Molecular Human Reproduction

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Molecular Human Reproduction, Vol. 8, No. 3, 221-227, March 2002
© 2002 European Society of Human Reproduction and Embryology

Testis and spermatogenesis

Purification and characterization of human seminal plasma -L-fucosidase

Sumpars Khunsook¹, Jack A. Alhadeff² and Barry S. Bean^1,3

¹ Department of Biological Sciences and ² Division of Biochemical Sciences, Department of Chemistry, Lehigh University, Bethlehem, PA 18015, USA

	Abstract

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Human seminal plasma -L-fucosidase (EC 3.2.1.51) has been purified 7100-fold to very high purity and specific activity (83 000 nmol/min/mg protein) by affinity chromatography on agarose--aminocaproyl-fucopyranosylamine. The purified -L-fucosidase appeared to contain a single subunit of 56–57 kDa (as determined by SDS–PAGE and Western analysis). Lectin blotting and N-glycanase treatment studies indicated that this subunit is N-glycosylated and contains sialic acid residues. Human seminal plasma -L-fucosidase was shown to contain three multimeric forms of 110, 236 and 314 kDa respectively (as determined by Sephadex^® G-200 chromatography) and therefore probably exists in dimeric, tetrameric and hexameric forms. Kinetic analysis with the 4-methylumbelliferyl--L-fucopyranoside (4MU-Fuc) substrate indicated a broad acidic optimum (pH 4.0–4.5) with a second neutral optimum (pH 6.4–7.4) with 60–80% of maximal activity. Apparent K_M and V_max values for the 4MU-Fuc substrate were determined to be 0.06 mmol/l and 92 µmol/min/mg protein respectively, using Lineweaver–Burk double reciprocal plots. Isoelectric focusing and neuraminidase treatment studies provided further evidence that the purified seminal plasma -L-fucosidase is a sialoglycoprotein with several isoforms between pI values 5–7. The acidic isoforms between pI values 5–6 appear to be related chemically to the more neutral isoforms by sialic acid residues since neuraminidase treatment converted the former into the latter isoforms.

glycosidase/glycoprotein/semen/sperm

	Introduction

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
Several groups have reported the presence of substantial levels of various glycosidases in the seminal plasma and/or sperm cells of several mammals, including humans (Bostrom and Ockerman, 1971; Ben Ayed et al., 1989; Alhadeff et al., 1999), rodents (Self et al., 1978; Avilés et al., 1996; Abascal et al., 1998), dogs (Dubé et al., 1985; Veeramachaneni et al., 1998), and cattle (Jauhiainen and Vanha-Perttula, 1986; Srivastava et al., 1986). Various lines of evidence suggest that these glycosidases may participate in molecular modifications of: (i) the surface of sperm cells during their life history (Töpfer-Petersen, 1999); (ii) the interactions of sperm with tissues of the female reproductive tract following the arrival of the ejaculate (Revah et al., 2000); or (i) the gametes themselves during sperm–oocyte interaction (Tulsiani et al., 1989; Wassarman, 1999). Some evidence suggests that the soluble glycosidases of the seminal plasma originate in the prostate (Dubé et al., 1985; Ben Ayed et al., 1989), but synthesis in other organs of the male reproductive system (e.g. seminal vesicle, epididymis, Cowper's gland) must remain under consideration (Eliasson and Johnsen, 1986). Isoforms of the soluble enzyme -1,4-glucosidase are found in human semen; the neutral isoform is considered a good marker for epididymal secretory function (Zöpfgen et al., 2000), while the acidic form is reported to originate primarily in the prostate (Paquin et al., 1984; Cooper et al., 1990). Our previous report (Alhadeff et al., 1999) showed that human semen contains very large amounts of -L-fucosidase activity, the great majority of which is in the seminal plasma. We demonstrated that the human seminal plasma -L-fucosidase is different from the sperm cell membrane -L-fucosidase. Others (Focarelli et al., 1997) have also provided evidence for different -L-fucosidases in seminal plasma and on sperm plasma membranes from the bivalve Unio elongatulus.

