Biol Reprod Email Content Delivery
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

BOR - Papers in Press, published online ahead of print October 17, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.004465
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
68/3/709    most recent
biolreprod.102.004465v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Khunsook, S.
Right arrow Articles by Alhadeff, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Khunsook, S.
Right arrow Articles by Alhadeff, J. A.
Agricola
Right arrow Articles by Khunsook, S.
Right arrow Articles by Alhadeff, J. A.
BIOLOGY OF REPRODUCTION 68, 709–716 (2003)
DOI: 10.1095/biolreprod.102.004465
© 2003 by the Society for the Study of Reproduction, Inc.

Gamete Biology

Purification and Characterization of Plasma Membrane-Associated Human Sperm {alpha}-L-Fucosidase1

Sumpars Khunsook3,a, Barry S. Bean2,a, Susan R. McGowana, and Jack A. Alhadeffb

a Department of Biological Sciences b Division of Biochemical Sciences, Department of Chemistry, Lehigh University, Bethlehem, Pennsylvania 18015


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Detergent and salt extraction studies, as well as cytochemical localization with fluorescein isothiocyanate-bovine serum albumin-L-fucose, have provided further evidence for the plasma membrane association of a novel human sperm, {alpha}-L-fucosidase. This {alpha}-L-fucosidase has been solubilized and purified 8600-fold to high specific activity (35 000 U/mg protein) by affinity chromatography on agarose-C24-fucosylamine. To our knowledge, this is the first report concerning the purification and characterization of a mammalian plasma membrane-associated {alpha}-L-fucosidase. Both SDS-PAGE and Western blot analysis indicated the {alpha}-L-fucosidase is highly purified and contains a single subunit with a molecular mass of 51 kDa. N-glycanase studies indicated the subunit contains N-glycans, and lectin blot analysis detected the presence of mannose, but no terminal galactose or sialic acid residues. Isoelectric focusing indicated the presence of two major {alpha}-L-fucosidase isoforms (pIs 6.5 and 6.7) and a possible minor isoform (pI 6.3). Treatment of {alpha}-L-fucosidase with neuraminidase did not change its isoform profile, providing further evidence for the enzyme's lack of sialic acid residues. Kinetic analysis with 4-methylumbelliferyl {alpha}-L-fucopyranoside indicated that sperm {alpha}-L-fucosidase has a pH optimum near 7, an apparent Km of 0.08 mM, and a Vmax of 6.8 µmol/min/mg protein. The unusual properties of human sperm {alpha}-L-fucosidase argue in support of a potentially important, but presently unknown, role for this enzyme in human reproduction.

fertilization, gamete biology, male reproductive tract, sperm, testis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Evidence is accumulating that sperm-egg interactions in many species are carbohydrate-mediated events that involve proteins on the sperm plasma membrane and glycoconjugates on the surface of oocytes (for review, see [1–4]). A number of sperm saccharide-binding proteins [5, 6], glycosyltransferases [7, 8], and glycosidases [9, 10] have been suggested as putative protein receptors for oocyte binding. The most detailed and definitive studies have been done with the mouse, in which oligosaccharides linked to two Ser residues (O-glycans) on the zona pellucida glycoprotein ZP3 have been shown to have a crucial role in binding to sperm proteins (e.g., sp56, ß-1,4-galactosyltransferase, and p95) [1, 4]. Mutagenesis of either of these Ser residues results in a form of ZP3 inactive for sperm binding [4].

A number of previous studies are consistent with the idea that a sperm-associated {alpha}-L-fucosidase may be involved as a protein receptor for oocyte binding. Plasma membrane-associated {alpha}-L-fucosidases have been found on sperm from bulls [11], rats [12, 13], the mollusk bivalve Unio elongatulus [14], and the ascidian Ciona intestinalis [15]. More recently, {alpha}-L-fucosidase has been demonstrated to be associated with sperm plasma membranes in the toad Bufo arenarum [16] and in humans [17]. The preliminary evidence for human sperm plasma membrane-associated {alpha}-L-fucosidase was provided by immunocytochemical and subcellular fractionation studies [17].

Further evidence that {alpha}-L-fucosidases may be involved in sperm-egg interactions comes from several studies. First, L-fucose and fucose-containing molecules have been shown to inhibit these germ cell interactions in hamsters [18], rats [19], mice [20], and humans [21–23]. Second, a fucosyl residue appears to be required on a zona pellucida ligand for high-affinity sperm binding in the mouse [24]. Third, L-fucosyl-binding sites have been found on intact [23, 25, 26] or acrosome-reacted [27] human sperm using the labeled neoglycoprotein fluorescein isothiocyanate-bovine serum albumin-L-fucose (FITC-BSA-Fuc; Sigma, St. Louis, MO). The binding of FITC-BSA-Fuc, primarily to the sperm head region, could be inhibited by a sulfated polymer of fucose, fucoidin [27], or by solubilized human zona pellucida proteins [26]. Finally, {alpha}-L-fucosidase treatment of eggs significantly decreases sperm binding [28].

In the present study, we provide further evidence for the plasma membrane association of human sperm {alpha}-L-fucosidase by detergent and salt extraction studies [29] and by cytochemical localization of fucosyl-binding sites on human sperm with the neoglycoprotein FITC-BSA-Fuc. In addition, the sperm plasma membrane {alpha}-L-fucosidase has been solubilized into the aqueous phase (and separated from more hydrophobic proteins) by Triton X-114 phase separation. The solubilized enzyme has been purified by affinity chromatography on agarose-C24-fucosylamine (Sigma) and characterized with regard to its stability properties, subunit and isoform compositions, multimeric forms, glycosylation, pH optimum, and kinetic properties. To our knowledge, the present study is the first investigation to purify and characterize a mammalian plasma membrane-associated {alpha}-L-fucosidase.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
General

{alpha}-L-Fucosidase assay {alpha}-L-Fucosidase was assayed at pH 7.0 (0.1 M NaH2PO4/Na2HPO4) using 1 mM 4-methylumbelliferyl-{alpha}-L-fucopyranoside (4-MU-Fuc; Sigma) as substrate, with subsequent determination of fluorescence and correction for tissue and substrate blanks [17]. The total assay volume was 150 µl, including 25 µl of sperm suspension or extract, 25 µl of buffer (0.1 M NaH2PO4/Na2HPO4, pH 7.0), and 100 µl of 1 mM 4-MU-Fuc (in the same buffer). One unit of enzyme activity is defined as the amount of enzyme that will hydrolyze 1 nmol/min of 4-MU-Fuc at 37°C. In some cases, 1% (w/v) L-fucose (Sigma), a competitive inhibitor of {alpha}-L-fucosidase [30], was present in the enzyme suspension; if so, L-fucose was diluted significantly or removed by dialysis before conducting enzyme assays.

