Proteolytic release and partial characterization of human sperm-surface glycopeptides

Sperm-surface glycopeptides were obtained from intact sperm membranes after proteolytic release by different enzymatic treatments such as autoproteolysis, trypsin, papain and pronase. Glycopeptides were isolated, their properties and composition were examined, and their monosaccharide and amino acid constituents were characterized. The monosaccharides identified were fucose, mannose, galactose, N-ac tylglucosamine, and N-acetylgalactosamine, which form part of more than one type of oligosaccharide units. Autoproteolytic treatment mainly provided O-glycosidic type oligosaccharides, while a mixture of Oand N-glycosidic oligosaccharides was obtained in variable proportions when treated with trypsin, papain or pronase. The highest degree of peptide cleavage was obtained with pronase. Despite the higher yields reached with trypsin, these glycopeptides contain the lowest percentage of oligosaccharide chains. Proteolytic treatment provides a simple, rapid procedure for the isolation of glycopeptides from the sperm surface. Correspondence


Introduction
Oligosaccharides are involved in the immunology of sperm and reproduction (1-7) and play a significant role in the spermoocyte interaction (8).Oligosaccharides constitute a singular source of biological relevant compounds for the knowledge of structural features of sperm cell membrane or any cell membrane.Despite their involvement in reproduction and immunological infertility (1), few studies have been carried out regarding the chemical structure of the carbohydrate constituents of the human sperm mem-brane glycoproteins and their nature and composition still remain unclear.Most of the efforts have been directed towards the identification of sperm antigens by immunochemical analysis of sperm cell membrane glycoproteins (2)(3)(4)(5).
Human sperm membrane from normal donors was examined and its macromolecular components were isolated and partially characterized (6).Autoproteolysis of normal human sperm produces peptides with oligosaccharide chains of the N-glycoside-linked type that are released from the surface-exposed parts of the glycoproteins.Cytochemi-cal characterization and localization of the N-glycoside-linked oligosaccharide receptors indicated that some are cryptic ones that become exposed by the action of proteolytic enzymes, and most receptors on the acrosome are eliminated when digestion is carried out in the presence of seminal plasma (6).
In the present investigation, the glycopeptides from human sperm membrane glycoproteins were studied in order to determine some of their chemical characteristics.These glycopeptides contain the carbohydrate moiety of the glycoproteins, and were obtained by the action of proteolytic hydrolases (autoproteolysis and extrinsic enzymes) on the sperm membrane.

Reagents
Pronase (Streptomyces griseus protease, type XXV), papain (from papaya latex), and trypsin (from bovine pancreas, type III) were obtained from Sigma Chemical Co.All chemicals used were of analytical grade from Merck.

Isolation of sperm
Sperm were obtained from healthy donors and isolated according to the method of Saji et al. (9).Semen was diluted with five volumes of phosphate buffered saline (PBS) and centrifuged at 840 g for 10 min.The supernatant was discarded and the cells were washed three times with the same buffer.

Proteolytic release of glycopeptides from sperm membranes
Washed sperm were suspended in PBS (for autoproteolysis), trypsin (0.8 mg/ml PBS), activated papain (10) (0.8 mg/ml PBS), or pronase (160 µg/ml PBS containing 4 mM CaCl 2 ), and then incubated at 37 o C. Release of glycopeptides was followed by periodic removal of aliquots (100 µl), centrifugation and assays for changes in the protein content and liberation of free amino acids and low molecular weight peptides.After incubation, the total supernatant containing the glycopeptide mixture was centrifuged at 2,840 g for 10 min at 4 o C, concentrated and freeze-dried.

Analytical methods
Protein analysis was carried out by the method of Lowry et al. (11).Total carbohydrate was determined by the phenol-sulfuric acid method (12), and low molecular weight peptides by the ninhydrin method (13).

Gas-liquid chromatography (GLC)
Monosaccharide analysis was carried out as described by Reinhold (14), employing the trimethylsilyl derivatives of the corresponding methyl glycosides.A Hewlett-Packard 5840 gas chromatograph was used.A W-HP column was eluted with nitrogen at 29 ml/min.Monosaccharides were obtained by trifluoroacetic acid treatment for 2 h at 121 o C, followed by methanolysis for 16 h at 65 o C. The ratio O-versus N-glycosidic linkages was estimated presuming that all GalNAc is present in O-glycosidic type chains, and is reported as mol GalNAc relative to Man = 3 (15,16), i.e., (O-/N-) = (GalNAc)/(Man/3).

Amino acid analysis
Amino acid analysis was performed as described elsewhere (17), using a Technicon Sequential Multi-Sample Amino Acids Analyzer TM S2 and the amino acids obtained by peptide hydrolysis with 6 M HCl at 105 o C for 24 h.Amino acids were purified with Amberlite IR-120 before analysis.

