitro capacitation and binds ⁴⁵ Ca on a 2-D gel, the latter effect being completely destroyed by dephosphorylation with alkaline phosphatase. CBP86 is the first illustration of a sperm protein that localizes to the fibrous sheath and the main part of human sperm ben hairs and both oligomerizes and acquires calcium binding capacity in a tyrosine phosphorylation-dependent manner during capacitation. is there.

Description

Detailed Description of the Invention

[0001] Field of the Invention: The present invention is referred to as a calcium-binding protein 86 (CBP86), novel polymorphic testis - directed to 86kDa isoforms of specific protein acidic (pi 4.0). This protein is phosphorylated on tyrosine during phosphorylation in vitro and after phosphorylation binds calcium. BACKGROUND OF THE INVENTION Fertilization capacity is acquired by sperm only after being in the defined microenvironment of the uterus or oviduct (depending on the species) for a period of time. The necessary series of changes, termed capacitation, was first described uniquely by Chang and Austin in the early to mid 1950s. Capacitation involves molecular changes in the sperm head and tail that allow the development of defined physiological endpoints, such as motor hyperactivity, whip-like sperm tail motility and regulated acrosome exocytosis. To do. Overactivation appears to enhance the ability of sperm to reach the oocyte and enhance sperm ovarian coat invasion by enhancing forward progression and lateral flagellar propulsion in their flagellar flexion amplitude and beat asymmetry. Is observed when increasing.

[0002]   The present inventor's understanding of the molecular mechanisms underlying fertility acquisition and hyperactivation is now at the rudimentary level.
Target, but Ca²⁺, CAMP and protein tyrosine phosphorylation are involved
There is evidence. Capacitation depends on protein sources such as albumin, NaHCO₃, CO ²⁺ And energy substrates such as glucose, pyruvate or lactate
With the cauda epididymis or ejaculated sperm incubated in defined medium
It can be achieved in vitro. The conditions that help in vitro fertility acquisition are:
And enhanced tyrosine phosphorylation of a subset of proteins in human sperm
Lead. Albumin, NaHCO from fertility acquisition medium₃, Or Ca²⁺Removal of tyrosine
Prevents the occurrence of both phosphorylation and capacitation. This capacitation-related phosphate
The two types of protein substrates for derivatization are the A kinase anchoring protein family
Lee, a component of the fibrous sheath of the sperm tail, AKAP4 (in mice
Therefore, it is originally called AKAP82 or Fsc1) and AKAP3 (originally AKAP85T, FSP95).
Or called AKAP110).

Cytoplasmic levels of cAMP are elevated during capacitation and pharmacological stimulators that enhance intracellular cAMP, such as phosphodiesterase inhibitors, caffeine and pentoxifylline, induce sperm hyperactivated motility. It enhances, enhances cervical mucus invasion, enhances strong binding to homologous zona and enhances fertility. calcium/
Calmodulin is an activator of both mammalian sperm adenylate cyclase (AC) and cyclic nucleotide phosphodiesterase, and sperm AC
HCO ₃ ^- is stimulated by anionic. Soluble testis adenylate cyclase has recently been cloned and has been shown to be sensitive to bicarbonate. Sperm protein tyrosine phosphorylation is promoted by cAMP agonists and PK
Antagonists of A inhibit tyrosine phosphorylation and capacitation. These and other observations, sperm protein tyrosine phosphorylation and capacitation are calcium and HCO ₃ ^- is activated by elevated cytosolic Zorureberu of anionic, cAMP / PK
Suggests that it is under the control of the A pathway. Mammalian sperm contain guanine nucleotide-binding regulatory proteins G _j of all three subtypes, and G proteins are specifically localized in the sperm tail, where protein kinases A and C have also been reported. Cyclic nucleotide gated Ca ²⁺ channels in the mammalian sperm plasma membrane have been reported, and N- and R-type Ca ²⁺ channels have been defined in mouse sperm.

The present invention is directed to targets at the intersection between calcium and protein tyrosine kinase signal transduction pathways in human sperm. In particular, the invention describes the isolation and characterization of sperm calcium binding proteins that are phosphorylated by tyrosine kinases. Definitions : In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.

As used herein, “nucleic acid”, “DNA” and like terms also include nucleic acid analogs, ie analogs having other than a phosphodiester backbone. For example, so-called "peptide nucleic acids" which are known in the art and have peptide bonds in the backbone instead of phosphodiester bonds are considered to be within the scope of the present invention.

[0006]   The term "peptide" includes a sequence of 3 or more amino acids, where
Amino acids are naturally occurring or synthetic (non-naturally occurring) amino acids.
Peptidomimetics include peptides with one or more of the following modifications:   1.1 or more peptidyl-C (O) NR-chains (bonds) are non-peptidyl chains,
For example -CH₂-Cardomate chain (-CH₂OC (O) NR-), phosphonate chain, -CH₂ -Sulfonamide (-CH₂−S (O)₂NR-) chain, urea (-NHC (O) NH-) chain, -CH ₂ -Secondary amine chain or alkylated peptidyl chain (-C (O) NR-)
Where R is C₁-C_FourA peptide which is substituted with (which is alkyl);   2. N-terminal is -NRR₁Group, -NRC (O) R group, -NRC (O) OR group, -NRS (O)₂R group, -NHC (
O) NHR group (where R and R₁Is hydrogen or C₁-C_FourAlkyl, with the proviso that R and R₁Is both
Not both hydrogen and hydrogen));   3. C-terminal is -C (O) R₂(Where R₂Is C₁-C_FourSelected from the group consisting of alkoxy
), And -NR₃R_Four(Where R₃And R_FourIs hydrogen and C₁-C_FourA group consisting of alkyl
Independently selected).

The naturally occurring amino acid residues in peptides are IUPAC-IUB Biochemical N
Shortened as recommended by the omenclature Commission as follows: Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile.
Or I; methionine is Met or M; norleucine is NIe; valine is Vat or V; serine is Ser or S; proline is Pro or P; threonine is Thr or T Alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine is Gln or Q; asparagine is Asn or N; lysine is Lys or K; asparagine Acid is Asp or D; Glutamic acid is Glu or E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; Glycine is Gly or G; and X Is any amino acid. Other naturally occurring amino acids include, for example, 4-hydroxypurine, 5-hydroxylysine and the like.

[0008] Synthetic or non-naturally occurring amino acids refer to amino acids that are not naturally occurring in vivo, but can be incorporated into the peptide structures described herein. The resulting “synthetic peptide” is 20 at one, two or more positions of the peptide.
Contains amino acids other than the naturally occurring genetically encoded amino acids. For example, naphthylalanine can be replaced with tryptophan to facilitate synthesis. Other synthetic amino acids that can be substituted in the peptide are L-hydroxypropyl, L
-3,4-dihydroxyphenylalanyl, α-amino acids such as L-α-hydroxylysyl and D-α-methylalanyl, L-α-methylalanyl, β-amino acids, and isoquinolyl. D amino acids and non-naturally occurring synthetic amino acids can also be incorporated into the peptide. Other derivatives include the replacement of the naturally occurring side chain of the 20 genetically encoded amino acids (or any L or D amino acid) with another side chain.

As used herein, the term “conservative amino acid substitution” refers to the following 5
Defined herein as an exchange within one of a group of species: I. Small aliphatic, non-polar or slightly polar residues: Ala, Ser, Thr, Pro, Gly; II. Polar negatively charged residues and their amides; Asp, Asn, Glu, Gln; III. Polar positively charged residues: His, Arg, Lys; IV. Large aliphatic non-polar residues: Met, Leu, Ile, Val, Cys; Large aromatic residues: Phe, Tyr, Trp.

As used herein, the term “purified” and like terms are substantially free of the contaminants normally associated with a molecule or compound in its native or natural environment. Isolation of a molecule or compound of

As used herein, the term “biologically active fragment” or “bioactive fragment” of a CBP86 polypeptide specifically binds to at least one natural ligand of the native CBP86 potipeptide. Can include natural or synthetic portions of SEQ ID NO: 2.

“Operably linked” refers to the juxtaposition in which the components are formed to perform their useful function. Thus, a control sequence or promoter operably linked to the coding sequence can provide for expression of the coding sequence.

As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers such as phosphate buffered saline, water and emulsions such as oil / water or water / oil. Includes emulsions and various types of wetting agents. SUMMARY OF THE INVENTION The present invention is directed to the isolation and characterization of a novel testis and sperm-specific calcium binding protein, CBP86, which is post-meiotically expressed and located in sperm flagella. This protein exhibits increased tyrosine phosphorylation during in vitro capacitation and increased calcium binding isoforms during capacitation. Specific Description of the Invention : Almost 50 years have passed since the independent discovery of capacitation by Chang and Austin, but the molecular functions that explain this method are not yet fully understood. The study of CBP86 provides features that add to the understanding of the molecular phenomena associated with capacitation in flagella. The observation that a novel calcium-binding protein (CBP86) is present in the sperm tail through the entire length of the main part associated with the fibrous sheath adds another possible molecular component to the calcium signaling pathway that is active during hyperactivation To do. CBP86 is involved in calcium sequestration and temporary opening and, therefore, can play a direct role in flagellar movement.

As reported herein, the 86 kDa ⁴⁵ Ca-binding isoform of CBP86 is composed of subunits. Their isoforms are elevated during in vitro capacitation, and dephosphorylation completely abolishes the calcium binding capacity and assembly of the 86 kDa isoforms. Those observations point to a role for capacitation-dependent phosphorylation in calcium signaling. Although the time course for capacitation in vitro varies from species to species, the median time for capacitation of human sperm in vitro is 3 hours. This time elapsed is CB
Similar to the course observed for P86 phosphorylation and assembly, oligomerization of CBP86 to its calcium-bound form is a capacitation-related phenomenon that requires tyrosine phosphorylation, and is required for this process. The time required leads to the hypothesis underlying the temporary requirements for capacitation and hyperactivation.

Furthermore, Northern and dot blot analysis of multiple panels of tissues define CBP86 in the category of sperm and testis-specific proteins. Immunohistochemical analysis of human testis reveals that the CBP86 gene becomes translated following meiosis, and that protein is only present in sperm cells, which migrate to the flagella during the final step of spermatogenesis I showed that. Tissue specificity of CBP86 targets calcium sequestration and signaling in sperm, in contrast to calmodulin, which sequesters sperm Ca ²⁺ but is present in many somatic cell types It can provide a unique opportunity. Furthermore, protein localization post-meiotic patterns and tissue specificity of gene expression was determined by antagonists of CBP86 during spermatogenesis.
Due to its potential to act selectively, we show that CBP86 should be considered as a candidate for targeted male contraception.

