Developmental Biology 247, 182–196 (2002)doi:10.1006/dbio.2002.0690

Arylsulfatase A Is Present on the Pig Sperm Surfaceand Is Involved in Sperm–Zona Pellucida Binding

Euridice Carmona,* Wattana Weerachatyanukul,* Tanya Soboloff,* ,†Arvan L. Fluharty,‡ Dawn White,* ,† Limthong Promdee,*Marc Ekker,* ,§ Trish Berger,¶ Mary Buhr,�and Nongnuj Tanphaichitr* ,† ,# ,1

*Hormones/Growth/Development Research Group, Ottawa Health Research Institute, Ottawa,Ontario, Canada, K1Y 4E9; †Department of Biochemistry, Microbiology and Immunology,§Department of Cellular and Molecular Medicine, and #Department of Obstetrics andGynecology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada, K1H 8M5;‡Mental Retardation Research Center, University of California, Los Angeles, California 90024;¶Department of Animal Science, University of California, Davis, California 95616; and�Department of Animal and Poultry Science, University of Guelph,Guelph, Ontario, Canada, N1G 2W1

We have previously described the affinity of a pig sperm surface protein, P68, to mammalian zonae pellucidae (ZP). In thisreport, we identified P68 as arylsulfatase A (AS-A) based on the presence of P68 tryptic peptide sequences in the pig testisAS-A cDNA sequence. Our objective was to demonstrate the presence of AS-A on the sperm surface and to elucidate its rolein ZP binding. Immunogold electron microscopy revealed the presence of AS-A on the sperm surface. Furthermore, live pigsperm and the extract of peripheral sperm plasma membrane proteins exhibited AS-A’s desulfation activity. Significantly,the role of pig sperm surface AS-A in ZP binding was demonstrated by dose-dependent decreases of sperm–ZP binding uponsperm pretreatment with anti-AS-A IgG/Fab, and by the binding of Alexa-430-conjugated sperm surface AS-A tohomologous ZP. ZP pretreatment with anti-pig-ZP3 antibody abolished AS-A binding, suggesting that ZP3, recognized asthe pig sperm receptor, was AS-A’s binding ligand. This was further confirmed by the ability of exogenous ZP3 tocompetitively inhibit AS-A–ZP binding. Similarly, purified ZP3�, a major sperm receptor component of ZP3, exhibited greatinhibitory effect on AS-A–ZP binding. All of these results designated a new function of AS-A in gameteinteraction. © 2002 Elsevier Science (USA)

Key Words: arylsulfatase A; sperm surface enzyme; sperm–egg interaction; desulfation activity; sulfoglycolipids; sulfatedglycans; fertilization; zona pellucida.

INTRODUCTIONBinding of sperm to the zonae pellucidae (ZP) is the first

step of mammalian sperm–egg interaction that culminatesin fertilization (Yanagimachi, 1994). Accumulated evidenceindicates that protein–carbohydrate interaction is one ofthe main mechanisms of sperm–ZP binding (Topfer-Petersen et al., 1997; Wassarman, 1999). Zonae pellucidaefrom all mammals invariably consist of three to four

1 To whom correspondence should be addressed at Ottawa HealthResearch Institute, 725 Parkdale Ave., Ottawa, Ontario, Canada, K1Y

4E9. Fax: (613) 761-5365. E-mail: ntanphaichitr@ohri.ca.

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polypeptides that are highly glycosylated (Prasad et al.,2000; Nakano and Yonezawa, 2001). In mice, ZP3 (ZPC) isthe primary sperm receptor and its O-linked oligosaccha-rides are important for this binding event (Wassarman andLitscher, 2001). In pigs, ZP3, consisting of hetero-oligomersof ZP3� (ZPB) and ZP3� (ZPC), is the sperm receptor withZP3� being a major player in sperm binding (Sacco et al.,1989; Yurewicz et al., 1993, 1998). Both N- and O-linkedoligosaccharides of pig ZPB/ZPC have been shown to me-diate sperm–ZP binding (Yurewicz et al., 1991; Noguchi etal., 1992). In addition, sulfated glycans present in both pig

and mouse ZP (Shimizu et al., 1983; Noguchi et al., 1992;

0012-1606/02 $35.00© 2002 Elsevier Science (USA)

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Nakano et al., 1996) have been suggested to be involved insperm–ZP binding (Jones, 1991; Chapman and Barratt, 1996;Takasaki et al., 1999).

To date, a number of sperm surface proteins have beenreported to recognize ZP (Wassarman, 1999; Dell et al., 1999),with several of them having lectin properties, including sper-madhesins (Dostalova et al., 1995; Calvete et al., 1996),zonadhesin (Gao and Garbers, 1998), Sp17 (Yamasaki et al.,1995), and sp56 (Cheng et al., 1994). In addition, a number ofZP-binding proteins on the sperm surface are glycoenzymes,such as �1,4-galactosyltransferase (Nixon et al., 2001),�-fucosyltransferase (Thaler and Cardullo, 1996), and �-D-mannosidase (Cornwall et al., 1991). These enzymes arebelieved to bind to their substrates, ZP sugar residues, but donot complete their catalytic reaction. This enzyme–substratebinding may be a basis of conjugation between sperm and theegg ZP. A sperm surface protein, called sulfolipidimmobiliz-ing protein 1 (SLIP1), has also been described by our group tohave ZP binding ability (Tanphaichitr et al., 1993). SLIP1 wasfirst isolated from rat testis homogenate by affinity chroma-tography using sulfogalactosylglycerolipid (SGG, HSO3-O-3Galp�1–3-sn-alkylacylglycerol, also known as seminolipid)(Lingwood, 1985) as the binding ligand. Like SGG (Murray andNarasimhan, 1990), SLIP1 is present selectively on male germcell plasma membranes (Law et al., 1988). SLIP1 was furthercharacterized to consist of a few proteins of similar molecularmass (68 kDa), including a ZP binding component, a heatshock protein 70 (HSP70), and albumin (Tanphaichitr et al.,1999; Boulanger et al., 1995). Using chromatofocusing, wehave purified P68, the ZP binding component of SLIP1, freefrom HSP70 and albumin, from the pig sperm peripheralplasma membrane extract. As expected, P68 also has affinityto SGG (Tanphaichitr et al., 1998).

Our recent results indicate high identity of three P68tryptic peptide sequences to the human testis arylsulfataseA (AS-A) sequence, suggesting that P68 is AS-A (Tanphai-chitr et al., 1999). AS-A (E.C. 3.1.6.8) is well recognized as alysosomal and acrosomal enzyme, and has been purifiedfrom several tissues and sperm (Allison and Hartree, 1970;Sarafian et al., 1982; Dudkiewicz, 1984; Fujii et al., 1992). Ithas a specific ability to desulfate various arylsulfates atthe pH optimum of �4.5–5, including small artificialsubstrates, such as p-nitrocatecholsulfate (NCS) andp-nitrophenylsulfate (Baum et al., 1959), and natural sulfo-glycolipid substrates, i.e., SGG and sulfogalactosylceramide(SGC) (present in brain, epithelial cells, and sperm of lowervertebrates and invertebrates) (Mehl and Jatzkewitz, 1968;Rahi and Srivastava, 1983). However, desulfation of thesesulfoglycolipids is only possible following their solubiliza-tion by saposin B (a coactivator) or a detergent making thelipids accessible to AS-A’s active site pocket (Fluharty etal., 2001). The physiological significance of AS-A in theneurological system has long been recognized. Individualsgenetically deficient in AS-A suffer from the disorder ofmetachromatic leukodystrophy due to the accumulation ofSGC in the myelin sheath (Kolodny and Fluharty, 1995).However, the enzyme’s functions in testes and sperm are

less clear despite the fact that AS-A is transcribed at thehighest level in testes (Kreysing et al., 1994b).