Trypan blue incubation
Aliquots of sperm suspensions were diluted with an equal volume of 2% Trypan blue in PBS, incubated at 37 o C for 15 min and centrifuged for 5 min at 900 g.The sperm pellet was washed in PBS until the supernatant became clear or pale blue (generally 2 washes), and then fixed for 30 min in formaldehyde.Formaldehyde was removed by centrifuging the sperm and washing the pellet in deionized water.Fixed sperm were placed on slides and air-dried.The slides were examined under a bright-field microscope at 1000X magnification, and at least 100 cells/sample were evaluated for each of 2 replicates.

Gel filtration chromatography
Fractions from proteolytic treatments were dissolved in water and loaded on a column (40 x 2 cm) of Bio-Gel P-6DG, exclusion limit 6 kDa (Bio-Rad Laboratories), equilibrated with water.Elution was performed with water at room temperature and 5-ml fractions were collected and monitored for carbohydrate and protein.The presence of salts was detected using the silver nitrate reagent.

Results
Sperm from normozoospermic donors were submitted to autoproteolysis and treatment with pronase, papain and trypsin to obtain glycopeptides from surface glycoproteins.Proteolysis was monitored over time and aliquots were analyzed for free amino acids and low molecular weight peptides, proteins, and carbohydrates.
The production of free amino acids and low molecular weight peptides varied slightly with the type of enzyme and/or sample.The protein/peptide release showed a regular pattern with an increased rate during the first 4 h followed by a concomitant decrease in the production of amino acids and low molecular weight peptides (see Figure 1, for trypsintreated samples).Autoproteolysis produced larger amounts in some samples and contin-ued without reaching a constant value as observed in the other treatments.Nevertheless, all samples showed the expected behavior with the enzymatic treatments, i.e., a decrease in protein content and an increase in amino acid amount against time.
The release of carbohydrate chains at different times is shown in Figure 2. Papain treatment caused the release of larger amounts of carbohydrates than trypsin or autoproteolysis at 4 h, although similar values were reached by later incubation with trypsin.Treatment of sperm with pronase produced a release of 115 µg carbohydrates per mg total sperm protein after 1 h.which caused lower levels of cell injury than the treatments with trypsin and papain (Figure 3).After 1-h incubation with pronase, the percentage of Trypan blue-positive sperm increased by 18%.Digestion of sperm for a prolonged time with no disintegration of cells was previously observed by Mazzini et al. (6).Samples obtained from the above treatments were isolated and studied.The glycopeptide mixtures released at 4 h (treatments with trypsin, papain or autolysis) or at 1 h (pronase treatment) were analyzed by size exclusion chromatography.A similar profile of molecular weight distribution was observed within two main fractions: the glycopeptide-containing fraction eluted with the void volume (exclusion limit 6 kDa) and a polydisperse fraction containing the free amino acids and low molecular weight peptides (data not shown).Some yields of the isolated sperm glycopeptide fractions are shown in Table 1.The carbohydrate content of the produced glycopeptides was monitored and calculated as shown in Table 1.
The carbohydrate composition of the oligosaccharide chains was analyzed.The monosaccharides found were fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine (Table 2).Galactose was always the main constituent, with variable proportions of the other monosaccharides.Glucose was identified mainly in autolyzed and trypsin-treated samples.
O-versus N-linked oligosaccharide ratios were estimated as described in Material and Methods.As shown in Table 2, autoproteolysis produced a molar ratio of O-glycosidic to N-glycosidic oligosaccharide chains of 10:1; trypsin and papain released elevated amounts of N-type oligosaccharides.When sperm were treated with pronase higher amounts of N-glycosidic chains were obtained (molar ratio O-/N-was 1:2).
Amino acid analysis was carried out on representative samples only.Aspartate, threonine, serine and glutamate were the main Table 1 -Release of glycopeptides from the surface of normal human sperm.Sperm were incubated as described in the legend to Figure 2 and the glycopeptides released at 4 h (trypsin, papain or autolysis) or at 1 h (pronase) were analyzed.Yield is reported as mg dry weight/20 normospermic samples containing 80-140 x 10 6 cells.a Non-dialyzable material after autoproteolysis: 21% amino acid/low molecular weight peptide content; small quantities of NeuNAc were also detected by the Warren assay (37).Glycopeptides of mass higher than 6 kDa were not detected by size exclusion chromatography on Bio Gel P-6DG.After prolonged incubation the cells became immotile, but were apparently intact by the criteria of light microscopy.To provide more evidence about the status of sperm, cell damage was evaluated using the vital stain Trypan blue.An increase in cell death was observed after 4 h for autoproteolysis constituents of the peptide moiety together with variable amounts of alanine, glycine, leucine, and lysine (Table 3).The percentages of the amino acids involved in the proteolysis breakdown, namely, tyrosine, lysine and arginine, were variable and usually low.Methionine was not detected.