Accordingly, the present invention is directed to therapeutic and diagnostic methods and compositions based on CBP86 proteins and nucleic acids. Antagonists of CBP86 function can be used to prevent vertebrate sperm capacitation and, therefore, can be used as contraceptives.
Furthermore, antibodies to the CBP86 protein can be used for the diagnosis of conditions or diseases characterized by expression or overexpression of CBP86, or in assays for monitoring patients treated with CBP86 agonists, antagonists or inhibitors.

In one aspect, the invention is directed to a purified polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that differs from SEQ ID NO: 2 by one or more conservative amino acid substitutions. . More preferably, the purified polypeptide comprises an amino acid sequence that differs from SEQ ID NO: 2 by 20 or less conservative amino acid substitutions, and more preferably by 10 or less conservative amino acid substitutions. . On the other hand, the polypeptide comprises an amino acid sequence which differs from SEQ ID NO: 1 by 1 to 5 alterations, wherein said alterations are independently selected from a single amino acid deletion, insertion or substitution.

One aspect of the present invention includes a truncated version of the polypeptide of SEQ ID NO: 2, wherein said polypeptide is at positions 343, 583, 631 and 652, respectively.
Translated from one of several other start codons located downstream from the first start codon. For example, the polypeptide may be the sequence of SEQ ID NO: 2, 3, 4, 5 or 6, or one or more conservative amino acid substitutions, more preferably 10 or less conservative amino acid substitutions. , 5 or 6 which comprises an amino acid sequence which is different.

The present invention also comprises various spliced forms of the CBP86 protein as shown in FIG. In particular, the invention is directed to a polypeptide comprising the sequence of SEQ ID NO: 15, or an amino acid sequence that differs from SEQ ID NO: 15 by one or more conservative amino acid substitutions. In another embodiment, the polypeptide comprises the sequence of SEQ ID NO: 16, or an amino acid sequence that differs from SEQ ID NO: 16 by one or more conservative amino acid substitutions.

The CBP86 protein also contains many binding motifs. Three of the six known motif catapases characteristic of the P-type ATPase cation transport superfamily are found in CR-A of CBP86: LKTLLEGISR (SEQ ID NO: 7); VSDNTGQEESGENSV (SEQ ID NO: 8). SGTSVKSSSGP (SEQ ID NO: 9); The N-terminus of CR-A contains the following three motifs among the four possible motifs that make up the SH3 domain: NQFAAAYFQEL (SEQ ID NO: 10); VEKWSEGTTP (SEQ ID NO: 10) 11); KTTQFPSVYAVPG (SEQ ID NO: 12); In addition, computer analysis found the following two (5 and 6) motifs of the eight possible progesterone receptor motifs: PSSPPPTAVSPEFAYVP (SEQ ID NO: 13); AEATALLSDTSLKGQPE (SEQ ID NO: 14); In one aspect, the present invention provides a method for screening for an agent, small molecule or protein that interacts with CBP86. The present invention screens small molecules, compounds, recombinant proteins, peptides, nucleic acids, antibodies, etc. that bind to CBP86 or modulate its activity, and thus are useful as therapeutic or diagnostic markers for fertility. In vivo and in vitro assays to

In one embodiment, the CBP86 polypeptide or bioactive fragment thereof is used to isolate a ligand that binds to the CBP86 polypeptide under physiological conditions. The method comprises contacting CBP86 with a mixture of compounds under physiological conditions, removing unbound and non-specifically bound material, and isolating compounds that remain bound to the CBP86 peptide. Comprising steps. Typically, the CBP86 polypeptide will be attached to a solid support using standard techniques to allow rapid screening of compounds. The solid support is selected from any surface that has been used to immobilize biological compounds, and includes but is not limited to polystyrene, agarose, silica or nitrocellulose. In one embodiment, the solid surface comprises functionalized silica or agarose beads. Screening for such compounds can be accomplished using libraries of pharmaceutical agents and standard techniques known to those of skill in the art.

The present invention also includes nucleic acid sequences that encode CBP86 polypeptides, and bioactive fragments and derivatives thereof. In particular, the present invention is directed to an amino acid sequence comprising the sequence of SEQ ID NO: 1, or a fragment thereof. In one aspect,
Provided is a purified nucleic acid comprising at least 8 contiguous nucleotides (ie, a hybridizable portion) that is identical to the 8 contiguous nucleotides of any of SEQ ID NO: 1. In other embodiments, the nucleic acid comprises at least 25 (consecutive) nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides or 500 nucleotides of SEQ ID NO: 1.
In one embodiment, the nucleic acid sequence comprises a 350 bp nucleic acid sequence that is identical to the contiguous 350 bp sequence of SEQ ID NO: 1. In another embodiment, the nucleic acid sequence comprises the sequence of SEQ ID NO: 25 or 26.

The present invention also includes a nucleic acid (under the conditions defined herein), or a complement thereof, to which all of the nucleotide sequence represented by SEQ ID NO: 1 hybridizes. The hybridizing portion of the hybridizing nucleic acid is typically at least 15 (eg 20, 25, 30 or 50) nucleotides in length. Hybridizing nucleic acids of the type described herein can be used, for example, as cloning probes, primers (eg PCR primers) or diagnostic primers. It is postulated that the DNA sequence of SEQ ID NO: 1 or a fragment thereof may be used as a probe to detect additional members of CBP86 and to detect homologous genes from other vertebrate tumors.

The stability of a nucleic acid overlap or hybrid depends on whether the nucleic acid overlap is a component of single-stranded DNA
It is expressed as the melting temperature or Tm, which is the temperature at which it separates into. This melting temperature is used to define the stringent conditions required. Typically, a 1% mismatch results in a 1 ° C decrease in Tm, and the temperature of the final wash in the hybridization reaction is consequently reduced (eg, two sequences show greater than 95% identity). If yes, the final wash temperature is 5 ° C, lowered from Tm). As a practical matter,
The change in Tm can be 0.5 ° C to 1.5 ° C per 1% mismatch.

The present invention provides a nucleic acid sequence of SEQ ID NO: 1 and its sequence under stringent or high stringent conditions (
(Or fragment) is directed to a nucleic acid sequence that hybridizes to. According to the present invention, high stringency conditions are defined as carrying out hybridization and washing conditions with a Tm not lower than -5 ° C. Stringent conditions are 5 x SSC / 5 x Denhardt
Hybridization at 68 ° C in 3'S solution / 1.0% SDS and 3 x SSC / 0.1% SDS
Includes a wash at 42 ° C with DS. Additional guidance on such conditions can be found in Samb
rook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Pre
ss, NY; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular B.
readily available in the industry by Biology, (John Wiley & Sons, NY), Unit 2.10.

In another aspect of the invention, the nucleic acid sequence encoding the CBP86 receptor is
It can be inserted into an expression vector and used to transfect cells in order to enhance the expression of their receptor on target cells. According to one embodiment, the nucleic acid sequence encoding CBP86, or a fragment or derivative thereof, is inserted into a eukaryotic expression vector in such a manner that the gene sequence is operably linked to appropriate regulatory sequences, and CBP86 is Expressed in eukaryotic host cells. Suitable eukaryotic host cells and vectors are known to those skilled in the art. In particular, the nucleic acid sequence encoding CBP86 can be added to cells in vitro or in vivo using delivery mechanisms such as liposomes, virus-based vectors or microinjection. Accordingly, one aspect of the invention is directed to transgenic cell lines containing recombinant genes that express CBP86.

Another aspect of the invention includes an antibody raised against CB86. The antibodies can be formulated with standard carriers and optionally labeled to prepare therapeutic or diagnostic compositions. Antibodies to CBP86 can be generated using methods well known in the art. Such antibodies include polyclonal, monoclonal, chimeric (ie "human adapted" antibodies), single chain (recombinant), Fab fragments, and fragments produced by Fab expression libraries, provided that It is not limited to only. Those antibodies are CB
It can be used as a diagnostic agent for the diagnosis of medical conditions or diseases characterized by the expression or overexpression of P86, or in an assay for monitoring patients treated with CBP86 receptor agonists, antagonists or inhibitors. Antibodies useful for diagnostic purposes may be prepared in the same manner as those described above for therapeutic agents. Antibodies can be used with or without modification, and can be labeled by covalently or noncovalently linking them to a receptor molecule.

According to one aspect, there is provided an antibody that specifically binds to the protein of SEQ ID NO: 2. More specifically, the antibody binds to the amino acid sequence of SEQ ID NO: 15.
On the other hand, the antibody specifically binds to the amino acid sequence of SEQ ID NO: 16. In one preferred embodiment, the antibody is a monoclonal antibody.

The invention also provides antibodies, such as anti-idiotypic antibodies, antagonists and agonists, and compounds or nucleotide structures that inhibit the expression of the CBP86 gene (transcription factor inhibitors, antisense and ribozyme molecules, or genes or regulatory sequences). Substitution structure) or a compound or molecular structure that promotes the expression of CBP86 (for example, an expression structure to which a CBP86 coding sequence is operably linked by an expression control element such as a promoter, a promoter / enhancer, etc.). ..

The present invention also provides compositions that can be placed in contact with sperm cells to inhibit the function of the CBP86 protein (ie, by inhibiting the expression of the CBP86 protein or by interfering with the function of the protein). Also includes. In particular, the composition comprises a peptide fragment of CBP86, or an analogue thereof, that is taken up by sperm cells and competes for binding to CBP86's natural ligand. Such inhibitory peptides can be modified to include fatty acid side chains that aid the peptide in invading the sperm cell membrane. A composition comprising a CBP86 inhibitor can be used to regulate fertility in an individual, and in one embodiment the inhibitor functions as a male contraceptive drug. According to one embodiment, there is provided a composition comprising an 8-15 amino acid sequence that is identical to the 8-15 contiguous amino acid sequence of SEQ ID NO: 2, and a pharmaceutically acceptable carrier. It

The CBP86 protein has a number of protein binding domains, such as the CR of the CBP86 gene.
It contains three SH3 domains located at the 5'end of -A. Furthermore, the 3'end of CR-A and the 5'end of CR-B are relatively proline rich. Both the SH3 domain and the proline-rich extension, referred to herein as the putative dimerization domain, are potential for interaction with other flagellar proteins (eg, AKAP). Provides CBP86 with site. According to one aspect of the invention, CBP86 polypeptides are used in an assay to screen for compounds that interfere with CBP's ability to bind AKAP. The assay comprises one or more potential inhibitors to monitor the ability of the inhibitor to interfere with AKAP binding to a CBP polypeptide and / or the ability of the inhibitor to disrupt the AKAP / CBP complex. Comprising the combination of CRP and AKAP in the presence of. Inhibitors of such binding interactions are utilized as contraceptives because of their ability to prevent sperm cell capacitation.