The functions of the -L-fucosidases of the seminal plasma and the sperm surface are not clear at present, but they may have important roles in reproduction. Several previous studies have suggested that L-fucose and/or L-fucose-containing molecules can inhibit mammalian sperm–oocyte interactions in hamsters (Ahuja, 1982), mice (Boldt et al., 1989; Johnston et al., 1998), rats (Shalgi, 1986) and humans (Lucas, 1994; De Cerezo et al., 1996). L-Fucose can also inhibit sperm binding to oviductal explants (Lefebvre and Suarez, 1997). Complex interactions between sperm and the tissues of the female reproductive tract, and associated sperm remodelling, involve recognition and modification of carbohydrate moieties (Suarez, 2001). Several studies have demonstrated the presence of a plasma membrane-associated -L-fucosidase on spermatozoa from bulls (Jauhiainen and Vanha-Perttula, 1986), rats (Avilés et al., 1996; Abascal et al., 1998) and humans (Alhadeff et al., 1999). In addition, -L-fucosidase from bull seminal plasma has been reported (Srivastava et al., 1986) to promote the acrosome reaction of guinea-pig spermatozoa in vitro.

To understand better the functions of -L-fucosidase from human seminal plasma, and to distinguish its different isoforms, we have purified and characterized the enzyme. The enzyme has been purified ~18000-fold in a 60–75% yield to high purity by affinity chromatography (Alhadeff et al., 1975), and the purified enzyme has been characterized with regard to its aggregated forms, subunit and isoform compositions, N-glycosylation and sialylation, pH optima, and kinetic properties.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
General
Enzyme activity for -L-fucosidase was assayed at pH 7.0 using 1 mmol/l 4-methylumbelliferyl--L-fucopyranoside (4MU-Fuc; Sigma Chemical Co., St Louis, MO, USA) as substrate, with subsequent determination of fluorescence emitted at 460 nm, as previously described (Alhadeff et al., 1999). One unit of enzyme activity is defined as the amount of enzyme that will hydrolyse 1 nmol of 4MU-Fuc per min at 37°C. In some cases, 1% L-fucose was present in enzyme suspensions; if so, L-fucose was diluted significantly or removed by dialysis prior to conducting enzyme assays.

Protein concentration was determined (Bradford, 1976) using Coomassie Brilliant Blue G-250 (BioRad, Hercules, CA, USA), following the standard assay and microassays (Rosenberg, 1996).

Enzyme purification
Human semen preparation
Semen samples were obtained from healthy male volunteers following protocols approved by the Lehigh University Institutional Review Board, including informed consent and protection of the identity of the donors. The sperm parameters were within the normal ranges for morphology, motility and numbers based on standard criteria (World Health Organization, 1999). After liquefaction (20–30 min) at room temperature, semen was diluted with Dulbecco's phosphate-buffered saline (PBS), pH 7.4 (1:1, v/v) and centrifuged at 600 g for 20 min. The supernatant seminal plasma was centrifuged at 105 000 g for 30 min at 4°C. The supernatant was dialysed against 100 volumes of 10 mmol/l sodium phosphate buffer, pH 5.5, containing 0.02% NaN₃ (w/v) three times for 1 h each and two more times, each overnight at 4°C. The dialysed seminal plasma was centrifuged at 10 000 g for 30 min to remove the proteins precipitated during dialysis.

Affinity chromatography
Substrate analogue chromatography was employed to purify -L-fucosidase (Alhadeff et al., 1975). Dialysed seminal plasma containing ~800-1250 units of -L-fucosidase was put onto the affinity column (14.5 ml) of agarose--aminocaproyl-fucosamine (ICN, Costa Mesa, CA, USA), equilibrated for ~20 min, and the flowthrough seminal plasma was collected and assayed for -L-fucosidase activity to identify the capacity of the column. The column was washed with sodium phosphate buffer, pH 5.5, containing 0.02% (w/v) NaN₃ until the absorbance at 280 nm stabilized at ~0.002. The -L-fucosidase was eluted from the column with 1% (60 mmol/l) L-fucose (Sigma) in the same buffer and eluted fractions (2.3 ml) were collected and assayed for enzyme activity. The fractions that contained -L-fucosidase were subsequently concentrated with a microconcentrator (Centricon-30; Millipore Corp., Bedford, MA, USA) or with a pressure ultrafiltration unit and ultrafiltration membrane (Diaflo 25YM10 or 25YM30, Millipore) under N₂ pressure (60 psi).

