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a Department of Immunology, the Bruce Rappaport Faculty of Medicine, Technion, Bat-Galim, Haifa 31096, Israel
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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gt11. 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 ß-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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isojima and associates [8, 9] described a human-mouse hybridoma secreting a sperm-agglutinating and -immobilizing monoclonal antibody (mAb) H6–3C4 directed to a carbohydrate epitope. Monoclonal antibody H6–3C4 recognized glycoprotein antigenic bands displaying molecular masses of 15–25 kDa in Western blots, of both sperm extracts and seminal fluids. Studies by Diekman, Herr, and associates [10] showed that a carbohydrate determinant on a sperm antigen designated by them as SAGA-1 is the target epitope of the sperm-agglutinating mAb S19 [11]. The S19 antibody induced a tangled pattern of agglutination, inhibited sperm penetration of cervical mucus, and affected sperm-zona pellucida binding. The SAGA-1 antigen has been shown to be a hydrophobic, highly acidic sperm glycoprotein, localized on the entire cell surface. Interestingly, the S19 antibody that competed with H6–3C4 binding to sperm antigens [8] reacted with a similar polymorphic set of sperm polypeptides (15–25 kDa). However, S19, unlike H6–3C4, did not react with seminal plasma antigens, thus raising the question whether these two sperm-agglutinating mAbs recognize an identical sperm glycoprotein or whether they are directed against different epitopes of the SAGA-1 antigen.
Recently, we have described [12] a mouse IgG1 antisperm mAb, designated A36, that exhibits a strong agglutinating activity toward motile human sperm. The mAb induced intensive head-to-head, tail-to-tail, and head-to-tail agglutination and recognized antigens located at the acrosomal cap and principal tail regions of noncapacitated human spermatozoa. Indirect immunofluorescent staining and flow cytometric assays revealed changes in both localization and levels of expression of A36-reactive epitope following capacitation and acrosome reaction. In Western blots of human sperm extracts, mAb A36 reacted with a highly polymorphic series of trypsin-sensitive protein bands, exhibiting molecular masses of 83 kDa to less than 29 kDa. Of the multiple polypeptides reactive with the antibody in sperm extracts from humans and from various animal species, a common 53-kDa antigen was identified. Monoclonal antibody A36 did not react, in immunofluorescent staining assays, with various somatic human blood cells and various cell lines including a line of mouse testicular Leydig cells.
In view of the strong sperm-agglutinating capacity of mAb A36, its broad species cross-reactivity, and its lack of reactivity with surface antigens on those somatic cells tested so far, we were interested to further characterize mAb A36 and its cognate sperm antigen. This study included cloning and sequencing of sperm antigen-36 (SA-36) cDNAs from a human testis cDNA expression library using mAb A36 as an immunoprobe.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female BALB\c mice were purchased from the animal breeding facility of the Hadassah Medical School, Jerusalem.
Monoclonal Antibody A36
Production and selection of the sperm-agglutinating mouse IgG1 mAb 2A36AF8 (designated A36) were described in a previous paper [12]. Culturing A36 hybridoma cells in tissue culture produced large quantities of mAb. Positive mouse antiserum to human sperm cells was collected from animals immunized with human sperm cells. Control NSO myeloma culture supernatants or preimmune sera collected prior to inoculation of mice with human sperm cells were used as negative controls. IgG from hybridoma culture fluids and sera were purified by affinity chromatography using Protein G Sepharose 4 Fast Flow (Pharmacia, LKB, Uppsala, Sweden) [13, 14]. Mouse IgG1 mAb to human progenitor cell antigen CD34 (no. 348050; Becton Dickinson, San Jose, CA) served as an isotypic control.
Cloning and Analysis of SA-36 cDNAs
Monoclonal antibody A36 was used as a probe for initial screening of a human testis 5'-stretch plus cDNA expression library in gt11 phage vector (no. HL 3024b; Clontech, Palo Alto, CA). The library was plated at a density 3 x 104 plaque-forming units (pfu) per 150-mm plate with Escherichia coli Y1090 as a host bacterium. After growth at 42°C for 3–4 h, plates were overlaid with dry nitrocellulose filters saturated with 10 mM isopropyl-ß-D-thiogalactoside (IPTG) and incubated at 37°C overnight. Filters were blocked with 4% nonfat dry milk in 10 mM Tris-buffered saline (pH 7.4) containing 0.05% Tween 20 and were screened with mAb A36 (10 µg/ml) at room temperature overnight with constant agitation. Specific antibody binding was detected as previously described [13, 14] using purified polyclonal rabbit anti-mouse immunoglobulins (1.6 µg/ml; Z-259; DakoPatts, Copenhagen, Denmark) as secondary antibodies followed by alkaline phosphatase-conjugated goat anti-rabbit immunoglobulins (Biomakor, Rehovoth, Israel) at a dilution of 1:50 000.