CBP86 polypeptides and splice derivatives thereof can also be used according to the invention as markers for determining the degree of capacitation of sperm cells present in a sperm sample. The assay is based on the premise that phosphorylation and formation of the 86 kDa isoform of CBP86 correlates with sperm cell capacitation. Therefore, measurement of phosphorylation or oligomerization of CBP86 serves as a marker of capacitation. Example Example 1: CBP86 protein isolation: Materials and Methods: Human Sperm Protein lysis and electrophoresis: the dissolution of the preparation and sperm protein semen sample, as described so far (such Naaby-Hansen. , 1997a) I went. For analytical two-dimensional electrophoresis, the detergent / urea extracted proteins were separated by isoelectric focusing (IEF) on an acrylamide tube gel, followed by a Protean II xi Multi-Cell instrument (Bio-R).
ad, Richmond, CA) or a large format (23 x 23 cm) gel (Investigator 2-D Electrophoresis System, E) that is also used for preparative 2D gel electrophoresis.
They were separated by two-dimensional gel electrophoresis (SDS-PAGE) performed on SA). Electrotransfer to the nitrocellulose membrane and subsequent visualization of the protein by gold staining was achieved as previously described (Naaby-Hansen et al., 1997), and the PVDF membrane (0
Electromigration to a .2 mm pore size, Pierce) was performed using a transfer buffer composition of Matsudaira (1987) (10 mM 3- [cyclohexyl] -1-propanesulfonic acid, 10% methanol, pH 11). Et al., (1993).
Staining the immobilized protein in a solution containing 0.1% Coomassie R250, 40% methanol and 0.1% acetic acid for 1 minute, followed by destaining in a solution of 10% acetic acid and 50% methanol for 3 × 3 minutes. Made clear by

In Vitro Capability Acquisition: Motile sperm were acquired by the swim up method of Bronson and Fusi (1990). Control samples were removed and immediately frozen (-70 ° C) and the remaining sperm were resuspended in one of the following media: Dulbecco's PBS, BWW, BWW + 3 mM b-.
Cyclodextran (Sigma), BWW + b-cyclodextran and 100 μM progesterone, human fallopian tube fluid [HTF] (Irvine) + HAS (30 mg / ml), HTF + HAS + 2,2
0 or 100 μM progesterone, HTF + HAS + 100 μM progesterone + 100, 200
Or 400 μM genestein or daidzein (Akiyama et al., 1987). Capacitation was achieved by incubating the sample at 37 ° C. in 5% CO ₂ with sperm removed at various time points and isolated by centrifugation.

Detection of Calcium Binding Proteins: Calcium binding proteins were demonstrated using a modified ⁴⁵ Ca overlay assay from the assay described by Maruyama et al., (1984). The experiment was repeated 4 times. Briefly, proteins separated by 2-D gel were loaded onto PVDF membrane (Jethmalani
Et al., 1994), and the membranes are washed with wash buffer (10 mM imidazole HCl, 60 mM).
KCl and 5 mM MgCl2, pH 6.8) for 3 x 20 minutes, and room temperature for 30 minutes,
Incubated with ₂ mCi / ml of ⁴⁵ CaCl ₂ in wash buffer. The membrane was rinsed continuously with distilled water for 2 minutes followed by 50% ethanol for 30 seconds and dried on filter paper for 15-20 minutes. The membrane is then dried with warm air from a hair dryer and a phospho-imaging screen (Molecular Dynami
cs) exposed for 10 days. Use of PVDF to shorten the final wash step, and phospho-
The use of imaging detection enhanced that ratio compared to the signal: noise ratio achieved by the method first proposed by Maruyama et al. (1984). Then PVDF
Some of the membranes were stained with Coomassie to place calcium binding proteins within the overall 2-D protein pattern, and other membranes were used for Western blot analysis as described below. Autoradiograms and computerized pattern analysis and densitometry of stained membranes were performed using 2D analyzer software (BioImage 2000).

Generation of gel-purified antiserum to CBP86: Coomassie-stained protein spots of 86 kDa were labeled with 3 of human sperm extract.
One piece was excised from a 1.5 mm thick 2-D SDS-PAGE gel. Gel column in 1 ml of PBS,
It was minced into a slurry and emulsified with an equal volume of complete Freund's adjuvant. 600 μl of this emulsion was injected subdermally into New Zealand white rabbits, followed by subcutaneous booster immunizations of similarly prepared antigens every 2 months with incomplete front adjuvant. Serum was collected 20 days after each booster injection.

Dephosphorylation of sperm proteins: To test the relationship between phosphorylation of the 86-kDa CBP86 form and calcium-binding capacity, sperm from four individuals were capacitated in HTF + albumin for 5 hours,
The sperm were then extracted into NP40 / urea and the extracts pooled. The lysate was split and one aliquot was used with 2 U / ml calf intestinal alkaline phosphatase (B
oehringer Manheim) at 37 ° C. for 0.5 h and the other aliquots remained untreated. Microsequencing of 86kDa Calcium-Binding Tyrosine Phosphorylated Protein: 86kDa Coomassie-stained protein spots, 1.5mm thick 2D SDS
-Dissected from polyacrylamide gel and fragmented into small sections. Proteins were destained in methanol, reduced in 10 mM dithiothreitol and alkylated in 50 mM iodoacetamide in 0.1 M ammonium bicarbonate. After removing the reagents, the gel pieces were washed with 50 mM ammonium bicarbonate,
Incubated overnight at 37 ° C. with 12.5 ng / ml trypsin. The peptide
Extracted from gel pieces into 50% acetonitrile in 5% formic acid and by tandem mass spectrometry and at the University of Virginia Biomolecular Research Facility.
Microsequenced by Edman degradation. The following five peptides were obtained by mass spectrometry: LVVPYGLK (SEQ ID NO: 7); TLLEGISR (SEQ ID NO: 18); TNPSNINQFAAAYFQELTMYR (SEQ ID NO: 19); KYSSVYMEAEATALLSDTSL (SEQ ID NO: 20); GQPEVPAQLLDAEGAI (SEQ ID NO: 21).

The distinction between leucine and isoleucine in the sequence was determined by HPLC-isolated peptides.
Determined by Edman sequencing.

Cloning, Sequencing and Analysis of cDNA: Denatured deoxyinosine-containing sense primer (5′-GGI-CAG-CCI-GAG-GTI
-CCI-GCI-CAA / G-C / TT-3 ') (SEQ ID NO: 22) was added to peptide number 5 (GQPEVPAQ
L; designed from SEQ ID NO: 23) and obtained from GIBCO BRL (Life Technologies, CA). Using this forward primer and adapter primer (API), 3'-
RACE (rapid amplification of cDNA ends) PCR was performed in a 26 μl assay system with 0.25
Forty cycles were performed with ng of human testis Marathon-prepared cDNA (CLONTECH, CA). Thermal cycling was performed in a MJ Research (Watertown, MA) thermal cycler (PTC-200 DNA engine) for 3 minutes at 94 ° C (1 cycle), followed by 94 ° C for 30 seconds, 60 ° C for 1 minute and 68 ° C for 2 minutes. This was done using a program of denaturation in minutes, annealing and extension (40 cycles). The PCR products were separated on a 1.7% NuSieve (FMC, ME) agarose gel and the unique 1.0 kb DNA fragment was reamplified, cloned into the pCR2.1-TOPO vector (Invitrogen, CA), and BioDyeo. Perkin-Elmer using fluorescent dye terminator chemistry and Taq DNA polymerase (Perkin-Elmer, NJ)
Sequencing was performed on an Applied Biosystems DNA sequencer. The 3'side clone is CR-A
And 1001 bp containing all of CR-B. The 5'end of the cDNA was also used as an adapter primer (AP1), and 26 bp downstream from the 5'end of the 1.0 kb 3'side clone.
3 bp antisense 3'gene-specific primer (5'-TTA-TTC-AGC-TG
T-TGA-TTC-CCC-TTC-TGG-TTC-AAT-TTC-TGG-3 ') (SEQ ID NO: 24) was used to amplify by 5'RACE PCR from the same template. A 1530 bp product was obtained and cloned into the pCR2.1-TOPO vector. The 5'clone displayed an untranslated region of 48 bp and an open reading frame of 1479 bp. cDNA clones were sequenced in both directions using vector-derived and insert-specific primers. Nucleotide and amino acid sequence data were collected. Cloning of other spliced forms of transcripts was performed with a 5'-extended λλDR2 human testis cDNA library according to the manufacturer's instructions with full length ³² P-labeled cDNA obtained through the RACE protocol. (Clontech, CA). The purified third plaques were transformed into their plasmid form, plated and grown in LB broth,
The plasmid DNA was then isolated with a Qiagen Mini-Kit column and then sequenced with both plasmid gene-specific primers.

Northern and dot blot analysis: Northern blot containing 2 mg of poly (A) ⁺ RNA from 8 selected human tissues,
And a standardized RNA dot blot containing 89-514 ng of mRNA from 50 different human tissues was obtained from Clontech. Northern blots were probed with ³² P-labeled 1479 bp DNA corresponding to bp 49-1527 of CR-A. Random oligonucleotide priming labeling (Feinberg and Vogelstein, 1983)
Was prepared by. Hybridizations were performed in ExpressHyb solution (Clontech) for 1 hour at 68 ° C., followed by washing 3 × with 2 × SSC, 0.05% SDS at room temperature, and 0.1 × SSC, 0.1% at 50 ° C. Washed twice with SDS. The blot was exposed to X-ray film for 60 hours at -70 ° C with two intensifying screens. Dot plots were probed with the same ³² P-labeled cDNA corresponding to coding region A. Blots of Ex with salmon sperm DNA and human placenta Cot-1 DNA
Hybridization was carried out in a presshHyb solution (Clontech) at 65 ° C. overnight. next,
The blot was washed 3 times at 65 ° C. with 2 × SSC, 1% SDS, followed by 0.1 × SSC,
It was washed twice more at 55 ° C with 0.5% SDS and then exposed to X-ray film at -70 ° C for 18 hours with two intensifying screens.

Human sperm protein reduction and carboxymethylation: Washed sperm samples (Naaby-Hansen et al., 1997) containing 2% NP40.
Extract into 8M urea in 0.36M Tris-HCl (pH 8.6) for 1 hour at 4 ° C. Supernatants were pelleted, washed twice with 80% ethanol (final) and reconstituted in NP40-free urea buffer. Aliquot 1.5 mg protein to 0.36M
Of Tris-HCl, pH 8.6, in 8 M urea, 0.2% EDTA, 119 mM mercaptoethanol at room temperature for 4 hours under nitrogen in screw cap tubes (Crestfield et al., 1963). Then the mixture was taken up in 1N NaOH,
15 minutes at room temperature with a freshly prepared solution of iodoacetic acid (final concentration of 0.111M)
Processed in the dark. After the reaction, the carboxymethylated protein was washed with ethanol and used for Western analysis. Expression and Purification of Recombinant Protein and Antibody Production: The cDNA encoding CR-A of CBP86 was purified by polymerase chain reaction from human purified Marathon-prepared cDNA (Clontech). The primers were designed to create an NcoI site at the 5'end and a NotI site at the 3'end of the polymerase chain reaction product. The amplified cDNA was cloned into the NcoI-NotI site of the pET286 expression vector (Novagen), and E. The E. coli strain NovaBlue (DE3) was transformed with the plasmid construct. The resulting construct added 6 histidine-tagged residues on the C-terminus of the protein. The expression plasmid construct was sequenced at the 5'and 3'ends,
The reading frame of the structure was confirmed.