Bradford protein assay Protein concentration was determined by the method of Bradford [31] using Coomassie brilliant blue (BioRad Labs, Hercules, CA) following the standard assay and microassays described by Rosenberg [32]. Bovine serum albumin (Sigma) was used for standard curves.

Semen preparation Human semen specimens were obtained from healthy, informed male volunteers following protocols approved by the Lehigh University Institutional Review Board. The sperm parameters were within the normal ranges for morphology, motility, and numbers based on World Health Organization criteria [33]. After liquefaction (20–30 min) at room temperature, semen was diluted with Dulbecco PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 1.5 mM KH2PO4; pH 7.4). The dilution factors and centrifugal forces were varied in some experiments as indicated.

Sperm preparation for {alpha}-L-fucosidase analysis Multiple semen samples were pooled, and 3.5 ml of PBS were added per milliliter of semen. The mixture was centrifuged at 1000 x g for 10 min at room temperature, the resulting supernatant was poured off, and the remaining pellet was washed two more times and used for experiments. Next, PBS (5–10 ml) was added to the pellet, which was recentrifuged at 1000 x g for 10 min at 20°C. The supernatant was decanted, the final pellet was resuspended in 0.5 ml of PBS, and both were assayed for total {alpha}-L-fucosidase activity.

Whole-Sperm Studies

Detergent and salt solubilization Extraction reagents (NaCl, KI, NaOH, Triton X-100, Triton X-114, n-octylglucoside, sodium deoxycholate, and sodium taurocholate) were purchased from Sigma. Sperm pellet suspensions (containing 0.5–1.0 U of {alpha}-L-fucosidase) in 15-ml conical tubes were extracted by the following reagents at the indicated concentrations: NaCl (0.34, 0.59, 1.06, and 2.09 M), KI (0.125, 0.25, 0.5, and 1.0 M), NaOH (1%), Triton X-100 (0.01%, 0.02%, 0.05%, 0.1%, 0.5%, and 1%), Triton X-114 (0.06%, 0.125%, 0.25%, 0.5%, and 1%), n-octylglucoside (6.5, 13, 20, 22, and 26 mM), sodium deoxycholate (0.5, 1, 2, and 4 mM), and sodium taurocholate (0.85, 1.7, 3.4, and 6.8 mM). A control tube contained 1 ml of PBS (without extraction agent). The tubes were placed on an oscillating platform at 75 rpm for 30 min at 21°C and then centrifuged for 20 min at 10 000 x g at 21°C. The {alpha}-L-fucosidase activity in the supernatant was expressed as a percentage of the total activity per tube and compared to the activity released to the supernatant by PBS alone.

Isoelectric focusing of extracts of whole-sperm and Percoll-prepared sperm {alpha}-L-fucosidase Sperm proteins from Triton X-100 or Triton X-114 (aqueous phase) extracts were loaded onto a 40-ml isoelectric focusing column and focused as previously described [17]. In some experiments, sperm cells were purified using 80% and 40%, or 45% and 30%, Percoll (Sigma) discontinuous density gradient centrifugation at 600 x g for 20 min at 21°C. By these methods, the mature sperm were separated from seminal plasma, immature germ cells, and nonsperm cells (mainly leukocytes). Liquefied semen was carefully loaded onto a discontinuous Percoll density gradient consisting of 80% (3 ml) beneath 40% (3 ml) isotonic Percoll solution prepared in PBS (pH 7.4) containing 3 mg/ml of BSA (Sigma). After centrifugation at 600 x g for 20 min at 21°C, the sperm pellet at the bottom of the 80% layer was collected and washed three times in PBS by centrifugation at 600 x g for 10 min. In some experiments, layers of 45% (3 ml) beneath 30% (3 ml) Percoll were used to obtain higher yields of purified sperm cells. All of the fractions, including interfaces, were carefully collected and analyzed for {alpha}-L-fucosidase activity and sperm count and were evaluated microscopically. The bottom pellet, containing purified sperm, was used for detergent extraction and isoelectric focusing experiments. The isoelectric focusing profiles were compared to those of purified sperm {alpha}-L-fucosidase.

Localization of fucose-binding sites on human sperm The FITC-BSA-Fuc was added to live sperm suspensions (20 x 106 sperm/ml) as previously described [23] at a final concentration of 40 µg/ml and then incubated for 40 min at 37°C. Competition studies were also done by pretreatment (1 h) and inclusion of L-fucose (50 mM) or D-glucose (50 mM; Sigma), fucoidin (10 µg/ml; Sigma), and goat anti-human liver {alpha}-L-fucosidase antibody (1:20 [v/v]) [34]. Following incubation, cells were washed by centrifugation at 500 x g for 10 min and evaluated by fluorescence microscopy. More than 100 cells were scored for the presence, intensity, and subcellular distribution of fluorescence, and photomicrographs were obtained using 1000 ASA film (Kodak, Rochester, NY) or digital imaging with a Nikon DMX1200 camera (Nikon, Melville, NY).

