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(The FASEB Journal. 1999;13:1303-1313.)
© 1999 FASEB

N-linked glycan of a sperm CD52 glycoform associated with human infertility

ALAN B. DIEKMAN, ELIZABETH J. NORTON, KENNETH L. KLOTZ, V. ANNE WESTBROOK, HIROAKI SHIBAHARA1, SØREN NAABY-HANSEN2, CHARLES J. FLICKINGER and JOHN C. HERR3

Center for Recombinant Gamete Contraceptive Vaccinogens, Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA

3Correspondence: Department of Cell Biology, University of Virginia Health Sciences Center, Box 439, Charlottesville, VA 22908, USA. E-mail: jch7k{at}virginia.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In a benchmark study, Isojima and colleagues established H6–3C4, the first successful heterohybridoma immortalized from the peripheral blood lymphocytes of an infertile woman who exhibited high sperm-immobilizing antibody titers. The present report demonstrates the identity between the glycoprotein antigens recognized by the human H6–3C4 monoclonal antibody (mAb) and the murine S19 mAb, generated in our laboratory to sperm agglutination antigen-1 (SAGA-1). Both mAb's recognize N-linked carbohydrate epitopes on the 15–25 kDa, polymorphic SAGA-1 glycoprotein that is localized to all domains of the human sperm surface. Treatment with phosphatidylinositol-specific phospholipase C demonstrated that SAGA-1 is anchored in the sperm plasmalemma via a GPI-lipid linkage. Immunoaffinity purification and microsequencing indicated that the core peptide of the SAGA-1 glycoprotein is identical to the sequence of CD52, a GPI-anchored lymphocyte differentiation marker implicated in signal transduction. Comparison of anti-SAGA-1 and anti-CD52 immunoreactivities revealed that the sperm form of CD52 exhibits N-linked glycan epitopes, including the epitope recognized by the infertility-associated H6–3C4 mAb, which are not detected on lymphocyte CD52. Thus, the two populations of the CD52 glycoprotein on lymphocytes and spermatozoa represent glycoforms, glycoprotein isoforms with the same core amino acid sequence but different carbohydrate structures. Furthermore, mAb's to the unique carbohydrate epitopes on sperm CD52 have multiple inhibitory effects on sperm function, including a cytotoxic effect on spermatozoa in the presence of complement. These results are the first to implicate unique carbohydrate moieties of a sperm CD52 glycoform as target epitopes in the anti-sperm immune response of an infertile woman. Furthermore, localization of CD52 on all domains of the sperm surface coupled with the multiple sperm-inhibitory effects of antibodies to its unique carbohydrate moieties suggest opportunities for immunocontraceptive development.—Diekman, A. B., Norton, E. J., Klotz, K. L., Westbrook, V. A., Shibahara, H., Naaby-Hansen, S., Flickinger, C. J., Herr, J. C. N-linked glycan of a sperm CD52 glycoform associated with human infertility.


Key Words: autoimmunity • isoimmunity • glycocalyx • lymphocyte • contraception


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ANTI-SPERM ANTIBODY TITERS in female patient sera and reproductive tract fluids have been shown to correlate with reduced fertility (1 2 3) . In a benchmark study of a human anti-sperm immune response, Isojima et al. (4) immortalized a human anti-sperm monoclonal antibody (mAb)4 by generating a human-mouse heterohybridoma with the peripheral blood lymphocytes of an infertile woman whose serum exhibited high sperm-inhibitory titers. The resulting human IgM mAb, H6–3C4, demonstrated sperm-agglutinating and complement-dependent sperm-immobilizing activities in vitro. These results implicated the H6–3C4 epitope as a target for antibodies involved in the etiology of clinical immune-mediated infertility and led Isojima and colleagues to pursue characterization of the H6–3C4 cognate antigen. The H6–3C4 mAb and related murine mAb's were subsequently shown to recognize N-linked oligosaccharide epitopes on a sperm surface glycoprotein identified as a series of 15 to 25 kDa immunoreactive bands on Western blots (5) . Furthermore, Tsuji et al. (6) demonstrated that the H6–3C4 mAb reacted with a carbohydrate epitope containing repetitive N-acetyl lactosamine. However, the precise structure of the carbohydrate side chains, as well as the core protein of the cognate H6–3C4 antigen, remained unknown.

Our laboratory recently described sperm agglutination antigen-1 (SAGA-1), a polymorphic (~15–25 kDa), highly acidic hydrophobic glycoprotein that is localized over the entire surface of the human spermatozoon (7) . SAGA-1 was characterized with S19, a murine mAb generated by the immunization of mice with human sperm homogenates that exhibited sperm-inhibitory actions in vitro including agglutination, inhibition of cervical mucus penetration, and inhibition of human sperm-zona pellucida binding (7 8 9) . Furthermore, Diekman et al. (7) demonstrated that the S19 mAb recognized a carbohydrate epitope on the SAGA-1 glycoprotein. Competitive inhibition studies, as well as similarities in reported mass, suggested that SAGA-1 and the H6–3C4 antigen represent the same sperm glycoprotein(s) (5 , 7 , 10) .