A single positive colony was inoculated into 1 L LB broth containing 30 mg / ml kanamycin and grown at 37 degrees until an A ₆₀₀ of 0.6 was reached. Next, recombinant protein expression was carried out using 1.0 mM IPTG (isopropyl-1-thio-bD-garaclopyranoside).
Was induced by the addition of and and growth was continued for an additional 3.0 hours. Cells were pelleted and IX binding buffer containing 0.1% NP40 (Sigma) and 0.1 mg / ml lysozyme (
Resuspended in 20 mM Tris-HCl, pH 7.9, 0.5 M NaCl, 5 mM imidazole) on ice for 30 minutes and sonicated briefly. The insoluble pellet resulting from centrifugation for 15 minutes at 15000 xg was dissolved in 6M urea in 1 x binding buffer for 1 hour on ice. After 15 minutes of re-centrifugation at 1500 × g, the urea soluble fraction was loaded onto a Ni ²⁺ -activated His-binding resin column (Novagen) and recombinant according to the manufacturer's protocol. Protein was added to 300 mM in 1X binding buffer containing 6 M urea.
Was eluted with imidazole. Affinity purified recombinant protein was used for immunization of female Lewis rats (200 μg / rat) in Freund's complete adjuvant. Animals were boosted with 200 μg of recombinant protein in incomplete Freund's adjuvant at 14-day intervals and collected 7 days after boosting individual sera.

Immunoblots: Western blots were prepared using a 1: 3500 diluted solution of rabbit antiserum raised against gel-purified CBP86 antigen and a 1: 2 dilution of rat antiserum corresponding to γCBP86.
This was done using 500 diluted solutions. Sperm proteins phosphorylated on tyrosine residues were treated with 1: 2500 in 10 mM Tris (pH 7.5), 0.1 M NaCl, and 0.05% Tween20.
It was identified by dilution with the horseradish peroxidase-conjugated anti-phosphotyrosine monoclonal antibody RC-20 (Transduction Laboratories) at 37 ° C for 20 minutes at dilution (Ruff-Jamisson et al., 1993).

Diagonal gel: Swim-up purified human sperm cells were placed in Laemmli sample buffer lacking β-mercaptoethanol and containing 2 mM PMSH and 5 mM EDTA.
℃ for 20 minutes, lysed (600 × 10 ⁶ cells / ml), inhibited protease activity.
The supernatant was heated and 50 L / lane was loaded on an SDS-PAGE gradient gel (5-12%) with 5% stacking gel. Then strip the gel (lane)
Cleavage, and some strips in reducing buffer (0.5% (w / v) DTT, 0
Incubated for 45 minutes at 37 ° C in .1% (W / V) SDS, 125 mM Tris, pH 6.8). Reduced and unreduced gel-strips were loaded with 7.5% SDS-PA.
Layered horizontally on top of GE gel and tested for protein. Proteins were transferred to nitrocellulose membranes and probed with anti-rec-CBP86 as described above.

Localization of CBP86 in the seminiferous epithelium of human testis: Testes were obtained from 3 patients undergoing selective orchiectomy. The testis is sliced once with a razor blade and neutral buffered formalin (4% solution (Sigma) for 1 hour,
Impregnated. The tissue was then minced and placed in fresh fixative overnight. Tissues were dehydrated in a graded series of ethanol, cleaned with xylene, and embedded in paraffin. 2.5 μm thick sections were excised, mounted on slides, deparaffinized, rehydrated and permeabilized with 100% methanol. Incubate in blocking solution containing 10% NGS in PBS,
In PBS containing 1% NGS (PBS-NGS), anti-γCBP86 antiserum or pre-immune serum (1
: 200), washed, incubated with FITC-labeled goat anti-rat IgG (1: 400; Jackson Immunoresearch) in PBS-NGS, washed, and Slow Fade (Molecular Probes, Eugene, OR). ) Containing DAPI II counterstain (Vysis, Downers Grove, IL). The sections were observed by epithelial fluorescence microscopy using a Zeiss microscope. Individual blue and green fluorescence images were acquired using a digital camera (Hamamatsu) and Openlab software (Improvis
ion Inc., Boston, MA).

Indirect Immunofluorescence of Human Sperm: For immunofluorescence studies, fresh human sperm were harvested on an intermittent 55% / 80% Percoll gradient and washed 3 times with Hams F-10 medium. Sperm were counted using a hemocytometer and diluted to a concentration of 1 x 10 ⁶ sperm / ml. 20 of sperm suspension
An aliquot of μl was added to wells on slides coated with poly-L-lysine (2 × 10 ⁵ sperm). Slides were dried at 40 ° C and then fixed with methanol for 10 minutes. In some experiments, no fixation was performed and sperm were briefly air dried on slides. After washing with PBS for 3 × 5 minutes, slides were frozen at −70 ° C. for 1 week. All subsequent incubations,
Performed in a humid chamber. Prepare the preparation with PBS containing 0.05% Tween-20 (PBS
-Tw) in 10% normal goat serum (NGS) for 30 minutes. Primary antisera, either rabbit anti-CBP86 antisera or rat anti-recombinant CBP86 and their pre-immune controls were diluted 400-fold with 10% NGS in PBS-tw and incubated at 4 ° C overnight. Incubated with specimen. The slides were then washed with PBS-tw for 3 × 5 minutes and conjugated with a second antibody, goat anti-rabbit IgG FITC (Jackson Im
munoResearch), or goat anti-rat IgG FITC conjugated (Jackson ImmunoRese)
arch) antibody was applied in PBS-tw in 10% NGS at 37 ° C for 1 hour at a dilution of 1: 200. Slides were washed 3x5 minutes with PBS-tw and Slow Fade-Li
The ght Antifade kit (Molecular Probes, Inc.) was used to reduce the rate of bleaching of fluorescein.

Electron Microscopy Positioning: Sperm from 4 donors were pooled and 2 by centrifugation at 550 × g in wash buffer (Ham's F10 nutrient mixture with 3% sucrose (Gibco / BRL)). Washed once. The washed sperm were resuspended in fixative consisting of 4% paraformaldehyde and 0.2% glutaraldehyde in wash buffer for 15 minutes at room temperature. After removing the fixative by centrifugation and washing three times with washing buffer, sperm were washed with 40
Dehydrated with a series of ethanol graded from 100% to 100%. Cells should be treated with Lowicryl K4M (Electron Microscopy Sciences, Ft. Washingt according to the manufacturer's recommendations.
on, PA) and embedded. The blocks were polymerized by UV light for 72 hours at −20 ° C. and 100 nm thick ultrathin sections were cut.

Non-specific sperm-antibody interactions were blocked by incubating the cleavage for 15 minutes at room temperature in undiluted normal goat serum and washing once with wash buffer. 1 rat antiserum and immature serum against rCBP86
Diluted 1:50 in wash buffer containing% normal goat serum, 1% bovine serum albumin and 0.05% Tween20. Lowicryl sections were incubated with diluted anti-rCBP86 or wash buffer alone for 16 hours at 4 ° C. After 4 washes with wash buffer, they were allowed to react for 1.5 h at room temperature with 5 nm gold-conjugated secondary antibody, goat anti-rat IgG (Goldmark Biologicals, Phillipsburg NJ) (1:35 with wash buffer). Diluted). Sections were washed with distilled water and stained with uranyl acetate before being examined by JEOL 100CX electron microscope.

In vitro phosphorylation of recombinant CBP86 by c-Src: Baculovirus-expressed c-Src was isolated from Upstate Biotechnology, Inc. (Lake P
I purchased from Lacid, NY). Recombinant CBP86 was treated with 0, 0.8, 0.16 or 0.03 μg of CBP86 in 50 mM HEPES, pH 7.4, 5 mM MnCl ₂ , 70 nM ATP, 10 Ci [ ³² P] ATP (6000 Ci / mmol
Phosphorylated by c-Src in an in vitro kinase assay incubated for 10 minutes in the presence or absence of 1 unit of c-Src in 50 μl of a reaction mixture containing The reaction was stopped with Laemlli SDS sample buffer and subjected to SDS-PAGE and autoradiography. Results: Identification and characterization of calcium-binding protein (CBP) in human sperm: Maruyama et al. (1983) ⁴⁵ Ca stacking technique with more than 20 calcium-binding protein spots (CBP) from 12.5 kDa to 115 kDa and 3.8. Used on 2-D blots of human sperm proteins to identify in the pI range of ˜5.3. The relative intensity of individual spots, which is an indication of the concentration of binding protein and / or its calcium binding capacity, was determined by computer densitometry. 90% or more of ⁴⁵ Ca was 86 / 4.0, 80.4 / 4.3, 60.5 / 4.2, 55 / 4.9, 55 / 5.25, 2 in 4 replicates.
6.5 / 5.2, 25 / 4.6, 24.7 / 4.75, 16.5 / 3.9, 15.8 / 4.7 and 14.5 / 3.95 MW (kDa) / pI
It was bound by 11 major CBPs that migrate in. The ⁴⁵ Ca lamination method performed at PH6.8 is
Human sperm CBP was not detected in the neutral and basic regions (pH 6.2-8.5) of the IEF / OAGE gel. ^The protein bound to the majority (60%) of ⁴⁵ Ca was 16.5 kDa and 3
It was identified as calmodulin (CaM) based on its electrophoretic migration with a pI of .9.

Migrate at 86 (84-88) kDa / 4.0 (3.9-4.1), 60.5 kDa / 4.2 and 26.5 kDa / 5.2 3
Species prominent calcium binding proteins were excised and CAD mass spectrometric (M
Microsequencing by S). Five internal peptide sequences and 15 N-terminal amino acids were obtained from 60.5 kDa CBP, and the protein was calreticulin (
CRT). The 26.5 kDa CBP has been previously identified as a human sperm surface protein by vector labeling with ¹²⁵ I and has also been detected in human semen. The 6 peptide sequences obtained by MS and the 22 N-terminal amino acids obtained by Edman degradation identified 26.5 kDa CBP as the serum amyloid P-component precursor (SAP). Calcium binding to CaM and SAP is EF-
Within the lateral motif, and the calcium binding of CRT is in a repetitive polyacidic C-terminal domain. The ability of those proteins to bind ⁴⁵ Ca is 2
The sensitivity and specificity of the ⁴⁵ Ca stacking method on -D gel was validated.