For studies on enzyme stability, purified seminal plasma -L-fucosidase, in the elution buffer above, was distributed in 10 µl quantities for storage at either 2–4°C or –20°C. Initially, and periodically thereafter, stored samples were tested for -L-fucosidase activity (for 5 min at 37°C).

Gel filtration chromatography
Sephadex^® G-200 (4 g) (Pharmacia LKB Biotechnology, Bromma, Sweden) chromatography was done using phosphate-buffered saline (PBS, 137 mmol/l NaCl, 2.7 mmol/l KCl, 8 mmol/l Na₂HPO₄^·7H₂O, 1.5 mmol/l KH₂PO₄, pH 7.0) containing 0.02% (w/v) NaN₃ (Sigma) as a bacteriostat in a pre-equilibrated column (98x0.5 cm). Standard proteins (0.5–2 mg each) included bovine thyroglobulin (670 kDa, Sigma), lactate dehydrogenase from rabbit muscle (150 kDa, Sigma), human serum albumin (67 kDa, Sigma), ovalbumin (45 kDa, ICN), and cytochrome C (12.4 kDa, Sigma). The void volume was determined as the volume required for passage of blue dextran (2000 kDa, Sigma). Approximately 0.5–1 ml samples (containing 20–50 units of -L-fucosidase) were chromatographed, and fractions (1.1 ml) were collected and assayed for protein (by absorbance at 280 nm) and for -L-fucosidase activity.

Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE)
This was carried out using conventional methods (Laemmli, 1970) as previously used for -L-fucosidase (Alhadeff et al., 1999). Briefly, 4% stacking and 10% separating gels were run at room temperature using 25 mmol/l tris, 0.2 mol/l glycine buffer, pH 8.3, containing 0.1% SDS and 2.5% (v/v) 2-mercaptoethanol for 1.5 h at 75 V and for 3 h at 150 V respectively. Molecular mass standards (BioRad) were run and stained as described (Alhadeff et al., 1999). For silver staining, additional molecular mass standards (Bio Rad low range for silver staining) were run and staining was accomplished with a standard method (Rosenberg, 1996).

Western analysis
The proteins from slab gels were electrotransferred to polyvinylidene difluoride membrane (Hybond-P; Amersham, Little Chalfont, Buckinghamshire, UK) as described (Alhadeff et al., 1999). The membrane was incubated for 1 h in a 200-fold dilution of the immunoglobulin G (IgG) fraction of anti-human liver -L-fucosidase polyclonal antibody from a goat (Andrews-Smith and Alhadeff, 1982), and washed three times with Tris-buffered saline–Tween-20 (TBST). The secondary antibody, rabbit anti-goat horse-radish peroxide-conjugated F(ab')₂ antibodies (Cappel; ICN), was diluted 10⁴-fold, incubated with the membrane for 1 h and washed three times with TBST. Development was accomplished using enhanced chemiluminescence (ECL) (Amersham, following instructions provided by the manufacturer) for 1 min, and the membrane was exposed to X-Omat LS film (Eastman Kodak, Rochester, NY, USA).

