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WO1996005305A1 - Utilisation de sequences peptidiques zrk pour la contraception - Google Patents

Utilisation de sequences peptidiques zrk pour la contraception Download PDF

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Publication number
WO1996005305A1
WO1996005305A1 PCT/US1995/010166 US9510166W WO9605305A1 WO 1996005305 A1 WO1996005305 A1 WO 1996005305A1 US 9510166 W US9510166 W US 9510166W WO 9605305 A1 WO9605305 A1 WO 9605305A1
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Prior art keywords
human
amino acids
seq
polypeptide
protein
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PCT/US1995/010166
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English (en)
Inventor
Patricia M. Saling
Deborah J. Burks
Lisette Leyton
Carl A. Dowds
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Duke University
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Priority to AU33628/95A priority Critical patent/AU3362895A/en
Publication of WO1996005305A1 publication Critical patent/WO1996005305A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/689—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to pregnancy or the gonads
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general

Definitions

  • Novel contraceptive strategies that are targeted to the sperm or eggs specifically, in the absence of any endocrine effect, could provide an effective means of fertility control, and may provide a variety of practical advantages.
  • An initial stumbling block to this approach is the identification of the appropriate gamete components to target. Ideal gamete components are those found only in sperm and/or eggs, those which are accessible to manipulation, and those which are essential to the fertilizing function of the gamete.
  • a method of inhibiting fertilization of human eggs comprises the step of: contacting human eggs with a polypeptide, said polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO:2 to human eggs.
  • polypeptide in another embodiment, consists of between eight and one hundred sixty-two consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, and wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO: 2 to human eggs.
  • a method is provided for screening agents for those which are candidate agents for inhibition of human egg fertilization.
  • the method comprises the steps of: contacting a first substance with a second substance in the presence and absence of an agent to be tested; wherein the first substance is selected from the group consisting of: (a) human eggs, (b) isolated zona pellucida, and (c) ZP3, and the second substance is selected from the group consisting of: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 2, (b) a polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO: 2 to human eggs, and (c) a fusion protein comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said fusion protein inhibits the binding of a human protein of SEQ ID NO:2 to human eggs; determining the amount of the second substance bound to the first substance in the presence and absence of the agent to be tested, an agent which inhibits the binding of the second substance to said first substance being
  • a method for testing agents for those which are candidate agents for inhibition of human egg fertilization.
  • the method comprises the steps of: contacting an agent to be tested with a polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO:2 to human eggs; determining whether the agent to be tested binds to said polypeptide, an agent which binds to said polypeptide being a candidate agent for inhibition of human egg fertilization.
  • a method of immunizing a human to achieve a contraceptive effect is provided.
  • the method comprises the step of: administering to a human a polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2 in an amount effective to induce antibodies which bind a 95 kD human sperm phosphoprotein which comprises the amino acid sequence of SEQ ID NO: 2.
  • a method of passively immunizing a human to achieve a contraceptive effect comprises the step of: administering to a human an antibody which specifically binds to an epitope formed by amino acids 1-161 of a 95 kD human sperm protein kinase, the amino acid sequence of said protein kinase being shown in SEQ ID NO:2.
  • a preparation of antibodies is provided.
  • the antibodies specifically bind to an epitope formed by amino acids 1-161 of a 95 kD human sperm protein kinase, the amino acid sequence of said protein kinase being shown in SEQ ID NO:2, said antibodies not binding to any other human sperm protein.
  • a vaccine preparation comprises: a polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO:2 to human eggs, and a pharmaceutically acceptable carrier or diluent for parenteral administration.
  • a medicament for contraception comprises: a polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO: 2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO:2 to human eggs, and a pharmaceutically acceptable carrier or diluent.
  • a method of producing a vaccine comprises the step of: mixing a polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO: 2 to human eggs, and a pharmaceutically acceptable carrier or diluent for parenteral administration.
  • a method of producing a medicament for contraception comprises the step of: mixing a polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO:2 to human eggs, and a pharmaceutically acceptable carrier or diluent.
  • a method for diagnosis of female infertility comprises the steps of: contacting (a) a body sample of a woman, with (b) a polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO: 2 to human eggs, and determining the presence of antibodies in said body sample which bind to said polypeptide.
  • test kit for determining the presence in a body sample of antibodies to human ZRK.