The 86 kDa region of the gel containing a series of protein spots that bound ⁴⁵ Ca easily,
It was named as calcium-binding protein 86 [CBP86]. Densitometry of the ⁴⁵ Ca stack showed that CBP86 was the second most intensely stained area on the 2-D image after calmodulin. MS microsequence data from 5 peptides (SEQ ID NOs: 17-21) obtained after tryptic digestion of excised 86 kDa spots matched with any known peptide sequence in any protein or gene bank I didn't. Silver staining showed several isoforms of CBP86 that differed slightly in mass and charge. The acidic isoforms of CBP86 bound calcium more than the more basic isoforms, even though the two differently charged groups of CBP86 showed similar staining with silver nitrate. The acidic CBP86 isoforms are poorly soluble when either of the basic 86 kDa isoforms is dissolved, but those acidic isoforms are only 20 seconds after dissolution in the nonionic surfactant / urea. As it seems to dissolve, it seems to dissolve more easily than the basic isoform.

The CBP86 mutant showed a shift in pI after dephosphorylation: the central dark part of the 86 kDa protein cluster was excised from several preparative 2-D gels and rabbit antiserum was removed. , Raised against gel-purified protein. On a 2-D immunoblot, this antiserum revealed a clear protein spot at 27-38, 38-42, 50-56, and 63-72, which showed an 86 kDa immunogen (and charge heterogeneity, respectively). Cluster). Western blots of sperm proteins lysed in the presence of calf intestinal alkaline phosphatase resulted in substantial elimination of the more acidic 86 kDa immunoreactive isoform, while the more basic isoform persisted. Furthermore, the isoforms in the 38-42 and 50-56 kDa clusters shift to a more basic pI after phosphatase treatment, which is partly due to their phosphorylation of CBP86-type charges. Suggest that there is.

Cloning of CB86 and its alternatively spliced variants: GQPEVPAQ, a modified inosine-containing forward primer designed from peptide no.
L (SEQ ID NO: 23) was used to amplify a 1.0 kb region of cDNA from human testis Marathon-Ready cDNA (Clontech, CA) by 3'-RACE PCR. Contains 48 bp untranslated region,
The 1530 bp 5'-cDNA fragment was amplified in the same manner and the standard 5'-RACE P
CR was used to clone with an antisense 3'reverse primer generated towards the sequence 263 bp downstream from the 5'-end of the 1.0 kb 3'-clone. 2228 bp compound C
The nucleotide sequence for the BP86 cDNA (SEQ ID NO: 1) was obtained by sequencing the two PCR fragments in both directions. This 2228 bp cDNA was the longest CBP86 cDNA obtained. This cDNA was labeled with ³² P and human testis λλDR25
'-Used to screen an extended cDNA library (Clontech, CA).
Phage isolates were digested with restriction endonucleases, grouped according to restriction fragment size and sequenced, some 2228 bp cDNA and Genbank under accession numbers AF295037, AF29038, AF295039, AF320634 and AF007205.
5 other splice variants deposited at

The five splice variants and the 2228 bp CBP86 cDNA are shown in FIG. 1 (VI-VII and X-XI).
Type I) indicated by an asterisk. Analysis of their sequences led to the initial conclusion that the CBP86 sequence was divided into two coding regions, CR-A and CR-B. CR-A has a stop codon TAA at bp 1528-30 that starts at bp49 and ends at bp1527 (codons 1-494) and acts as a positive stop codon for CR-A. CR
-A encodes a predicted protein of 493 amino acids with a mass of 52.8 kDa and a pI of 4.5. The 18 in-frame nucleotides [1531-1546] then separate CR-A from the ATG start codon [1547-1550] of CR-B. CR-B [nucleotides 1547-2145] encodes a peptide that acts as the carboxy terminus on some CBP86 variants. Bp49-51 [clone VI, VII, and XII], bp343-345
Splice variants containing alternative start codons were sequenced with [clone XI], bp583-585 [clone IX] or bp652-654 [clone X]. If the start codon at bp 1528-30 was functional, then those splice variants were labeled with CR-B [clone V
I, VII], the small N-terminal region of CR-A [clone VII], the major part of CR-A [clone VIII and XI] and all the large domains spanning CR-A and B [clone XII]. Included.

To determine whether the splice variant results in a translated product,
Human sperm proteins were reduced and carboxymethylated, separated on 1-D gels and Western blotted with antisera raised against recombinant CR-A. Twelve immunoreactive CBP86 peptides in the apparent mass range of 79-24 were identified. The isoforms at 67, 59 and 51 kDa were the most immunoreactive.
Importantly, from their mass deduced either from coding region A, or from variants containing a deletion of coding regions A or B, or variants with splice bonds into coding region B. Also detected CBP86 protein with high mass. CR
This observation, coupled with the fact that intervening nucleotides between -A and CR-B co-exist, concludes that translational read-out of the UAA translation termination signal at the end of CR-A often exists. Led. This translational read-out explains the 12 translated peptides observed in vivo from the 6 mutant DNAs.

The CBP86 transcript is testis-specific: A ³² P-labeled cDNA probe, corresponding to CR-A, from several tissues was used.
It was used for Northern analysis of mRNA (Figure 2A) and dot blots containing mRNA from 50 different human tissues (Figure 2B). Interestingly, two broad bands of approximately 2.4 and 1.4 kb were shown in testis mRNA (Fig. 2A, lane 4), which shows several CBP86 messages of different sizes, namely above. The findings regarding the cloning and sequencing of 6 DNAs, including the 5 splice variants described, were expressed in human testis. The 2.4 kb transcript detected in pooled human testis mRNA (FIG. 2A) has an untranslated region of approximately 200 bp, and splices of forms I-VI and IX (2228 bp cDNA or VII (2173 bp)). It can be explained by the variant, and the 1.4 bp transcript can be explained by Form VIII (1270 bp) or Form X (1088 bp) An mRNA of about 0.9-1.0 kb is predicted for clone XII. Only the weak message was detected on the overexposed Northern blots, suggesting that clone XII mRNA and the 24 kDa protein are present at relatively lower abundance than other CBP86 mRNAs and proteins. Importantly, the CBP86 transcript was expressed in testis (FIG. 2A, lane 4 and FIG. 2B, spot D1) but not in other human tissues.

Motif analysis of splice variants revealed MAP4, RII dimerization, and extension domains: six of the six sequences sequenced to date, assuming translational read-out of the stop codon that terminates CR-A. Analysis of the deduced amino acid sequence from the CBP86 variant showed 24-74.7 kDa.
Proteins in the following mass range were predicted (Fig. 1). Two of the predicted proteins (V and VI forms; Figure 4) are nearly identical in mass (52.8 and 52.9 kDa). The masses for the 12 putative proteins are several kDa lower than the masses observed for the 12 reduced and blocked CBP86 translated proteins, which indicates that
Suggests that there are some post-translational modifications. Post-several kDa by translational modification
, The number and pattern of apparent masses of CBP86 detected in the reduced and carboxymethylated sperm protein extracts were both the number and mass of proteins predicted from the 6 variants. Corresponding to.

All five tryptic peptides microsequenced by MS from the original 86 kDa spot excised from the 2-D gel were regenerated in the predicted amino acid sequence of CR-A. This finding confirmed that the cDNA originally cloned as a Ca ²⁺ binding protein and corresponding to an 86 kDa protein spot excised from a preparative 2D gel was cloned.

Computer analysis to confirm the functional domain of CBP86 was performed at amino acid 94
It was shown that -493 has 25% identity with amino acids 308-717 of human microtubule associated protein 4 (MAP4). However, its homologous region had neither the microtubule-binding domain of MAP4 nor the 18-mer reactant specificity of the microtubule-binding domain of MAP present in CR-A or B. 98 amino acid extension at the N-terminus of CBP86 (residues 10-10
8) had 30% identity to the testis-specific protein SP17. Importantly, because it is embedded within this domain, it showed no sequence similarity to the regulatory subunit of the type II cAMP-dependent protein kinase. In particular, Val ¹⁰ −L
eu ⁴⁴ had 40% and 57% similarity to the amino acid 7-41 of RIIαα respectively (such Newlon., 1999).

This amino-terminal region of RII contains both the RII dimerization domain and the AKAP binding domain. This region also contains one domain and one SH3 motif with similarity to catapase. P-type ATPases 3 of the 6 known motifs of catapases, which are characteristic for the cation transport superfamily, are CBP8.
Shown in CR-A of 6. A subfamily of this superfamily, like CBP86, is a Ca ²⁺ -pump ATPase with Ca ²⁺ -binding activity.

[0060] Motif analysis was used to screen a set of proteins with weak global homology to CBP86 for those proteins with known interactions with calcium. When analyzed by this means, the third C-terminus of CR-A overlaps, but shows similarity to a cation transporter in a distinct segment, eg 98 residues of Gln ³⁶⁷ -Gly ^463. The region shows 25% identity and 45% conserved homology with β-3 regulatory subunits from L-type voltage-gated calcium channels [Fugu rubripes]; whereas 67 of Ser ⁴²⁸ -Gln ⁴⁹³ . The residue region of 34 shows 34% identity and 42% conserved homology with the Na-Ca + K exchanger [Bos t
aurus]; and the 57 residue region of Gln ³³¹ -Leu ³⁸⁷ showed 19% identity and 50% conserved homology with the central domain of the ion-channel forming colony 1A toxin [E. Stiffness].

The N-terminus of CR-A contained 3 of the 4 possible motifs that make up the SH3 domain. Such an Src homology-3 domain acts as a site for intermolecular protein binding, which interacts with a wide range of signaling and proline-rich sequences on cytoskeletal proteins. Three PXXP consensus motifs, a cognate site for SH3 interaction, are present in CR-A (aa 396-399,
471-474 and 473-476), and three motifs were present in CR-B (aa 211-
214, 214-217, 326-329). The extended helical domain or transmembrane domain was not clear within CR-A or B. However, CBP8
In view of the fact that 6 undergoes oligomerization (see below), it has similarities to the 7 element fingerprints for G-protein coupled receptors.
Four elements, each consisting of 22 amino acids in length, are located at positions 185-206, 204-225.
It should be noted that it is shown in DR-A at 295-316 and 455-476. C
Oligomerization of BP86 can confer function on those elements.