For lectin blotting, polyvinylidene difluoride membranes (containing electrotransferred proteins) were blocked overnight in 5% (w/v) bovine serum albumin (BSA; Sigma) in TBST containing 20 mmol/l Tris, 0.9% (w/v) NaCl and 0.1% (v/v) Tween-20. The membranes were washed twice (10 min each) with TBST and once for 10 min with TBS (without Tween-20) containing 1 mmol/l MnCl₂, 1 mmol/l MgCl₂ and 1 mmol/l CaCl₂. The membranes were incubated with biotinylated lectins [Galanthus nivalis agglutinin (GNA), Sambucus nigra agglutinin (SNA), or Maackia amurensis lectin II (MAL II); Vector Laboratories, Inc., Burlingame, CA, USA] at concentrations of 1 µg of biotintylated GNA/ml of TBST (1% BSA), 2 µg of biotinylated SNA/ml of TBST (1% BSA), and 1 µg of biotinylated MAL II/ml of TBST (1% BSA), for 1 h at 21°C. The membranes were washed three times in TBST (10 min/wash) and subsequently incubated with avidin and biotinylated peroxidase macromolecular complex (Vectastain Elite ABC Kit, Vector, following the manufacturer's recommendations, except that the ABC mix was prediluted 1:2, v/v and the final mixture contained 1% BSA) for 30 min. The membranes were washed three times with TBST (0.1% Tween-20) and developed using ECL (Amersham). Correspondence of reactivity with that of authentic human liver -L-fucosidase, and consistency with previously published carbohydrate specificities (Argade et al., 1988; Alhadeff, 1998; Alhadeff et al., 1999) confirmed the predicted specificity of lectin binding.

N-glycanase treatment
Purified seminal plasma -L-fucosidase (74 ng) and purified human liver -L-fucosidase (0.5 µg) (as a positive control) were denatured at 100°C for 3 min in the presence of 0.5% (w/v) SDS and 2.5% (v/v) 2-mercaptoethanol. The samples were diluted with pH 8.6, 0.55 mol/l Na₂HPO₄ buffer to reach final concentrations of 0.2 mol/l Na₂HPO₄, 1.25% (v/v) Nonidet P-40 (Sigma), 0.1% SDS and 1.1% 2-mercaptoethanol. N-glycanase (Genzyme Corp., Framingham, MA, USA) was added to give final concentrations of 10 or 15 units/ml, and the reaction mixtures were incubated at 37°C for 17 or 20 h. Controls that contained all constituents except N-glycanase were run, and N-glycanase was also run alone. The reaction mixtures were evaluated by SDS–PAGE and silver staining (as described above).

pH optimum and kinetic studies
The pH activity curves for purified seminal plasma -L-fucosidase were determined as described (Abascal et al., 1998) using duplicate assays for 10 min at 37°C, and actual pH values of a third set of mock (lacking substrate) tubes were recorded. Apparent K_M values were determined graphically from a double reciprocal plot (Lineweaver and Burk, 1934) using 4MU-Fuc (0.003–0.66 mmol/l) as substrate. The purified enzyme was dialysed against 10 mmol/l sodium phosphate buffer, pH 5.5, for 2–3 h, with 2–3 changes of buffer, to remove L-fucose, and assayed in 0.1 mol/l citric acid/trisodium citrate, pH 4.3, for 5 min.

Isoelectric focusing
Isoelectric focusing was performed on a 40 ml column with 2% (v/v) ampholine ampholytes (pH range 5–8) (Pharmacia LKB Biotechnology, Bromma, Sweden), essentially as described (Abascal et al., 1998) on 4–25 units of -L-fucosidase activity. Electrofocusing was conducted at 600 V (3–3.5 mA starting amperage) for 15–20 h after which time 0.25–0.35 ml fractions were collected. The pH of each fraction was determined, each fraction was assayed for -L-fucosidase activity, and data were plotted as -L-fucosidase activity versus isoelectric point (pI).

Neuraminidase treatment
Purified seminal plasma -L-fucosidase (15 units) was incubated with 1.5 units of neuraminidase (Clostridium perfringens, type x; Sigma) for 6 h or 17.5 h at 37°C in 0.1 mol/l citric acid/citrate buffer, pH 5.0. Negative controls were incubated in parallel without neuraminidase. Activities of -L-fucosidase were determined before and after incubation, and the samples were subjected to isoelectric focusing (see above) to determine if neuraminidase treatment altered the -L-fucosidase isoform profile.

	Results

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
An effective procedure was developed for the purification of -L-fucosidase from human seminal plasma. The process involves low and high speed centrifugation, dialysis, and affinity chromatography as detailed in Materials and methods, and subsequent concentration by ultrafiltration or centrifugal concentration. This procedure typically resulted in a purification of ~7100-fold to a very high specific activity of ~83 000 units/mg protein and yields in the range of 60–75%, as summarized in Table I.