In this report, the identity of P68 as AS-A was establishedby the presence of three P68 tryptic peptide sequences inthe pig testis AS-A sequence, obtained by molecular clon-ing. This finding suggested that AS-A existed on the spermsurface (besides the acrosome) and was involved in ZPbinding. To validate this postulation, we demonstrated thepresence of AS-A on the pig sperm surface by immunogoldelectron microscopy, as well as its affinity to the ZP. Theseresults herein uncover a new attribute of AS-A in mamma-lian fertilization.

MATERIALS AND METHODS

Production of Rabbit Polyclonal Antibodiesagainst Purified Human Liver AS-A (Anti-AS-A)and P68 Peptide (Anti-P68pep)

Two different antigens were used to immunize the rabbits:human liver AS-A, purified previously (Sarafian et al., 1982), and aP68 peptide, AQLDAAVTFGPSQVAR (Tanphaichitr et al., 1999),chemically synthesized by Dr. Ajoy Basak (Protein ChemistryUnit, Ottawa Health Research Institute, Ottawa, ON, Canada). Astandard regimen was followed to produce rabbit polyclonal anti-bodies (Harlow and Lane, 1988). Approximately 0.5 mg of theantigen, human liver AS-A, or P68 peptide, plus Freund’s adjuvantwas used for each of the three sequential intramuscular injections.The titers of the antibodies produced were assessed by immuno-blotting using purified human liver AS-A and pig sperm P68.

The IgG fraction of anti-AS-A was prepared by ammoniumsulfate precipitation and further purified by using an Affinity PakImmobilized Protein A Kit (Pierce, Rockford, IL). Anti-AS-A Fabfragments were generated from the purified IgG fraction by usingthe Immuno Pure Fab Preparation Kit (Pierce). Affinity purifiedanti-AS-A IgG was prepared, as previously described (Ahnonkitpa-nit et al., 1999), by adsorbing 1 ml of the IgG antibody (10 �g) to 1�g purified human liver AS-A band electroblotted onto the nitro-cellulose membrane. The adsorbed antibody was then eluted with1 ml of 100 mM glycine–HCl, pH 2.5, washed, and concentrated to100 �l in CM (capacitation medium, see below) by Microcon 30(Amicon, Beverly, MA). The nonadsorbed IgG solution (termedAS-A-nonadsorbed IgG) was also collected and used as a negativecontrol of affinity purified anti-AS-A IgG. The anti-AS-A IgGsolution of the same concentration and volume, used for affinitypurification, was also incubated with a blank blot (no protein)under the same conditions described above. Since this anti-AS-AIgG solution was not expected to adsorb to the blot, it wasrecovered and concentrated to 100 �l in CM (i.e., its concentrationbecame 100 �g/ml), and used in comparison with affinity purifiedanti-AS-A IgG in the in vitro sperm–egg binding experiments.

Molecular Cloning of Pig Testis AS-A

A pig testis UniZAP cDNA library was constructed according tothe manufacturer’s instructions (Stratagene, La Jolla, CA). Approxi-mately 800,000 recombinant plaques were screened for their hy-bridization with a full-length human testis AS-A cDNA (kindlyprovided by Dr. V. Gieselmann, University of Bonn, Germany)randomly labeled with [�-32P]dCTP. Positive clones were plaque

183AS-A Exists on the Pig Sperm Surface and Binds to ZP

© 2002 Elsevier Science (USA). All rights reserved.

purified, and the selected phages were in vivo excised. Plasmidswere then purified and subjected to long-range nucleotide sequenc-ing (ThermoSequenase; Amersham Pharmacia Biotech, Piscat-away, NJ) of the cDNA insert, using an automated Li-Cor DNASequencing Analyzer Image 4000 and fluorescent M13 forward andreverse primers (Li-Cor Biosciences, Lincoln, NE). The sequencesobtained were analyzed for their identity to known sequencesavailable in the GenBank data bank through NCBI’s Blastn ser-vices. The sequence similarity of the pig testis AS-A to human andmouse testis AS-A was assessed by Clustal W (1.81) MultipleSequence Alignments software.

Immunolocalization of AS-A on Live Pig Sperm

Indirect immunofluorescence. Live ejaculated pig sperm,washed free of seminal plasma by centrifugation (350g, 10 min,25°C), were incubated with affinity purified anti-AS-A (30 min,room temperature). Following two washes in PBS, the sperm wereincubated (30 min, room temperature) with 50 �g/ml Alexa-488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR) andthen washed twice with PBS. The sample was mounted onto a glassslide under a coverslip and viewed under a Zeiss epifluorescent IM35 inverted microscope.

Transmission electron microscopic immunoprotein-A gold la-beling. Live ejaculated pig sperm were incubated with affinitypurified anti-AS-A (30 min, room temperature). Following twowashes in PBS, the sperm were incubated (30 min, room tempera-ture) with protein A-gold (8 nm) solution, prepared as described bySlot and Geuze (1985) and provided by Dr. F. Kan (Queen’sUniversity, Kingston, ON, Canada). The sperm were then washedtwice in PBS and fixed (1 h, room temperature) with a mixture of4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphatebuffer, pH 7.4, containing 0.2 M sucrose. The fixed samples werethen serially dehydrated in ethanol and embedded in LR White(London Resin Co., Berkshire, UK). Thin sections of these spermwere stained with saturated uranyl acetate and Reynold’s leadcitrate, and were viewed with a Hitachi 7100 transmission electronmicroscope at 75 kV. A negative control sample was prepared inparallel by using 100 �g/ml preimmune rabbit serum (PRS) IgGinstead of affinity purified anti-AS-A.

Preparation of an AS-A Crude Extract from PigSperm Plasma Membranes

The method previously used for P68 (Tanphaichitr et al., 1998)was applied here. Briefly, ejaculated pig sperm, washed free ofseminal plasma, were treated with an AES solution (320 mMsucrose containing 1 mM ATP, 1 mM EDTA, and 0.2 mM TLCK(N-�-p-tosyl-L-lysine chloromethylketone) for 1 h at 4°C. Followingcentrifugation (350g, 15 min, 25°C) to pellet the AES-treatedsperm, the supernatant containing AS-A activity, assayed as de-scribed below, was ultracentrifuged (100,000g, 1 h, 4°C) to removecellular debris. The final supernatant obtained was used for furtherpurification of the enzyme (see below). To verify that only theplasma membrane was removed from sperm by AES treatment, theAES-treated sperm were processed for electron microscopy, follow-ing the same procedures described above, except that the steps ofantibody and gold labeling were omitted.

Purification of Pig Sperm Surface AS-A

The presence of AS-A throughout the purification steps wasdetected by NCS desulfation activity, following the method de-scribed by Fluharty and Edmond (1978). The reaction was per-formed at 37°C for 30 min in 250 �l of 250 mM sodium acetate, pH5.0, containing 5 mM NCS, 0.25 mM Na4P2O7, 0.1% bovine serumalbumin (BSA), and 5% NaCl, and was stopped by adding 0.8 ml of1 M NaOH. The reaction product, p-nitrocatechol, was quantifiedby its absorbance at 515 nm. One unit of activity was defined as 1�mol of NCS hydrolyzed in 1 h.

The AES extract containing AS-A activity was subjected tochromatofocusing (Polybuffer exchanger 94; Sigma, St. Louis, MO)following the same method described for P68 (Tanphaichitr et al.,1998). Proteins were eluted with Polybuffer 74 (Sigma), followed bya NaCl gradient (0–1 M) and 1 M HCl. The salt fractions containingAS-A activity were pooled, dialyzed against 20 mM ammoniumbicarbonate, pH 7.4, lyophilized, and resuspended in 0.15 M NaCl,50 mM Tris–HCl (pH 7.0) for subsequent Sephacryl S-200 (Amer-sham Pharmacia Biotech) size-exclusion chromatography (columndimension: 85 � 1.5 cm). Proteins were eluted by using the sameTris–salt buffer. Calibration of the Sephacryl S-200 column wasperformed by chromatographing molecular mass standards, includ-ing rabbit IgG (150 kDa), BSA (65 kDa), and ovalbumin (45 kDa).The molecular mass of pig sperm AS-A was estimated from thelinear plot of the standards’ molecular masses vs their peak elu-tion volumes. Sperm surface AS-A was further characterized byimmunoblotting using anti-AS-A and anti-P68pep antibodies, andthen used for studies of its kinetic properties and binding ability tothe ZP (see below).