Six potential phosphorylation sites for PKC, two phosphorylation sites for CKII and four tyrosine residues are present at the C-terminus of CBP86 CR-A. Suggest that this region may be regulated by phosphorylation. Interestingly, the C-terminal domain encompassing the two cataptase sites was deleted in clones VII, XI and XII, which indicates that full-length CBP86 was associated with their splice variants. Suggest that they are functionally different.

Further computational analysis found motifs of 2 (5 and 6) of the 8 possible progesterone receptor motifs. The region encompassing residues 17-102 is the helix domain 9, 10, 11 and 1 of the progesterone receptor binding domain.
2 shared 20% identity. 63% of the residues in this region were either identical to, or conservatively replaced by, the progesterone receptor binding domain. A potential N-linked glycosylation site (residues 50, 109 and 237) and two potential O-glycosylation sites (residues 258-261; 467-468) were also found in CBP.
It was detected within CR-A of 86 sequences. The 5'region of CR-B is rich in proline,
And it contains two prolymplets, and overall CR-B contains three cysteine residues.

The BLAST study showed that the best single alignment was 40% similarity (25% identity) between ORF-B 225-329 and the proline-rich extended glycoprotein found in the plant cell wall. The evaluation was shown (Keller and Lamb, 1989). The extension is a member of the hydroxyproline-rich glycoprotein family (HRGP), and CR-B
In includes a characteristic pentapeptide repeats Ser-Pro _4, which may be represented by the modified Ser-Pro ₃ domain at aa212-215 (Chen and Varner, 1985) . Interestingly, Ser-Pro ₃ motif similar is present in CR-A at position 155-158.

Western analysis with antiserum to recombinant CBP86 shows protein polymorphism and oligomerization: The cDNA sequence encoding CBP86 ORF-A was cloned into the bacterial expression vector pET28b and NovaBlue (DE3) cells. Introduced in. Recombinant protein, Ni ²⁺ −
Purified by immobilized metal affinity chromatography using Sepharose. Subsequently, purified antisera to rCBP86 were raised in female rats. Nonspecific rat antisera to rCBP86, like the gel-purified rabbit antisera to CBP86, also recognized multiple protein spots on the 2D Western blot of human purified protein. The immunoreactive species have the following five sizes, based on size:
Migrated in major groups of species: 1) 27-38kDa; 2) 38-47kDa; 3) 50
-56kDa; 4) 63-72kDa; 5) 81-87kDa. The discovery of a similar pattern of CBP86 isoforms on 2D gels probed with antisera directed against both gel-purified and recombinant CBP86 revealed that other splice variants identified as cDNA during cloning were comparable. It was confirmed that CBP86 was expressed at the protein level resulting in heterogeneity. As a further pool of rat and rabbit antisera specificity for CBP86, immunoblots of purified recombinants were probed with the two antisera. Both antisera are indicative of oligomerization of recombinant proteins. The same MW form of the recombinant protein was recognized, including high molecular weight complexes above 140 kDa.

The relationship between CBP86 isoforms is shown on a 1-D Western blot of SDS extracts of human sperm electrophoresed under reducing conditions, where CBP8 at 31, 43 and 72 kDa.
Three major immunoreactive forms of 6 were shown along with a few minor antigenic bands at 51 and 90-102 kDa. Western blots of unreduced samples were observed on the reduced gel along with prominent immunoreactive bands at 64 and 86 kDa and lower immunoreactive bands at 34 kDa, 45 kDa, 76 kDa. Same amount of 31
, 43 and 72 kDa species, and several high molecular weight forms. The discovery of an additional CBP86 form on unreduced gel is used for S-S cross-linking or heterodimerization between the LMW CBP86 form and an unknown partner protein, i.e. 2-D gel electrophoresis. The presence of a complex composed of a low molecular weight form, which was stabilized by interactions that were not sufficiently dissociated by the relatively weak dissolution method, was shown. Further evidence for CBP86 oligomerization is that only one major high molecular weight [HMW] complex reduces human sperm protein dissolved in 0.2% DOC and 1% NP40 in the absence of reducing agent. Shown when detected on an immunoblot obtained from a native 1-D PAGE gel that was not run.

Laemmli extract of sperm was analyzed by 1D SDS-PAGE in the non-reduced one dimension, and then the immunoblot diagnostic gel in the reduced two dimensional fractions of several high molecular weight CBP86 species. Showed disaggregation. The running of the protein at 86 kDa on the unreduced gel was shown in the reducing dimension to be composed of 43 kDa monomer. Similarly, a 76 kDa protein (predominantly 72 kDa on unreduced gel)
Migrating above the protein) appeared to be composed of 43 and 31 kDa monomers, and a 64 kDa protein was composed of 31 kDa monomers.
The 43 kDa and 31 kDa subunits did not dissociate in the reducing dimension and migrated with the same mass in the reduced and unreduced 1-D gels. From their immunoblots of diagnostic gels it can be concluded that the two major CBP86 forms running on gels reduced at 31 and 43 kDa are involved in the HMW complex by both homodimerization and heterodimerization.

The 86-kDa Form of CBP86 Enhances Capacitation: Comparison of capacitated spermatozoa in vitro with extracts from freshly ejaculated human sperm for 5 hours shows that capacitation follows capacitation. It showed a substantial increase in the amount of the 86 kDa CBP86 isoform that could be formed. Furthermore, CBP86 type groups 2 and 3 (MW38-4
Acidic proteins from 2 and 50-56 kDa) were also more prominent in the capacitated sperm, eg the phosphorylated form of Group 2 shown previously.

Localization of CBP86 in seminiferous epithelium: Immunofluorescent localization of CBP86 in human testis using an antibody against recombinant CBP86 is an indication of post-meiotic pattern of expression of CBP86, seminiferous epithelium And staining of round and elongated spermatids in testicular sperm within the lumen of tubules. The staining pattern showed a gradual transfer of CBP86 protein from diffuse cytoplasmic localization in round spermatids to the posterior pole of early spermatids and then to the flagella as a tail formed. Testes from 3 patients showed the same localization pattern.

Localization of CBP86 to the main part of mature human sperm flagellum by immunofluorescence and immuno-electron microscopy: Antibodies raised against rCBP86 were ejaculated by indirect immunofluorescence microscopy with methanol. Recognizing the full length of the fixed sperm body, both the mid and end pieces showed a weaker staining pattern. CBP86 immunofluorescence was not shown in human sperm heads in their uncapacitated sperm. Importantly, immunofluorescent staining was not observed on viable motile sperm, suggesting that the CBP86 epitope was inaccessible on the plasma membrane. A similar staining pattern was achieved with antisera raised against gel-cleaved CBP86.

When the distribution of CBP86 in freshly purified human sperm was examined by electron microscopy immunocytochemical staining, gold particles were distributed over the surface of the elongated columns and over the fibrous sheath compartment containing the ribs. A few gold particles were present in the space around the cord. CBP8
6 was not detected in the annulus or mitochondrial sheath, and there was no evidence for CBP86 localization within the dye thread either at the main part or distal to the end of the outer dense fiber. .

CBP86 is Tyrosine Phosphorylated during In Vitro Capacitation: Proteins phosphorylated on tyrosine residues during capacitation are newly ejaculated with sperm 2-D immunoblots. Or by staining with the monoclonal anti-phosphotyrosine antibody RC-20 for 3 or 6 hours. After 3 and 6 hours of in vitro capacitation, a significant increase in tyrosine phosphorylation of AKAP3 (fibrous sheath protein 95) and several sperm proteins including the 64 and 86 kDa forms of CBP86 was observed. Following 3 hours of capacitation, the major acid tyrosine phosphorylated component was a 64 kDa protein. However, after 6 hours of capacitation, the strength of the 64 kDa protein was reduced, and the predominant tyrosine phosphorylation in that region was the 86 kDa form of CBP86. Furthermore, the 53 kDa protein showed weak tyrosine phosphorylation after 3 hours of capacitation, and a further increase in phosphorylation of this CBP86 group was observed during the following 3 hours.

The 86-kDa form of tyrosine phosphorylation of CBP86 varied with the composition of the capacitation medium. The 86 kDa form of tyrosine phosphorylation of CBP86 in human oviduct fluid + albumin was higher than that observed in Dulbecco's PBS. Interestingly, addition of 100 μM progesterone to capacitation medium containing HTF + albumin further enhanced phosphorylation of the most acidic CBP86 isoform. 86kDa CBP86
Capacitance-induced tyrosine phosphorylation of isoforms was inhibited in a concentration-dependent manner by treatment with a tyrosine kinase inhibitor, genistein, but at similar concentrations, i.e., soybean zein.
Had an inhibitory effect on the phosphorylation of CBP86, but not on PSP95 (AKAP3). As further proof that CBP86 could act as a substrate for tyrosine kinase, recombinant CBP86 was phosphorylated using an in vitro assay with purified baculovirus-expressed c-Src. An increase in tyrosine phosphorylation of CBP86 is autophosphorylated, as expected, with a constant amount of c
-Shown as the concentration increased in the presence of Src. The data show that the acidic tyrosine phosphorylated 86kDa form of CBP86 immunoreacts with anti-rCBP86, and that the acidic immunoreactive form of CBP86 after capacitation in the presence of human oviduct fluid and progesterone. I confirmed it was there.

[0074] Comparison of the ⁴⁵ Ca-binding to normal and alkaline phosphatase-treated sperm protein, ⁴⁵ Ca is (phosphorylated) acidic CBP86 in sperm extract not treated were readily bind to isoform However, in the extract treated with alkaline phosphatase prior to electrophoresis, the 86 kDa form of CBP86 was found to completely abolish the calcium-binding capacity, whereas calcium binding to calreticulin was unaffected. A comparison of immunoreactive forms of CBP86 present before and after treatment with alkaline phosphatase showed that alkaline phosphatase reduced the presence of the acidic 86 kDa isoform. Taken together, those observations are CB
We show that both the 86 kDa ⁴⁵ Ca binding capacity and assembly of P86 are dependent on phosphorylation. Discussion: CBP86 is a highly polymorphic protein derived from alternatively spliced messages and translational readthrough: 6 cDNAs for CBP86 have been cloned and sequenced, where 5 The species is a splice variant that lacks the domain of coding region A or B. On the Northern blots, broad bands of 2,4 and 1.4 bp were shown, representing polymorphic CBP86 mRNA. Immunoblots of reduced and carboxymethylated sperm protein extracts on 1-D gels with antiserum to recombinant CBP86 showed 24-79 kDa CB
Twelve translated forms of P86 are shown. The four CBP86 peptides are larger than the predicted molecular weight (about 53 kDa) from CR-A or a splice variant, eg, part of CR-B (forms VI-VIII and X-XII in Figure 1). Has been found, CR-A
Problems arise with the efficiency of the UAA translational stop signal at the ends of.