  • the test kit comprises: a polypeptide bound to a solid support, wherein said polypeptide comprises at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO:2 to human eggs.
  • a method for diagnosing male infertility comprises the steps of: comparing ZRK genes of a man being tested to wild-type ZRK, a mutation in at least one of said genes of said male indicating infertility due to the inability of ZP3 to bind to sperm of said male.
  • a different method for diagnosing male infertility comprises the steps of: contacting sperm of a male being tested with an antibody which specifically binds to an epitope formed by amino acids 1-161 of a 95 kD human sperm protein kinase, the amino acid sequence of said protein kinase being shown in SEQ ID NO:2; and determining binding of said antibody to the sperm of the male being tested, failure of the sperm to bind to said antibody indicating male infertility.
  • the present invention provides the art with a non-hormonal, non- mechanical-barrier means of contraception which prevents an early step in the conception process, preventing the formation of a zygote.
  • Figure 1 Human sperm contain a 95 kD protein that becomes tyrosine- phosphorylated during capacitation and is recognized by the mAb 97.25. Human sperm were obtained from fresh ejaculates that were allowed to liquify at RT for 1 hr and collected by centrifugation (800 xg) through a 47%/90% Percoll gradient. 25 The resulting pellet was washed twice by centrifugation (400 xg, 10 min) in TN (20 mM Tris, 130 mM NaCl, pH 7.4) buffer.
  • TN 20 mM Tris, 130 mM NaCl, pH 7.4
  • Capacitation was achieved by incubating sperm in Ham's F12 medium, supplemented with 20 mM NaHCO 3 and 7.5% serum, at a concentration of 5 x 10 6 cells/ml for 6 hrs at 37°C in 5% CO 2 .
  • Fig. la Non-capacitated (lane 1) or capacitated (lane 2) sperm were solubilized in SDS sample buffer, the proteins separated under reducing conditions on 8% polyacrylamide gels 26 and transferred 27 to nitrocellulose membranes (Schleicher and Schuell, Keene, NH). Non-specific reactivity was blocked by incubation with 3% gelatin (cold water fish skin) in 100 mM NaCl, 50 mM Tris (pH 7.6), 0.05% Tween-20, 0.1 % NaN 3 for 1 hr. The blots were then incubated for 1 hr at RT with anti-phosphotyrosine antibody PY20 (ICN, Irvine, CA) (1 g/ml).
  • PY20 anti-phosphotyrosine antibody
  • PY20 reactivity was visualized autoradiographically using peroxidase-conjugated goat anti-mouse IgG (KPL, Rockville, MD) followed by enhanced chemiluminescence detected (ECL Western Blot Kit, Amersham International).
  • ECL Western Blot Kit ECL Western Blot Kit, Amersham International.
  • the reactive bands appear specific for PY since use of PY20 that was preincubated with 40 mM o- phospho-DL-tyrosine eliminated immunoreactivity with the same sperm samples (data not shown).
  • Each lane contains 2 x 10 6 sperm.
  • Pre-stained molecular weight markers x 10 '3 ; BRL were used to estimate relative molecular mass, and are indicated to the left of lane A.
  • Fig. lb Proteins from capacitated human sperm were extracted (10 6 cells/ l) in RIPA buffer (50 mM Tris HC1, pH 7.5, 150 mM NaCl, 1 % Nonidet P-40, 1 % deoxycholate, 0.1 % SDS) 28 in the presence of 500 KlU/ml aprotinin, 1 mM PMSF, and 500 ⁇ M Na 3 VO 4 . Extracted proteins were separated from insoluble material by centrifugation and then incubated overnight at 4°C with PY20 conjugated directly to Sepharose beads (10 mg IgG/ml beads; ICN).
  • Figure 2(A) Deduced amino acid sequence for hu9.
  • An unamplified ⁇ gtl l human testis library (Clontech, Palo Alto, CA) was screened separately with PY20 and with mAb 97.25 as described. 29"31 The library was plated to achieve a plaque density of - 50,000 pfu/150 mm dish. Following IPTG induction, plaques were transferred to nitrocellulose filters (Schleicher & Schuel) and incubated with antibody; both PY20 and mAb 97.25 were used at ⁇ l ⁇ g/ml, the former as purified IgG and the latter as tissue culture supernatant. Positive clones were detected using an 125 I-labeled second antibody.
