Discovery, Primary, and Crystal Structures and Capacitation- related

Discovery, Primary, and Crystal Structures and Capacitation-related Properties of a Prostate-derived Heparin-bindingProtein WGA16 from Boar Sperm*

Received for publication, December 24, 2014 Published, JBC Papers in Press, January 8, 2015, DOI 10.1074/jbc.M114.635268

Estelle Garenaux‡1, Mayumi Kanagawa§, Tomoyuki Tsuchiyama‡¶, Kazuki Hori‡¶, Takeru Kanazawa‡¶, Ami Goshima‡¶,Mitsuru Chiba�, Hiroshi Yasue**, Akemi Ikeda§, Yoshiki Yamaguchi§, Chihiro Sato‡¶, and Ken Kitajima‡¶2

From the ‡Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan, the §RIKEN Structural GlycobiologyTeam, Saitama 351-0198, Japan, the ¶Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan,the �Hirosaki University Graduate School of Health Sciences, Hirosaki 036-8564, Japan, and the **National Institute ofAgrobiological Sciences, Tsukuba 305-8602, Japan

Background: Mechanisms of glycoprotein redistribution during sperm capacitation remain unclear.Results: Prostate-specific ZG16-like lectin WGA16 was discovered in sperm lipid rafts. Its primary and crystal structure andGalT- and heparin-binding properties were characterized.Conclusion: Attachment to sperm via surface GalT and capacitation-induced detachment involve unique N-glycans and theheparin-binding domain.Significance: This is the first demonstration of a glycan-mediated mechanism for transient existence of seminal plasma glyco-protein on the sperm surface.

Mammalian sperm acquire fertility through a functional mat-uration process called capacitation, where sperm membranemolecules are drastically remodeled. In this study, we found thata wheat germ agglutinin (WGA)-reactive protein on lipid rafts,named WGA16, is removed from the sperm surface on capaci-tation. WGA16 is a prostate-derived seminal plasma protein thathas never been reported and is deposited on the sperm surface inthe male reproductive tract. Based on protein and cDNA sequencesfor purified WGA16, it is a homologue of human zymogen granuleprotein 16 (ZG16) belonging to the Jacalin-related lectin (JRL) fam-ily in crystal and primary structures. A glycan array shows thatWGA16 binds heparin through a basic patch containing Lys-53/Lys-73 residues but not the conventional lectin domain of the JRLfamily. WGA16 is glycosylated, contrary to other ZG16 members,and comparative mass spectrometry clearly shows its uniqueN-glycosylation profile among seminal plasma proteins. It hasexposed GlcNAc and GalNAc residues without additional Gal res-idues. The GlcNAc/GalNAc residues can work as binding ligands

foraspermsurfacegalactosyltransferase,whichactuallygalactosyl-ates WGA16 in situ in the presence of UDP-Gal. Interestingly, sur-face removal of WGA16 is experimentally induced by either UDP-Gal or heparin. In the crystal structure, N-glycosylated sites and apotential heparin-binding site face opposite sides. This geography oftwo functional sites suggest that WGA16 is deposited on the spermsurface through interaction between its N-glycans and the surfacegalactosyltransferase, whereas its heparin-binding domain may beinvolvedinbindingtosulfatedglycosaminoglycansinthefemaletract,enabling removal of WGA16 from the sperm surface.

During natural fertilization in mammals, sperm relies on helpfrom the female tract to acquire fertilizing capacity through a func-tional maturation process called capacitation (1, 2). Before beingable to fertilize oocyte, sperm has to undergo several maturationprocesses in the male reproductive tract, followed by functionalmodifications in the female tract. Sperm leave testes morphologi-cally complete but lack motility as well as crucial proteins involvedin oocyte binding and fertilization. Key proteins related to motilityor later to interaction with the egg surface are added to spermmembrane in epididymis (3). It is then crucial that sperm do notbecome fertile too quickly. At the time of ejaculation, seminalplasma proteins, present in secretions from male accessory glands(seminal vesicles, prostate, and bulbourethral glands) coat thesperm surface and stabilize the plasma membrane, inhibiting fer-tilization ability (4). While migrating through the female genitaltract, sperm acquire a series of functional modifications to be ableto reach the egg, interact with the zona pellucida (ZP),3 and finally

* This research was supported in part by Grants-in-Aid for Scientific Research (B)22380187 and 25292216; Grant-in-Aid for Japan Society for the Promotion ofScience (JSPS) fellows relating to JSPS Postdoctoral Fellowship for ForeignResearchers 21-09722 (to K. K. and E. G.); Grant-in-Aid for Scientific Research(C) 23570133 (to C. S.); the JSPS Strategic Young Researcher Overseas VisitsProgram for Accelerating Brain Circulation (to K. K.) from the Ministry of Edu-cation, Science, and Sports; the CNRS and Japan Science and TechnologyAgency (JST) Strategic Japanese-French Cooperative Program on MarineGenome and Marine Biotechnology (to K. K.); and a grant from the Naito Founda-tion Subsidy for Promotion of Specific Research Projects in 2012 (to E. G. and K. K.).