View this table: [in this window] [in a new window]   Table I. Purification summary for human seminal plasma -L-fucosidase

 
The stability of purified seminal plasma -L-fucosidase was evaluated following periods of storage at 2–4°C and –20°C, at pH 5.5 in the presence of 1% L-fucose. Samples of the frozen material retained 100% of enzyme activity for >6 weeks. Samples kept refrigerated showed slow but progressive loss of -L-fucosidase activity, with 77% of initial activity remaining after 6 weeks of storage. Incidental observations also showed that 100% enzyme activity remained following aseptic storage at room temperature (~21°C) for 3 days. Control conditions for the neuraminidase experiment (described below) showed that the enzyme retains 100% activity following 6 h of incubation at 37°C. Enzyme activity appeared to be more stable (i) in solutions containing higher concentrations of enzyme, (ii) in solutions containing higher concentrations of other proteins such as albumin, and (iii) in the presence of L-fucose.

Figure 1 illustrates the pattern of migration of crude seminal plasma proteins (lane 3) and purified seminal plasma -L-fucosidase (lane 4) in denaturing polyacrylamide gel electrophoresis. The SDS-denatured purified enzyme appears to be a single protein band corresponding to ~56–57 kDa, about the same size as the large M_r subunit (56 kDa) found in human liver -L-fucosidase (lane 5) (Alhadeff, 1998), and consistent with the Western blotting pattern previously reported for crude seminal plasma -L-fucosidase (Alhadeff et al., 1999). Seminal plasma -L-fucosidase was readily detected (lane 7) in Western blot analysis with goat antibody prepared against human liver -L-fucosidase (Andrews-Smith and Alhadeff, 1982) and the band co-migrated with the protein band in lane 4. Western blots of purified seminal plasma -L-fucosidase showed only one broad band. As also evident in Figure 1, the seminal plasma -L-fucosidase appeared to be highly purified or homogeneous, even when detected by the very sensitive Ag-staining method. Staining with Coomassie Blue showed no contaminating protein bands (data not shown). Several specimens obtained from post-vasectomy donors showed the same major seminal plasma -L-fucosidase enzyme activity and the same major glycoprotein band in electrophoretic profiles as that obtained from intact donors (data not shown).

View larger version (82K): [in this window] [in a new window]   Figure 1. Electrophoretic analysis of crude and purified human seminal plasma -L-fucosidase. SDS–PAGE followed by silver staining (lanes 1–5) and Western-blot analysis (lanes 6–8) of: lane 1, sample buffer; lane 2, protein standards (Bio-Rad, low range); lanes 3 and 6, crude seminal plasma (40 µg protein); lanes 4 and 7, purified seminal plasma -L-fucosidase (30 ng protein; ~56 kDa); lanes 5 and 8, purified human liver -L-fucosidase (50 ng protein; ~51 kDa). See Materials and methods for details.

 
The results of lectin blotting of purified seminal plasma -L-fucosidase (lanes 2, 4, 6) compared to purified human liver -L-fucosidase (lanes 1, 3, 5) are shown in Figure 2. Both fucosidases were recognized strongly by GNA (lanes 1, 2), recognized less strongly by SNA (lanes 3, 4) and not recognized by MAL II (lanes 5, 6). These results suggest that purified seminal plasma -L-fucosidase is similar in its glycosylation to human liver -L-fucosidase (Argade et al., 1988) and contains oligomannoside-type structures as well as -2,6 (but not -2,3) sialylated N-acetyllactosaminyl-type structures.

View larger version (105K): [in this window] [in a new window]   Figure 2. Lectin blot analysis of purified human seminal plasma -L-fucosidase. Lanes 1, 3 and 5 each contain 62 ng purified human liver -L-fucosidase; lanes 2, 4 and 6 each contain 56 ng purified human seminal plasma -L-fucosidase; lanes 1 and 2 were incubated with biotinylated GNA (1 µg/ml of TBST); lanes 3 and 4 were incubated with biotinylated SNA (2 µg/ml of TBST); and lanes 5 and 6 were incubated with biotinylated MAL II (1 µg/ml of TBST). See Materials and methods section for details.