Discussion
The sperm-surface glycopeptides from normospermic donors were examined.Sperm antigen preparations are difficult to deal with because of the somewhat rigid sperm cell membrane that resists the usual solubilization methods.For the isolation of glycopeptides we used autoproteolysis, a technique which has been previously demonstrated to be useful (6).Other types of proteolytic enzymatic degradation were also investigated, such as trypsin and papain which have a more defined action, and pronase which has a wide specificity of action, cleaving glycosylated proteins of the membrane surface (18).
Acrosin, the sperm-specific protease involved in several aspects of fertilization (19,20), and trypsin possess similar proteolytic activity and specificity for peptide bonds which involve the basic amino acids lysine and arginine.Pronase is less specific (hydrolyzes arginine bonds and others) and differs from trypsin and other proteases since there are no known pronase inhibitors in the human sperm.Papain is commonly used for the release of ectoenzymes from membranes without incurring any loss of activity (21,22).Proteolytic enzymes other than acrosin have been extracted from sperm but are not well characterized, and activities of fibrinolytic enzymes (seminin), plasminogen activators, pepsin-like proteinase, aminopeptidase and dipeptidase have been described in seminal plasma (23).Treatments with extrinsic enzymes also involve intrinsic proteases activated by the hydrolytic conditions which characterize the autoproteolytic treatment.
The results of the enzymatic treatment,  despite showing a similar pattern for the release of the glycoconjugates, indicate some important differences and also the accessibility or exposure of the carbohydrate chains on the sperm membrane to the action of the different enzymes.The differences observed in the monosaccharide composition of the sperm membrane glycopeptides released after different enzymatic treatments indicate that important variations in the nature of the glycopeptides occur according to the mode of action of the enzyme(s).The monosaccharides galactose, fucose, mannose, N-acetylglucosamine and Nacetylgalactosamine, that are present in the sugar moiety of human sperm as indicated by lectin studies (24,25), were identified in the present study.Glucose found in considerable relative proportions in some cases is not a usual monosaccharide in glycoproteins (26), and therefore may indicate release of a glycolipid component from the membrane in the case of prolonged treatment (autolysis, trypsin and papain), but not in the case of degradation with pronase which is fast and effective (30-60 min) (27).The carbohydrate composition of these glycopeptides is unlike that of either the N-glycosyl or the Oglycosyl type, since it contains GalNAc as well as Fuc and Glc, in addition to Man, Gal and GlcNAc residues.However, in the peptide moiety, a significant content of Asp and/ or Thr/Ser suggests a predominant proportion of both N-and O-glycosylated chains in the original glycoprotein.Under mild conditions, trypsin caused the removal of glycoprotein from the surface of Macaca radiata sperm (28) and from human semen (29), albeit of higher molecular weight, results similar to those obtained in the present investigation.
The Asp (Asp + Asn) and Ser + Thr contents of the autolysis products were 10.6 and 18.0 mol%, respectively.Hence, the O-/ N-ratio can only be 10:1 if the N-glycosylated Asn is 1.8 mol%, or 17% of Asp content of the amino acid analysis.Extending the same rationale to the other treatments, the N-glycosylated Asn is 36% for trypsin and 41.5% for papain.In the case of pronase treatment, the O-/N-ratio was estimated as 1:2, indicating a relative higher number of N-linked carbohydrates.
The specific shape of complex oligosaccharide-protein structures located on the extracellular side of membrane-bound peptides is thought to be responsible for their function as carriers of significant biological information and cellular recognition (30)(31)(32).The importance of these oligosaccharides in reproductive physiology has been demonstrated by several observations.The human sperm cell membrane antigen gp12, which is highly specific for sperm and seminal plasma, has been isolated from a plasma membrane fraction (9).The antigenic determinant of this molecule, recognized by the corresponding antibody, should be located in the sugar portion of the glycoprotein (33).On the other hand, antisperm monoclonal antibodies, reacting to glycosylated epitopes, presented very strong properties of sperm agglutination and/or immobilization (24).Finally, gamete interaction is mediated by protein-carbohydrate recognition (34) and the clustering of sperm receptors to the zona pellucida oligosaccharides has been proposed as a mechanism of acrosomal exocytosis induction (35,36).In conclusion, the present study provides relevant information about the chemical properties of human sperm-surface glycopeptides.

Figure 1 -Figure 2 -
Figure 1 -Time course of protein ( • ) and amino acid (à) re- lease by proteolytic treatments.The figure presents data for trypsin-treated normozoospermic samples as a typical pattern of peptide release.Sperm (90 x 10 6 ) were incubated at 37 o C in PBS containing 0.8 mg/ml trypsin for different times.Assays for amino acids/ low molecular weight peptides, and proteins were carried out by the ninhydrin and Lowry methods, as described in Material and Methods.

Table 2 -
Monosaccharide composition of human sperm-surface glycopeptides released by different proteolytic enzymes.Sperm were incubated as described in the legend to Figure2and the glycopeptides released at 4 h (trypsin, papain or autolysis) or at 1 h (pronase) were analyzed.Results are reported as mol%.The molar ratio of O-glycosidic to N-glycosidic oligosaccharide chains was estimated as described in Material and Methods.

Table 3 -
Amino acid composition of human sperm glycopeptide fractions obtained by proteolytic treatment of normal sperm samples.