The efficiency of translation termination is sensitive to the 5'and 3'sequences flanking the stop codon. A strong bias in the relative frequency of the 3'-flanking bases was observed in highly expressed E. coli.
Found in the E. coli gene. For UAA, the 3'-flanking base preference is U >>G>A> C, and for UGA it is U >>A>G> C (Bra
wn et al., 1990). The affinity of open factors for stop codons is probably enhanced by specific interactions with the 3'base (Pedersen et al., 1991). Furthermore, although the last 5 amino acids in proteins are generally not important for protein function, the last 2 amino acids have a synergistic major effect on translation termination (Bjornsson et al., 1996). Regarding the two amino acid residues, their acidic / basic and hydrophobic / hydrophilic properties are important for termination efficiency. For UAA, one preferred amino acid is E. coli. In the E. coli gene, basic residues,
Ricin (Brown et al., 1990). In UGA, the two neutral amino acids provide an effective stop if they have a hydrophilic side chain, and an ineffective stop if the side chain is hydrophobic. Adjacent to UAA in CBP86
Examination of the 3'and 5'sequences revealed that the 3'adjacent nucleotides (G instead of the preferred U) and the two amino acids on the 5'side (hydrophilic residues alanine instead of hydrophilic residues). And a single amino acid on the 5'side (which is an acidic residue, ie glutamic acid instead of a preferred basic residue such as lysine), effectively stops translation of CBP86 in CR-A. I showed that I don't like it.

Thus, CR-A or splice variants (eg VI, VII, VIII, X, XI and XII
One explanation for the presence of CBP86 peptides larger than those peptides deduced from the form) assumes translational readthrough of the UAA stop signal encoded by nucleotides 1528-30. Stop codon suppression in mammalian cells is associated with the translation of proteins of different sizes from viral genomes, such as HIV and MLV (Hatfield et al., 1992), and the Drosophila kelch gene (
Well-proven in Robinson and Cooley, 1997), CBP86 appears to be the first example of a translational readthrough of a normal human gene. To date, translational readthrough has been shown in fibroblasts of patients with aspartylglucosamineuria having a mutation that creates an immature stop codon in the second exon of the aspartylglucosaminidase [AGA] gene. The identification of normal endogenous human genes that show translational readthrough can lead to a better understanding of both spermatogenesis and HIV susceptibility.

Relationship between the different forms of CBP86: Immunoblots of gels carried out under various conditions showed an oligomerization of CBP86 and a relationship between the high and low molecular weight forms. When the native gel was run and immunoblotted, CBP86 migrated as one major high molecular weight complex. On blots of unreduced 1-D SDS-PAGE gels, more than 10 CBP86 bands of different immunoreactivity were resolved, for example, prominent bands at 86, 72, 64, 43 and 31 kDa. . Reduced, but unblocked sperm extract on 1-D SDS-PAGE gels showed CBP86 immunoreactive forms at 72, 51, 43 and 31 kDa, and on unreduced gel. The 86kDa and 64kDa that are present in are no longer present. This indicated that the 86-kDa form of the calcium-binding form and the 64-kDa form consisted of mercaptoethanol-sensitive subunits. Immunoblot analysis of diagnostic gels ran in the non-reduced dimension, and then the reduced dimension showed subunit relationships. One 43kDa immunoreactive subunit was observed when the 86kDa form of CBP86 was reduced, which indicates that the 85kDa form is a homodimer of the protein that moves at 43kDa, or between this CBP86 form and an unknown partner. It consists of a heterodimer. Similarly, the 31 kDa immunoreactive form of CBP86 was cleaved from the reduction of the 64 kDa form, which shows additional homodimerization.

On Western blots of 2-D IEF-PAGE gels, five major immunoreactive groups were shown: 1) 27-38 kDa; 2) 38-42 kDa: 3) 50-56 kDa; 4 ) 63
-72 kDa; and 5) 81-87 kDa, where the individual groups show considerable charge heterogeneity in the overall range of pI4-6. The masses deduced from the CBP86 cDNA are reduced and different from the apparent mass of the immunoreactive CBP86 form on gels of sperm proteins that are carboxymethylated, thus affecting both charge and mass. Post-translational modifications of proteins encoded by the body are probably present. Gel-purified 86 kDa spots and antibodies raised against rCBP86 showed similar immunoreactive groups of proteins on 2-D gels, confirming the heterogeneous nature of the relevant isoforms. , And other cloned and sequenced spliced testis mRNAs provide the translated CBP86 protein.

Calcium Binds Elevated Acidic 86 kDa Isoforms of CBP86 After In Vitro Capacitation: Of the ⁴⁵ Ca-binding human sperm proteins, only calmodulin is 86 kPa of CBP86.
It provided a stronger signal than the phosphorylated isoform of Da. Although other immunoreactive forms of CBP86 reveal a wide range of masses and charges on 2-D immunoblots, calcium binding has a phosphorylated, oligomerized 86 kDa isoform with approximately 3.8-4.0 pI. Seems to be a unique property to. Computer analysis shows no typical EF-side motif or typical polyacid extension in the CBP86 sequence, and the calcium-binding domain of CBP86 remains unmapped. The oligomerization of the 43 kDa subunit, shown by diagnostic gel analysis, is probably associated with the acquisition of calcium binding capacity of the 86 kDa complex.

Elevated amounts of the 86 kDa acidic isoform of CBP86, and phosphorylated members of other forms of CBP86 (groups 2 and 3) were observed after in vitro capacitation. Since the 86-kDa CBP86 isoform consists of a 43-kDa immunoreactive subunit, this increase in the 86-kDa isoform is, to the knowledge of the present inventor, the first human sperm protein that undergoes oligomerization during capacitation. Provide proof.

Tyrosine phosphorylation of the acidic 86 kDa form of CBP86 occurs during in vitro fertility acquisition: Enhanced tyrosine phosphorylation of the acidic 86 kDa and 64 kDa isoforms of CBP86 is
It occurred during fertility acquisition. The 64 kDa form of CBP86 was significantly phosphorylated 3 hours after capacitation. Six hours of capacitation was required for the 86 kDa isoform, which is highly phosphorylated. The acidic isoform of CBP86 (pI of 4.0) is the bound form of ⁴⁵ Ca, and calcium binding was not observed in that area if the sample was dephosphorylated prior to electrophoretic separation. It is noteworthy that calreticulin acts as a positive control in this experiment. Because the sperm extract did not impair the calcium binding capacity after treatment with alkaline phosphatase prior to electrophoretic separation. Interestingly, after protein transfer, treatment of PVDF membrane with alkaline phosphatase resulted in 86 kD
It did not completely destroy the calcium binding properties of the CBP86 complex of a. This suggests that phosphorylation is not essential for calcium binding once the 86 kDa complex is formed.

Taken together, the observations point to a model involving the continuous phosphorylation, assembly and acquisition of the 86 kDa form of CBP86 during capacitation. CBP86 provides the first example of calcium binding to human sperm proteins that are tyrosine phosphorylated during capacitation. The characterization of CBP86 is a protein component of the fibrous sheath and has been shown to be phosphorylated during in vitro capacitation. AKAP4 (originally called AKAP82 or Fsc1 in mouse) and AKAP3 (originally AKAP95T, FSP)
95 or AKAP110) is provided with a third protein substrate.

Motif at CBP86: CBP86 has a putative motif for self-assembly and progesterone and AKAP binding, and it has extended homology. Three of the four possible motifs of the SH3 fingerprint are present at the N-terminus of CBP86, and their three SH3 domains are conserved in I, VI, VII, VIII and XII forms, and cloned
II and XI carry one SH3 domain. Therefore, the eight predicted CBP86 forms have at least one SH3 domain. The SH3 domain is present in many proteins and appears to act as a protein binding structure and may be involved in the linking signal transmitted from the cell surface by protein tyrosine kinases. The crystal structure of several SH3 domains has been determined, and the SH3 domain interacts with a cognate proline-rich region containing the consensus sequence Pro-X-X-Pro (PXXP) to allow the partner protein to interact. The views that facilitate binding to are revealed. The three SH3 domains are located at the 5'end of CR-A of the CBP86 gene, and the 3'end of CR-A and the 5'end of CR-B are relatively proline rich. Three PXXP consensus motifs are present in CR-A (aa396-399,471-
474 and 473-476), and three motifs are present in CR-B (aa529-532,532).
-535,643-646), and provides structural modules to illustrate CBP86 self-assembly. Both the SH3 domain and the proline-rich extension, referred to herein as the putative dimerization domain, are also associated with other flagellar proteins (
For example, it provides CBP86 with a potential site for interaction with AKAP).

CBP86 with similarity to sperm protein SP17 (Richardson et al., 1994)
The domain in CR-A includes amino acids 10-108. A region of 40% identity and 57% similarity to the type II regulatory chain of the cAMP-dependent protein kinase is embedded within this SP17 domain at amino acids 10-44. This homologous region of the mouse and human RII subunits is N-terminally located between amino acids 7-41 and contains both the dimerization domain and the AKAP binding region (Newlon et al., 1999). The dimerization domain is the site responsible for the interaction between the individual RII subunits, and the required AKAP binding region dimerized for assembly is the A-kinase anchor protein (AKAP). Mediate the binding of. The region of RII required for high affinity binding to AKAP is an extended hydrophobic face, a dimerization motif present in another type of signaling molecule, S100 protein S100B
And within the X-type 4-helix bundle dimerization motif with calcyclin. By anchoring PK-A to specific areas within the cell, AKAP directs and directs PK-A action. The RII subunit of PK-A consists of a fibrous sheath, AKAP3 and A
It has been shown to bind to two components of KAP4. AKAP3 was localized to the ribs of the fibrous sheath in human sperm, and mouse AKAP4 was localized to the ribs and elongated columns. The presence of the RII regulatory chain dimerization domain in CBP86 suggests that this region of CBP86 can interact with AKAP3 and / or AKAP4, which contribute to the supramolecular structure of the fibrous sheath. It also shows that CBP86 is phosphorylated by PK-A.

CR-B is an interesting line with an extended family of hydroxyproline-rich glycoproteins (HRGPs) expressed in specific subpopulations in plant roots that appear to strengthen the cell wall against mechanical pressure. Including alignment. CBP86 includes two Ser-Pro ₃ similar to Ser-Pro ₄ repeats characterizing the extension. In terms of its structural similarity to elongation, localization of CBP86 throughout the entire body plays a functional role in the elongation of the cytoskeleton-structure of the fibrous sheath to withstand the opportunistic forces of microtubule slides. Suggest.