 
The results of N-glycanase treatment of purified seminal plasma -L-fucosidase (as determined by SDS–PAGE) are shown in Figure 3. After treatment for 20 h using 15 units N-glycanase/ml, two protein-staining bands are seen at 51 and 45 kDa (lane 6) rather than the ~56 kDa band seen after comparable treatment without N-glycanase (lane 5). Under less drastic digestion conditions (17 h, 10 units N-glycanase/ml), the 51 and 45 kDa bands were seen along with a light, protein-staining band remaining at 56 kDa (data not shown). The combined results suggest that the 51 and 45 kDa bands represent partially and fully N-deglycosylated -L-fucosidase respectively. Treatment of human liver -L-fucosidase with N-glycanase (20 h, 15 units N-glycanase/ml) led to a 45 kDa (presumably N-deglycosylated) protein-staining band (lane 4), compared to ~51 kDa band for the heat-treated control (lane 3). The protein-staining bands at 34 kDa (lanes 4, 6) correspond to N-glycanase (as determined in other experiments; data not shown). No evidence of significant O-glycosylation was obtained, so further investigation of this possibility was not pursued (see Discussion).

View larger version (80K): [in this window] [in a new window]   Figure 3. SDS–PAGE of purified human seminal plasma -L-fucosidase before and after N-glycanase treatment. Lane 1, sample buffer; lane 2 standard protein molecular-mass markers for silver staining; lane 3, 75 ng of human liver -L-fucosidase; lane 4, human liver -L-fucosidase treated for 20 h at 37°C with N-glycanase; lane 5, 24 ng of purified seminal plasma -L-fucosidase; lane 6, purified seminal plasma -L-fucosidase treated for 20 h at 37°C with N-glycanase. See Materials and methods for details.

 
To evaluate the multimeric composition of the seminal plasma -L-fucosidase in its native state, purified seminal plasma -L-fucosidase was subjected to gel filtration on Sephadex^® G-200. Effluent fractions were monitored for enzyme activity and protein content, and the approximate mol. wts of the peak fractions were calculated by comparison with the elution positions of known protein standards. The results of three gel filtration profiles were consistent, and showed a major peak with a calculated mol. wt of 236 ± 1 kDa (mean ± SD for three experiments), and two other peaks with approximate mol. wts of 314 ± 1.7 kDa, and 110 ± 6.5 kDa respectively.

Activity of the purified -L-fucosidase of human seminal plasma was determined at different pH values, as detailed in Materials and methods. A typical pH optimum curve is illustrated in Figure 4, showing substantial activity over a broad range of pH values, with an optimum in the acid range (pH 4.0–4.5), but with retention of 60–80% of maximal activity into the neutral range (pH 6.4–7.4). Overlapping values of pH were achieved using the different buffer systems described in Materials and methods, and no significant buffer effects were evident in the regions of overlap.

View larger version (12K): [in this window] [in a new window]   Figure 4. pH–activity curve for purified human seminal plasma -L-fucosidase. See Materials and methods for details.

 
Isoelectric focusing profiles for purified human seminal plasma -L-fucosidase were conducted on several preparations. Profiles of different preparations, including those from different semen donors, consistently resolved several isoforms with pI values of 5.0–7.0, as illustrated in Figure 5A. This profile is similar to the profile published previously for the -L-fucosidase activity within crude seminal plasma (Alhadeff et al., 1999). There were significant changes in the focusing profile for purified seminal plasma -L-fucosidase following treatment with neuraminidase, as shown in Figure 5B,C. A heat-treated negative control sample of -L-fucosidase (data not shown), incubated in the absence of neuraminidase, gave a profile similar to that of Figure 5A. Following 6 h (Figure 5B) or 17.5 h (Figure 5C) digestion with neuraminidase at 37°C, isoforms with pI < 6.2 were markedly reduced, with proportionate increase in peaks of pI > 6.2. The untreated sample had 55% of its enzyme activity associated with acidic isoforms of pI 6, while the 6 h and 17.5 h neuraminidase-treated samples showed corresponding reductions of isoforms of pI < 6 to 15.6% and 0% respectively.