The atomic coordinates and structure factors (codes 3WOB and 3WOC) havebeen deposited in the Protein Data Bank (http://wwpdb.org/).

The nucleotide sequence(s) reported in this paper has been submitted to the Gen-BankTM/EBI Data Bank with accession number(s) AB851481

1 Present address: Clermont Universite, “Microbe intestin inflammation etSusceptibilite de l’Hote,” UMR1071 INSERM/Universite d’Auvergne M2iSH,63000 Clermont Ferrand, France.

2 To whom correspondence should be addressed: Bioscience and Biotechnol-ogy Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan. Tel.:81-52-789-4297; Fax: 52-789-5228; E-mail: [email protected].

3 The abbreviations used are: ZP, zona pellucida; GalT, galactosyltransferase;GS-IB4, G. simplicifolia-IB4; JRL, Jacalin-related lectin; NCM, noncapacitat-ing medium; WGA, wheat germ agglutinin; CAPS, 3-(cyclohexylamino)pro-panesulfonic acid; GAG, glycosaminoglycan; LacNAc, N-acetyllactosamine;LacdiNAc, N,N-diacetyllactosamine; HexNAc, N-acetylhexosamine.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 290, NO. 9, pp. 5484 –5501, February 27, 2015© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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fuse with the oocyte plasma membrane. This acquisition processor capacitation only occurs after sperm have spent a period of timein the female reproductive tract (5) and is completed when sper-matozoa acquire the ability to respond to ZP proteins, leading tothe acrosome reaction (6). Main events include the removal ofstabilizing factors that spermatozoa have taken up in the seminalplasma and the remodeling of the sperm plasma membrane, as aprelude to the acrosome reaction and fertilization (4, 6). Capacita-tion corresponds in vitro to a cAMP-dependent rise in tyrosinephosphorylation and is associated with changes in both the headand tail that prepare the sperm to undergo a regulated acrosomereaction and hyperactivated motility (7, 8). Finally, capacitation-related events include the loss, modification, and redistribu-tion of molecules on the sperm surface (9), in particular theredistribution of sulfogalactosylglycerolipid at the spermsurface (10). The sperm membrane is a complex mosaic oflipids, glycolipids, and glycoproteins that is totally remod-eled during capacitation. Albumin and high density lipopro-teins present in the female reproductive tract will induce therelease/efflux of cholesterol from the sperm plasma mem-brane (11, 12). This cholesterol efflux leads to a globalincrease in membrane fluidity in capacitated sperm, which isbeneficial to the ZP-induced sperm acrosome reaction andthe sperm-egg plasma membrane fusion (13). Capacitationcan be obtained in vitro by mimicking the physiological con-ditions inducing capacitation in vivo, usually in the presenceof Ca2� and bovine serum albumin (BSA) under a 5% CO2

atmosphere (14). It has been demonstrated that lipid rafts,isolated as low density detergent-insoluble or detergent-re-sistant membranes from capacitated pig sperm had highaffinity for the ZP (10). Involvement of lipid rafts as plat-forms of cell adhesion and signaling molecules has beenestablished earlier in fertilization (15–17), medaka fishembryogenesis (18, 19), and other cellular processes (20 –23). Thus, we believe that the remodeling of lipid rafts duringcapacitation is of particular importance.

We have focused on prefertilization events on sperm andhave sought to find unique glycoproteins redistributed in thelipid rafts during capacitation. It was found that at least twoWGA epitope-bearing glycoproteins, named WGA16 andWGA-gp, were enriched in the pig sperm rafts (24). WGA-gp isa highly glycosylated 15–25-kDa glycoprotein that persists inthe raft before and after capacitation. Therefore, it is notdirectly related to capacitation but is implicated in intracellularCa2� regulation of sperm (24). On the other hand, WGA16,which is also highly glycosylated, was decreased from the spermsurface and even lost from the rafts after capacitation. Ourobjective was thus to identify and characterize WGA16 to gainnew insight into the capacitation phenomenon at the molecularlevel. Here we report the discovery, purification, determinationof chemical and crystal structures, and functional characteriza-tion of WGA16. We found that WGA16 is a novel sperm lectinthat has never been reported in sperm and that the attachmentand detachment of WGA16 on the sperm surface before andafter capacitation can be explained by its unique glycans and thelectin activity.