Purification of sperm {alpha}-L-fucosidase After liquefaction, semen was diluted with one volume of PBS and centrifuged at 600 x g for 20 min. The sperm pellet was transferred to a new tube, resuspended with 5 ml of PBS (pH 7.4), and centrifuged at 600 x g for 20 min at 21°C two more times. The resulting sperm pellet was used for enzyme purification or frozen at -20°C for future use. Sperm suspensions (containing ~100 x 106 sperm/ml in cold PBS, pH 7.4) were mixed with Triton X-114 (11%, v/v) to obtain a final concentration of 2% (v/v) and subjected to phase partitioning using a procedure modified after that described by Bordier [35]. The mixtures were shaken for 1 h in an ice bath at 75 rev/min, vortexed for 10 sec, and subsequently centrifuged at 105 000 x g for 1 h at 4°C to remove the insoluble residue and to recover solubilized plasma membrane-associated sperm {alpha}-L-fucosidase. The supernatant, containing the {alpha}-L-fucosidase, was subjected to phase separation by incubation at 37°C in a water bath for 3–5 min. During this treatment, the clear detergent solution became cloudy because of the condensation of Triton X-114 micelles. The sample was centrifuged at 1000 x g for 5 min at room temperature to separate the aqueous (top) and detergent (bottom) phases. The aqueous phase was carefully removed and put on ice, and the detergent phase was again subjected to phase separation. The second detergent-depleted aqueous phase and the first aqueous phase were pooled, dialyzed two times (1 h each) against 100 volumes of 10 mM sodium phosphate buffer (pH 5.5) at 4°C, and dialyzed one more time overnight. The dialysate was centrifuged at 10 000 x g for 20 min at 4°C to remove precipitate, and the supernatant (which contained {alpha}-L-fucosidase activity) was put on 4.3 ml of fucosylamine attached to 4% cross-linked beaded agarose (24-carbon spacer arm; Sigma) in a 0.7 x 20-cm column at a flow rate of 2.5 ml/h. The column was washed with 10 mM sodium phosphate buffer (pH 5.5) until the absorbance at 280 nm stabilized near 0.002 [30]. The sperm {alpha}-L-fucosidase was eluted with 1% L-fucose in the same buffer, collected in fractions (1.1 ml) at 21°C, assayed for {alpha}-L-fucosidase activity, and concentrated with a centrifugal concentrator (Centricon-YM30; Millipore, Bedford, MA) at 4°C (following the instructions of the manufacturer) or by ultrafiltration (25 YM-30; Millipore) at 60 psi of N2 at 21°C. Protein concentration was determined using the method of Bradford [31].

Characterization of Purified Sperm {alpha}-L-Fucosidase

Stability study The stability of purified sperm {alpha}-L-fucosidase (in 10 mM sodium phosphate buffer, pH 5.5, containing 1% L-fucose with or without 0.015% Triton X-100) was assessed after storage at either 2–4°C or -20°C. Initially, and periodically thereafter, stored samples (10 µl) were tested for {alpha}-L-fucosidase activity.

SDS-PAGE and Western blot analysis Purity of the purified sperm {alpha}-L-fucosidase was assessed by slab SDS-PAGE as previously described for {alpha}-L-fucosidase [36]. Proteins were visualized with Coomassie blue (BioRad Labs) or by silver staining using the method of Rosenberg [32].

Western blot analysis was done as previously described for {alpha}-L-fucosidase [36] with goat anti-human liver {alpha}-L-fucosidase polyclonal antibody [34] and horseradish peroxidase-conjugated rabbit anti-goat F(ab')2 (ICN/Cappel, Aurora, OH) as the secondary antibody. Development was accomplished using enhanced chemiluminescence (ECL) as previously described [36].

Lectin blot analysis was performed as previously described [36] using polyvinylidene difluoride membranes (containing the electrotransferred proteins) and biotinylated lectins (Galanthus nivalis agglutinin [GNA], Sambucus nigra agglutinin [SNA], and peanut agglutinin [PNA]; Vector Laboratories, Inc., Burlingame, CA). The membranes were washed, incubated with avidin and biotinylated peroxidase macromolecular complex containing 1% BSA for 30 min, rewashed three times, and developed using ECL [36].

N-glycanase treatment N-glycanase treatment was performed as previously described [36] on purified sperm {alpha}-L-fucosidase (42 ng), purified human liver {alpha}-L-fucosidase (115 ng; as a positive control), and purified seminal fluid {alpha}-L-fucosidase (42 ng; for comparison purposes). N-glycanase (Genzyme Corp., Framingham, MA) was added to give a final concentration of 10 U/ml, and the reaction mixtures were incubated at 37°C for 17 h. Controls were run that contained all constituents except N-glycanase, 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 crude and purified sperm plasma membrane-associated {alpha}-L-fucosidases were determined essentially as previously described [12] using duplicate assays for 10 min at 37°C, and actual pH values of a third set of mock (i.e., lacking substrate) tubes were recorded. Assays were done with 10-µl enzyme aliquots, 40 µl of appropriate buffer, and 100 µl of 4-MU-Fuc substrate (1 mM) in H2O.

Neuraminidase treatment and isoelectric focusing studies Purified sperm {alpha}-L-fucosidase (15 U) was incubated at 37°C in 0.1 M citric acid/sodium citrate buffer (pH 5.0) with either 1.5 U of neuraminidase (Clostridum perfringens, type x; Sigma) for 6 h or 3.8 U of neuraminidase for 17.5 h. Sham-treated controls were run in parallel but without neuraminidase. Activities of {alpha}-L-fucosidase were determined before and after incubation, and the isoform composition of each determination was investigated by isoelectric focusing as previously described [17].

Gel filtration chromatography Sephadex G-200 (Amersham Biosciences, Piscataway, NJ) chromatography was performed on a 98 x 1.0-cm glass column as previously described [36] using standard proteins for calibration. The void volume was determined using blue dextran (2000 kDa; Sigma). Approximately 0.5- to 1.0-ml samples (containing 20 U of {alpha}-L-fucosidase) were loaded onto the column and eluted with PBS (pH 7.0) containing 0.02% (w/v) NaN3, and fractions (1.1 ml) were collected and assayed for protein (by absorbance at 280 nm) and for {alpha}-L-fucosidase activity.

Kinetic studies Apparent Km and Vmax values were determined graphically by Lineweaver-Burk double reciprocal plots [37] using 4-MU-Fuc as substrate. Aliquots of enzyme were incubated in 12 concentrations of substrate (from 0.003 to 0.66 mM) in 0.1 M NaH2PO4/Na2HPO4 (pH 6.7) for 10 min.

Reproducibility All experiments reported here have been obtained with comparable results on a minimum of at least three different occasions and using several different semen donors.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Two complementary studies were initially done to provide further evidence for the plasma membrane association of human sperm {alpha}-L-fucosidase. Several reagents (i.e., salts, NaOH, and detergents) were used that have been employed previously for determining the plasma membrane localization of proteins [29] and by other investigators [38, 39] to study plasma membrane-associated glycosidases. These reagents were used at various concentrations (see Materials and Methods) to determine their effectiveness in releasing {alpha}-L-fucosidase activity from the sperm to the supernatant fluid after incubation and centrifugation. High concentrations of NaCl and Kl, as well as NaOH, did not release any {alpha}-L-fucosidase activity into the supernatant fluid over the control level with PBS alone. All five detergents, at or above the indicated concentrations (0.1% Triton X-100, 0.06% Triton X-114, 10 mM n-octylglucoside, 1 mM sodium deoxycholate, and 1.7 mM sodium taurocholate), released essentially all (93–98%) of the {alpha}-L-fucosidase activity to the supernatant fluid and stimulated this activity by 3.0- to 5.0-fold. Control studies indicated that none of the above detergents had any effect on stimulating the activity of soluble human seminal plasma {alpha}-L-fucosidase (data not shown), which is the major {alpha}-L-fucosidase found in human semen [17, 36].