In the current study, we provide evidence that the SAGA-1 antigen identified in our laboratory and the cognate antigen of the human H6–3C4 and related murine mAb's developed by the Isojima group are the same human sperm glycoprotein. Significantly, microsequence analysis of immunoaffinity-purified SAGA-1 demonstrated that the protein core of the SAGA-1/H6–3C4 antigen is identical to that of CD52, a glycophosphatidylinositol (GPI) -anchored lymphocyte differentiation marker implicated in signal transduction. Furthermore, immunochemical studies revealed N-linked carbohydrate epitopes present on sperm CD52 that are absent on lymphocyte CD52, indicating that these two populations of CD52 are differentially glycosylated. We further demonstrate that the S19 mAb to SAGA-1 immobilizes human spermatozoa in the presence of complement. These findings identify sperm CD52 as one of the few well-defined sperm surface glycoproteins implicated in human antibody-mediated infertility.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All studies involving human semen donors were approved by the University of Virginia's Human Research Committee and informed consent was obtained from each participant after explanation of the nature and possible consequences of the studies.

Monoclonal antibodies
The S19 mAb [IgG1; alternate nomenclature: MHS-8 (8) ] was generated by the immunization of mice with human spermatozoa and ascites fluid containing the S19 mAb was generated in mice as described (7) . For use in the sperm immobilization test (SIT), immunofluorescence analysis, and immunohistochemical analysis, the S19 mAb was isolated by ammonium sulfate precipitation of supernatants collected from in vitro culture of the MHS-8 hybridoma (7) . The H6–3C4 (human IgM), 2B6 (mouse IgG3), 2C6 (mouse IgM), and 2E5 (mouse IgG3) mAb's (5) were generously provided by Dr. Koji Koyama, Hyogo Medical College, Nishinomiyz, Japan. The CAMPATH-1M mAb (rat IgM) was purchased as clarified ascites from Serotec (Raleigh, N.C.) and Chemicon (Temecula, Calif.).

Extract preparation
Spermatozoa obtained from donated ejaculates were washed with Ham's F-10 medium and pelleted by centrifugation at 400 x g. Sperm and spleen samples were extracted with H2O/methanol/chloroform (3:8:4) following the procedure of Svennerholm and Fredman (11) . Spermatozoa and minced spleen tissue were homogenized in 3 volumes H2O. Subsequently, 8 volumes methanol and 4 volumes chloroform were added, and homogenates were vortexed after each addition. Extracts were centrifuged at 12,000 x g to remove insoluble debris and the supernatants were dried under vacuum. For electrophoresis, extracted material was resuspended in 1% sodium dodecyl sulfate (SDS) and mixed with Laemmli buffer (12) .

Electrophoresis and immunoblot analysis
For 1-dimensional immunoblots, protein samples were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) through 15% acrylamide and electroblotted onto nitrocellulose as described by Towbin et al. (13) . Blots were blocked in phosphate-buffered saline (PBS) containing 0.05% Tween-20 and 5% non-fat dry milk and incubated with primary antibody diluted in PBS/0.05% Tween-20/0.5% non-fat dry milk. Dilution factors for the S19 mouse ascites, CAMPATH-1M rat ascites, and H6–3C4, 2B6, 2C6, and 2E5 hybridoma supernatants are indicated in the figure legends. Negative controls included secondary antibodies alone. Blots were washed three times with PBS/0.05% Tween-20 and incubated in horseradish peroxidase-conjugated goat anti-mouse, human, or rat immunoglobulin (1:5000; Jackson ImmunoResearch Laboratories, West Grove, Pa.). Blots were washed twice in PBS/0.05% Tween-20, once in PBS, and developed with TMB reaction substrate (Kirkegaard & Perry, Gaithersburg, Md.).

Two-dimensional electrophoresis was performed following the method of O'Farrell (14) with modification (15) . Spermatozoa were separated from seminal plasma and contaminating cells by centrifugation over a discontinuous (55%–80%) Percoll density gradient (Pharmacia, Piscataway, N.J.) (7) . Sperm proteins were extracted in an octyl-ß-glucoside solubilization buffer and separated on an isoelectric focusing gel with a pH 2.5 to 10.0 ampholine range as described previously (7 , 15) . Proteins were then separated through a second dimension over a 9%–16% acrylamide gradient by SDS-PAGE, electroblotted onto nitrocellulose, and immunostained with the S19 or CAMPATH-1M ascites.

Linkage analysis
To cleave N-linked carbohydrate side chains, methanol/chloroform-extracted sperm proteins were incubated with 20 U/ml N-glycanase (Boehringer Mannheim, Indianapolis, Ind.) in 0.1% SDS, 0.5% octyl-ß-glucoside, 20 mM sodium phosphate, pH 7.0 at 37°C for 20 h. A reaction mixture lacking N-glycanase was included as a negative control. Additional controls included enzymatic cleavage of purified bovine fetuin (Sigma, St. Louis, Mo.). Reactions mixtures were subjected to 1-dimensional SDS-PAGE and blotted to nitrocellulose. Immunoblot analysis of the sperm protein reactions was performed with the S19 mAb. Purified fetuin was visualized by Amido black staining.

Immunoaffinity purification and microsequencing
Hydrophobic sperm proteins were prepared by Triton X-114 detergent phase partitioning (16) and acetone precipitation or by extraction in chloroform/methanol (11) as described above. Hydrophobic protein pellets were resuspended in 0.5% octyl-ß-glucoside. To prepare an immunomatrix, the S19 mAb was chemically cross-linked to protein-G Sepharose beads (Pharmacia) with dimethyl pimelimidate (17) . SAGA-1 was purified from the pool of hydrophobic sperm protein by immunoaffinity column chromatography (18) and deglycosylated with N-glycanase (Boehringer Mannheim) following the manufacturer's instructions. After 1-dimensional SDS-PAGE, purified deglycosylated SAGA-1 was transferred to polyvinylidene difluoride (PVDF) membrane, excised as a single band, and microsequenced by Edman degradation (18) .