Positive clones were plaque purified by additional plating at 500–800 and then at 100–200 pfu/90-mm plate. Phage cDNAs were isolated from liquid lysates, digested with EcoRI, and electrophoresed on 1% agarose gels. Complementary DNA inserts were cut from gel and isolated using DNA Isolation Kit (Biological Industries, Beit-Haemek, Israel). A 2.5-kilobase (kb) cDNA insert from a mAb A36-positive clone (designated SA-36–3), which was nonreactive with either NSO myeloma IgG or with an isotypic mouse IgG1 mAb control, was digested with Pst1. Of the resulting cDNA fragments, separated by agarose gel electrophoresis, a fragment of 383 base pairs (bp) (SA-36-3-f 1) corresponding to the 5' end of SA-36–3 was purified. Labeling was performed by the random hexamer method [15] using [32P]dCTP (3000 Ci/mmol; NEN, Life Sciences Products, Boston, MA) and a kit from Biological Industries. Labeled probe was used to rescreen the human testis gt11 library as described by Sambrook et al. [16] for clones representing the 5' end of SA-36 cDNA. Isolated clones (SA-36-4 and SA-36-5) were plaque purified and their cDNA inserts isolated.
Isolated cDNA inserts were subcloned into pUC57 plasmid (MBI Fermentas Molecular Biology, Vilnius, Lithuania). Plasmids were subjected to DNA sequencing at the DNA sequencing biological services of the Weizmann Institute (Rehovot, Israel) by automated dye terminator cycle sequencing method with AmpliTaq DNA polymerase. Sequences of both strands were determined using universal primers and oligonucleotide primers designed on the basis of sequencing data obtained with previous primers. Primers were synthesized at Bio-Technology General (Kiriat Weizmann, Rehovot, Israel).
Expression of Recombinant Fusion Proteins
ß-Galactosidase fusion proteins were prepared from gt11 lysogens in E. coli Y1089 as described by Snyder et al. [17]. Briefly, a culture of E. coli Y1089 was infected with selected gt11 clones for 20 min at room temperature and then plated and incubated at 30°C. Lysogens were selected according to their ability to grow at 30°C but not at 42°C.
Protein lysates were prepared from lysogens grown at 30°C with constant shaking and then induced by shifting the temperature to 44°C for 15 min, followed by induction with 2 mM IPTG for 4 h at 37°C. After incubation, cells were centrifuged and quickly suspended in gel electrophoresis sample buffer containing 5% mercaptoethanol and boiled for 4 min.
Generation of Polyclonal Monospecific Antiserum to ß-Galactosidase/SA-36-3 Fusion Protein
ß-Galactosidase/SA-36-3 fusion protein was isolated from E. coli Y1089 cell lysate by gel electrophoresis according to Hager and Burgess [18]. Proteins were separated by 7.5% SDS-PAGE under reducing conditions and stained with 0.25 M KCl; the ß-galactosidase fusion protein band was excised from the gel, crushed, and used for immunization. A group of three female BALB/c mice were injected i.p. and s.c. with 0.5 ml of the gel slurry (50–100 mg of protein) emulsified in complete Freund's adjuvant (Difco Laboratories, Detroit, MI). Intraperitoneal booster injection with the same amount of antigen in incomplete Freund's adjuvant was administered 4 wk and 6 wk after initial immunization. Eight days after the last injection, mice were bled, and their serum was tested for reactivity with ß-galactosidase fusion proteins. Removal of antibodies cross-reactive with ß-galactosidase was performed according to Sambrook et al. [16]. For this purpose, mouse antiserum to ß-galactosidase/SA-36-3 fusion protein was incubated with an acetone extract of E. coli Y1089, expressing ß-galactosidase at a final concentration of 1% (w:v). The suspension was mixed carefully and incubated for 2 h at room temperature with gentle agitation. The powder was removed by centrifugation at 10 000 x g for 10 min at 4°C. Control mouse polyclonal monospecific antibodies to ß-galactosidase prepared against protein expressed by a gt11 clone without insert were treated in the same manner.