The FITC-BSA-Fuc was used to evaluate the presence of fucose-binding sites on sperm cells, and competitors were introduced to demonstrate the specificity of binding. Washed, living sperm were incubated with the labeled neoglycoprotein FITC-BSA-Fuc, and typical fluorescence microscopy results are shown in Figure 1. As used, treatment with FITC-BSA-Fuc (40 µg/ml) generates detectable labeling of approximately 60% of sperm. The FITC-BSA-Fuc fluorescence appears primarily on the postacrosomal region, with some heterogeneity for staining of the acrosome, equatorial region, neck, and midpiece (Fig. 1, A and B). Binding of FITC-BSA-Fuc (40 µg/ml) was significantly reduced in both intensity of labeling and number of cells on which signal could be detected by pretreatment and inclusion of each of the following: polyclonal antibodies against human {alpha}-L-fucosidase (goat anti-human fucosidase [34], 1:20 dilution) (Fig. 1, C and D), L-fucose (50 mM) (Fig. 1, E and F), or fucoidin, a sulfated polymer of fucose [27] (10 µg/ml) (Fig. 1, G and H). By contrast, only slight reduction of staining by FITC-BSA-Fuc resulted from treatment with D-glucose (50 mM; data not shown). Under these conditions of labeling, the sperm population is 85–90% acrosome intact.



View larger version (71K):
[in this window]
[in a new window]
  		FIG. 1. Localization of fucose-binding sites on human sperm by fluorescence microscopy. Fluorescence (A, C, E, and G) and corresponding bright-field images of the same microscopic fields (B, D, F, and H, respectively) are shown. All specimens were treated with FITC-BSA-Fucose. A and B) No pretreatment with competitors. C and D) Pretreatment with polyclonal goat anti-human fucosidase. E and F) Pretreatment with L-fucose. G and H) Pretreatment with fucoidin. Bar = 20 µm

The detergent studies described above indicated that 1% Triton X-100 was useful for extracting and solubilizing {alpha}-L-fucosidase from the sperm plasma membrane. This detergent extract was subjected to Triton X-114 phase separation to remove the more hydrophobic membrane proteins into the detergent phase. The great majority (93%) of sperm plasma membrane {alpha}-L-fucosidase activity partitioned into the aqueous phase, which could then be used for purifying the solubilized {alpha}-L-fucosidase by affinity chromatography. Before affinity chromatography, {alpha}-L-fucosidase extracted from Percoll-prepared sperm was subjected to isoelectric focusing (Fig. 2) to determine its prepurification isoform profile. Two major isoforms with pI values of 6.7 and 6.9 were found.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 2. Isoelectric focusing profile of {alpha}-L-fucosidase extracted from Percoll-prepared human sperm. See Materials and Methods for details

The purification scheme, which employed ultracentrifugation, Triton X-114 phase separation, dialysis, and affinity chromatography on fucosylamine-agarose, resulted in an 8600-fold increase in sperm {alpha}-L-fucosidase specific activity (to 35 000 U/mg protein) with a yield of 16%. The SDS-PAGE analysis (Fig. 3) illustrates the migration pattern of crude sperm proteins from the dialyzed aqueous phase of Triton X-114 phase separation (lane 3) and purified human sperm {alpha}-L-fucosidase (lane 4). The purified enzyme appears to be a single protein band corresponding to approximately 51 kDa (as determined by the migration of standard proteins in lanes 2 and 6). The purified sperm {alpha}-L-fucosidase is smaller than purified human seminal fluid {alpha}-L-fucosidase (lane 5) [36], which has a molecular mass (Mr) of approximately 56–57 kDa and can be barely seen after Ag (silver) staining. However, the seminal fluid {alpha}-L-fucosidase can be easily visualized (lane 9) by Western blot analysis. Purified sperm {alpha}-L-fucosidase was readily detected (lane 8) at Western blot analysis (with goat antibody prepared against human liver {alpha}-L-fucosidase), and a major antigenic band comigrated with the single protein band in lane 4. The human sperm {alpha}-L-fucosidase (lane 4) appears to be highly purified, even when detected by the very sensitive Ag-staining method.



View larger version (104K):
[in this window]
[in a new window]
  		FIG. 3. SDS-PAGE (lanes 1–6) and Western blot analysis (lanes 7–9) of crude and purified human sperm {alpha}-L-fucosidase. Lane 1: sample buffer; lanes 2 and 6: protein standards (BioRad Broad Range); lanes 3 and 7: crude sperm extract (dialyzed aqueous phase after Triton X-114 phase partitioning); lanes 4 and 8: purified human sperm {alpha}-L-fucosidase (0.3 µg/lane); lanes 5 and 9: purified human seminal fluid {alpha}-L-fucosidase (10 ng/lane). See Materials and Methods for details

The stability of purified plasma membrane-associated human sperm {alpha}-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. One group of aliquots was mixed with 0.015% Triton X-100. Samples of the frozen enzyme in the presence of 0.015% Triton X-100 retained 100% of {alpha}-L-fucosidase activity for at least 3 mo. Comparable samples lost all activity in only 2 wk in the absence of detergent. At 2–4°C, in the presence and absence of 0.015% Triton X-100, the enzyme retained 100% of its activity for 2 wk. After 2 wk, the samples without detergent lost all their enzyme activity. In the presence of detergent, the enzyme activity gradually decreased to approximately 20% of the starting activity after 6 wk. In the presence of 0.015% Triton X-100, the enzyme exhibited enhanced activity at least twice that of the activity without detergent.