Immunofluorescence
Human spermatozoa were isolated from semen by the swim-up method (19) . Procedures for immunofluorescence followed those of Diekman et al. (7) . Spermatozoa were fixed in 4% paraformaldehyde/PBS and air-dried onto slides. Slides were blocked in 5% normal goat serum (NGS)/PBS and incubated at room temperature with the S19 (purified from hybridoma supernatant, 1:50) or CAMPATH-1M (1:20) mAb's in 1% NGS/PBS for 1.5 h. Slides were washed in 1% NGS/PBS, incubated for 1.5 h with goat anti-mouse or rat FITC-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories), washed in PBS, and mounted with Slow Fade (Molecular Probes, Eugene, Oreg.). Secondary antibodies alone were included as negative controls. Results were visualized with a Zeiss Axioplan microscope (Carl Zeiss, Inc., Thornwood, N.Y.) equipped for epifluorescence and DIC microscopy.

GPI anchor analysis
To cleave GPI anchor structures on the human sperm surface, 1 x 108 Percoll-harvested spermatozoa were incubated in suspension with or without 3 U Bacillus cereus phosphatidylinositol-specific phospholipase C (PI-PLC) (Molecular Probes) for 20 min at room temperature. Heat-inactivated PI-PLC and PI-PLC minus reactions were included as negative controls. Spermatozoa were removed from suspension by centrifugation at 600 x g for 30 min. After addition of 2 mM PMSF and 5 mM EDTA, supernatants were dialyzed against H2O and concentrated by lyophilization. Two-dimensional immunoblots of the reaction supernatants were incubated with the S19 mAb, followed by horseradish peroxidase-conjugated antibody to mouse immunoglobulin G. Immunoreactivity was visualized by chemiluminescence (Boehringer Mannheim).

Immunohistochemistry
Surgical samples of human epididymis and spleen were obtained from the University of Virginia Hospital. After fixation in 10% neutral buffered formalin, tissues were paraffin-embedded, sectioned, and mounted onto slides. Slides were dewaxed and rehydrated following the procedures of Southgate and Trejdosiewicz (20) . Sections were treated with 0.6% H2O2/methanol for 10 min to abolish endogenous peroxidase activity, placed in 0.1% CaCl2/trypsin to expose antigens, and blocked in PBS/10% NGS for 30 min. Sections were incubated overnight at 4°C with the S19 mAb purified from hybridoma supernatant (1:100), followed by 1 h incubation at room temperature with horseradish peroxidase-conjugated antibody to mouse immunoglobulin. Negative controls included secondary antibodies alone. Immunoreactivity was visualized with the TrueBlue reaction substrate (Kirkegaard & Perry).

Sperm immobilization test
Guinea pig complement was preincubated with washed human spermatozoa to absorb endogenous sperm-immobilizing factors. For a negative control, preabsorbed guinea pig complement was inactivated by incubation at 56°C for 30 min. Motile human spermatozoa were obtained from ejaculated semen by the swim-up procedure (19) . The SIT was performed as described by Isojima and Koyama (21) . Briefly, 25 µl of S19 mAb (purified from hybridoma supernatant) serially diluted two-fold, 5 µl guinea pig complement, and 2.5 µl motile sperm suspension (20 x 106 spermatozoa/ml) were gently mixed and delivered to spermatozoa counting chambers (Humagen Fertility Diagnostics, Charlottesville, Va.). Counting chambers were sealed with vacuum grease and incubated at 32°C for 1 h. Spermatozoa in each sample were scored microscopically for motility.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Immunological identity between SAGA-1 and the H6–3C4 cognate antigen
Isojima et al. (4) demonstrated that the S19 mAb to SAGA-1 inhibited the sperm-binding activity of H6–3C4, a human mAb immortalized from the peripheral blood lymphocytes of an infertile woman with anti-sperm antibody titers. In our laboratory, the murine S19 mAb, the human H6–3C4 mAb, and related murine mAb's (2B6, 2C6, and 2E5) recognized a similar pattern of ~15 to 25 kDa immunoreactive bands on duplicate, 1-dimensional immunoblot strips of human sperm extracts (data not shown). To provide a formal proof that these antibodies recognize the same sperm antigen, SAGA-1 was affinity purified using an S19 immunomatrix and immunoblot analysis was performed with the S19, H6–3C4, 2B6, 2C6, and 2E5 mAb's. For each of the described mAb's, immunoreactivity with SAGA-1 was demonstrated by identification of a ~15 to 25 kDa series of SAGA-1 bands on 1-dimensional immunoblot strips of affinity-purified material (Fig. 1 ). This result demonstrated that SAGA-1 is a cognate antigen recognized in common by the S19, H6–3C4, 2B6, 2C6, and 2E5 mAb's.