Immunoblotting of Purified Pig Sperm Surface andHuman Liver AS-A with Anti-AS-A and Anti-P68pep Antiserum

Purified pig sperm surface AS-A and human liver AS-A weresubjected to SDS–PAGE (10% polyacrylamide) as described byLaemmli (1970), followed by silver staining using Bio-Rad Silver-Stain Reagent (Bio-Rad Laboratories, Hercules, CA) or by electro-blotting to a nitrocellulose membrane (0.45 �m; Bio-Rad Labora-tories) (Towbin and Gordon, 1984). For immunoblotting, blockingof nonspecific binding was performed by using TBS (20 mMTris–HCl, 137 mM NaCl, pH 7.4) supplemented with 5% milk(TBS–milk). The immobilized proteins were incubated (1 h, roomtemperature) with anti-AS-A antiserum (1:500 dilution in TBS–milk), followed by secondary antibody anti-rabbit IgG conjugatedwith horseradish peroxidase (Bio-Rad Laboratories) (1:3000 dilutionin TBS–milk). Protein–antibody recognition was detected by usingan ECL Western Blotting Detection Kit (Amersham PharmaciaBiotech). The bound anti-AS-A was then stripped from the blot byincubation (55°C, 30 min) with 2% SDS, 0.8% mercaptoethanol in50 mM Tris–HCl, pH 6.8. The blot was washed with TBS andreprobed with anti-P68pep antiserum (1:500 dilution in TBS–milk)using the same conditions as those for anti-AS-A.

Enzymatic Properties of Purified Pig Sperm SurfaceAS-A and Human Liver AS-A

pH profile. Desulfation activity of purified pig sperm surfaceAS-A and human liver AS-A was measured in 250 mM sodiumacetate buffer at various pH values (3.3–6.5) by using NCS (5 mM)as a substrate (see purification section above).

184 Carmona et al.

© 2002 Elsevier Science (USA). All rights reserved.

Enzyme kinetics. Experiments were performed at 37°C and pH4.5 using 1.2 �g purified sperm surface AS-A or 0.4 �g human liverAS-A. Kinetic properties of the enzymes were determined for bothartificial (NCS) and natural (sulfoglycolipids) substrates. The assayfor NCS was as described above, using various substrate concen-trations (0.1–10 mM). For the sulfoglycolipids, the assay wasperformed with 0.02–0.2 mM of SGG or SGC in 50 �l of 250 mMsodium acetate buffer, pH 4.5, containing 0.1% BSA and 0.1%taurodeoxycholate (Fluharty and Edmond, 1978). SGG was purifiedfrom ram testes following our previously described method (Tupperet al., 1994), whereas SGC was purchased from Sigma. Afterincubation, the reaction mixture was subjected to lipid extractionaccording to the modified (Kates, 1986) Bligh and Dyer’s method(1959). The extracted sulfoglycolipids (SGG or SGC) and thedesulfated glycolipids (GG, galactosylglycerolipid, or GC, galacto-sylceramide) were concentrated under a stream of N2 and separatedfrom each other by HPTLC (200 �m mesh size, dimension: 10 � 10cm; Whatman Inc., Kent, UK) using chloroform/methanol/water(65:25:4, v/v/v) as a solvent system (Levine et al., 1976). TheHPTLC plate was stained with 0.2% orcinol in 75% H2SO4 at100°C (Kates, 1986) and poststained with 0.03% Coomassie blueG250 in 30% methanol, 100 mM NaCl (Nakamura and Handa,1984). The amount of the glycolipid, GG or GC, generated fromSGG or SGC, respectively, by AS-A was determined by densito-metric analysis, using ScionImage software for Windows (ScionCorporation, Frederick, MD). A standard curve of GG (a generousgift from Dr. B. Gadella, Utrecht University, prepared by aciddesulfation of SGG) (Alvarez et al., 1990) or GC (Sigma) wasconstructed from the co-chromatographed glycolipid standards.Kinetic parameters (Km and Vmax) for desulfation of the threesubstrates (NCS, SGG, or SGC) by AS-A were calculated from theMichaelis–Menten equation using Grafit 4.0 software for Windows(Erithacus Software Ltd., Surrey, UK).

AS-A Activity of Intact Pig Sperm

Pig sperm washed free of seminal fluid were incubated (30 min,37°C) at a concentration of 3 � 107/ml in 200 �l of 0.1 M sodiumacetate buffer, pH 5.0, containing 10 mM NCS, 0.5 mM Na4P2O7,and 0.6% NaCl, as described by Chang and Moudgil (1984).Following centrifugation (350g, 10 min, 28°C), sperm were resus-pended in saline and assayed for their viability by propidium iodide(Molecular Probes) (used at 1 �g/ml) exclusion. The supernatantwas then measured for p-nitrocatechol, the reaction product ofNCS desulfation catalyzed by sperm surface AS-A, following themethod described above.

In Vitro Sperm–ZP Binding Assay

Ejaculated pig sperm, washed free of seminal plasma by centrif-ugation (350g, 10 min, 25°C), were resuspended in capacitationmedium (CM: 0.1 M NaCl, 0.36 mM NaH2PO4, 8.6 mM KCl, 0.5mM MgCl2, 11 mM glucose, 10 mM NaHCO3, 2 mM CaCl2, 5 mMpyruvate, 23 mM Hepes, pH 7.6) supplemented with 0.3% BSA(CM-BSA) (Melendrez et al., 1994) and subjected to two-step(35%/70%) Percoll-gradient centrifugation to select motile sperm,which sedimented as a pellet. Percoll-gradient centrifuged (PGC)sperm were washed once in CM-BSA, resuspended in the samemedium at a concentration of 2 � 107 sperm/ml, and furtherincubated (2 h, 37°C, 5% CO2) to complete capacitation. Capaci-tated pig sperm at 4 � 106/ml were then incubated (45 min, 37°C,5% CO2) with various concentrations of anti-AS-A IgG/Fab or with

affinity purified anti-AS-A IgG. Sperm treated with 100 �g/ml PRSIgG/Fab or with AS-A-nonadsorbed IgG (see the section on prepa-ration of affinity purified anti-AS-A IgG) served as controls. Afterwashing with CM-BSA, 1 � 105 sperm were incubated (30 min,37°C, 5% CO2) in 0.5 ml of CM-BSA with 10–15 cumulus-freeZP-intact oocytes, which were isolated from frozen pig ovariesaccording to the method of Hedrick and Wardrip (1987). At the endof the incubation, sperm–egg complexes were washed free ofunbound sperm through four droplets of CM-BSA using a drawnPasteur pipet having 250-�m bore size. The number of spermbound to the egg ZP was counted under a microscope at 100�magnification. Since a high number of sperm bound to each ZP,only sperm that bound to the periphery of the ZP in the diameterfocal plane were counted. Data of anti-ASA-treated sperm sampleswere expressed as percentages of the control values. Under theseexperimental conditions, the number of sperm bound per egg in thecontrol samples (untreated or treated with PRS IgG/Fab or AS-A-nonadsorbed IgG) was always �30. Differences between the num-bers of sperm bound per egg for the control and anti-ASA-treatedsamples were analyzed by Student’s t test.

SDS–PAGE of Isolated ZP, Purified ZPSulfoglycoproteins, and Deglycosylated ZP

Pig ZP, isolated from homogenized ZP intact oocytes as previ-ously described, were solubilized at 70°C for 1 h in 20 mMTris–HCl, pH 7.4 (Hedrick and Wardrip, 1986). Pig ZP3 (55 kDa,consisting of ZP3� and ZP3�) and partially deglycosylated ZP3� (46kDa) and ZP3� (42 kDa), both generated from ZP3 treated withendo-�-galactosidase, were produced as previously described(Yurewicz et al., 1987a) and were gifts from Dr. E. Yurewicz (WayneState University, Detroit, MI). Total solubilized ZP were deglyco-sylated by trifluoromethanesulfonic (TFMS) acid treatment(Hedrick and Wardrip, 1987). All of these ZP sulfoglycoproteins andZP polypeptide cores were subjected to SDS–PAGE and silver-staining as described above for AS-A, except that the electrophore-sis was performed under a nonreducing condition. Densitometricanalysis was performed to estimate the distribution of ZP1 (86 kDa)and ZP3 in total solubilized ZP, as well as that of deglycosylated ZPpolypeptides [ZP1 (65 kDa), ZP3� (37 kDa), and ZP3� (32 kDa)] inTFMS-treated solubilized ZP.