Kinetic analysis of purified plasma membrane-associated human sperm {alpha}-L-fucosidase was also undertaken. Figure 4, which shows a pH optimum curve typical of three experiments, indicates sperm {alpha}-L-fucosidase has substantial activity over a broad range of pH values. The optimum was always in the neutral range (pH 6.8–7.3) but with retention of approximately 80% of maximal activity at pH 5.5–6.0 and at pH 4.0–4.5. Slight (10%) buffer effects were noted in the acidic region (pH 3.2–3.6), where oxalate buffer gave slightly higher activity than citrate buffer. Lineweaver-Burk double reciprocal plots (using 4-MU-Fuc as substrate) were done three times and closely approximated straight lines. The purified human sperm {alpha}-L-fucosidase has an apparent Km of 0.08 ± 0.04 mM (mean ± SD) and an apparent Vmax of 6.8 ± 1.3 µmol/min/mg protein (mean ± SD). The kinetic data must be interpreted with caution, however, because the analyses were done in the presence of 0.015% Triton X-100.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 4. pH Activity curve of purified plasma membrane-associated human sperm {alpha}-L-fucosidase. See Materials and Methods for details

A typical isoform profile of purified plasma membrane-associated human sperm {alpha}-L-fucosidase, as depicted in Figure 5A, indicates two major peaks at pIs 6.5 and 6.7 and a possible minor isoform near pI 6.3. This profile indicates that the two major fucosidase isoforms (seen in crude extracts) were purified by our scheme. After treatment of purified {alpha}-L-fucosidase at 37°C with 1.5 U of neuraminidase for 6 h (Fig. 5B) or under more drastic conditions with 3.75 U of neuraminidase for 17.5 h (Fig. 5C), two isoforms are still present at pIs 6.5 and 6.7, comparable to the untreated {alpha}-L-fucosidase. Heat-treated {alpha}-L-fucosidase (without neuraminidase) gave isoform profiles comparable to those in Figure 5 (data not shown). These results suggest that the isoforms of sperm {alpha}-L-fucosidase do not appear to contain terminal sialic acid residues.



View larger version (15K):
[in this window]
[in a new window]
  		FIG. 5. Isoelectric focusing profiles of purified plasma membrane-associated human sperm {alpha}-L-fucosidase. A) Untreated {alpha}-L-fucosidase (15 U). B) {alpha}-L-Fucosidase (15 U) treated with 1.5 U of neuraminidase for 6 h at 37°C. C) {alpha}-L-Fucosidase (15 U) treated with 3.75 U of neuraminidase for 17.5 h at 37°C. See Materials and Methods for details

Gel filtration chromatography of purified plasma membrane-associated human sperm {alpha}-L-fucosidase on Sephadex G-200 yielded the profile depicted in Figure 6. The sperm enzyme, in the presence of 0.015% Triton X-100, appears to contain a major form with an approximate Mr of 86 kDa and a minor form with an approximate Mr of 230 kDa (as determined by calibrating the column with standard proteins). The presence of Triton X-100 (which was necessary to keep the fucosidase soluble in aqueous buffer) makes it difficult to interpret the gel filtration data.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 6. Gel filtration profile of purified plasma membrane-associated human sperm {alpha}-L-fucosidase on Sephadex G-200. See Materials and Methods for details

The N-glycosylation of purified plasma membrane-associated human sperm {alpha}-L-fucosidase was investigated by SDS-PAGE/Ag-staining analysis before and after treatment with N-glycanase, an enzyme that removes N-linked glycans [40]. Purified human liver {alpha}-L-fucosidase (as a positive control) and purified human seminal fluid {alpha}-L-fucosidase (for comparison to sperm fucosidase) [36] were also analyzed in the same experiment. The fucosidases were incubated at 37°C for 17 h with or without (i.e., mock-treated) N-glycanase (10 U/ml). Figure 7, which depicts the experimental results, indicates that purified sperm {alpha}-L-fucosidase treated with N-glycanase (lane 8) migrates farther toward the anode (to a Mr of ~45 kDa) than mock-treated sperm {alpha}-L-fucosidase (lane 7), which migrates (as in Fig. 3) to a Mr of approximately 51 kDa. Treatment of human liver {alpha}-L-fucosidase (lane 4) and human seminal fluid {alpha}-L-fucosidase (lane 6) with N-glycanase led to two faster-migrating protein bands (Mr, ~50 and ~45 kDa) compared to mock-treated samples (lanes 3 and 5). The 50- and 45-kDa protein bands are similar to those found previously [41] and might represent partially and more-fully N-deglycosylated {alpha}-L-fucosidases, respectively. The protein-staining band at 34 kDa (lanes 4, 6, and 8) corresponds to authentic N-glycanase (as determined in other experiments; data not shown). The band seen near 60 kDa in all lanes (including sample buffer lanes 1 and 10) appears to be an artifact.



View larger version (97K):
[in this window]
[in a new window]
  		FIG. 7. SDS-PAGE of purified plasma membrane-associated human sperm {alpha}-L-fucosidase before and after treatment with N-glycanase (10 U/ml) for 17 h at 37°C. Lanes 1 and 10: sample buffer; lanes 2 and 9: protein standards (BioRad Broad Range); lane 3: purified human liver {alpha}-L-fucosidase (120 ng); lane 4: purified human liver {alpha}-L-fucosidase (120 ng) treated with N-glycanase; lane 5: purified human seminal fluid {alpha}-L-fucosidase (40 ng); lane 6: purified human seminal fluid {alpha}-L-fucosidase (40 ng) treated with N-glycanase; lane 7: purified plasma membrane-associated human sperm {alpha}-L-fucosidase (40 ng); lane 8: purified plasma membrane-associated human sperm {alpha}-L-fucosidase (40 ng) treated with N-glycanase. See Materials and Methods for details.

Once it was established that purified plasma membrane-associated human sperm {alpha}-L-fucosidase contained N-glycans, lectin blot analysis (Fig. 8) was undertaken to investigate the monosaccharides that might be present in the sperm enzyme (lanes 3, 6, and 9). Purified human liver {alpha}-L-fucosidase (lanes 1, 4, and 7) and purified human seminal fluid {alpha}-L-fucosidase (lanes 2, 5, and 8) [35] were also studied. A lectin with specificity for mannose, GNA reacted strongly with all three fucosidases (lanes 1–3). A lectin with specificity for sialic acid linked {alpha}2–6 to galactose or N-acetylgalactosamine, SNA recognized human liver fucosidase slightly (lane 4) and seminal fluid fucosidase strongly (lane 5) but did not recognize human sperm fucosidase (lane 6). A lectin with specificity for galactose, PNA did not recognize any of the fucosidases (lanes 7–9). The SNA results, indicating that no sialic acid residues are detectable in the sperm fucosidase, are consistent with the neuraminidase-treatment studies (Fig. 5), which gave no evidence for the presence of sialic acid residues on plasma membrane-associated human sperm {alpha}-L-fucosidase.