Analysis of the CBP86 sequence revealed that 2 out of 8 possible progesterone receptor motifs
The species motif was detected. Residues 17-102 share 20.0% identity with the helix domains 9, 10, 11 and 12 of the progesterone receptor binding domain, and a local alignment shows that 63% of the residues in this region are identical or It was shown to be either a conservative substitution. Helix domains 11 and 12 are directly involved in ligand binding. This observation is noteworthy due to the oligomerization of CBP86 and the potential for assembly of the CBP86 progesterone domain. CBP8 in the sperm tail
For the localization of 6, either progesterone receptor bound to it is
It can resemble isoform C, which lacks the highly conserved DNA-binding domain, so that perfect compatibility with known progesterone receptor fingerprints is not expected. It should be noted that two progesterone receptor motifs are present in the other spliced domain of CR-A, so that forms with and without this motif are possible. . Progesterone is
It induces hyperactivation in human sperm and capacitation of the acrosome reaction. By stimulating a well-characterized receptor that appears to be distinct from the nuclear progesterone receptor, progesterone causes elevated levels of free intracellular calcium in human sperm, followed by phosphatidylinositol 4,5-biphosphate. It induced a rapid influx of extracellular calcium leading to hydrolysis. Progesterone-induced calcium fluctuations in human sperm are characterized by an early, transient peak followed by a sustained plateau phase lasting several minutes. Both of these effects depend on extracellular calcium as they are not present in calcium-free media. Progesterone has also been shown to promote tyrosine phosphorylation of human sperm proteins. It should be noted that the addition of progesterone to the in vitro capacitation medium leads to phosphorylation of tyrosine residues on the acidic CBP86 isoform. The progesterone receptor appears as a heterodimer similar to the heteropolymeric complex formed by CBP86 (Nishikawa et al., 19
95).

Example 2: Isolation of mouse CBP86 homologue: A mouse testis λTriplEx cDNA library was screened using human CBP85 cDNA as a probe. Approximately 80% against positive clones, ie human CBP86-2
A 5'truncated, bimodally spliced product with similarities and the same splice sites was obtained (designated clones 1-7). This clone was then used to screen a mouse λEIX II germ library.

The mCBP86 genomic insert was approximately 12 kb in size. The full-length mouse cDNA and genomic sequence of CBP86 remained unknown. For further analysis of the mCBP86 genomic clone, it was necessary to obtain full-length and other spliced cDNA sequences for mCBP86. The mouse λTriplEx cDNA library was rescreened with the clone 1-7 cDNA probe. Ten positive clones were obtained. Restriction map and DNA sequencing analysis of those clones showed that no full-length cDNA was obtained. A comparison of the cDNA sequences of mCBP86 clones 1-7 hCBP86 reveals that about 80% between them.
Showed that there was a similarity.

PCR was performed using Marathon mouse testis cDNA template and designed forward primer and 1
Performed with the -7R3, 1-7R2 'reverse primer and gave a band of the expected size. Two bands on the agarose gel, the forward primer and 1
Appeared in the PCR product system amplified by the -7R2 'reverse primer. The high band followed a band of the expected size, and the low band was similar in size to the band amplified by the forward primer and the 1-7R3 reverse primer.
It makes sense to assume that this band is a PCR product of other spliced sequences similar to hCBP86-5.

Sequencing analysis of the above PCR products was present in the four alternatively spliced products of mCBP86, Gamouse, and they had the same splice sites as their corresponding hCBP86 mutants. I showed that. A cDNA and amino acid sequence comparison between human and mouse CBP86 is as follows: The cDNA sequence of mouse CBP86 is 78% (CBP86-4) to 81% that of human CBP86.
It had similarities and identities to (CBP86-1 and CBP86-2). Regarding the amino acid sequence, mCBP86-2 had the highest similarity (80%) and identity (76%) with hCBP86; then it was mCBP86-4 (78% similarity and 72%). Identity). mCBP86
-1 had 72% similarity and 66% identity with hCBP86.

2. Deleted region in mCBP86-2 cDNA of mCBP86-1 669 bp (human 590 bp) ~
The 1493 bp (human 1456 bp) had many large gaps compared to the sequence of human CBP86. Splice Site-Before 668 bp (human 589 bp) and Splice Site-
The sequence after 1494 bp (human 1457 bp) is the intermediate region 669 bp (human 590) to 1493b of CBP86.
It was more conserved than p (human 1546 bp).

3. MCBP86 according to Northern blot, library screening and PCR
It is possible that −2 is the most abundant variant of the alternatively spliced product of mCBP86.

4. mCB86-1 is calculated MW48.28kD and PI 4.48 (hCBP86-1 MW52.75, PI4.
51): mCBP86-2 has MW41.25kD and PI6.55 (hCBP86-2 MW41kD, PI8.65); mCBP86-3 has MW23.93, PI6.45 (hCBP86-3 MW23.96, PI 7.68).

5. The sequence comparison of motif SH3, progesterone receptor and CATATPASE is mCBP
It was shown that there was a high similarity between 86 and hCBP86, except for the 61aa-71aa CATATPASE of hCBP86 located on the long gap region of mCBP86.

[Sequence list]

[Brief description of drawings]

FIG. 1 is a schematic of possible translational variants of CBP86. The 12 predicted CBP86 forms are indicated by Roman numerals (I-XII, respectively). The I-V forms are predicted through other start sites and readings between amino acids 493-499. Splice variants VII, VIII and X-XII, indicated by an asterisk, were cloned and sequenced from a cDNA library. Clone VI was first amplified from human testis adapter-ligated cDNA and confirmed by cDNA library cloning. The predicted number of amino acids, pI and molecular weight (MW), and the observed MW calculated from the reduced and carboxymethylated sperm peptides are shown for each form. Individual CBP86 code regions are shown as blocked regions.
The speckled region of variant XI represents a sequence not found in any other CBP86 cDNA sequence. The oblique region of variant IV represents the read region. The splice junctions found in individual variants are numbered, and the contiguous amino acid sequence at the start and end of the splice site is shown below each junction.

FIG. 2A shows Northern blots of multiple tissues where the CBP86 cDNA corresponding to CR-A.
Was radiolabeled with ³² P and hybridized to 2 μg of poly- (A) + mRNA, which shows 2.4 and 1.4 kb messages only in testis RNA. The size of the molecular weight markers is shown on the left, and lanes 1-8 show spleen, thymus, prostate, testis,
Contains poly- (A) + mRNA isolated from ovary, small intestine, colon, lymphocytes. The lower panel of FIG. 2A shows the same blot probed with β-actin cDNA as a positive control. B shows that Northern blots of tissue-mRNA probed with ³² P-labeled CBP86 cDNA showed hybridization only in testis (D1). Standardized (100-500ng) poly- (A) + mRNAs present on the grid were isolated from various tissue sources: A1-8 were complete brain, tonsil, caudate, cerebrum, cerebral cortex, respectively. , Anterior lobe, hippocampus, medulla oblongata; B1-7 are occipital lobe, putamen, substantia nigra,
Temporal lobe, thalamus, hypothalamic nucleus, spinal cord; C1-8 represents heart, aorta, skeletal muscle, colon, bladder, uterus, prostate, stomach; D1-8 represents testis, ovary, respectively
Pancreas, pituitary, adrenal gland, brain gland, salivary gland, mammary gland; E1-8 represents kidney, liver, small intestine, spleen, thymus, peripheral leukocyte, lymph muscle, bone marrow; F1-4 represents appendix, respectively. Represents lung, trachea, placenta; G1-7 represents brain, heart, kidney, liver, spleen, thymus, lung (all fetuses); and H1-8 represents 100 ng of total yeast RN, respectively.
A, 100 ng yeast tRNA, 100 ng E. E. coli rRNA, 100 ng of E. coli. E. coli DNA, 100 ng poly r (
A), 100 ng of Cot1 human DNA, 100 ng of human DNA, 500 ng of human DNA.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 1/21 C12Q 1/02 5/10 G01N 33/15 Z C12Q 1/02 33/50 Z G01N 33 / 15 C12N 15/00 ZNAA 33/50 5/00 A (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC , NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, T, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH , GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN , YU, ZA, ZW (72) Inventor Buar, Sen United States, Georgia 30047, Lilburn, Wood Springs Trace 3220 (72) Inventor Mandar, Alabinda United States, Virginia 22903, Charlottsville, Brandon Avenue 600 , # 32 (72) Inventor Volkovitz, Michael USA, Virginia 22901, Charlottesville, Hydraulic Road 2607 E. (72) Inventor Narby-Hansen, Soren London, England NDA 71 DEE, Grant Claus 70 F Term (reference) 2G045 AA40 CB14 DA12 DA13 DA14 DA36 FB01 4B024 AA01 AA11 CA02 4B063 QA05 QQ08 QR50 QS40 4B065 CA01 BA02 CA02 CA46 4H045 AA10 AA11 AA30 BA10 CA40 EA26 EA50

Claims (15)

[Claims] 1. An amino acid sequence of SEQ ID NO: 2; an amino acid sequence different from SEQ ID NO: 2 by one or more conservative amino acid substitutions;
Or a purified polypeptide comprising an amino acid sequence which differs from SEQ ID NO: 2 by a single mutation which represents a single amino acid deletion, insertion or substitution. 2. A purified or recombinant polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 15 and 16. 3. The polypeptide according to claim 2, wherein the amino acid sequence is SEQ ID NO: 15 or 16. 4. A nucleic acid sequence comprising the sequence of SEQ ID NO: 1, 25 or 26. 5. A nucleic acid sequence that hybridizes under stringent conditions to a fragment of 100 nucleotides of SEQ ID NO: 1. 6. A transgenic host cell comprising the nucleotide sequence of claim 5. 7. A nucleic acid sequence comprising a 25 bp nucleic acid sequence that is identical to the contiguous 25 bp sequence of SEQ ID NO: 1. 8. A method for screening for potential human therapeutic agents, comprising contacting a CBP86 protein with a candidate compound; and whether the candidate compound selectively binds to the CBP86 protein. A method comprising determining. 9. The method of claim 8, wherein the CBP86 protein is expressed on the surface of cells. 10. An antibody that specifically binds to the protein of SEQ ID NO: 2. 11. An antibody that specifically binds to the protein of SEQ ID NO: 15. 12. An antibody that specifically binds to the protein of SEQ ID NO: 16. 13. A method for detecting a compound that inhibits capacitation, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 15 and 16. Combining the polypeptide and AKAP under conditions that allow specific binding of AKAP to said polypeptide; contacting said polypeptide with a potential inhibitor; and of AKAP bound to said polypeptide A method comprising the step of measuring an amount. 14. A method for determining sperm capacitation in a sample comprising sperm cells containing the 86 kDa isoform of CBP86, comprising the step of measuring phosphorylation of said 86 kDa isoform. How to be. 15. A method for determining sperm capacitation in a sample comprising sperm cells containing the 86-kDa isoform of CBP86, which comprises the 86-kDa isoform of CBP86 relative to other isoforms of CBP86. A method comprising the step of measuring formation.