  • nucleotide sequence has been deposited in Genbank (accession umber L08961). Predicted sequences are indicated as follows: bold type, signal sequence; 34 underline, potential glycosylation sites; double underline, putative transmembrane domain; 18 shading, invariant (or highly conserved) PTK sequences; 13 and underlined Y, potential autophosphorylation sites. 13
  • Fig. 2(B) Predicted structure of Hu9.
  • SP signal peptide
  • EC extracellular domain
  • TM transmembrane region
  • A potential autophosphorylation sites. Numbering indicates amino acid positions.
  • Fig. 2(C) Comparison of Hu9 catalytic domain with that of c-Eyk. Sequence comparison was performed using FASTA. 33 Identities are denoted by shading.
  • RNA was isolated from the human tissues indicated using the guanidium monothiocyanate/LiCl method. 35 Ten ⁇ g of each sample were subjected to electrophoresis in 1 % agarose containing formaldehyde, transferred to Nytran membranes, and probed with radiolabeled hu9 cDNA. 32 The gel was stained with ethidium bromide to ascertain equal loading and transfer of the RNA samples. Hybridization was in 6X SSC, 50% formamide, 50 ⁇ g/ml denatured salmon sperm DNA, and 0.1X Denhardt's at 48°C.
  • RNA marker RNA
  • T testis
  • L lung
  • Li liver
  • K kidney
  • S spleen
  • B brain
  • O ovary
  • Fig. 3(b) Cellular localization of hu9 mRNA by in situ hybridization. Frozen tissue sections were prepared using testis collected from mature mice. Following fixation in 4% paraforrnaldehyde and delipidation, tissue sections were treated with acetic anhydride. Slides were prehybridized for 1 hr at 55 °C.
  • 35 S- riboprobes were transcribed from hu9 plasmid DNA using T3 (sense strand) and T7 polymerase (antisense strand). Probes were subsequently hydrolyzed to yield a final probe length of 200 base pairs. Slides were hybridized with either the sense or antisense probe overnight at 55°C in 50% formamide, 10% dextran sulfate, 4X SSC, IX Denhardt's, 0.5 mg/ml salmon sperm DNA and 10 mM DTT. Following treatment with RNAse A, slides were subjected to a stringent washing procedure including a final 30 min wash in 0.1X SSC, 10 mM EDTA at 65 °C.
  • Fig. 3b The testis section in Fig. 3b was hybridized with the antisense probe.
  • Hu9 RNA transcripts detected with the antisense probe are exclusively localized within the seminiferous tubules.
  • the density and cellular distribution of the grains observed with the antisense probe suggest postmeiotic expression of hu9.
  • Fig. 4 An antibody generated using a synthetic peptide corresponding to residues 539-553 of the deduced sequence encoded by hu9 recognizes a 95 kD tyrosine-phosphorylated protein in human sperm.
  • the KLH-conjugated peptide was used to immunize a rabbit, and resulted in an ELISA titer of 1: 125,000 against the peptide and 1:5,000 against KLH. Proteins from capacitated human sperm were reduced and separated on SDS gels, transferred to nitrocellulose and probed with pre-immune serum (1 :200 dilution; lane A), anti-Hu9 539 .
  • Lanes B-D represent the same nitrocellulose sheet which has been stripped between treatments and subsequently re-probed in the order presented.
  • Fig. 5 Competitive hemi-zona assay identifies peptide sequences involved in human sperm-zona pellucida interaction. Excess unfertilized human eggs, which had not been exposed to sperm and would normally have been discarded, were snap frozen in Ham's F-12 medium containing 7.5-10% human serum with liquid nitrogen and maintained at ⁇ -70°C. At the time of use, vials were thawed rapidly in a 37°C bath and eggs removed directly into PBS containing 2 mM EGTA and 5% serum at 4°C. Oocytes were completely non-viable following this procedure.
  • Zona pellucida were bisected with a micro- scalpel, and matched zp halves were used subsequently. Following bisection, zp were washed (x3) and each half- zp placed pair-wise in matched 40 ⁇ l drops of Ham's F-12 medium containing 7.5% serum. For each zp used, one half served as an internal control to which results from the other half were compared: Half A was preincubated for 30 min at 37°C with Ham's F-12 containing 7.5% serum, whereas Half B was preincubated with either Ham's F-12 containing 7.5 % serum (control samples) or with peptides 1 , 2, or 3 to achieve a final concentration of 10 ⁇ M.