Binding of Pig Sperm Surface AS-A to Pig ZP

ZP-intact oocytes, washed in TBS–0.1% PVP (polyvinylpyrroli-done), were forced through a Pasteur pipet having 100-�m bore sizeto generate free ZP, which were then cleaned by pipetting througha series of TBS–0.1% PVP droplets. The absence of egg remnantswas verified in ZP samplings by the lack of autofluorescence underthe Zeiss epifluorescent microscope (using a rhodamine filter). Theisolated ZP were then incubated (45 min, room temperature) withpig sperm surface AS-A (0.14 �M, molarity calculated from themolecular mass of AS-A monomer, i.e., 68 kDa) conjugated withAlexa 430 (Molecular Probes) (conjugation was performed accord-ing to the manufacturer’s instructions). Unbound Alexa-430 AS-Awas removed by pipetting the ZP through a series of the TBSdroplets, and the ZP were viewed under the Zeiss epifluorescentmicroscope (using a fluorescein filter). ZP incubated with Alexa-430-conjugated ovalbumin (0.28 �M) served as a negative control.In another set of experiments, isolated pig ZP were pretreated (1 h,room temperature) with rabbit polyclonal anti-pig ZP3 antiserum(1:500 dilution in CM-BSA), produced (Yurewicz et al., 1987b) and

185AS-A Exists on the Pig Sperm Surface and Binds to ZP

© 2002 Elsevier Science (USA). All rights reserved.

kindly provided by Dr. E. Yurewicz, or with preimmune rabbitserum (1:500 dilution in CM-BSA) before incubation with 0.14 �MAlexa-430 AS-A.

Kinetics of AS-A binding to both intact and solubilized pig ZPwas also determined. For the assay of AS-A binding to intact ZP, 30pig ZP, individually isolated from zona-intact oocytes as describedabove, were incubated with various concentrations (0–0.8 �M) ofAlexa-430 AS-A in TBS droplets by using the same incubation andwashing conditions described above. These 30 ZP from a singleincubation were then placed in each well of a black 96-wellpolystyrene plate (Corning Inc., New York, NY) for fluorescenceintensity reading with a Spectramax GeminiXS spectrofluorometer(Molecular Devices, Sunnyvale, CA) at the excitation and emissionwavelengths of 425 and 520 nm, respectively. The same number ofZP incubated with 0.8 �M Alexa-430 ovalbumin was used as ablank. The amount of AS-A bound to 30 ZP in each well wasdetermined from the Alexa-430 AS-A standard curve. The dataobtained were analyzed for the Kd value of AS-A–ZP binding byScatchard plotting using Graffit 4.0 software for Windows.

For the assay of AS-A binding to solubilized pig ZP, a black96-well polystyrene was coated with solubilized ZP (2 �g/well) byovernight incubation at 4°C in the presence of 100 mM sodiumcarbonate buffer, pH 9.6. The plate was blocked with 1% BSA inTBS at room temperature for 1 h and washed four times with TBS.Various concentrations (0–0.4 �M) of Alexa-430 AS-A in 50 �l TBSwere then added to different wells of the plate and incubated atroom temperature for 1 h. Unbound Alexa-430 AS-A was thenremoved by washing the plate four times in TBS. Fluorescenceintensity of Alexa-430 AS-A bound to solubilized pig ZP was thenmeasured, and the Kd value of AS-A-solubilized ZP binding wasobtained following the same methods described for intact ZP.Alexa-430 ovalbumin was likewise used as a negative control.

To determine whether pig ZP3, ZP3�, and ZP3�, all known toparticipate in sperm–ZP interaction (Yurewicz et al., 1993, 1998),were involved in AS-A–ZP binding, these ZP sulfoglycoproteinswere assessed for their ability to compete with solubilized ZP inAS-A binding. A competitive binding assay was performed byadding various concentrations (0–4 �M) of each of these purifiedZP sulfoglycoproteins, solubilized ZP (containing all ZP sulfogly-coproteins), or ovalbumin (negative control) together with 0.15 �Mof Alexa-430 AS-A in 50 �l TBS to each well of the plate precoatedwith 2 �g of solubilized ZP. Following incubation for 1 h at roomtemperature, the plate was washed and measured for fluorescenceintensity of Alexa-430 AS-A bound to ZP in the plate wells.Competitive binding curves were plotted as percentage of thecontrol fluorescence intensity values (i.e., from incubations ofAlexa-430 AS-A with ZP in the wells without any competitors) vsmolarity of the competitors. Molarity of total ZP sulfoglycopro-teins in solubilized ZP samples was calculated based on the 1:4molar distribution of ZP1 to ZP3 (determined as described above).In an alternate experiment, the ability of total ZP polypeptide cores(prepared by TFMS treatment of solubilized ZP) to compete withAS-A–ZP binding was assessed. Total ZP polypeptides were addedwith Alexa-430 AS-A to the ZP wells. The 1:1.6:2.4 molar distri-bution of the three ZP polypeptides, ZP1, ZP3�, and ZP3�, wasused for calculating molarity. Significant differences of the abilityof a pair of ZP protein samples (total ZP sulfoglycoproteins vs totalZP polypeptide cores, and ZP3� vs ZP3�) to compete with Alexa-430 AS-A–ZP binding at a specific concentration were analyzed byStudent’s t test. In addition, the levels of AS-A binding to ZP of thecontrol incubations (no competitors added) vs those included with

competitive ZP proteins were analyzed for significant differencesby using ANOVA.

RESULTS

Molecular Cloning of Pig Testis AS-A

Previous work from our group described a pig spermsurface protein, P68, with binding ability to ZP (Tanphai-chitr et al., 1998). Sequences of three tryptic P68 peptidesrevealed high identity (81–100%) to human AS-A, stronglysuggesting that P68 was AS-A (Tanphaichitr et al., 1999).This prompted us to obtain the pig testis AS-A cDNAsequence. Screening 800,000 plaques of our UNIZAP pigtestis cDNA library with radiolabeled human AS-A cDNAyielded 9 positive clones after tertiary plaque purification.These clones, having various insert sizes (i.e., 2.0, 1.5, and1.2 kb), overlapped with each other, and all showed simi-larity to human (Stein et al., 1989) and mouse (Kreysing etal., 1994a) AS-A cDNA. Two clones were complete, con-taining the entire coding sequence flanked by 5� and 3�untranslated regions, and were used to construct the pigtestis AS-A cDNA sequence (GenBank Accession No. AF316108) that contained 1917 nucleotides, with an openreading frame of 1512 bp (nucleotides 262-1773), a poly(A)tail (19 A’s) (nucleotides 1899–1917), and an adenylationsequence (nucleotides 1876–1880).

The open reading frame encoded 503 amino acids begin-ning with a putative signal peptide of 17 residues. Thededuced amino acid sequence (Fig. 1) contained three po-tential N-glycosylation sites and 16 cysteines, and wascolinear (100% matched) with chemically determinedamino acid sequences for the three tryptic P68 peptides(amino acids 58–70, 368–380, and 460–475). The pig testisAS-A sequence showed 86 and 82% identity to human andmouse AS-A, respectively (Fig. 1,*). The positions of theN-glycosylation sites of AS-A were conserved in all threespecies. The pig sequence contained all 14 cysteine residuespresent in common in the mouse and human sequences,and had two additional cysteines at positions 134 and 340.Like the mouse sequence, pig testis AS-A did not possessthe cysteine residue at position 38 in the human sequence.All of the amino acid residues forming the AS-A’s activesite pocket (i.e., D28, C68, K122, H124, S148, H226, D278,K299), as well as the three amino acid pairs (i.e., H325/P41,S42/S428, and Y435/T404), presumably involved in inter-molecular hydrogen bonding important for the enzymedimerization (Lukatela et al., 1998), were conserved in allthree species (Fig. 1).