View larger version (89K):
[in this window]
[in a new window]
  		FIG. 8. Lectin blot analysis of purified plasma membrane-associated human sperm {alpha}-L-fucosidase. Lanes 1, 4, and 7: purified human liver {alpha}-L-fucosidase (50 ng/lane); lanes 2, 5, and 8: purified human seminal fluid {alpha}-L-fucosidase (56 ng/lane); lanes 3, 6, 9: purified plasma membrane-associated human sperm {alpha}-L-fucosidase (30 ng/lane). Lanes 1–3: detection with biotinylated GNA; lanes 4–6: detection with biotinylated SNA; lanes 5–9: detection with biotinylated PNA. See Materials and Methods for details


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In previous studies, evidence was provided for a novel plasma membrane-associated {alpha}-L-fucosidase on rat sperm [12, 13]. Later studies provided preliminary evidence (from immunocytochemical and subcellular fractionation analyses) for the localization of {alpha}-L-fucosidase to the human sperm plasma membrane [17]. The plasma membrane-associated human sperm {alpha}-L-fucosidase was different in its structural and kinetic properties from the more abundant, soluble seminal fluid {alpha}-L-fucosidase. In the present study, further evidence has been provided for the plasma membrane association of human sperm {alpha}-L-fucosidase, and the membrane-associated enzyme has been extracted, to our knowledge purified for the first time, and more fully characterized. The almost complete release of {alpha}-L-fucosidase from sperm cells by detergents and the significantly decreased release by high salt concentrations and NaOH suggests that sperm {alpha}-L-fucosidase is not a peripheral membrane protein but, instead, is an integral membrane protein [29]. In this respect, our results are similar to those of recent studies, which have provided good evidence for the presence of an integral plasma membrane {alpha}-L-fucosidase on human red blood cells [39] and on several human hematopoietic, epithelial, and mesenchymal cells [42]. The observations that significant fucosidase activity could be released by PBS and concentrated salt solutions and that {alpha}-L-fucosidase was found in the aqueous fraction after phase partitioning are consistent with {alpha}-L-fucosidase being a single-pass membrane protein [29]. The function of these novel plasma membrane-associated {alpha}-L-fucosidases is not yet known.

The labeled neoglycoprotein FITC-BSA-Fuc was also employed in the present study to demonstrate the plasma membrane association of sperm {alpha}-L-fucosidase. The fucosidase was primarily found on the postacrosomal region, and labeling with the neoglycoprotein could be competitively inhibited by L-fucose, fucoidin, and antibodies against {alpha}-L-fucosidase. These results, which suggest that FITC-BSA-Fuc was binding to the sperm surface {alpha}-L-fucosidase, extend our earlier results [17] and confirm those of several other investigators [23, 25–27] who reported localization of {alpha}-L-fucosidase to the plasma membrane of human sperm cells.

The human sperm plasma membrane-associated {alpha}-L-fucosidase has been extracted, solubilized, and separated from the more hydrophobic plasma membrane proteins by Triton X-114 phase separation of fucosidase to the aqueous fraction. Without this phase separation (which may be useful in separating other loosely associated plasma membrane proteins from more hydrophobic proteins), we were unable to purify {alpha}-L-fucosidase by affinity chromatography on fucosylamine-agarose, presumably because of "coating" of the resin by the more hydrophobic proteins. The {alpha}-L-fucosidase in the aqueous phase was readily purified by the affinity resin and represents, to our knowledge, the first purification of a mammalian plasma membrane-associated {alpha}-L-fucosidase. The purified enzyme, in the presence of 0.015% (v/v) Triton X-100 and 1% (w/v) L-fucose, was completely stable for at least 3 mo at -20°C and for at least 2 wk at 2–4°C. Isoelectric focusing indicated the purified sperm fucosidase contained the two major isoforms (and a possible, more-acidic minor isoform) found for {alpha}-L-fucosidase extracted from Percoll-prepared sperm. This indicates that both isoforms were purified by our scheme, a necessary confirmation because of the low yield obtained in purification. Both SDS-PAGE and Western blot analysis indicated that the sperm {alpha}-L-fucosidase was highly purified, if not homogeneous, and contained a major protein band of 51 kDa. N-glycanase treatment caused the purified fucosidase to migrate farther toward the anode (Mr, ~45 kDa), indicating the presence of N-glycans in the human sperm enzyme. Lectin blot analysis indicated the presence of mannose but a lack of detectable galactose and sialic acid residues. Neuraminidase treatment of sperm {alpha}-L-fucosidase, followed by isoelectric focusing analysis, yielded no change in isoform profile and provided further complementary evidence for an absence of sialic acid residues. This finding differs from the observation of sialic acid residues on purified human liver and human seminal fluid {alpha}-L-fucosidases as analyzed in parallel studies.

Previous studies concerning human liver {alpha}-L-fucosidase indicated the presence of two subunits of 56 and 51 kDa, both of which were glycosylated, but only the 56-kDa subunit contained sialic acid residues [41]. In addition, the acidic fucosidase isoforms contained enriched amounts of the 56-kDa subunit [41]. Purified human seminal plasma {alpha}-L-fucosidase contains a 56-kDa subunit, is sialylated, and has several acidic fucosidase isoforms [36]. Results of the present study indicate purified sperm {alpha}-L-fucosidase contains a 51-kDa subunit, is not sialylated, and has only neutral fucosidase isoforms. In addition, the seminal plasma and sperm {alpha}-L-fucosidases are both cross-reactive with antibodies prepared against human liver {alpha}-L-fucosidase [34]. Taken together, these results suggest that the 56-kDa seminal plasma subunit and the 51-kDa sperm subunit may be comparable to the 56- and 51-kDa subunits, respectively, of human liver {alpha}-L-fucosidase.

Gel filtration studies of purified human sperm {alpha}-L-fucosidase provided evidence for two enzymatically active structures, a major form at 86 kDa and a minor form at 230 kDa. However, these results must be interpreted with caution, because the gel filtration studies were done in the presence of Triton X-100 to keep the purified {alpha}-L-fucosidase both soluble and stable. This nonionic detergent could interact with hydrophobic domains on {alpha}-L-fucosidase and affect the multimeric structure of this membrane-bound enzyme.

Kinetic analysis with the 4-MU-Fuc substrate indicated that purified sperm {alpha}-L-fucosidase has a neutral pH optimum near 7, which is very different than the more acidic optimum (pH 4.0–4.5) found for most previously studied mammalian {alpha}-L-fucosidases [43]. Lineweaver-Burk double reciprocal plot analysis of the purified sperm {alpha}-L-fucosidase indicated an apparent Km of 0.08 mM and a Vmax of 6.8 µmol/min/mg protein for 4-MU-Fuc. These kinetic values are similar to those previously found for a number of mammalian {alpha}-L-fucosidases [43] and suggest that the presence of 0.015% Triton X-100 in the purified sperm fucosidase is not affecting the kinetic results.