View larger version (15K): [in this window] [in a new window]   Figure 5. Isoelectric focusing profiles for purified human seminal plasma -L-fucosidase: (A) untreated purified human seminal plasma -L-fucosidase; (B) purified human seminal plasma -L-fucosidase following treatment at 37°C with neuraminidase for 6 h; (C) purified human seminal plasma -L-fucosidase following treatment at 37°C with neuraminidase for 17.5 h. See Materials and methods for details.

 
The kinetics of purified seminal plasma -L-fucosidase were examined in three separate experiments using 4MU-Fuc as substrate. In all cases, data reflected the expected simple kinetic profiles as have been reported previously for human liver -L-fucosidase (Alhadeff, 1998), with Lineweaver–Burk double reciprocal plots closely approximating a straight line. For the purified human seminal plasma -L-fucosidase, an apparent K_M of 0.06 ± 0.004 mmol/l, and a V_max of 92 ± 7.5 µmol/min/mg protein (means ± SD for three experiments) were found.

	Discussion

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
In the present investigation, human seminal plasma -L-fucosidase has been purified by affinity chromatography to a specific activity considerably higher than the values previously found for human -L-fucosidases purified from liver (24 200 units/mg protein) (Alhadeff et al., 1975), brain (7510 units/mg protein) (Alhadeff and Janowsky, 1977), and serum (16 000 units/mg protein) (Alhadeff and Janowsky, 1978), and from rat epididymis (6500–15 600 units/mg protein) using the p-nitrophenylfucoside substrate (Carlsen and Pierce, 1972; Wright et al., 1976; Jain et al., 1977). The purified seminal plasma -L-fucosidase appears to contain a single subunit (near 56 kDa), consistent with our previous findings by Western blotting analysis of crude seminal plasma (Alhadeff et al., 1999). Only one subunit (51 kDa) has been found for human brain -L-fucosidase (Alhadeff and Janowsky, 1977), but two non-identical subunits have been found for human -L-fucosidases from liver (51 and 56 kDa) (Alhadeff et al., 1999) and from serum (54 and 56.5 kDa) (Alhadeff and Janowsky, 1978). Purified rat epididymal -L-fucosidase has been shown to contain one subunit of 54 kDa (Wright et al., 1976) or two subunits of 47 and 60 kDa (Carlsen and Pierce, 1972) or 50 and 57 kDa (Jain et al., 1977).

As reported here, lectin blotting and N-glycanase treatment studies of purified seminal plasma -L-fucosidase indicated that it is N-glycosylated and contains sialic acid residues linked -2,6 to Gal or GalNAc. It is likely that seminal plasma -L-fucosidase is similar in its glycosylation to human liver -L-fucosidase (Argade et al., 1988) and contains both oligomannoside-type and -2,6 sialylated N-acetyllactosamine-type structures. Gel filtration studies of purified human seminal plasma -L-fucosidase indicated the presence of three multimeric forms. These results, in conjunction with the SDS–PAGE results, suggest the presence of dimeric, tetrameric and hexameric forms of the enzyme. These results are similar to multimeric forms found previously for human liver (230 kDa), serum (296 kDa), placenta (200 and 305 kDa) and spleen (100 kDa) -L-fucosidases, and for rat epididymal (210–220 kDa) and rat liver (160, 217 and 300 kDa) -L-fucosidases (Alhadeff, 1998; Table I).

Isoelectric focusing indicated that the purified seminal plasma -L-fucosidase consisted of multiple isoforms, similar to what we have reported for crude seminal plasma -L-fucosidase (Alhadeff et al., 1999), and suggesting that all isoforms of fucosidase were purified by our scheme. The results for the seminal plasma fucosidase are similar to the isoform profiles reported for numerous mammalian -L-fucosidases (Alhadeff, 1998: Table 1), but different from the one isoform at pI 6.3 reported for rat epididymal -L-fucosidase (Carlsen and Pierce, 1972). Results of treatments with neuraminidase constituted strong evidence that human seminal plasma -L-fucosidase is a sialoglycoprotein, and may occur in different isoforms depending on the number of terminal sialic acid residues present on the glycoprotein molecule. The more acidic isoforms appeared to be converted to more neutral isoforms by progressive treatment with neuraminidase (Figure 5). These results are similar to previous results on the chemical relationship of the isoforms of human liver, serum, placenta, and spleen -L-fucosidases (Alhadeff, 1998).

The possibility of O-glycosylation has not been addressed experimentally in this investigation. The properties of the seminal plasma -L-fucosidase are very similar to those of the human liver enzyme (Alhadeff, 1998), for which there is no evidence of O-glycosyl components. In addition, the seminal plasma -L-fucosidase is reduced to a mol. wt of ~45 kDa following treatment with N-glycanase (Figure 3), consistent with the size of the polypeptide that corresponds to the 439 amino acids for the final human FUCA1 gene product reported by one study (Kretz et al., 1992). Together, these considerations suggest that there is little, if any, O-glycosylation of the seminal plasma -L-fucosidase.

Kinetic analysis of purified human seminal plasma -L-fucosidase indicated an acidic pH optimum with a second, somewhat smaller neutral optimum. These results are comparable to those found for human liver -L-fucosidase (Alhadeff et al., 1975), but the acidic optimum is somewhat lower than the pH optima found for most mammalian -L-fucosidases (Alhadeff, 1998: Table II), and significantly lower than the pH 6.3 optimum found for rat epididymal -L-fucosidase (Carlsen and Pierce, 1972). As seen with other mammalian -L-fucosidases, the seminal plasma enzyme did not exhibit any buffer effects at the same pH value. The apparent K_M of seminal plasma -L-fucosidase for the 4MU-Fuc substrate (0.06 mmol/l) is comparable to values found for human liver (0.06 mmol/l) (Kress et al., 1980), human brain (0.07 mmol/l) (Hopfer et al., 1990), and mouse liver (0.05 mmol/l) (Laury-Klientop et al., 1985) -L-fucosidases. Purified rat epididymal -L-fucosidase had an apparent K_M of 0.27 mmol/l (0.06 mmol/l) for the p-nitrophenylfucoside substrate (Jain et al., 1977). However, the V_max of seminal plasma -L-fucosidase for 4MU-Fuc (92 µmol/min/mg protein) is 3–9-fold greater than those values previously reported for mammalian -L-fucosidases using 4MU-Fuc and p-nitrophenylfucopyranoside (PNP-Fuc) substrates (Alhadeff, 1998: Table II).

The seminal plasma -L-fucosidase is present in substantial amounts in semen, constituting typically 1–3% of the total protein of the seminal plasma. The seminal plasma -L-fucosidase originates in one or more of the male accessory organs, and not in the testis or epididymis, as indicated by the normal representation of the predominant seminal plasma -L-fucosidase isoforms in specimens from post-vasectomized donors (data not shown). Thus, this fucosidase is freshly added to the seminal concoction at the time of ejaculation, and a substantial dose of this enzyme is delivered with the ejaculate. It may have significant functions in sperm transport, survival, storage, or maturational remodelling within the female reproductive tract. In particular, it is possible to envision an important role of the seminal plasma -L-fucosidase in the penetration of sperm through the cervical mucus. In humans, terminal fucose residues are present in the cervical mucins, and are known to vary during the menstrual cycle (Chantler and Scudder, 1984). Since the pH of the cervical mucus changes over the length of the cervical canal (Chantler and Scudder, 1984), it is possible that the broad pH spectrum of the seminal plasma -L-fucosidase could improve the likelihood for sperm penetration of the mucus, especially during the periovulatory period.

	Acknowledgements

Top Abstract Introduction Materials and methods Results Discussion Acknowledgements References

 
This research was supported in part by a grant for Undergraduate Education in Biological Sciences from the Howard Hughes Medical Institute. We gratefully acknowledge the technical assistance of Brian Hwang.

	Notes

 
³ To whom correspondence should be addressed at: Biological Sciences, 111 Research Drive, Lehigh University, Bethlehem,PA 18015, USA. E-mail: bb\|[Oslash]\|\|[Oslash]\|{at}lehigh.edu

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