EXPERIMENTAL PROCEDURES

Sperm Preparation—Ejaculated sperm from Sus scrofa(Duroc, male, 2 years old) were provided by Ishikawa Pig Farm(Aichi, Japan). Sperm capacitation was performed as describedpreviously (10). Briefly, ejaculated semen samples collected frommature fertile boars were diluted in Beltsville Thawing Solution(0.2 M glucose, 3 mM EDTA, 20 mM sodium citrate�2H2O, 15 mM

NaHCO3, 10 mM KCl, pH 7.4). Sperm and seminal plasma wereseparated by centrifugation (200 � g, 28 °C, 10 min). Seminalplasma was further centrifuged at 9,500 � g for 30 min to removeresidual sperm and debris. Sperm were washed twice with thenon-capacitation medium (NCM; 100 mM NaCl, 0.36 mM

NaH2PO4�2H2O, 8.6 mM KCl, 23 mM HEPES, 0.5 mM

MgCl2�6H2O, 11 mM glucose, pH 7.6) (25). Finally, sperm wereresuspended at 1 � 108 cells/ml in NCM. Motile sperm popula-tions were prepared by density gradient on Percoll (GE Health-care) diluted in NCM. The uncapacitated sperm was applied onthe top of the tube containing 35–70% Percoll and centrifuged at28 °C at 200 � g for 5 min and then at 800 � g for 25 min to removedead sperm and seminal gel particles (26). The Percoll gradientcentrifuged uncapacitated sperm was obtained as a pellet. Percollgradient centrifuged sperm was preincubated in NCM (30 min,39 °C, 5% CO2) and then suspended in the capacitation medium(containing all components of NCM plus 10 mM NaHCO3, 2 mM

CaCl2�2H2O, 5 mM pyruvate, and 3 mg/ml BSA, pH 7.6) finally at2.0 � 107 cells/ml (2 h, 38 °C, 5% CO2) to obtain capacitatedsperm. Sperm was then washed three times by NCM (150 � g,28 °C, 10 min). Capacitation status was monitored visually undermicroscope to check whether sperm attained hyperactivatedmotility or monitored biochemically by Western blotting (seebelow), showing a specific phosphorylation pattern, typical ofcapacitated sperm (12, 28).

Preparation of Low Density Detergent-insoluble Mem-branes—The low density detergent-insoluble membrane frac-tion was prepared under 4 °C as described previously (16, 29). Inbrief, sperm (uncapacitated and capacitated sperm, 200–300 �l assperm pellet) were suspended in 1.0 ml of 10 mM Tris-HCl, pH 7.5,150 mM NaCl, 5 mM EDTA (TNE) containing 1% Triton X-100, 1mM PMSF, and protease inhibitor mixture (1 �g/ml leupeptin, 2�g/ml antipain, 10 �g/ml benzamidine, 1 �g/ml pepstatin, 0.9�g/ml aprotinin). The mixture was stood on ice for 20 min andhomogenized with 10 strokes by a Dounce homogenizer. Afterremoval of the pellet by centrifugation at 1,300 � g for 5 min, thesupernatant was mixed with an equal volume of 85% (w/v) sucrosein TNE. The mixture was layered successively with 6 ml of 30%(w/v) sucrose in TNE and with 3.5 ml of 5% (w/v) sucrose in TNEand centrifuged at 200,000 � g for 18 h (Beckman L-70K centri-fuge, SW 41 It rotor). After ultracentrifugation, 1 ml each of 13fractions was collected from the top to the bottom of the tube.Each fraction was dialyzed against PBS (137 mM NaCl, 8.1 mM

Na2HPO4, 2.68 mM KCl, and 1.47 mM KH2PO4, pH 7.4), deter-mined for protein amount by a BCA assay kit (Pierce) using BSA asa standard, and monitored by Western and lectin blotting.