These results identified the sperm surface P68 as AS-Aand suggest a new location, the cell surface, for AS-A,previously recognized as a lysosomal/acrosomal enzyme(Allison and Hartree, 1970; Sarafian et al., 1982; Dud-kiewicz, 1984; Fujii et al., 1992).

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Localization of AS-A to the Pig Sperm Surface

Immunolocalization of AS-A was performed at both lightand electron microscopic levels, using affinity purifiedanti-AS-A IgG and Alexa-488-conjugated secondary anti-body or protein A-gold, respectively. Since live spermwould exclude antibody entry into the cytoplasm, positivesignals would represent only the antigen entity on thesurface. Figure 2A (a) shows the presence of immunofluo-rescent staining of pig sperm at the head anterior region, thesite of ZP binding (Burkin and Miller, 2000). In contrast,control sperm exposed to PRS IgG did not show anystaining (Fig. 2A, c). The presence of AS-A on the spermhead surface was ultimately demonstrated by electron mi-

croscopic immunogold labeling. The gold particles werelocalized to the sperm head surface, on top of the plasmamembrane (Fig. 2B, a and b), which appeared as an electronlucent (i.e., white in the image) layer, since the spermsample was not subjected to osmication (which fixes lipids).A negative control sample, which was exposed to PRS IgG,had only a minimal number of gold particles on the surface(Fig. 2B, c), indicating that the positive results obtainedwith sperm incubated with anti-AS-A IgG were specific toAS-A. Furthermore, sperm treated with the AES solutionhad their plasma membrane sporadically removed (Fig. 2C,b), as compared with untreated sperm possessing intactplasma membrane (Fig. 2C, a). This AES extract contained

FIG. 1. Amino acid sequence of pig testis AS-A shown in comparison with the human and mouse sequences. The deduced amino acidsequence of pig testis AS-A was obtained from the sequences of pig testis cDNA clones, positively screened with 32P-labeled human AS-AcDNA probe. Amino acid sequences of P68 tryptic peptides (100% matched) are in bold font underneath a line. Open boxes enclose theconsensus sites for N-linked glycosylation. Gray boxes enclose the amino acids forming the active site pocket. Black boxes enclose extracysteine residues present on the pig sequence. Comparison of the pig (GenBank Accession No. AF 316108), human (GenBank Accession No.X 52151), and mouse (GenBank Accession No. X 73230) testis AS-A was performed by the Clustal W (1.81) multiple sequence alignmentprogram. Identical residues are indicated by asterisks, conserved substitutions by colons, and semiconserved substitutions by dots.

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AS-A desulfation activity on NCS (Table 1). Therefore, theintensity of the immunofluorescent staining of AS-A inthese AES-treated sperm was markedly diminished, asexpected (Fig. 2A, b), compared with untreated sperm (Fig.2A, a). This staining presumably represented AS-A on theresidual sperm plasma membrane and exposed acrosomalmembrane of AES-treated sperm. Finally, the presence ofsurface AS-A was illustrated by the NCS desulfation activ-ity of intact pig sperm, i.e., 0.15 � 0.06 mU/106 sperm (n �5). Although this assay was performed at AS-A’s pH opti-mum of 5, all of the sperm were still viable, as shown by thelack of propidium iodide staining in these cells.

Purification and Biochemical Characterization ofPig Sperm Surface AS-A

Possessing all amino acids that form the active sitepocket (Fig. 1), pig testis/sperm AS-A was expected tocontain arylsulfate desulfation activity in solution. Toverify this, we monitored desulfation activity of the puri-fied pig sperm AS-A, at pH 5.0 (the pH optimum of anAS-A), using an artificial substrate, NCS (Fluharty andEdmond, 1978). As described for P68 (Tanphaichitr et al.,1998), chromatofocusing was used as the initial purificationstep of AS-A from the pig sperm AES extract. An additionalstep, Sephacryl S-200 gel filtration, was included to increaseAS-A’s purity, as well as to determine its native molecularmass. NCS desulfation activity was present in the AESextract and in the eluted fractions (110–130 ml) from thechromatofocusing column (Fig. 3A), with an 11-fold in-crease in specific activity, relative to the AES extract (Table1). When the chromatofocused fractions were pooled andsubjected to Sephacryl S-200 chromatography, a peak ofNCS-desulfation activity was obtained (Fig. 3B), with 122%yield and specific activity of 141.86 U/mg, i.e., a 39-foldincrease from the corresponding value of the AES extract(Table 1). The increase in activity yield may reflect removalof inhibitors as observed in AS-A purification from humanliver (Draper et al., 1976) and rabbit sperm acrosome (Yangand Srivastava, 1974). All of these findings revealed that pigsperm AS-A was an active peripheral plasma membraneenzyme. The molecular mass of this purified sperm AS-A inthe neutral pH solution was estimated to be 135–140 kDa(Fig. 3B). The AES extract and the peaks containing NCSdesulfation activity, eluted from both chromatofocusingand Sephacryl S-200 columns, reacted with both anti-AS-Aand anti-P68pep antibodies. Reactivity with these twoantibodies was also observed with purified human liverAS-A, as expected (Figs. 3D and 3E).

Significantly, the final purification by Sephacryl S-200yielded pig sperm AS-A with high purity, appearing as themajor band on silver-stained SDS-polyacrylamide gel (ap-parent molecular mass of 68 kDa; Fig. 3C), consistentlymigrating slightly slower than the major band of humanliver AS-A (apparent molecular mass of 63–66 kDa). Usingthis highly purified pig sperm AS-A for kinetic studies, itspH optimum for NCS desulfation was shown to be in the

FIG. 2. Localization of AS-A to the pig sperm surface. (A)Indirect immunofluorescence: Live pig sperm washed with PBS(a) or treated with AES solution (b) were incubated with affinitypurified anti-AS-A IgG followed by Alexa-488-conjugated goatanti-rabbit IgG. A negative control was the PBS-washed spermsample that was incubated with PRS-IgG instead of affinitypurified anti-AS-A IgG (c). Bar, 10 �m. (B) Transmission electronmicroscopic immunogold labeling: Live pig sperm washed withPBS were incubated with affinity-purified anti-AS-A IgG (a, b) orPRS-IgG (c) followed by protein A-gold (8 nm). The sperm werepostfixed in aldehyde, dehydrated, and embedded in LR White.Bars, 0.2 �m. (C) Transmission electron microscopy of AES-treated pig sperm: Control sperm (a), washed with PBS, pos-sessed an intact plasma membrane (PM), outer acrosomal mem-brane (OAM), and the acrosomal matrix (AC). In contrast, spermexposed to AES (b) showed scattered detachment of the plasmamembrane, while the outer acrosomal membrane and the acro-somal matrix remained intact. N, nucleus. Bars, 0.2 �m.

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range of 4.5–5.0 (data not shown), similar to that observedfor human liver AS-A (Fluharty and Edmond, 1978). SimilarKm values were also obtained for both pig sperm AS-A andhuman liver AS-A, using either a soluble artificial sub-strate, NCS, or detergent-solubilized natural sulfoglycolip-ids, SGG and SGC (Table 2). On the other hand, Vmax valuesof pig sperm AS-A were consistently lower than those ofhuman liver AS-A for all three substrates used (i.e., 23, 3,and 12% for NCS, SGC, and SGG, respectively).

Role of AS-A in Pig Sperm–ZP Interaction

Our identification of P68 as AS-A and localization ofAS-A to the sperm head surface strongly suggested theinvolvement of AS-A in sperm–ZP binding. Figure 4 (leftpanel) shows that pig sperm pretreated with anti-AS-A IgGhad reduced ability to bind to the egg ZP in a dose-dependent manner, with a marked inhibition of �70% atthe antibody concentration of 100 �g/ml. The affinitypurified IgG antibody showed a similar inhibitory effect(65% inhibition) as the original anti-ASA IgG (100 �g/ml,see Materials and Methods). In contrast, IgG in the originalanti-AS-A IgG solution that was not adsorbed to AS-A(AS-A-nonadsorbed IgG) did not show any inhibition ofsperm–ZP binding. The results suggested that the inhibi-tion observed with sperm pretreatment with anti-AS-A IgGwas specifically attributed to the masking of sperm surfaceAS-A. This inhibition was not from the steric hindrancedue to the bivalent nature of IgG, since anti-AS-A-Fabfragments also exerted similar inhibitory effects (Fig. 4,right panel). However, when compared with IgG concentra-tions of the same antigen-binding valency, the degree ofinhibition was less pronounced with Fab fragments (i.e.,41% inhibition for 35 �g/ml anti-AS-A Fab vs 48% for 50�g/ml anti-AS-A IgG; and 58% inhibition for 70 �g/mlanti-AS-A Fab vs 70% for 100 �g/ml anti-AS-A IgG). Thelower degree of inhibition of sperm–ZP binding caused byanti-AS-A Fab may be due to its lower affinity for AS-A,relative to anti-AS-A IgG.

The participation of AS-A in ZP binding was furtherdemonstrated by its direct binding to pig ZP. Figure 5A (a)shows that intact pig ZP, incubated with 0.14 �M Alexa-430 AS-A, possessed uniform fluorescent staining. In con-

trast, pig ZP exposed to 0.28 �M Alexa-430 ovalbuminshowed background fluorescent staining (Fig. 5A, b). Anexcess amount (100�) of the unlabeled enzyme, present inthe coincubate of pig ZP with Alexa-430 AS-A, also abro-gated the fluorescent staining of the ZP (data not shown).When increasing amounts of Alexa-430 AS-A were incu-bated with intact ZP, the fluorescence intensity of theZP-bound Alexa-430 AS-A increased and approached aplateau at 0.8 �M of free Alexa-430 AS-A (Fig. 5B). Notably,the amount of Alexa-430 AS-A bound to the intact ZP wasonly 1/500–1/120 of the free Alexa-430 AS-A in the ZPincubation mixture. Scatchard plot analysis revealed one Kd

of 0.57 �M. A similar Kd value (0.50 �M) was obtainedwhen solubilized ZP (coated onto the plate well) were usedin place of intact ZP (Fig. 5C). All of these results stronglysuggested specific binding of Alexa-430 AS-A to ZP.

Pretreatment of pig ZP with anti-pig ZP3 antibody, spe-cific to pig ZP3 sulfoglycoprotein (Yurewicz et al., 1987b),abolished the binding of Alexa-430 AS-A to the ZP (Fig. 5A,c). In contrast, control ZP pretreated with PRS showed thesame fluorescent staining of Alexa-430 AS-A, as seen withuntreated ZP (see Fig. 5A, a). The results suggested thatZP3, recognized as the pig sperm receptor (Sacco et al.,1989; Yurewicz et al., 1998), was the binding ligand ofAS-A.

To further investigate specificity of pig ZP componentsthat bound to AS-A, competitive binding assays were per-formed in a microtiter plate. As expected, solubilized pig ZPcontaining total ZP sulfoglycoproteins, added together withAlexa-430 AS-A, effectively inhibited Alexa-430 AS-A bind-ing to the ZP immobilized in the plate well, showing an IC50

of 0.35 �M (Fig. 6A, �—�). However, the maximuminhibition was 74%. The inability of the added solubilizedZP to further inhibit Alexa-430 AS-A–ZP binding might bedue to incomplete availability of the AS-A binding ligand(s)of solubilized ZP in solution. On the other hand, theseligands may be well exposed when the solubilized ZP wereattached to the plate well. When 4 �M deglycosylated ZPwas present in the Alexa-430-ZP incubation, Alexa-430AS-A–ZP binding was also significantly reduced as com-pared with control incubations without any competitor(P � 0.05). However, the degree of competitive inhibition bydeglycosylated ZP (Fig. 6A, �—�) was much lower than

TABLE 1Purification of Pig Sperm AS-A

Total protein (mg) Total activity (�mol/h) Yield (% activity) Specific activity (U/mg)a Purification fold

AES extract 6.83 24.84 100 3.64 1Chromato-focusing 0.850 36.1 145 42.47 11Sephacryl S-200 0.215 30.5 122 141.86b 39

a AS-A enzymatic activity was assayed at pH 5.0, 37°C, using the artificial substrate NCS (5 mM). Protein was quantified by using theBioRad Protein Assay. One enzymatic unit was defined as 1 �mol of substrate hydrolyzed in 1 h.

b The final specific activity obtained after Sephacryl S-200 chromatography was reproducible throughout different rounds of purification(n � 5).

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that observed with total ZP sulfoglycoproteins (Fig. 6A,�—�), i.e., 15, 17, and 43% vs 67, 71, and 75% at 1, 2, and4 �M, respectively. In contrast, ovalbumin up to 8 �M,added together with Alexa-430 AS-A to the ZP well, did notshow any inhibition on Alexa-430 AS-A–ZP binding (datanot shown). These results suggested that the carbohydratemoieties of ZP sulfoglycoproteins were important for AS-A

binding. The ZP polypeptide cores also participated inAS-A–ZP binding, but to a lesser extent than the ZPcarbohydrate moieties.

Purified pig ZP3 also competed effectively with Alexa-430 AS-A–ZP binding, revealing an IC50 of 0.18 �M andmaximum inhibition of 90% (Fig. 6B, ‚—‚). This result isin agreement with the observation that anti-ZP3 antibodyblocked AS-A binding to the ZP (Fig. 5A, c). ZP3�, one ofthe two ZP3 components, also exhibited strong competitiveinhibition on Alexa-430-AS-A binding, similar to that ob-served with ZP3, with IC50 of the same range, i.e., 0.50 �M(Fig. 6B, Œ—Œ). The maximum inhibition induced by ZP3�was also as high as that observed with ZP3 (i.e., 84%). Incontrast, exogenous ZP3� (Fig. 6B, f—f) possessed a muchlower inhibitory effect than ZP3�. At 0.5, 1, 2, and 4 �M ofZP3�, competitive inhibition of Alexa-430 AS-A–ZP bind-ing was at 12, 21, 29, and 45% (vs 50, 63, 82, and 87%,respectively, observed with ZP3�). The results indicatedgreater significance of ZP3� in AS-A–ZP binding. ZP3� hasalso been recognized to be a major player in pig sperm–ZPbinding, as compared with ZP3� (Yurewicz et al., 1993,1998). Therefore, our results suggested that AS-A is part ofthe sperm machinery involved in ZP binding.

DISCUSSION

We have described a pig sperm surface protein, P68,which possesses affinity to both ZP and SGG, and alsoparticipates in sperm–ZP interaction (Tanphaichitr et al.,1998). In this report, molecular cloning revealed the pres-ence of three P68 tryptic peptides in the pig testis AS-Asequence (Fig. 1), indicating that P68 was AS-A, an enzymepreviously shown to be present in the somatic cell lyso-some and sperm acrosome (Allison and Hartree, 1970;Sarafian et al., 1982; Dudkiewicz, 1984; Fujii et al., 1992).This result, therefore, uncovered two new physiologicalattributes of AS-A, i.e., its existence on the sperm surfaceand its adhesive property to the egg extracellular matrices,the ZP.

Immunolocalization was the first approach used to dem-onstrate the presence of AS-A on the pig sperm surface.Indirect immunofluorescence of live pig sperm revealedAS-A staining in the anterior head region (Fig. 2A), the siteof ZP binding (Burkin and Miller, 2000), and this localiza-tion was further confirmed by electron microscopic immu-nogold labeling (Fig. 2B). Furthermore, live sperm possessedNCS desulfation activity at pH 5.0, typical of an AS-A.Finally, AES treatment of pig sperm yielded a solubleextract with NCS desulfation activity, whereas the treatedsperm pellet showed only sporadic plasma membrane de-tachment (Fig. 2C). AS-A purified from this sperm AESextract, through a series of column chromatography, in factpossessed a number of enzymatic properties (i.e., pH opti-mum of 4.5, substrate specificity, and Km values for sulfo-glycolipids and NCS) very similar to those of human liverAS-A (Table 2). In addition, the purified sperm surface AS-A

FIG. 3. Purification of pig sperm AS-A. Pig sperm surface proteinswere extracted by using an AES solution (1 h, 4°C). Extracts weresubjected to chromatofocusing (A), and fractions with AS-A activ-ity were pooled and chromatographed on a Sephacryl S-200 column(B). Column fractions were assayed for the amount of proteins atA230 (�—�) and for AS-A desulfation activity of NCS at A515

(F—F). pH of each chromatofocusing fraction was also measured(‚—‚). The vertical arrow in (A) indicates where the salt gradientstarted, whereas the arrowheads in (B) denote the elution positionsof protein standards, i.e., rabbit IgG (150 kDa), bovine serumalbumin (65 kDa), and ovalbumin (45 kDa). (C) Silver-stainedSDS–polyacrylamide gel of the AES extract (lane 1), pooled frac-tions from chromatofocusing (lane 2) and Sephacryl S-200 chroma-tography (lane 3), which possessed AS-A activity, and purifiedhuman liver AS-A (lane 4). Std, molecular mass markers. Immuno-blotting using anti-AS-A (D) or anti-P68pep (E) antibody wasperformed with the same blot. The horizontal arrows indicate theposition of AS-A band on SDS–polyacrylamide gel.

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appeared as a dimer in solution at neutral pH (Fig. 3B) likehuman liver AS-A (Sarafian et al., 1982), and as a single68-kDa band on SDS–PAGE, which reacted with the anti-body produced against human liver AS-A (Figs. 3C and 3D).Our work using various approaches conclusively revealedthe existence of AS-A on the pig sperm surface. AlthoughAS-A was previously implied to be on the rabbit spermsurface, indirect immunofluorescence was the only ap-proach used in this study (Nikolajczyk and O’Rand, 1992).

Previous reports indicate transcriptional expression ofAS-A in spermatogenic cells (Kreysing et al., 1994b) and the

presence of AS-A in the sperm acrosome (Allison andHartree, 1970; Dudkiewicz, 1984; Nikolajczyk and O’Rand,1992). However, since AS-A does not contain any trans-membrane domain (Fig. 1), its trafficking from the cyto-plasm to the surface of male germ cells needs an explana-tion. While a postulation has been made that acrosomalproteins may be transported to the sperm plasma mem-brane through dynamic fusion pores across the acrosomalmembranes (Monck and Fernandez, 1996; Foster et al.,1997), our recent results suggest an alternative mechanismfor the presence of AS-A on the mature sperm surface(Weerachatyanukul et al., 2001b). Immunolocalization atboth light and transmission electron microscopic levelsreveal the absence of AS-A on the surface of mouse sper-matogenic cells and testicular sperm. However, surfaceAS-A increasingly appears in sperm transiting through theepididymis, due to the adsorption of the enzyme, present inthe epididymal fluid, onto the sperm surface. This adsorp-tion is via AS-A’s affinity to SGG already present intesticular sperm (Weerachatyanukul et al., 2001b) andwould likely be part of the events necessary for spermmaturation (Tulsiani and Abou-Haila, 2001).

The involvement of sperm surface AS-A in sperm–ZPbinding was demonstrated in the present work by twoapproaches. In the first, capacitated pig sperm pretreatedwith anti-AS-A IgG/Fab antibody were shown to havereduced ability to bind to the ZP, in a dose-dependentmanner. The fact that inhibition was observed with themonovalent Fab fraction of anti-AS-A to a similar extent asanti-AS-A IgG (Fig. 4) indicated that the inhibition ofsperm–ZP binding was due to specific masking of AS-A, andnot from steric hindrance induced by the bivalent nature ofthe anti-AS-A IgG antibody. Nonspecific inhibition causedby other immunoglobulins that do not recognize AS-A wasalso excluded since affinity purified AS-A inhibitedsperm–ZP binding to the same extent as the original anti-AS-A IgG solution from which it was purified (Fig. 4),whereas AS-A-nonadsorbed IgG did not show any inhibi-tion on gamete binding (data not shown). In the secondapproach, Alexa-430 AS-A was shown to have direct andspecific binding to both solubilized ZP and intact ZP, withsimilar Kd values (0.50 and 0.57 �M, respectively) (Fig. 5).

FIG. 4. Inhibitory effects of sperm pretreatment with anti-AS-Aon sperm–ZP binding. Sperm were pretreated with anti-AS-A IgG(left panel) or its Fab fragments (right panel) at various concentra-tions or with affinity purified anti-AS-A IgG before coincubationwith eggs. Control sperm were pretreated with 100 �g/ml PRS IgGfor the anti-AS-A IgG experiments or with 70 �g/ml PRS Fab for theanti-AS-A Fab experiments, and similarly coincubated with eggs.The number of sperm bound to egg ZP was counted and expressedas percent control. The number of PRS IgG/Fab-treated sperm(control) bound to eggs was assigned as 100%. Data were expressedas mean � SD of percent control of means from three experimentsor more, except for the experiments using affinity purified anti-AS-A IgG-treated sperm. These experiments were done twice andthe plot represents an averaged percent control from these twoexperiments. *, denotes significant difference (P � 0.05) from thecontrol samples, as determined by ANOVA of the raw data (i.e.,number of sperm bound/egg).

TABLE 2Km and Vmax for the Hydrolysis of an Artificial Substrate (NCS) and Natural Sulfoglycolipids (SGC and SGG)by Pig Sperm and Human Liver AS-A

NCS SGC SGG

AS-A Km (mM) Vmax (U/mg) Km (mM) Vmax (U/mg) Km (mM) Vmax (U/mg)

Pig sperm 7.02 � 2.84 387.5 � 85.97 0.121 � 0.061 0.525 � 0.109 0.091 � 0.052 0.72 � 0.19Human liver 6.90 � 1.53 1712 � 205 0.124 � 0.056 23.04 � 5.51 0.092 � 0.040 6.15 � 1.15

Note. Kinetic experiments (n �3) were carried out at pH 4.5, 37°C using 1.2 �g of purified pig sperm AS-A or 0.4 �g of purified humanliver AS-A.

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Furthermore, the intensity of ZP-bound Alexa-430 AS-Aincreased and approached saturation with increasingamounts of free Alexa-430 AS-A, and the ZP-bound Alexa-430 AS-A was only �1/120 of free Alexa-430 AS-A. Inaddition, Alexa-430 ovalbumin did not show binding to theZP.

Scatchard plot analysis yielded only one Kd of 0.50 �Mfor AS-A binding to solubilized pig ZP, which was seventimes higher than a Kd described for the binding ofsolubilized mouse ZP to homologous sperm (Thaler andCardullo, 1996; Kerr et al., 2001). This discrepancy couldbe due to species differences. In addition, the synergisticaction of sperm surface molecules in ZP binding mayaccount for the lower Kd of the binding of solubilizedmouse ZP to intact sperm. In the case of sperm surfaceAS-A, this could be SGG, which also has ZP-bindingability (White et al., 2000; Weerachatyanukul et al.,2001a). AS-A and SGG have high affinity to each other(Carmona et al., 2002) and they are localized to the sameregion on the surface of mature sperm (White et al., 2000;Weerachatyanukul et al., 2001a; Tantibhedhyangkul etal., 2002). In addition, our results indicated that AS-Abound to ZP3, the pig sperm receptor (Sacco et al., 1989),since pretreatment of eggs with anti-ZP3 inhibited theAS-A–ZP binding (Fig. 5A) and since ZP3 could competi-tively inhibit AS-A–ZP binding (Fig. 6B). The competitivebinding assay also revealed that ZP carbohydrate moi-eties were important for AS-A binding (Fig. 6A). In thesame assay, ZP3� inhibited AS-A–ZP binding at a lowIC50 (0.50 �M) in contrast to ZP3� showing a much lesserextent in inhibiting AS-A–ZP binding (Fig. 6B). Both ZPcarbohydrate moieties and ZP3� have been shown to besignificant for interaction between ZP and pig spermplasma membranes (Yurewicz et al., 1991, 1993; Noguchiet al., 1992). Therefore, all of our results strongly suggestthat AS-A is an integral component of the sperm plasmamembrane machinery for ZP binding. Like the situationwith �-galactosyltransferase (Nixon et al., 2001) and�-mannosidase (Tulsiani and Abou-Haila, 2001), the con-tribution of AS-A in sperm–ZP binding is not speciesspecific, since AS-A also exists on the mouse spermsurface and is involved in ZP binding (Tantibhedhy-angkul et al., 2002).

AS-A is known to desulfate glycosulfates, includingdetergent-solubilized sulfoglycolipids (Fluharty and Ed-mond, 1978; Fluharty et al., 2001) and sulfated monosac-charides (Mehl and Jatzkewitz, 1968; Rahi and Srivastava,1983). Recently, we have shown that AS-A, in the absenceof a detergent, also binds tightly to SGG and SGC withoutexerting its desulfation activity (Carmona et al., 2002).Nonetheless, the glycosulfate moiety of SGG/SGC is stillessential for this binding (Carmona et al., 2002). Based onthese results, we speculate that AS-A might bind to sulfatedsugar residues of the acidic ZP glycans, including thosepresent in pig ZP3� (i.e., in both the glycan antennae andthe reducing end; Mori et al., 1998). Notably, sperm AS-Ahad low Vmax values for desulfation of SGG, SGC, and NCS,

FIG. 5. Binding of pig sperm AS-A to the ZP. (A) Pig ZP wereincubated with 0.14 �M Alexa-430-conjugated purified pigsperm AS-A (a) or 0.28 �M Alexa-430-conjugated ovalbumin (b).After washing the excess unbound fluorescent protein, the ZPwere viewed under an epifluorescent microscope. In (c), pig ZPwere pretreated with anti-pig ZP3 antibody before incubationwith Alexa-430 AS-A. Control pig ZP, pretreated with PRS IgGand similarly incubated with Alexa-430 AS-A, showed the samebinding as in (a). Bar, 10 �m. (B) Binding of Alexa-430-conjugatedAS-A to intact ZP. Thirty pig ZP were incubated with 0– 0.8 �MAlexa-430 AS-A, and the fluorescent intensity of the ZP wasmeasured spectrofluorometrically by using ZP preincubatedwith 0.8 �M Alexa 430-ovalbumin as a blank. The amount ofAlexa-430 AS-A bound to the ZP was determined from theAlexa-430 AS-A standard curve. Open circles represent indi-vidual sample points. Linear Scatchard plot analysis (inset)revealed a Kd of 0.57 �M. (C) Binding of Alexa-430 to solubilizedZP. Alexa-430 AS-A (0 – 0.4 �M) was incubated with solubilizedpig ZP (2 �g), attached to each microtiter plate well, and thebinding of Alexa-430 AS-A to ZP was measured as described in(B). Kd was likewise analyzed to be 0.50 �M.

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and this may also be the case for ZP sulfated glycans, shouldthey bind to AS-A’s active site. The initial binding betweensulfated sugars of the acidic ZP glycans may then beretained due to the low capacity of sperm AS-A to hydrolyzetheir substrates. Subsequently, desulfation of the ZP gly-cans’ sulfated sugars may occur, and this may weaken thebinding between sperm surface AS-A and ZP glycans. To-gether with other mechanisms involved in disrupting theinteraction between sperm and the ZP (e.g., hydrolysis ofperipheral sugars of the ZP glycans involved in spermbinding; Dell et al., 1999), sperm could migrate furtherthrough the ZP layer. Interestingly, the concept that spermsurface hydrolases repeatedly bind to their substrates on theegg vestment (e.g., ZP) and surface, and then exert theirenzymatic activity, as part of sperm passage processthrough these egg entities, has been previously described(Primakoff and Myles, 2000). Our results demonstrating thesignificance of AS-A on the sperm surface in ZP binding areconsistent with the recent report revealing the role ofsurface N-acetyl glucosamine sulfatase in cell adhesion andsignaling during quail embryo patterning (Dhoot et al.,

2001). The postulation that sulfated sugars of the ZPglycans participate in binding to sperm surface molecules(in our case, AS-A) (Huang et al., 1984; O’Rand et al., 1988;Jones, 1991; Oehninger et al., 1992) is also in accordancewith recent observations implicating (poly)sulfated glycansin cell adhesion (Vilela-Silva et al., 2002; Talevi and Gual-tieri, 2001; Su et al., 2001; Pinzon-Ortiz et al., 2001;Shikano et al., 2001). Attempts to elucidate the molecularmechanisms of AS-A interaction with the ZP sulfoglycop-roteins are ongoing in our laboratory.

ACKNOWLEDGMENTS

We thank Mr. Ali Shoushtarian, Ms Jennifer Miles, and Ms.Barbara J. Nitta for their valuable technical assistance; and Ms.Terri Van Gulik for help in manuscript preparation. We alsoappreciate the permission from the United Leukodystrophy Foun-dation to use the human liver arylsulfatase A in this study. Thiswork was supported by the Canadian Institutes of Health Research(MT-10366) and the Rockefeller Foundation (both to N.T.), Na-tional Science and Technology Developmental Agency of Thailand

FIG. 6. Competitive inhibition of Alexa-430 AS-A binding to pig ZP by ZP proteins. Solubilized ZP, coated in microtiter plate wells (2�g/well), were incubated with 50 �l of 0.15 �M of Alexa-430 AS-A in the presence of various concentrations (0–4 �M) of each purified ZPprotein, total ZP sulfoglycoproteins, total ZP polypeptides, or ovalbumin (negative control). Binding of Alexa-430 AS-A to ZP in the wellswas then measured spectrofluorometrically. Competitive binding curves were plotted as percentage of control values (i.e., fluorescenceintensity of Alexa-430 AS-A bound to ZP in the plate when incubations were performed without any competitors) vs molarity of the pigZP sulfoglycoprotein competitors. (A) Inhibition by total pig ZP sulfoglycoproteins (�—�) and TFMS-deglycosylated ZP proteins (�—�).SDS–PAGE patterns of total ZP sulfoglycoproteins (lane 1) and TFMS-deglycosylated ZP proteins (lane 2) are shown on the right of thepanel. *, denotes significant difference (P � 0.05) of the AS-A–ZP binding levels compared between incubations with total ZPsulfoglycoproteins and with deglycosylated ZP proteins, at the same molar concentration, as determined by Student’s t test. The AS-A–ZPbinding levels observed in incubations with total ZP sulfoglycoprotein incubations at all concentrations were significantly lower from thelevels of control incubations (without any competitors), as determined by ANOVA. In contrast, a significant decrease in AS-A–ZP bindingwas observed at only 4 �M of deglycosylated ZP. (B) Inhibition by ZP3 (‚—‚), ZP3� (Œ—Œ), and ZP3� (f—f). SDS–PAGE on the rightdisplays electrophoretic patterns of ZP3 (lane 1), ZP3� (lane 2), and ZP3� (lane 3). *, denotes significant differences (P � 0.05) of theAS-A–ZP binding levels compared between ZP3�- and ZP3�-containing incubations, at the same molar concentration, as determined byStudent’s t test. The AS-A–ZP binding levels at all concentrations of ZP3 and ZP3� and at 2 and 4 �M of ZP3� were significantly lowerfrom the control values, as determined by ANOVA.

193AS-A Exists on the Pig Sperm Surface and Binds to ZP

© 2002 Elsevier Science (USA). All rights reserved.

(graduate study scholarship to W.W.), OMAFRA, and Ontario Pork(to M.B.) and USDA (NRICGP 99-02240) (to T.B.).

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Received for publication September 20, 2001Revised March 12, 2002Accepted April 8, 2002

Published online May 31, 2002

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