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  		Figure 1. Identity between the S19 and H6–3C4 cognate antigens. Immunoaffinity-purified SAGA-1 was subjected to preparative SDS-PAGE and immunoblot strips were incubated with the S19 ascites (1:20,000) and H6–3C4 (1:50), 2B6 (1:50), 2C6 (1:200), and 2E5 (1:200) hybridoma supernatants. Each mAb detected a similar polymorphic pattern (~15–25 kDa) demonstrating recognition of the SAGA-1 glycoprotein.

S19 immunoreactivity with an N-linked carbohydrate epitope
The H6–3C4, 2B6, 2C6, and 2E5 mAb's recognize N-linked carbohydrate epitopes on the SAGA-1 glycoprotein (5) . The S19 mAb was shown previously by our group to identify a carbohydrate epitope (7) . Furthermore, competitive inhibition of H6–3C4 binding to human spermatozoa by the S19 mAb suggested that the S19 carbohydrate epitope is also N-linked (10) . In the current study, linkage analysis was performed by incubating extracted human sperm proteins with N-glycanase to cleave N-linked glycan side chains. N-glycanase treatment abolished S19 immunoreactivity with the SAGA-1 glycoprotein, whereas incubation in the absence of enzyme had no effect on S19 immunoreactivity (Fig. 2 A). Enzymatic cleavage of N-linked carbohydrate side chains from purified fetuin, included as a positive control, was evident by increased mobility in SDS-PAGE (Fig. 2B ). This result demonstrates that the S19 mAb identifies an N-linked carbohydrate epitope on the SAGA-1 glycoprotein.



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  		Figure 2. S19 immunoreactivity with an N-linked carbohydrate epitope. A) The S19 ascites (1:10,000) reacted with SAGA-1 (~15–25 kDa) on an immunoblot of untreated, methanol/chloroform-extracted human sperm proteins (lane 1). N-glycanase treatment of the sperm extract abolished S19 immunoreactivity (lane 2), indicating that the S19 mAb reacts with an N-linked carbohydrate epitope. B) Amido black stained control blot of untreated (lane 1) and N-glycanase-treated (lane 2) bovine fetuin. Enzymatic cleavage of N-linked carbohydrate side chains from fetuin was evident by increased mobility in SDS-PAGE.

Microsequence analysis
Although the S19, H6–3C4, 2B6, 2C6, and 2E5 mAb's were shown previously to identify carbohydrate epitopes on the SAGA-1/H6–3C4 antigen, the precise structure of the carbohydrate side chain(s) and the core protein of the glycoprotein remained unknown. Furthermore, the lack of an antibody against a SAGA-1 peptide epitope obviated characterization of the SAGA-1 core protein and elucidation of its amino acid sequence by standard techniques for screening recombinant protein expression libraries. Therefore, we used a strategy that encompassed affinity purification of the native glycoprotein and microsequence analysis to characterize the SAGA-1 core protein. Native SAGA-1 was purified from human spermatozoa by the partitioning of hydrophobic proteins and immunoaffinity chromatography with the S19 mAb. After deglycosylation of N-linked glycans and excision from a 1-dimensional PVDF blot as a single band, the affinity-purified SAGA-1 was microsequenced at its NH2 terminus by Edman degradation. The first seven amino acids of SAGA-1 were assigned confidently as GQDDTSQ; the eighth amino acid could not be determined and amino acids 9 to 11 were deduced as SSP (peptide A, Fig. 3 A). A second peptide sequence was theoretically deduced with an asparagine residue substituted for aspartate 3 (peptide B, Fig. 3B ) since deamination of the asparagine side chain during deglycosylation of N-linked glycans can convert glycosylated asparagine to aspartate (22) . This substitution creates a consensus motif for N-linked glycosylation (ND[T or S]) at position 3 in peptide B.



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  		Figure 3. Microsequence analysis of the SAGA-1 core peptide. A) Peptide A: eleven amino acid sequence determined by Edman degradation. Capital letters indicate the highest probability sequence. The eighth amino acid, designated X, could not be determined. B) Peptide B: asparagine assigned at position 3 rather than aspartate due to the probable deamination of glycosylated asparagine to aspartate during deglycosylation. C) Peptide B and the 12 amino acid peptide core of the mature CD52 glycoprotein are identical at all known positions.

Sequence identity to CD52
Amino acid residues 1–7 and 9–11 of peptide B were identical to the amino acid sequence of CD52 (23) , a differentiation marker on the surface of human lymphocytes (Fig. 3C ). Comparison with the 12 amino acid mature core peptide of CD52 indicated that peptide B represents nearly the full-length sequence of the SAGA-1 core peptide with the twelfth amino acid implicated as serine. The assignment of asparagine, rather than aspartate, at position 3 of peptide B corresponds to asparagine 3 of CD52. This assignment was further confirmed by identification of this residue as the only appropriate site for the N-linked glycosylation that was detected biochemically for SAGA-1 in this study and for CD52 (24 , 25) . The unknown amino acid (X) at position 8 in SAGA-1 corresponds to a threonine residue in CD52; thus, the identification of a corresponding threonine in SAGA-1 may have been inhibited by O-linked glycosylation of this amino acid residue.