  • Capacitated human sperm were added to each drop (10 6 cells/ml, final cone). After 30 min, each half-zp was recovered, washed gently to remove sperm that were not tightly bound, fixed in 2% glutaraldehyde. examined with phase contrast optics and the number of sperm bound to each half-zp determined. Sperm binding levels are expressed as percentages, with the number of sperm bound to zp half B normalized to those bound to zp half A. Data were analyzed by ANOVA and paired t-test using PSPLOT software. Each treatment was evaluated using 9-10 independent hemi-zp pairs. Pepl corresponds to residues 57-71; pep2, to residues 152-166; and pep3, to residues 94-105. All peptides were made as 1 mM stocks in Ham's F-12. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
  • ZRK zona receptor kinase
  • human ZRK is a sperm protein that is fundamental to fertilization serving two essential functions in gamete interaction: it is responsible for specific recognition of the egg and it is responsible for stimulating acrosomal exocytosis in sperm. Fertilization will not occur if defects in either of these events occur.
  • Polypeptides derived from the sequence predicted by the hu9 cDNA have been found to inhibit binding of ZRK to the zona pellucida, the extracellular matrix of eggs. These polypeptides are particularly useful for contraception because the polypeptide is specific to sperm, is not expressed in women, and is involved in two inter- related and essential events of the fertilization process. Moreover, the polypeptides of the invention are derived from a domain of ZRK which bears no homology with any other reported protein. The polypeptides disclosed herein, as well as other similar polypeptides which can be found using the screening method described herein, are thus suitable for use as contraceptive vaccines and contraceptive medicaments.
  • antibodies specific for the epitopes of ZRK corresponding to the disclosed polypeptides can be used as a means of passive immunization against sperm.
  • DNA encoding the polypeptides of the invention can be used to produce the polypeptides, either in recombinant host cells, or in women who have been "vaccinated” with the DNA. (See Wolff et al., Hum. Mol. Genet.
  • polypeptides comprise at least eight and preferably at least ten, twelve, or fourteen consecutive amino acids of amino acids 1-161 of SEQ ID NO: 2, i.e. , the amino terminal domain of ZRK.
  • the peptide inhibits the binding of a human protein of SEQ ID NO:2, i.e. , ZRK, to human eggs.
  • Particularly useful segments of this sequence for forming polypeptides according to the invention are amino acids 40-151, and more particularly useful are amino acids 57-71 and amino acids 94-105.
  • candidate polypeptides for inhibition of human egg fertilization are identified by contacting isolated zona pellucida with ZRK, a protein having an amino acid sequence as shown in SEQ ID O:2, in the presence and absence of the candidate polypeptide. The amount of binding of ZRK to the zona pellucida is determined.
  • a candidate polypeptide for contraceptive activity is identified if the polypeptide significantly inhibits the binding of ZRK to human zona pellucida.
  • the inhibition is greater than 50% of the control binding, and more preferably greater than 65% or 75%.
  • the polypeptide of the invention may be covalently attached to another substance, such as keyhole limpet hemocyanin (KLH) in order to stimulate a larger immune response.
  • KLH keyhole limpet hemocyanin
  • the polypeptide may also contain repeated units of active portions of ZRK, in order to stimulate a larger response than a single unit provides.
  • DNA encoding the polypeptides of the invention can be used to produce the polypeptides in recombinant host cells. Techniques for producing expression vectors and transfectants are well known in the art. Suitable host cells are also known and can be selected according to the desired purpose and cell characteristics. While any DNA which encodes the polypeptides can be used, one may desire to use the nucleotide sequence as it occurs in humans. The human sequence is shown in SEQ ID NO: l. Antisense constructs can also be used in order to inhibit the expression of ZRK in males. Production of antisense constructs are well known in the art. It may also be desirable to use the nucleotide sequence encoding a suitable peptide according to the invention for "vaccination".
  • DNA encoding a particular polypeptide is administered to a recipient and the recipient expresses the protein, against which the recipient also has an immune response.
  • immunizations are typically intramuscular, but may also be intraperitoneal, intravenous, or subcutaneous.
  • Immunization with polypeptide vaccines is also contemplated by the present invention. Such immunizations may be intramuscular, intraperitoneal, intravenous or subcutaneous. Polypeptide vaccines are typically formulated in a pharmaceutically acceptable carrier or diluent for parenteral administration.
  • polypeptides of the invention may also be used as a medicament rather than as a vaccine. That is, the polypeptides can be used as a contraceptive medicament which is administered at or near the time of intercourse.
  • the polypeptides thus function as active competitors of the fertilization reaction.
  • Medicaments for such uses are typically formulated from a pharmaceutically acceptable carrier or diluent for vaginal delivery.
  • the polypeptides of the present invention can also be used diagnostically. They can be used to screen women for certain anti-sperm immune responses which cause or contribute to infertility or low fertility.
  • the polypeptide of the invention is coated on a solid support, such as a microtiter plate.
  • a body sample, such as serum, from the woman being tested is applied to the solid support coated with polypeptide. If there are anti-ZRK antibodies in the body sample they will bind to the polypeptide.
  • Such antibodies can be detected, e.g. , using anti-human immunoglobulin antibodies which are detectably labeled.
  • kits which can be conveniently used by physicians or clinical laboratories.
  • the kit may contain a solid support, such as a plate or beads, which has been pre-coated with polypeptide of the invention.
  • Anti-human immunoglobulin antibodies labeled or not, may also be included.
  • Instructions for carrying out the assay and calibration curves may also be included.
  • male causes of infertility may also be detected according to the findings of the present invention.
  • Sperm can be screened using antibodies which specifically bind to the polypeptides of the invention.sperm which fail to bind to such antibodies would indicate a mutation in or affecting ZRK.
  • the ZRK gene of a man can be examined to determine whether a mutation has occurred which affects ZRK expression or function. Mutations can be determined by comparison to the wild-type sequence shown in SEQ ID NO: 1.
  • first substance is selected from the group consisting of: (a) human eggs, (b) isolated zona pellucidae, and (c) ZP3
  • second substance is selected from the group consisting of: (a) a protein having an amino acid sequence as shown in SEQ ID NO: 2, (b) a polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO:2 to human eggs, and (c) a fusion protein comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said fusion protein inhibits the binding of a human protein of SEQ ID NO: 2 to human eggs.
  • the amount of the second substance bound to the first substance in the presence and absence of the agent to be tested is determined.
  • An agent which inhibits the binding of the second substance to said first substance is identified as a candidate agent for inhibition of human egg fertilization.
  • Techniques for determining binding of one protein to another are well known in the art and any such technique as is convenient may be used.
  • an agent to be tested can be contacted with a polypeptide comprising at least eight consecutive amino acids of amino acids 1-161 of SEQ ID NO:2, wherein said polypeptide inhibits the binding of a human protein of SEQ ID NO:2 to human eggs.
  • the binding of the agent to be tested to said polypeptide is then determined.
  • An agent which binds to said polypeptide is identified as a candidate agent for inhibition of human egg fertilization. Examples Example 1
  • This example demonstrates the existence of a human sperm protein that becomes tyrosine-phosphorylated during capacitation.
  • Regulated exocytosis of the sperm acrosome also called the acrosome reaction (AR)
  • AR acrosome reaction
  • Tyrosine phosphorylation in sperm appears important for fertilization, since inhibition of protein tyrosine kinase (PTK) activity prevents acrosomal exocytosis, 5 and blocks fertilization.
  • PTK protein tyrosine kinase
  • this receptor is a member of the PTK receptor family: i) it contains phosphotyrosine (PY), the level of which increases upon exposure to zp proteins, 3 and ii) PTK activity is stimulated by direct exposure of the isolated receptor to ligand. 5 Further analogy with PTK receptors includes the finding that ZP3 receptor aggregation is an initiating signal in the cascade leading to acrosomal exocytosis. 6 Given these characteristics, we referred to this mouse 95 kD protein as zona receptor kinase (ZRK).
  • ZRK zona receptor kinase
  • This example shows the sequence of a human tyrosine kinase cloned from testis DNA.
  • the predicted amino acid sequence of hu9 contains motifs found in all PTKs 13 (Fig. 2), including the highly conserved ATP-binding site (GEGEKG, residues 249-254) and highly conserved sequences from the eleven major catalytic subdomains of these molecules.
  • Two potential autophosphorylation sites lie within 20 residues upstream of the Ala-Pro-lle consensus triplet in subdomain VIII of Hu9.
  • Hu9 has little homology to other PTKs in subdomains X and XI, which are the least conserved catalytic domains.
  • 13 Subdomain X often consisting of approximately 20 residues in other PTKs, 13 is represented by 30 residues in Hu9.
  • Hu9 contains other structural features common to PTK receptors (Fig. 2B), including a sequence of hydrophobic amino acids (residues 162-182) capable of serving as a transmembrane region 18 and an amino terminal extracellular domain that could possess the ligand binding site(s).
  • Fig. 2B PTK receptors
  • Hu9 extracellular domain contains several potential glycosylation sites and is cysteine-rich but the 9 Cys residues present in Hu9 are not arranged in typical clusters, as observed in other PTK receptors. 19
  • Example 3 Example 3
  • This example shows the tissue distribution and cellular localization of hu9 expression.
  • This example shows that the sperm protein encoded by hu9 is not hexokinase.
  • a polyclonal antibody was prepared using a synthetic peptide that corresponds to a unique portion of the intracellular domain of the deduced polypeptide (residues 539-553).
  • This antibody recognized a 95 kD human sperm protein that contains PY (Fig. 4, lanes B, C).
  • the protein that is recognized by anti-Hu9 S39-553 as well as anti-Py antibodies is distinct from hexokinase (Fig. 4, lane D).
  • This example shows that selected portions of the ⁇ «9-encoded polypeptide are able to inhibit the binding of sperm to human zona pellucida.
  • the putative receptor function of the ⁇ wP-encoded polypeptide was addressed in a competitive sperm-zp binding assay, using a hemi-zona assay design.
  • 21'24 Human zp were preincubated with synthetic peptides corresponding to different regions of the predicted extracellular domain and were then challenged with sperm to determine the potential of these sequences to compete with sperm for binding sites (Fig. 5).
  • Two of the peptides tested (pepl [residues 57-71] and pep3 [residues 94-105]) caused significant inhibition of sperm-zp interaction, blocking binding by 69% and 80%, respectively.
  • a third peptide pep2 [residues 152-166] produced no effect on binding levels.
  • hu9 encodes a transmembrane PTK expressed exclusively in sperm and spermatogenic cells of the testis, that antibodies directed against a unique portion of the deduced intracellular domain recognize a 95 kD tyrosine-phosphorylated human sperm protein, and that peptides corresponding to specific regions of the amino terminal domain effectively inhibit human sperm interaction with the zona pellucida.

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Abstract

On décrit des séquences peptidiques qui correspondent au domaine de la terminaison amino d'une tyrosine kinase spécifique du sperme. Ces peptides peuvent bloquer la liaison du sperme humain à la membrane pellucide humaine, la matrice extracellulaire spécifique de l'ovule, et ils bloquent donc l'interaction entre le sperme et l'ovule. Ces peptides s'utilisent dans des stratégies contraceptives.
PCT/US1995/010166 1994-08-11 1995-08-10 Utilisation de sequences peptidiques zrk pour la contraception WO1996005305A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001032008A1 (fr) * 1999-11-04 2001-05-10 Pig Improvement Co (Uk) Ltd. Procedes
US7019114B2 (en) 1998-02-19 2006-03-28 Eastern Virginia Medical School Recombinant, biologically active human zona pellucida protein 3 (HZP3) to test male fertility
US7037663B2 (en) 1998-02-19 2006-05-02 Eastern Virginia Medical School Human zona pellucida protein 3 and uses thereof
US7148021B2 (en) 2001-08-02 2006-12-12 Eastern Virginia Meical School Human zona pellucida proteins and methods of their use in diagnosing male infertility

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7019114B2 (en) 1998-02-19 2006-03-28 Eastern Virginia Medical School Recombinant, biologically active human zona pellucida protein 3 (HZP3) to test male fertility
US7037663B2 (en) 1998-02-19 2006-05-02 Eastern Virginia Medical School Human zona pellucida protein 3 and uses thereof
WO2001032008A1 (fr) * 1999-11-04 2001-05-10 Pig Improvement Co (Uk) Ltd. Procedes
US7148021B2 (en) 2001-08-02 2006-12-12 Eastern Virginia Meical School Human zona pellucida proteins and methods of their use in diagnosing male infertility

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