Motile Sperm Preparations
Sperm samples were obtained from fertile volunteers with normal semen parameters [13]. After liquefaction for 30 min at 37°C, sperm cells were collected by centrifugation at 450 x g for 10 min and washed twice in Ham's F-10 medium (Biological Industries) supplemented with 1% human serum albumin (HSA). Aliquots of spermatozoa (0.5 ml) were carefully overlaid with 1.5 ml of Ham's F-10 1% HSA medium, and motile sperm cells were allowed to swim up during a 60-min incubation period at 37°C, in 5% CO2, 95% air. Motile spermatozoa were collected, pooled, and washed three times in Ham's F-10 1% HSA medium. Samples were placed on slides, air dried, and subsequently fixed with acetone for 5 min.
Sperm Antigenic Extracts
Human sperm cells obtained from at least 10 individuals were washed twice in Dulbecco's modified PBS (D-PBS; Biological Industries). Cells were subjected to freezing and thawing followed by sonication, in the presence of protease inhibitors (0.05 mM leupeptin, 5 mM benzamidine, 0.03 mM pepstatin A, 1 mM PMSF, and 1 mM EDTA) as previously described [12,13].
Sperm Agglutination Test
The assay was based on the tray agglutination test described by Friberg [5]. Motile sperm cells were diluted with D-PBS to 40 x 106 cells/ml. Sperm suspension (5 µl) was mixed with 20 µl of affinity-purified IgG antibodies (2 mg/ml) or with equal doses of negative and positive IgG controls in 96-well round-bottom microplates. The plates were incubated for 60 min at 37°C and examined under an inverted microscope. Agglutination patterns were observed and recorded according to the criteria previously defined by Rose et al. [6] to reflect head-to-head, tail-to-tail, tail tip-to-tail tip, mixed, and tangled agglutination.
Immunofluorescent Staining
Immunofluorescent staining was performed as previously described [12] using motile human spermatozoa collected by the swim-up technique and acetone fixed.
Electrophoresis and Western Blotting
SDS-PAGE was performed, under reducing conditions, as previously described [13]. Antigens suspended in dissociation buffer were subjected to 4 min of boiling and loaded onto gels at 20 µg/lane. Molecular weight standards were run in parallel. Separated antigens were transferred onto nitrocellulose membranes and assayed by Western blots as previously described [12, 14].
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A human testis 5'-stretch plus cDNA expression library in gt11 phage vector was first screened with the sperm-agglutinating mouse IgG1 mAb designated mAb A36 [12]. Four positive clones were initially isolated and plaque purified. Subsequent immunoscreening and Western blot analysis using purified mAb A36 and the ß-galactosidase fusion proteins produced by these four clones yielded one positive immunoreactive cDNA clone. As demonstrated in Figure 1, the positive cDNA clone, designated SA-36-3, encoded a 122-kDa ß-galactosidase fusion protein that was specifically recognized by purified mAb A36 while being nonreactive with either control NSO myeloma IgG or with an isotypic control of mouse IgG1 mAb to human CD34. The insert of SA-36-3 cDNA, which displayed a size of 2.5 kb by agarose gel electrophoresis, was subcloned into pUC57 plasmid and subjected to nucleotide sequencing. The 2485 bp of SA-36-3 cDNA contained an open reading frame (ORF) of 1347 bp encoding 449 deduced amino acids and a 3' untranslated region of 1138 bp. SA-36-3 cDNA and amino acid sequences were searched for nucleotide and amino acid homologous sequences in GenBank, EMBL, DDBJ, and PDB, SwissProt, SP update, and PIR databases, using the advanced BLAST computer programs of the National Center of Biotechnology Information [19]. It was found that nucleotides (nt) 1–1552 of clone SA-363 displayed 99% homology to bases 343–1894 of Homo sapiens glucose phosphate isomerase (GPI) mRNA/human neuroleukin (NLK) mRNA (accession no. NM 000175 derived from gb K03515) [20]. To obtain the complete sequence of SA-36 mRNA, including its 5' region, SA-36-3 cDNA was subjected to digestion with Pst1 (Fig. 2). This restriction enzyme cleaved SA-36-3 cDNA at nt 383 and 1363, which correspond to nt 725 and 1705 of human GPI/NLK mRNA. The resulting cDNA fragments designated SA-36-3-f 1 (nt 1–383, 383 bp), SA-36-3-f 2 (nt 384–1363, 980 bp), and SA-36-3-f 3 (nt 1364–2485, 1122 bp) were separated by agarose gel electrophoresis. Fragment SA-36-3-f 1 (Fig. 2) at the 5' end of SA-36-3 was purified and used to prepare a labeled probe by the random hexamer method [15]. Labeled probe was used to reprobe the gt11 5'-stretch plus human testis cDNA expression library. Two additional gt11 clones with cDNA inserts homologous to SA-36-3f 1 (designated SA-36-4 and S-A36-5) were isolated and purified. Restriction digestion of phage DNAs with EcoRI followed by agarose gel electrophoresis showed that clone SA-36-4 contained a cDNA insert of 1037 bp and that clone SA-36-5 had an insert of 1223 bp (Fig. 2). The ß-galactosidase fusion proteins encoded by clones SA-36-4 and SA-36-5 (data not shown) were specifically recognized by mAb A36 and did not react with negative controls of NSO myeloma IgG and the isotypic control of mouse anti-human CD34. It was concluded that these two clones also coded for mAb A36-related proteins. Purified cDNA inserts were subcloned into pUC57 and subjected to sequencing of both strands. Alignment of the nucleotide sequences obtained for SA-36-3, SA-36-4, and SA-36-5 yielded the complete sequence of 3046 bp comprising SA-36 cDNA and its deduced amino acids (Fig. 3 and GenBank accession no. AF187554). SA-36 cDNA contained a 5' untranslated region of 234 nt, 1674 nt in an ORF, and a 3' untranslated region of 1138 nt. Analysis of the complete SA-36 cDNA sequence revealed > 99% homology of bases 230–2113 to human GPI/NLK mRNA (bases 11–1894) (accession no. NM 000175.1 derived from gb K03515) [20].
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Interestingly, nt 191–356 at the 5' end of SA-36 cDNA were 100% identical to nt 364–529 described for Exon1 of human GPI gene (gb M55538) [21], including the consensus eukaryotic initiation sequence CCGCC [22] found upstream from the translational start codon ATG. SA-36 cDNA also contained the consensus polyadenylation sequence 5'-AATAAA at position 1907 [23] and mRNA degradation sequences (5'-ATTTA) at positions 2127, 2485C, and 2807C within the 3' untranslated region [24]. The 558 amino acids deduced from SA-36 cDNA (bases 235–1908) exhibited > 99% homology to the 558 amino acids [20, 25] of human GPI/NLK (NP 000166 derived from K03515). In fact, only three SA-36 deduced amino acids (at positions 158, 426, and 436) were nonidentical to those of human GPI/NLK. Two of these nonidentical amino acids (positions 426 and 436) were related residues (valine in SA-36 and leucine in GPI). Glycine residue found at position 158 of SA-36 differed from the valine reported at the same position of GPI/NLK in GenBank databases (accession no. NP 000166 derived from K03515). All four cysteine residues of GPI/NLK [26] were also present in SA-36. SA-36 also exhibited high homology to GPI obtained from other sources such as the pig (92%), the mouse (88%), Cricetulus griseus (84%), and Sus scrofa (83%).
Characterization of the GPI/NLK-Like Sperm Antigen
The sequencing data suggesting that mAb A36 cognate sperm antigen is in fact a GPI/NLK homologous polypeptide were confirmed by Western blot analysis using mAb A36 and commercial preparations of glucose GPI (E.C. 5.3.1.9). As can be seen in Figure 4, mAb A36 antibody reacted (Fig. 4, lane 1) with a highly polymorphic series of protein bands in human sperm extracts, including a 53-kDa polypeptide, as previously reported [12]. Furthermore, mAb A36 recognized GPI prepared from rabbit muscle (Fig. 4, lane 2) and GPI from baker's yeast (Fig. 4, lane 3), which were shown [20, 27] to exhibit relative molecular masses of 55–56 kDa under reducing conditions. Additional verification that our cDNA clones, isolated using the sperm-agglutinating mAb A36, encode a sperm surface antigen [12] was obtained in experiments using polyclonal monospecific antibodies raised in mice against ß-galactosidase/SA-36-3 fusion protein and preadsorbed with ß-galactosidase. Figure 5 clearly demonstrates that these antibodies stained human sperm cells, whereas a negative control of mouse polyclonal antibodies to ß-galactosidase, which were also pretreated with ß-galactosidase, showed only background staining of human spermatozoa.