In summary, the present investigation has provided further evidence for the presence of a novel form of {alpha}-L-fucosidase associated with the plasma membrane of human sperm. In addition to its unusual membrane location, presumably as a single-pass protein, human sperm {alpha}-L-fucosidase is unusual in that the highly purified enzyme has a neutral pH optimum and does not appear to contain sialic acid residues on its carbohydrate moieties. These unusual properties argue for a potentially important, but presently unknown, function for human sperm {alpha}-L-fucosidase in human reproduction.


   ACKNOWLEDGMENTS
 
William Magilton and Brian Hwang are gratefully acknowledged for technical assistance.


   FOOTNOTES
 
1 Supported in part by a grant for Undergraduate Education in Biological Sciences from the Howard Hughes Medical Institute. S.K. was supported as a scholar of the Thai Government Ministry of University Affairs. Back

2 Correspondence: Barry Bean, Lehigh University, 111 Research Drive, Bethlehem, PA 18015. FAX: 610 758 4004; e-mail: bb00{at}lehigh.edu Back

3 Current address: Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen, Thailand 40002 Back

Received: 15 March 2002.

First decision: 9 April 2002.

Accepted: 7 August 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Shalgi R, Raz T. The role of carbohydrate residues in mammalian fertilization. Histol Histopathol 1997 12:813-822[Medline]
  2. Benoff S. Carbohydrates and fertilization: an overview. Mol Hum Reprod 1997 3:599-637[Abstract/Free Full Text]
  3. Dell A, Morris HR, Easton RL, Patankar M, Clark G. The glycobiology of gametes and fertilization. Biochim Biophys Acta 1999 1473:196-205[Medline]
  4. Wassarman PM, Litscher ES. Towards the molecular basis of sperm and egg interaction during mammalian fertilization. Cells Tissues Organs 2000 168:36-45
  5. Huang TTF, Ohzu E, Yanagimachi R. Evidence suggesting that L-fucose is part of a recognition signal for sperm-zona pellucida attachment in mammals. Gamete Res 1982 5:355-361[CrossRef]
  6. Cheng A, Le T, Palacios M, Bookbinder LH, Wassarman PM, Suzuki F. Sperm-egg recognition in the mouse: characterization of sp56, a sperm protein having specific affinity for ZP3. J Cell Biol 1994 125:867-878[Abstract/Free Full Text]
  7. Ram PA, Cardullo RA, Millette CF. Expression and topographical localization of cell surface fucosyltransferase activity during epididymal sperm maturation in the mouse. Gamete Res 1989 22:321-332[CrossRef][Medline]
  8. Miller DJ, Macek MB, Shur BD. Complementarity between sperm surface ß-1,4-galactosyltransferase and egg-coat ZP3 mediates sperm-egg binding. Nature 1992 357:589-593[CrossRef][Medline]
  9. Tulsiani DRP, Skudlarek MD, Orgebin-Crist M-C. Novel {alpha}-D-mannosidase of rat sperm plasma membrane: characterization and potential role in sperm-egg interactions. J Cell Biol 1989 109:1257-1267[Abstract/Free Full Text]
  10. Brandelli A, Miranda PV, Tezon JG. Human sperm ß-glucuronidase is poorly extractable by Triton X-100. Cell Biol Int 1996 20:351-354[CrossRef][Medline]
  11. Jauhiainen A, Vanha-Pertula T. {alpha}-L-Fucosidase in the reproductive organs and seminal plasma of the bull. Biochim Biophys Acta 1986 880:91-95[Medline]
  12. Aviles M, Abascal I, Martinez-Menarguez JA, Castells MT, Skalaban SR, Ballesta J, Alhadeff JA. Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum {alpha}-L-fucosidase from rat testis and epididymal spermatozoa. Biochem J 1996 318:821-831
  13. Abascal I, Skalaban SR, Grimm KM, Aviles M, Martinez-Menarguez JA, Castells MT, Ballesta J, Alhadeff JA. Alteration of the isoform composition of plasma- membrane-associated rat sperm {alpha}-L-fucosidase during late epididymal maturation: comparative characterization of the acidic and neutral isoforms. Biochem J 1998 333:201-207
  14. Focarelli R, Cacace MG, Seraglia R, Rosati F. A nonglycosylated, 68-kDa {alpha}-L-fucosidase is bound to the mollusc bivalve Unio elongatulus sperm plasma membrane and differs from a glycosylated 56-kDa form present in the seminal fluid. Biochem Biophys Res Commun 1997 234:54-58[CrossRef][Medline]
  15. Hoshi MR, DeSantis R, Pinto MR, Cotelli F, Rosati F. Is sperm {alpha}-L-fucosidase responsible for sperm-egg binding in Ciona intestinalis?. In: Andre J (ed.), The Sperm Cell. The Hague, The Netherlands: Martines Nijhoff; 1983: 107–110
  16. Martinez ML, Martelotto L, Cabada MO. Purification and biological characterization of N-acetyl ß-D-glucosaminidase from Bufo arenarum spermatozoa. Mol Reprod Dev 2000 57:194-203[CrossRef][Medline]
  17. Alhadeff JA, Khunsook S, Choowongkomon K, Baney T, Heredia V, Tweedie A, Bean B. Characterization of human semen {alpha}-L-fucosidases. Mol Hum Reprod 1999 5:809-815[Abstract/Free Full Text]
  18. Ahuja KK. Fertilization studies in the hamster. The role of cell-surface carbohydrates. Exp Cell Res 1982 140:353-362[CrossRef][Medline]
  19. Shalgi R, Matityahu A, Nebel L. The roles of carbohydrate in sperm-egg interactions in rats. Biol Reprod 1986 34:446-452[Abstract]
  20. Boldt J, Howe AM, Parkerson JB, Gunter LE, Kuehn E. Carbohydrate involvement in sperm-egg fusion in mice. Biol Reprod 1989 40:887-896[Abstract]
  21. Oehninger S, Clark GF, Acosta AA, Hodgen GD. Nature of the inhibitory effect of complex saccharide moieties on the tight binding of human spermatozoa to the human zona pellucida. Fertil Steril 1991 55:165-169[Medline]