Biochemical/immunochemical identity with CD52
Characterized predominantly in the spleen, CD52 is a GPI-anchored, low molecular weight glycoprotein composed of a 12 amino acid core peptide with N-linked carbohydrate side chains (24) . Significantly, anti-CD52 mAb's (CAMPATH-1G and CAMPATH-1M) were previously shown to react with a low molecular weight protein on the human sperm surface and inhibit sperm function in vitro (26 , 27) . In the current study, the identity of sperm CD52 and SAGA-1 was confirmed further by biochemical analysis and by comparing the immunoreactivities of anti-CD52 and anti-SAGA-1 mAb's. For example, S19 and CAMPATH-1M, an IgM mAb that reacts with an epitope spanning the carboxyl-terminal tripeptide of CD52 and its GPI anchor (25) , both demonstrated immunofluorescence over the entire sperm membrane (Fig. 4 A), indicating that the S19 and CAMPATH-1M cognate antigens exhibit colocalization on human spermatozoa.



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  		Figure 4. Biochemical and immunochemical identity between SAGA-1 and CD52. A) The S19 mAb to SAGA-1 and the CAMPATH-1M mAb to CD52 immunolocalized over all human sperm domains by indirect immunofluorescence. Scale bar: 20 µM. B) The S19 mAb (ascites, 1:1000) detected at least 22 charge and mass variants of the SAGA-1 antigen (~15–25 kDa and ~pI 2.5–3.5) on high-resolution, 2-dimensional immunoblots of sperm proteins. On a duplicate immunoblot, the CAMPATH-1M mAb (1:500) identified at least 18 immunoreactive spots that comigrated with SAGA-1. To facilitate comparison, the identified immunoreactivities are enlarged and only the low molecular weight, acidic region of the immunoblots are shown.

To further examine the immunochemical identity of SAGA-1 and CD52 in spermatozoa, sperm proteins were subjected to high-resolution 2-dimensional electrophoresis specifically formulated for separation of acidic and low molecular weight proteins (Fig. 4B ). The S19 mAb detected a pattern of highly polymorphic, immunoreactive SAGA-1 spots (~15–25 kDa and pI ~2.5–3.5) comprised of six isoelectric variants, each of which exhibited three to five mass variants, giving a total of at least 22 isoforms. On a duplicate immunoblot, the CAMPATH-1M mAb identified at least 18 spots that colocalized with the SAGA-1 charge and mass variants detected with the S19 mAb. This anti-CD52 mAb also reacted with immunoaffinity-purified SAGA-1 on 1-dimensional immunoblots (data not shown). Based on immunoreactivity with CD52, the CAMPATH-1M mAb apparently reacts with an epitope composed of the carboxyl-terminal tripeptide (SPS) and GPI anchor on the sperm SAGA-1 glycoprotein (25) . Thus, high-resolution, 2-dimensional immunoblot analyses and immunofluorescent microscopy confirmed that the S19 and CAMPATH-1M mAb's react with the same sperm glycoprotein(s) localized over the entire sperm surface.

SAGA-1 was previously shown to exhibit hydrophobicity characteristic of lipid association (7) . To investigate whether SAGA-1 is a GPI-anchored glycoprotein on the sperm surface, viable human spermatozoa were incubated with PI-PLC to cleave GPI anchor structures. SAGA-1 was identified in the PI-PLC reaction supernatant as a series of mass and charge variants (~15–25 kDa and ~pI 2.5–5.2) by 2-dimensional immunoblot analysis with the S19 mAb (Fig. 5 A). S19 immunoreactivity was not observed in supernatants that contained heat-inactivated PI-PLC (Fig. 5B ) or from which PI-PLC was omitted. The observed increase in SAGA-1 pI is likely due to cleavage of the GPI anchor. The demonstration that SAGA-1 is cleaved by PI-PLC indicates that SAGA-1, similar to CD52, is anchored in the sperm plasmalemma via a GPI-lipid linkage.



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  		Figure 5. SAGA-1 is a GPI-anchored glycoprotein, as is CD52. A) SAGA-1 was identified by 2-dimensional immunoblot analysis with the S19 mAb (ascites, 1:1000) as a series of charge and mass isoforms (~15–25 kDa and ~pI 2.5–5.2) in the reaction supernatant that contained proteins liberated from the sperm surface by PI-PLC cleavage. B) S19 immunoreactivity was absent in negative control supernatant containing heat-inactivated PI-PLC.

Tissue of origin
CD52 expression in the human epididymis was demonstrated previously by cDNA analysis and in situ hybridization (28) . Immunohistochemical analysis identified the GPI-anchored CD52 protein in the cytoplasm of epithelial cells and in the lumen of the human epididymis (29) . Likewise, H6–3C4 and 2C6 immunoreactivities were demonstrated in the epididymal epithelium but not in the testis (30 , 31) . In the current study, immunohistochemical analysis identified S19 immunoreactivity in the apical regions, primarily the stereocilia, of the principal cells in the human epididymal epithelium and with the contents of the epididymal lumen (Fig. 6 A). S19 immunoreactivity was not detected in the testis (data not shown). These results suggest that although the CD52 GPI-anchored glycoprotein was identified throughout the epididymal epithelium and lumen (29) , the S19 carbohydrate epitope is restricted to CD52 molecules in the apical region of the epithelial cells and in the epididymal lumen.



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  		Figure 6. Unique carbohydrate epitopes are detected on the sperm glycoform of CD52. A, B) Immunohistochemical analysis of human epididymis and spleen. S19 immunoreactivity was identified on the stereocilia of epithelial cells surrounding the epididymal lumen and on the luminal contents (A: scale bar: 250 µM). S19 immunoreactivity was not detected in human spleen (B: scale bar: 400 µM). C, D) Immunoblot analysis of human sperm and spleen chloroform/methanol extracts with the anti-SAGA-1 and anti-CD52 mAb's (dilution factors: CAMPATH-1M, 1:100; S19 ascites, 1:1000; H6–3C4, 1:50; 2B6, 1:50; 2C6, 1:100; 2E5, 1:100). These mAb's identified a similar ~15–25 kDa polymorphic pattern of protein bands in the sperm extract C). The lowest molecular weight form was identified exclusively by the CAMPATH-1M mAb. In the spleen extract (D), the 2B6, 2C6, 2E5, and CAMPATH-1M mAb's detected a series of ~18–22 kDa bands, a polymorphic pattern distinct from that identified in the sperm extract, while neither S19 nor H6–3C4 immunoreactivity was detected.

Unique CD52 glycoform on spermatozoa
In contrast to immunoreactivity identified in the epididymis and spermatozoa, S19 immunoreactivity was not detected on human spleen sections (Fig. 6B ) or on isolated peripheral blood lymphocytes (data not shown), suggesting that the S19 carbohydrate epitope is not present on the CD52 lymphocyte antigen. To investigate the potential tissue-specific N-linked glycosylation of the sperm and lymphocyte CD52 glycoproteins, immunoblot analysis was performed with the S19, H6–3C4, 2B6, 2C6, and 2E5 anti-carbohydrate mAb's and the CAMPATH-1M mAb, and the identification of their cognate carbohydrate epitopes in sperm and spleen extracts was compared. Human spleen was used as the source of the lymphocyte antigen since previous structural studies focused primarily on CD52 isolated from this tissue (23 , 24) . In the sperm extract, a similar ~15–25 kDa series of protein bands was identified with each of the six mAb's (Fig. 6C ). CD52 was detected in the spleen extract by a subset of the mAb's, CAMPATH-1M, 2B6, 2C6, and 2E5 as an ~18–22 kDa series of protein bands (Fig. 6D ). This polymorphic pattern of CD52 on spleen immunoblots was narrower in molecular weight and distinct from that identified in the sperm extract. Significantly, neither S19 nor H6–3C4 immunoreactivity was detected on the spleen immunoblot. Thus, the S19 and H6–3C4 N-linked carbohydrate epitopes on spermatozoa were not identified on CD52 or other glycoprotein/glycolipid antigens in the spleen extract. These immunochemical differences and the variation in apparent molecular weight between sperm and spleen extracts indicate that while the sperm and lymphocyte CD52 core proteins are identical, their N-linked carbohydrate side chains contain structural differences.

Sperm-immobilizing activity of anti-SAGA-1 antibody
The SIT (21) was performed to examine the sperm-immobilizing activity of the S19 mAb purified from hybridoma supernatant. In the presence of guinea pig complement, the S19 mAb demonstrated a dose-dependent immobilizing effect on spermatozoa from four individuals (Fig. 7 ). Within the parameters of this assay, total sperm immobilization was observed with S19 mAb concentrations of 0.75 mg/ml and above. In the presence of heat-inactivated complement, the sperm-immobilizing effect was not observed over the same antibody range, demonstrating the complement dependency of the immobilization. Additional negative controls, which included the SIT performed with active or inactive complement but in the absence of antibody, demonstrated sperm motility consistent with that observed with inactivated complement. These results indicate that the binding of antibody to SAGA-1 carbohydrate epitopes has a complement-dependent, cytotoxic effect on human spermatozoa.



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  		Figure 7. Complement-dependent immobilizing effect of antibody binding to the S19 carbohydrate epitope as demonstrated by the sperm immobilization test. In the presence of complement (open circles), the S19 mAb had a dose-dependent effect on sperm motility indicative of cytotoxicity. This cytotoxic effect was not observed in the presence of heat-inactivated complement (filled circles). Each of four tests was performed with spermatozoa from a different individual. Bars indicate standard error of the mean.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present report demonstrates that the murine S19 mAb, generated in our laboratory, and the human infertility-associated H6–3C4 mAb recognize carbohydrate epitopes on an identical antigen, the highly acidic glycoprotein SAGA-1 that is localized to all domains of the human sperm surface. Immunoreactivity of the H6–3C4 mAb with affinity-purified SAGA-1 suggests that the SAGA-1 glycoprotein and its carbohydrate side chains represent targets for antibodies involved in clinical immune-mediated infertility. Numerous investigators have described the characterization of sperm antigens using anti-sperm antibodies from infertile patients (32 33 34 35 36 37 38 39 40) . However, SAGA-1 was not directly identified in these previous studies. Sperm antigen identification was limited to 1-dimensional SDS-PAGE in most studies, and extrapolation from the multiple reactivities detected is difficult due to the variability in apparent molecular weight of small, heavily glycosylated antigens under various electrophoretic conditions. Furthermore, studies that used 2-dimensional electrophoresis did not encompass the acidic pI range where SAGA-1 would be detected. Thus, a combination of monospecific antibodies and modified 2-dimensional electrophoresis was required to identify the SAGA-1/H6–3C4 cognate antigen as a potential immuno-infertility antigen.

Immunoaffinity purification and microsequencing of the S19/H6–3C4 cognate antigen provided the first formal proof that the peptide core of this glycoprotein is identical to that of the CD52 lymphocyte differentiation marker, a GPI-anchored glycoprotein that has also been identified on the human sperm surface. Moreover, our microsequence and immunochemical data confirm that the sperm surface glycoprotein recognized by an anti-CD52 mAb contains the CD52 core peptide. In addition to identity in amino acid sequence, the SAGA-1 glycoprotein exhibits several biochemical and immunochemical similarities to CD52. As demonstrated in this report, SAGA-1 is a GPI-anchored glycoprotein on the human sperm surface similar in apparent molecular weight and pI to the sperm CD52 glycoprotein, and is expressed in the human epididymis.

The N-linked carbohydrate structure of the CD52 lymphocyte antigen is partially composed of sialic acid and poly-N-acetyl-lactosamine (24) , a common carbohydrate moiety of tissue-specific N-linked glycans (41) . Although the oligosaccharide structure(s) of CD52 on spermatozoa has not been precisely defined, immunoreactivity of the 2B6, 2C6, and 2E5 anti-N-linked carbohydrate mAb's with both sperm and lymphocyte CD52 indicate a partial identity in N-linked glycan composition. Further, the acidic pI (~2.5 to 3.5) of the sperm CD52 is indicative of anionic moieties such as sialic acid; Tsuji et al. (6) demonstrated reactivity of the H6–3C4 mAb with an epitope on polysaccharide chains partially composed of poly-N-acetyl-lactosamine.

Although similarities in carbohydrate structure were apparent, the sperm CD52 glycoprotein exhibits N-linked glycan epitopes, including the epitope recognized by the infertility-associated H6–3C4 mAb, that are not detected on lymphocyte CD52. These results represent the first evidence for glycosylation differences between CD52 identified on spermatozoa and lymphocytes, indicating that the N-linked glycans of sperm SAGA-1 and lymphocyte CD52 are structural variants with similar carbohydrate frameworks. Therefore, the lymphocyte CD52 antigen and the sperm SAGA-1 protein represent glycoforms, glycoproteins with the same core peptide but with differing carbohydrate structures (42) . Furthermore, our identification of distinctions between N-linked CD52 glycans in the epididymis and spleen indicates that CD52 is differentially glycosylated in a tissue-specific manner.

Figure 8 summarizes our results and conclusions regarding the unique glycoforms of CD52 found in spermatozoa, designated by us as SAGA-1, and CD52 in the spleen. Both glycoforms are composed of a 12 amino acid, GPI-anchored glycopeptide with a single N-linked carbohydrate side chain. The N-linked glycans of the sperm and spleen glycopeptides are partially composed of similar carbohydrate moieties. However, the sperm CD52 glycan contains carbohydrate moieties that are not present in the spleen CD52 glycoform. Whether the spleen CD52 glycoprotein also contains unique N-linked carbohydrate epitopes has not been determined. Although the core peptide sequence of the SAGA-1 antigen exhibits identity with CD52, a nomenclature for the sperm and lymphocyte CD52 glycoforms must await detailed analysis of the SAGA-1 glycan composition and sequence.



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  		Figure 8. Model comparing the sperm and spleen glycoforms of CD52. Each glycoform is composed of a 12 amino acid core peptide that is bound to a lipid (orange) by a GPI anchor (blue). The CAMPATH-1M mAb (blue) recognizes an epitope comprised of the GPI anchor and the last three amino acid residues of the core peptide. A single consensus site for N-linked glycosylation (ND[T or S]) is present at asparagine 3 in the core peptide of each glycoform. The N-linked glycans of the sperm and spleen glycopeptides are partially composed of similar carbohydrate moieties (red), but the sperm CD52 glycoform contains unique carbohydrate epitopes that are not present in the spleen glycoform (green). N-linked carbohydrate epitopes common to both sperm and spleen CD52 were identified with the 2B6, 2C6, and 2E5 mAb's (red). The S19 and H6–3C4 mAb's reacted with N-linked carbohydrate epitopes specific to the sperm CD52 glycoform (green). The presence of N-linked carbohydrate moieties specific to the spleen glycoform (purple) is inferred in this model. The plasma membrane is represented as a gray shaded box.

Differential glycosylation provides an explanation for the molecular weight differences observed for the sperm and spleen CD52 glycoproteins. In addition, the identification of a single core peptide for CD52/SAGA-1 indicates that the molecular weight and charge polymorphism of SAGA-1 observed in spermatozoa are not due to expression of multiple core peptides but to structural/compositional variations in the N-linked glycan and/or GPI anchor. Variable length and number of poly-N-acetyl-lactosamine chains could give rise to multiple molecular weight forms, while differences in sialylation and sulfation could produce multiple charge variants. Furthermore, modifications in the GPI anchor may also contribute to molecular weight and charge polymorphism as exhibited by the increase in pI of SAGA-1 after PI-PLC cleavage of the GPI anchor. Additional studies will be required to specifically define the structural factors that generate the multiple variants of the SAGA-1 sperm glycoprotein.

After initial characterization in the rat epididymis (43 , 44) , CD52 homologs were identified in the epididymis of multiple species including the human, mouse, macaque, and dog (29) . In the present report, the S19 mAb identified a unique CD52 glycoform in both the epithelium and lumen of the human epididymis. Thus, our results support the conclusions of previous studies (45 , 46) that in the species described, CD52 is secreted by the epididymal epithelium with its GPI anchor intact and is then inserted into the sperm membrane during epididymal transport. However, whereas the CAMPATH-1M mAb identified the CD52 GPI-anchored protein in the human epididymis over the entire cytoplasm of epithelial cells (29) , S19 immunoreactivity was restricted to apical regions of these cells. This variation in immunohistochemical localization may reflect the differential temporal and spatial expression of the S19 and CAMPATH-1M cognate epitopes. Multiple posttranslation modifications contribute to expression of CD52 in the epididymis. Human CD52 is initially expressed as a 61 amino acid pro-protein that is then proteolytically cleaved and glycosylated to generate the mature 12 amino acid, GPI-anchored CD52 glycopeptide (23) . The CAMPATH-1M mAb reacts with an epitope spanning the carboxyl-terminal tripeptide of CD52 and its GPI anchor (25) ; thus, immunohistochemistry with the CAMPATH-1M mAb indicated that GPI-anchored CD52 is present throughout the cytoplasm of epididymal epithelial cells (29) . Apical localization of S19 immunoreactivity suggests that whereas the GPI-anchored CD52 peptide is present throughout the epididymal epithelium, the N-linked S19 carbohydrate epitope is expressed on CD52 by a posttranslational modification that occurs after attachment of the GPI anchor and just before and/or during secretion of CD52 into the epididymal lumen. Additional studies will be required to further define the posttranslational events encompassed in the expression of CD52 by the epididymis.

The acquisition of CD52 is one example of modifications that occur to the sperm membrane and glycocalyx during epididymal transport and capacitation (47) . However, the role of these modifications in sperm function and fertilization has not been completely elucidated. Characterization of the CD52 sperm glycoform and its unique carbohydrate epitopes provides the means to study specific glycocalyx alterations that are potentially significant for sperm maturation and fertilization. Furthermore, identity of the SAGA-1 core peptide with that of lymphocyte CD52 suggests potential function(s) of the unique CD52 glycoform of spermatozoa. This GPI-anchored glycoprotein is implicated in lymphocyte signal transduction pathways. Aggregation of CD52 on the lymphocyte surface was shown to induce intracellular calcium fluxes and T cell activation (48) . In addition, CD52 was identified on the lymphocyte membrane in large, noncovalently bound protein complexes that include intracellular tyrosine kinases (49) . Thus, inhibition of human sperm-zona binding in vitro by anti-SAGA-1 mAb's (9) suggests that the CD52 sperm glycoform may be involved in signal transduction during sperm capacitation or gamete interactions.

Although CD52 had previously been reported on the human sperm surface (26 , 29) , expression in lymphocytes had largely discounted its consideration as a candidate for immunocontraceptive development due to potential autoimmunity. However, the identification of unique N-linked carbohydrate epitopes on sperm CD52, the multiple sperm-inhibitory effects (including complement-dependent cytotoxicity) of antibodies to these unique epitopes, and localization of the sperm CD52 glycoform on all domains of the sperm surface suggest opportunities for immunocontraceptive development. Contraceptive approaches could include use of anti-sperm CD52 mAb's as an intra-vaginal spermicide or active immunization with carbohydrate epitopes specific to the sperm CD52 glycoform. Recent advances in oligosaccharide synthesis for vaccine development indicate the feasibility of the latter approach (50 , 51) .

In conclusion, our data demonstrate that a sperm glycoform of the CD52 lymphocyte differentiation marker is the cognate antigen identified by the first clinically relevant, sperm-inhibitory mAb to be immortalized from an infertile woman. In vitro, anti-SAGA-1/CD52 mAb's cause sperm agglutination, foster complement-dependent lysis and immobilization, and block sperm-zona interactions. The sperm glycoform of CD52, localized over all surface domains, is one of only a few known sperm surface isoantigens implicated in immunological infertility in humans. Additional studies are indicated to examine the apparent structural and immunological variation in N-linked glycosylation between the sperm and lymphocyte CD52 glycoforms and to investigate significance of this variation in the etiology of antibody-mediated infertility.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. John Shannon of the Microsequencing Core, Biomolecular Research Facility, University of Virginia, the staff of the Lymphocyte Culture and Tissue Procurement Cores at the University of Virginia, Leigh Ann Bush, Angela Rinker, and Melissa Bevard for technical assistance, and Dr. Tod McCauley for critical review of the manuscript. Supported by NIH HD U54 29099, P30 28934, U54 HD 28934, T32 HD 07382, F32 HD 08002, T32 DK 07642, D43 TW/HD 00654 from the Fogarty International Center, R43 HD 35771 to ContraVac, Inc., Virginia's Center for Innovative Technology Award BIO-98–002, Contraceptive Research and Development (CONRAD) Award CIG-97–15, and the Andrew W. Mellon Foundation.


   FOOTNOTES
 
1 Current address: Department of Obstetrics and Gynecology, Hyogo Medical College, Hyogo 663 Japan.

2 Current address: Ludwig Institute for Cancer Research, University College Branch, London W1P 8BT United Kingdom.

4 Abbreviations: GPI, glycophosphatidylinositol; mAb, monoclonal antibody; NGS, normal goat serum; PBS, phosphate-buffered saline; PI-PLC, phosphatidylinositol-specific phospholipase C; PVDF, polyvinylidene difluoride; SAGA-1, sperm agglutination antigen-1; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SIT, sperm immobilization test.

Received for publication September 14, 1998. Revision received February 18, 1999.
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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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