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Further experiments revealed that the polyclonal monospecific anti-ß-galactosidase/SA-36-3 caused extensive tangled agglutination [6] of motile human spermatozoa while negative control antibodies did not.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In an attempt to isolate sperm antigen involved in sperm agglutination, we have employed a potent sperm-agglutinating mAb. This study comprised the cloning and sequencing of cDNAs encoding a GPI/NLK-like sperm antigen, designated SA-36, that is involved in sperm agglutination. A cDNA clone was isolated from a human testis cDNA expression library in gt11 with use of the sperm-agglutinating mAb A36 [12] as an immunoprobe. A fragment of 383 bp obtained from the original SA-36-3 cDNA (isolated by immunoscreening) was further used, as a labeled probe, to obtain additional clones that together yielded the entire composite of SA-36 cDNA sequence. Authenticity of cDNAs was confirmed by specific recognition of their fusion proteins by mAb A36. Furthermore, polyclonal, monospecific antibodies raised against ß-galactosidase/SA-36-3 protein that were preadsorbed with ß-galactosidase caused intensive agglutination of motile human spermatozoa, similar to that induced by mAb A36 [12]. These antibodies also stained, in immunofluorescent assays, methanol-fixed sperm cells. On the other hand, human sperm cells were not agglutinated and were only slightly stained by control antibodies produced against ß-galactosidase, expressed by a gt11 clone devoid of insert, that were preadsorbed with ß-galactosidase. These latter observations provided additional proof that SA-36 cDNA encodes mAb A36 cognate sperm surface antigen involved in sperm agglutination. Sequencing data of SA-36 cDNA revealed > 99% homology with published (accession no. NM 000175.1 derived from gb K03515) sequences of GPI/NLK mRNA. The protein deduced from SA-36 had 555 of 558 amino acids identical to those of human GPI, with two of the nonidentical amino acids at positions 426 and 436 being related [20]. Indeed, mAb A36 produced against human spermatozoa recognized in Western blots commercial preparations of GPI from rabbit muscle and from baker's yeast, thus confirming that the sperm antigen encoded by SA-36 is homologous to GPI/NLK.
The glycolytic enzyme GPI, also known as phosphohexose isomerase, is a ubiquitous enzyme that catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate. The 50-kb gene for human GPI, located on the long arm of chromosome 19, has been cloned [39, 40]; and its structure and organization have been elucidated. Interestingly, it has been reported [25, 41] that GPI mRNA shares high sequence homology to the mRNA encoding NLK, which is a soluble neurotrophic mediator for spinal and sensory neurons, associated with motor neuron disease [20, 26]. NLK is also secreted by lectin-activated T lymphocytes and promotes secretion of immunoglobulins by cultured human peripheral blood mononuclear cells [20, 26]. Another activity attributed to this T cell-secreted cytokine is the induction of differentiation and maturation of myeloid leukemia cells and thus action as a maturation factor [42, 43]. Elucidation of the coding sequence of GPI gene strengthened the notion that the same gene [41, 44, 45] probably encodes GPI and NLK. More recent data suggest that GPI/NLK, which displays enzymatic, neurotrophic, and cytokine activities, is also secreted by tumor cells and acts as an autocrine motility factor [27, 46]. It appears that while GPI/NLK RNA is expressed in normal nonstimulated cells, elevated secretion of protein is restricted to malignant tumors or to stimulated cells [27, 42, 46, 47].
These multiple activities of GPI/NLK are also reflected in the variable isoforms of this protein. Whereas mature active GPI from normal tissues is usually a homodimer of two identical subunits [48], additional subunits were detected in normal and in cancerous tissues. These variable subunits may form homodimers or heterodimers, thereby yielding the GPI isoforms [47, 49, 50]. It has been postulated that posttranslational modifications lead to formation of these isoforms [49]. Although potential glycosylation sites exist in GPI, the isoforms are not due to glycosylation [26, 49] but rather represent deamination of specific sequences [49, 51]. Unlike the dimeric GPI, NLK has been reported to be a monomer polypeptide of 56 kDa composed of 558 amino acids and encoded by an mRNA of about 2 kb [20]. It has been suggested that specific intracellular enzymatic cleavage of GPI leads to its conversion to NLK [47].
Pertinent to the data reported by us on the presence of GPI/NLK homologous protein on the surface of sperm cells are the observations of Buehr and McLaren [50] demonstrating that a unique isoform of GPI, absent from somatic tissues, exists in mouse sperm cells and mouse testes, but only after puberty. Evidently, further studies will be needed to elucidate the relationships between this unique sperm isoform of GPI/NLK and SA-36 described in this report, to evaluate the biological functions of SA-36, and to elucidate the expression of SA-36 during spermatogenesis at transcriptional and posttranscriptional levels.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This research was supported in part by grant (No. 181-903) from the Israel Ministry of Health (the committee for state inherited funds) and the Fund for promotion of research at the Technion and by grant (No. 2400066) from the Mars Pittsburgh funds for medical and surgical research, ATS, USA. 
2 Correspondence: Yehudith Naot, Department of Immunology, the Bruce Rappaport Faculty of Medicine, Technion, P.O. Box 9649, Bat-Galim, Haifa 31096, Israel. FAX: 972 4 8342106; naot{at}tx.technion.ac.il
Accepted: November 29, 1999.
Received: September 27, 1999.
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