  22. Lucas H, Bercegeay S, Pendu J, Jean M, Mirallie S, Barriere P. A fucose-containing epitope potentially involved in gamete interaction. Hum Reprod 1994 9:1532-1538[Abstract/Free Full Text]
  23. DeCerezo JMS, Marquinez AC, Sarchi MI, Cerezo AS. Fucosylated glycoconjugates of the human spermatozoon-comparison of the domains of these glycoconjugates with the {alpha}-fucosyl binding-sites, and with lactosaminic glycoconjugates and ß-D-galactosyl binding-site domains. Biocell 1996 20:11-20[Medline]
  24. Johnston DS, Wright WW, Shaper JH, Hokke CH, Van den Eijnden DH, Joziasse DH. Murine sperm-zona binding, a fucosyl residue is required for a high affinity sperm binding ligand. A second site on sperm binds a nonfucosylated, ß-galactosyl-capped oligosaccharide. J Biol Chem 1998 273:1888-1895[Abstract/Free Full Text]
  25. Jones RC. Identification of carbohydrate-binding proteins in mammalian spermatozoa (human, bull, boar, ram, stallion and hamster) using [125I]fucoidin and [125I]neoglycoprotein probes. Hum Reprod 1989 4:550-557[Abstract/Free Full Text]
  26. Tesarik J, Mendoza C, Ramirez J-P, Moos J. Solubilized human zona pellucida competes with a fucosylated neoglycoprotein for binding sites on the human sperm surface. Fertil Steril 1993 60:344-350[Medline]
  27. Francavilla S, Gabriele A, Properzi G, Cordeschi G, DeStefano C, Giammateo M. Carbohydrate binding sites in human sperm heads during capacitation, acrosome reaction, and zona pellucida interaction. In: Motta PM (ed.), Microscopy of Reproduction and Development:. A Dynamic Approach. Rome: Antonio Delfino Editore; 1997: 23–28
  28. Blithe DL. Biological functions of oligosaccharides on glycoproteins. Trends Glycosci Glycotech 1993 5:81-98
  29. Findlay JBC. The isolation and labelling of membrane proteins and peptides. In: Findlay JBC, Evans WH (eds.), Biological Membranes: A Practical Approach. Washington: IRL Press; 1987: 179–218
  30. Alhadeff JA, Miller AL, Wenaas H, Vedvick T, O'Brien JS. Human liver {alpha}-L-fucosidase. Purification, characterization, and immunochemical studies. J Biol Chem 1975 250:7106-7113[Abstract/Free Full Text]
  31. Bradford MM. A rapid and sensitive method for quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976 72:248-254[CrossRef][Medline]
  32. Rosenberg IM. Electrophoretic techniques. In: Protein Analysis and Purification: Benchtop Techniques. Boston: Birkhauser; 1996: 55–98
  33. World Health Organization. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interactions, 4th ed. Cambridge, U.K.: Cambridge University Press; 1999
  34. Andrews-Smith GL, Alhadeff JA. Radioimmunoassay determination of decreased amounts of {alpha}-L-fucosidase protein in fucosidosis. Biochim Biophys Acta 1982 715:90-96[Medline]
  35. Bordier C. Phase separation of integral membrane proteins in Triton X-114 solution. J Biol Chem 1981 256:1604-1607[Abstract/Free Full Text]
  36. Khunsook S, Alhadeff JA, Bean BS. Purification and characterization of human seminal plasma {alpha}-L-fucosidase. Mol Hum Reprod 2002 8:221-227[Abstract/Free Full Text]
  37. Lineweaver H, Burk D. The determination of enzyme dissociation constants. J Am Chem Soc 1934 56:658-666[CrossRef]
  38. Pasini ME, Cattaneo F, Pinto MR, De Santis R, Perotti ME. Plasma membrane association and preliminary characterization of Drosophila sperm surface glycosidases. Mol Reprod Dev 1999 52:166-173[CrossRef][Medline]
  39. Goi G, Bairati C, Massaccesi L, Lovagnini A, Lombardo A, Tettamanti G. Membrane anchoring and surface distribution of glycohydrolases of human erythrocyte membranes. FEBS Lett 2000 473:89-94[CrossRef][Medline]
  40. Hirani S, Bernasconi RJ, Rasmussen JR. Use of N-glycanase to release asparagines-linked oligosaccharides for structural analysis. Anal Biochem 1987 162:485-492[CrossRef][Medline]
  41. Johnson SW, Piesecki S, Wang RF, Damjanov I, Alhadeff JA. Analysis of purified human liver {alpha}-L-fucosidase by Western-blotting with lectins and polyclonal and monoclonal antibodies. Biochem J 1992 282:829-834
  42. Cordero OJ, Merino A, Paez de la Cadena M, Bugia B, Nogueira M, Vinuela JE, Martinez-Zorzano VS, de Carlos A, Rodriguez-Berrocal FJ. Cell surface human alpha-L-fucosidase. Eur J Biochem 2001 268:3321-3331[Medline]
  43. Alhadeff JA. Structure and function of mammalian {alpha}-L-fucosidases. Trends Comp Biochem Physiol 1998 4:105-118



This article has been cited by other articles:


Home page
 J. Cell Sci.Home page
P. C. N. Chiu, M.-K. Chung, R. Koistinen, H. Koistinen, M. Seppala, P.-C. Ho, E. H. Y. Ng, K.-F. Lee, and W. S. B. Yeung
Glycodelin-A interacts with fucosyltransferase on human sperm plasma membrane to inhibit spermatozoa-zona pellucida binding
J. Cell Sci., January 1, 2007; 120(1): 33 - 44.
[Abstract] [Full Text] [PDF]

Home page
 GlycobiologyHome page
F. Cattaneo, M. E. Pasini, J. Intra, M. Matsumoto, F. Briani, M. Hoshi, and M. E. Perotti
Identification and expression analysis of Drosophila melanogaster genes encoding {beta}-hexosaminidases of the sperm plasma membrane
Glycobiology, September 1, 2006; 16(9): 786 - 800.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
P.C.N. Chiu, H.Y. Tsang, R. Koistinen, H. Koistinen, M. Seppala, K.F. Lee, and W.S.B. Yeung
The Contribution of D-Mannose, L-Fucose, N-Acetylglucosamine, and Selectin Residues on the Binding of Glycodelin Isoforms to Human Spermatozoa
Biol Reprod, June 1, 2004; 70(6): 1710 - 1719.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
68/3/709    most recent
biolreprod.102.004465v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions