Molecular cloning and sequencing of cDNA encoding for a novel testis-specific antigen

cDNA encoding for a sperm antigen, designated NZ-1, was cloned and sequenced from murine testis cDNA-λgt11 expression library using antibodies to human sperm surface antigens belonging to 14–18 kD molecular region. These sperm antigens are involved in zona pellucida binding and have tyrosine phyosphorylation activity. Computer generated translation analysis of 1395-bp cDNA yielded an open reading frame (ORF) of 152 aa with first ATG, Met start codon at nt 32 and the stop codon TGA at nt 487. The translated protein has a calculated molecular weight of 17.9 kD and a potential tyrosine phosphorylation site at aa 46–54, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced aa sequence indicated it to be a membrane-anchored peptide with a hydrophobic NH₂-terminus that is characteristic of a signal peptide. Extensive computer search in the GenBank, NBRF, and Swiss sequence banks, indicating it to be a novel protein. Northern blot analysis indicated testis-specific expression of NZ-1 antigen. The NZ-1 cDNA was subcloned into pGEX-1λT vector and expressed in glutathione-S-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with the original antibodies raised against the native 14–18 kD sperm proteins. These findings suggest that the sperm-specific recombinant NZ-1 may find applications in the development of a contraceptive vaccine, and in studying the normal and abnormal sperm function and the signal transduction mechanism. Mol. Reprod. Dev. 48:449–457, 1997. © 1997 Wiley-Liss, Inc.     

Cloning and sequencing of cDNA encoding for a human sperm antigen involved in fertilization

A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-λgt11 expression library by using antibodies to human sperm surface antigens belonging to 14–18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5′ and 139-bp 3′ noncoding regions. The translated protein has a calculated molecular weight of ∼19 kD, and has two casein kinase II (CK-2) sites at aa 94–97 and 149–152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935–76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of ∼20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The ∼20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans. Mol. Reprod. Dev. 51:176–183, 1998. © 1998 Wiley-Liss, Inc.     

Novel human prostate-specific cDNA: molecular cloning,expression, and immunobiology of the recombinant protein

The differential display-polymerase chain reaction technique was employed to obtain a prostate-specific approximately 300-bp cDNA fragment. On screening the human prostate-lambdagt10 library with this fragment, a full-length approximately 1.5-kb cDNA encoding for a prostate antigen, designated as human novel prostate-specific antigen (hNPSA), was found. Extensive database searches revealed that the hNPSA cDNA is a novel sequence. It has an open reading frame (ORF) of 735-bp encoding for 245 amino acids (aa), with a calculated molecular mass of approximately 27kDa. Hydrophilicity analysis of the deduced aa sequence indicated that hNPSA is a membrane-anchored peptide. Analysis for tissue-specificity by Northern blot and RT-PCR-Southern blot procedures indicated that hNPSA is specifically expressed only in human prostate. The hNPSA (ORF) was subcloned into pET22b(+) vector and expressed using the histidine-tagged gene fusion system. The recombinant (r) protein of approximately 27kDa was purified and antibodies (Ab) were raised in rabbits. The rhNPSA Ab recognized a specific protein band of approximately 35kDa in solubilized human prostate tissue and not in any of the other 10 human tissues tested in the Western blot procedure. The hNPSA expression is upregulated 2.5- to 3-fold, both at the mRNA and protein levels in androgen-dependent LNCaP cells, as compared to normal whole prostate tissue. Antisense, but not the sense, phosphothiorate-conjugated oligonucleotides based on the hNPSA cDNA sequence significantly (p<0.001) inhibited proliferation of LNCaP cells in a concentration-dependent manner. Thus, the novel hNPSA, which has prostate-specific expression and seems to be involved in carcinogenesis, may have applications in the specific diagnosis and treatment of prostate cancer.     

Effect of antibodies to sperm-specific recombinant contraceptive vaccinogen (rCV) on murine fertilization: search for an animal model to examine its contraceptive potential

Recently, we cloned and sequenced a sperm-specific antigen, designated as Contraceptive Vaccinogen (rCV), from human testis (Naz et al., 2001). The present study was conducted to examine its proteomic homologue and function in murine sperm, in order to find out whether or not the mouse can provide a suitable model for examining its immunocontraceptive effects. This was examined by using purified antibodies (Ab) raised against the recombinant (r) human CV antigen of approximately 44 kD. In the Western blot procedure, rCV antibodies recognized a specific protein band of approximately 64 +/- 5 kD in murine testis and murine sperm extracts, the band similar to that found in human testis and human sperm. In the immunoprecipitation procedure, rCV Ab immunoprecipitated a protein band of similar size from murine sperm and murine testis extracts. The immunocytochemical (ICT), immunoscanning electronmicroscopic (ISEM) and the immunobead binding technique (IBT) revealed the subcellular localization of CV antigen on the surface of acrosome and tail regions of the noncapacitated and capacitated murine sperm cell. In functional bioassays, rCV Ab inhibited the acrosome reaction as well as sperm-egg binding in vitro. These data indicate that the CV antigen is expressed in murine sperm and has a biological role in sperm function and sperm-egg binding. In vitro inhibition of capacitation/acrosome reaction and sperm-zona binding suggest that the mouse can provide a suitable model to examine the immunocontraceptive effects of CV antigen in actively-immunized animals.     

Cloning and characterization of a novel sperm-associated isoantigen (E-3) with defensin- and lectin-like motifs expressed in rat epididymis

In the present study we report the identification of a novel epididymis-specific secretory glycoprotein, E-3, which is a sperm-associated isoantigen containing defensin- and lectin-like motifs. E-3 was detected in rat epididymal fluid and in sperm extracts by two-dimensional (2-D) Western blotting using rat hyperimmune sera raised against rat sperm. The immunoreactive spot of approximately 28 kDa with an isoelectric point (pI) of 3.5 was cored from silver-stained gels. Microsequencing by tandem mass spectrometry and database searches revealed several peptides to be novel sequences. Degenerate deoxyinosine-containing primers corresponding to the novel peptides were used in rapid amplification of cDNA ends and polymerase chain reaction to clone E-3 from a rat epididymal cDNA library. A 449-base pair nucleotide sequence was subsequently obtained consisting of a complete open reading frame (ORF) of 111 amino acids, which showed similarity to the defensin and lectin families. The first 21 amino acids constituted a putative signal peptide, suggesting that E-3 is a secretory protein. Mature E-3 protein corresponding to amino acids 22-111 was expressed in E. coli, and chickens were immunized with recombinant E-3 (rE-3). The resulting anti-rE-3 antisera recognized the recombinant immunogen as well as a "native" protein of 28 kDa, pI 2.5-3.5 in both epididymal fluid and in sperm extracts on 2-D Western blots. Northern hybridization indicated that E-3 mRNA was present in the epididymis but not in testis or other tissues, and that E-3 mRNA was predominantly expressed in the corpus and cauda of the epididymis, but not in the initial segment or caput. Similarly, Western blots detected the E-3 protein only in the epididymal fluid and sperm from the corpus and caudal regions. Finally, indirect immunofluorescence localized E-3 on the entire tail, and with less intensity on the head of the sperm. These observations indicate that E-3 is a secreted epididymal protein that becomes associated with the sperm as it transits through the corpus and cauda. The presence of a defensin-like motif suggests that E-3 may play a role in protecting the sperm from microbial infections in the epididymis and in the female reproductive tract.     

A novel asparaginase-like protein is a sperm autoantigen in rats

A novel asparaginase-like protein (ALP) of spermatozoa was cloned from rat and human testis cDNA libraries on the basis of reactivity with antibodies produced after vasectomy. Although obstruction of the male reproductive tract is known to cause an immunologic response, few of the sperm antigens responsible for the generation of autoantibodies have been characterized. We are identifying proteins of interest by coring autoantigenic protein spots from two-dimensional (2-D) gels of rat sperm extracts and microsequencing them by mass spectrometry. The peptide sequences from ALP, a 28 kDa, pI 5.7 protein, matched to a single partial length rat EST. These peptide sequences were used to clone a cDNA encoding a novel 333 amino acid open reading frame. The new protein had a similarity to portions of L-asparaginases of plants (43%) and to glycosylasparaginases in animal cells (32%). Human ALP cDNA was subsequently cloned. It showed 77% identity to the rat ALP sequence and the gene, ASRGL1 (asparaginase-like 1), mapped to chromosome locus 11q12.3. Purified recombinant rat ALP (rALP), expressed in E. coli, was used to raise polyclonal antiserum in guinea pigs. Two observations verified that the correct protein had been cloned: 1) the anti-rALP antibody reacted with both rALP and rat sperm; and 2) post-vasectomy sera bound rALP. Anti-rALP antibody stained the midpiece of rat and human sperm coincident with staining by MitoTracker Green FM, suggesting that ALP is associated with the mitochondria. Northern analysis revealed that rat ALP message was abundantly expressed in the testis but was also present in heart, brain, liver, skeletal muscle, and kidney.     

Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination

The mouse monoclonal antibody (mAb) A36 produced by us and shown to induce extensive, "tangled" sperm agglutination was used to isolate cDNAs encoding its cognate antigen. Three overlapping cDNA clones specifically recognized by the mAb were isolated from a human testis cDNA expression library in lambdagt11. Sequencing of these cDNAs yielded the complete nucleotide sequence of a 3-kilobase cDNA that encodes the mAb-related polypeptide, designated sperm antigen-36 (SA-36), composed of 558 deduced amino acids. SA-36 cDNA contained a 5' untranslated region of 234 nucleotides (nt), an open reading frame of 1674 nt, and a 3' untranslated region of 1138 nt. SA-36 cDNA displayed > 99% homology to glucose phosphate isomerase (GPI)/neuroleukin (NLK) mRNA. This surprising homology was confirmed in Western blots demonstrating that mAb A36 reacted specifically with GPI obtained from rabbit muscle and from baker's yeast. Moreover, polyclonal, monospecific antibodies produced against beta-galactosidase/SA-36-3 fusion protein stained human spermatozoa and caused intensive agglutination of these cells in a manner similar to that with the mAb. Taken together, the data presented here demonstrated that mAb A36 cognate sperm surface antigen, encoded by SA-36 cDNA, is a GPI/NLK-like protein involved in sperm agglutination.     

小鼠睾丸特异表达基因TSEG-1的克隆及序列分析

从表达序列标签(expressed sequence tags, ESTs)数据库ZooDDD中获得小鼠正常睾丸表达的EST, 通过dbEST数据库检索出与其高度同源的EST序列, 构建EST叠加群(contigs), Biolign软件拼接, GeneScan软件预测contigs对应的基因组序列中的外显子、内含子; 针对开放阅读框设计引物序列, 采用RT-PCR从小鼠睾丸组织中克隆新基因的cDNA, 分析该基因在小鼠各脏器中的mRNA表达, 并对测序结果进行生物信息学分析。结果表明: 在小鼠X染色体的1 668~2 011 kb间克隆出一新基因TSEG-1, 全长为510 bp, 开放阅读框为336 bp, 编码111氨基酸, 分子量12.84258 kDa, 等电点11.4000。RT-PCR证实该基因开放阅读框正确, 在小鼠睾丸组织中特异性表达, 且与小鼠其他cDNA 无同源性, 获得GenBank 登录号EU079024。功能区分析发现TSEG-1蛋白可能为一种跨膜蛋白, 跨膜区位于第41~61氨基酸残基。TSEG-1基因与人类睾丸特异性组蛋白2a变异体基因有较高同源性, 在TSEG-1基因5′-端非编码侧翼预测发现存在1个启动子区域, 范围为680 bp。 TSEG-1蛋白可能有4个抗原性位点, 2个特异性蛋白激酶的磷酸化位点, 其亚细胞定位可能位于线粒体。小鼠睾丸特异性基因TSEG-1的克隆为进一步研究其生物学功能和表达调控奠定了基础。     

Molecular and developmental studies of a sperm acrosome antigen recognized by HS-63 monoclonal antibody.

A conserved mouse sperm antigen (MSA-63) recognized by a monoclonal antibody (HS-63) was isolated from mouse testes by single-step immunoaffinity chromatography. Isolated MSA-63 preparation was shown to be a group of proteins ranging from 24-84 kDa and with isoelectric points (pIs) ranging from 4.0-6.0 when analyzed by two-dimensional (2-D) gel electrophoresis. Microsequencing techniques were employed to determine the relationships of various protein spots on 2-D gels. Partial amino acid sequences of some protein spots in isolated MSA-63 preparation were shown to be homologous to mouse actins, while others revealed homology only to the SP-10 protein. Rabbit antisera raised against isolated MSA-63 antigen preparation were used to immunoscreen a mouse testis cDNA library. Isolated cDNA clones carrying a 1.2-kb insert were used to obtain nucleotide sequences containing open-reading frames and to deduce the corresponding amino acid sequence of MSA-63. A high degree of homology was observed between MSA-63 and a known human sperm antigen, SP-10, at DNA/protein levels. Amino acid sequences of tryptic peptides derived from protein spots of 24-47 kDa and pIs of 4.2-4.4 were found to be identical to those deduced from isolated cDNA clones. The gene expression of MSA-63 during spermatogenesis in mice was studied using a specific cDNA probe as well as HS-63. It was observed that MSA-63 was not expressed until the postmeiotic stages of spermatogenesis.     

SAMP32, a testis-specific, isoantigenic sperm acrosomal membrane-associated protein

To identify novel human sperm membrane antigens, we analyzed two-dimensional gels of sperm extracts containing hydrophobic proteins that partitioned into Triton X-114. Four protein spots with isoelectric points (pIs) ranging from 4.5 to 5.5 and apparent molecular weights from 32 to 34 kDa were sequenced by mass spectrometry and found to contain common peptide sequences. Cloning the corresponding cDNA revealed that these protein spots were products of a single gene (SAMP32), encoding a protein of 32 kDa with a predicted pI of 4.57. SAMP32 has a potential transmembrane domain in the carboxyl terminus and is phosphorylated in vivo on serine 256. Northern blotting of eight human tissues and RNA dot blotting of 76 human tissues showed that SAMP32 expression was testis specific. SAMP32 contained an amino terminal domain homologous to the major malarial circumsporozoite surface protein and a domain similar to that of Krp1 from Schizosaccharomyces pombe in its carboxyl terminus. The SAMP32 locus consists of seven exons on chromosome 6q15-16.2. Antiserum against recombinant SAMP32 recognized protein spots originally cored from a two-dimensional gel. This antiserum strongly stained the equatorial segment and faintly stained the acrosome cap of ejaculated human spermatozoa by immunofluorescence. Immunoelectron microscopy showed that SAMP32 was associated with the inner acrosomal membrane in the principal and the equatorial segments of the sperm acrosome. By immunostaining enzyme-dissociated testicular cells, SAMP32 was localized to Golgi phase round spermatids and subsequent stages of acrosome biogenesis. Recombinant SAMP32 reacted with serum from an infertile man, suggesting that it is isoantigenic. Antibodies against recombinant SAMP32 inhibited both the binding and the fusion of human sperm to zona-free hamster eggs.     

Identification and Characterization of Putative Receptors for Sperm-Activating Peptide I (SAP-I) in Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus1

We characterized putative receptors specific for sperm-activating peptide I (SAP-I: GFDLNGGGVG) in spermatozoa of the sea urchin Hemicentrotus pulcherrimus, using both binding and crosslinking techniques. Analysis of the data obtained from the equilibrium binding of a radioiodinated SAP-I analogue [GGGY(¹²⁵I)-SAP-I] to H. pulcherrimus spermatozoa showed the presence of two classes of receptors specific for SAP-I in the spermatozoa. The incubation of intact spermatozoa as well as sperm tails or sperm membranes prepared from H. pulcherrimus spermatozoa with GGGY(¹²⁵I)-SAP-I and a chemical crosslinking reagent, disuccinimidyl suberate, resulted in the radiolabelling of a 71 kDa protein. The protein appears to be associated with a 220 kDa wheat germ agglutinin (WGA)-binding protein. A cDNA encoding the 71 kDa protein was isolated from a H. pulcherrimus testis cDNA library. The cDNA was 2443 bp long and an open reading frame predicted a protein of 532 amino acids containing a 30-residue amino-terminal signal peptide, followed by the same sequence as the N-terminal sequence of the 71 kDa protein. The amino acid sequence of the matured 71 kDa protein is strikingly similar to the 77 kDa protein of Strongylocentrotus purpuratus (95.5% identical) and also similar to cysteine rich domain of a human macrophage scavenger receptor. Northern blot analysis demonstrated that mRNA of 2.6 kb encoding the 71 kDa protein was expressed only in the testis.     

Molecular cloning of an acrosomal sperm antigen gene and the production of its recombinant protein for immunocontraceptive vaccine

A monoclonal antibody, HS-63, which reacts specifically with a highly conserved sperm acrosome antigen, was shown to inhibit in vitro fertilization of mouse and human. The corresponding sperm antigen designated as MSA-63 was purified to homogeneity from mouse testes and used as an immunogen to generate polyclonal antisera in rabbits. The cDNA fragments of MSA-63 gene were cloned from mouse testis cDNA library by an immunoscreening method using polyclonal antisera specific for MSA-63. Using the established cDNA clone as a probe, the gene encoding for MSA-63 protein was found to be conserved among different mammalian species. Only one specific mRNA 1.5 kb in size was identified from the adult mouse testis among different mouse tissues. The recombinant fusion protein containing MSA-63 protein fragment was produced in Escherichia coli and used to immunize female mice. Similar to the original HS-63 monoclonal antibody, the antisera thus produced reacted only with the sperm acrosome and revealed significant inhibition to the in vitro fertilization of mouse oocytes. The results of this preliminary study suggest that it is feasible to mass produce sperm-specific antigens or their antigenic fragments by recombinant DNA technology for the development of sperm antigen-based immunocontraceptive vaccines.