Purification of WGA16 from Sperm and Seminal Plasma—Unless otherwise stated, all of the experimental procedureswere performed under 4 °C. Sperm (10 –25 ml of cell pellet) wasmixed with lysis buffer (10 mM Tris-HCl, pH 7.5, 1% Triton

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X-100, 150 mM NaCl, 5 mM EDTA, protease inhibitor mixture),stood on ice for 1 h, and centrifuged at 15,000 � g for 30 min.Supernatant was collected and applied onto a CM-Toyopearl650 M cation exchange column (Tosoh Biosciences, 1.2 � 88cm) equilibrated with 0.1% Triton X-100, 10 mM Tris-HCl, pH7.5. The column was extensively washed with 0.1% TritonX-100, 10 mM Tris-HCl, pH 7.5, and eluted with a linear gradi-ent of NaCl (0 – 0.6 M). At every purification step, the elutionprofile was monitored by SDS-PAGE visualized by lectin blot-ting and silver staining. WGA16-positive fractions were pooledand dialyzed two times at 4 °C against WGA-agarose equilibra-tion buffer: 10 mM Tris-HCl, pH 7.5, 150 mM NaCl (membranesize 36, Wako Chemicals). Dialyzed sample was applied on aWGA-agarose column (Seikagaku Biobusiness Corp.) over-night at 4 °C, washed with the equilibration buffer, and succes-sively eluted by 100 mM GlcNAc and 1 M GlcNAc in the equil-ibration buffer. Remaining contaminants were removed bypassage on Sephacryl S-100 (GE Healthcare; 1.0 � 150 cm)equilibrated in 0.1% Triton X-100, 10 mM Tris-HCl, pH 7.5, 150mM NaCl. Finally, WGA16-positive fractions were subjected toa 1.25-ml CM-Toyopearl 650 M column equilibrated with 0.1%Triton X-100, 10 mM Tris-HCl, pH 7.5, to remove any remain-ing contaminants. Elution was performed by a salt gradient(from 0 to 0.6 M NaCl, 20 ml in total). The same procedureswere used to purify seminal plasma WGA16.

SDS-PAGE/Lectin Blotting and Western Blotting—Spermsamples were solubilized in a lysis buffer containing proteaseand phosphatase inhibitors (50 mM Tris-HCl, pH 7.5, 1% TritonX-100, 1% deoxycholate, 1 mM EGTA, 150 mM NaCl, 0.4 mM

EDTA, 1 mM PMSF, 0.2 mM Na3VO4, 2.5 mg/ml leupeptin, andaprotinin) on ice for 20 min and then sonicated. Pellets wereremoved by centrifugation at 6,000 � g for 5 min, and superna-tant protein concentration was assessed by a BCA assay kit.Samples were incubated with Laemmli buffer containing 10%mercaptoethanol at 100 °C for 3 min. Proteins were separatedby SDS-PAGE (15% polyacrylamide gel), followed by either sil-ver staining (Silver Stain Kit II, Wako Chemicals) or electro-blotting onto polyvinylidene difluoride (PVDF) membrane(Millipore, Bedford, MA) using a semidry blotting apparatus forlectin blotting or immunoblotting. One percent of BSA in PBST(10 mM PBS, pH 7.2, 0.15 M NaCl, 0.05% Tween 20) was used fordiluting antibodies and blocking nonspecific binding to PVDFmembrane. Capacitation was monitored by Western blottingusing a monoclonal antibody 4G10 (1:10,000 dilution; SantaCruz Biotechnology, Inc.) that specifically recognizes phospho-tyrosine and a specific second antibody (American Qualex, LaMirada, CA; HRP-conjugated goat anti mouse IgG � IgM,1:5,000 dilution). WGA-lectin epitope-bearing glycoproteinswere detected by lectin blotting using WGA lectin (Sigma; 1mg/ml, 1:2,500 dilution) and anti-WGA lectin serum (Sigma-Aldrich; 1:3,000 dilution), finally detected by the HRP-conju-gated anti-rabbit IgG (Seikagaku; 1:4,000 dilution). For immu-noblotting for WGA16, the blotted membrane was probed withthe primary antibody (anti-WGA16 (see below); 1:1000 dilu-tion) and the HRP-conjugated anti-mouse IgG � IgM (1:5,000dilution; American Qualex). Visualization in lectin and West-ern blotting were processed using enhanced chemilumines-cence reagent (Amersham Biosciences). In addition to WGA,

Maackia amurensis agglutinin (Sigma-Aldrich), Ricinus com-munis agglutinin (Sigma-Aldrich), peanut agglutinin (J-OilMills, Inc.), and Griffonia simplicifolia-IB4 (GS-IB4; Sigma-Al-drich) were also used at 1 �g/ml, followed by incubation withmouse serum to each lectin (1:1,000 dilution) that was preparedin our laboratory.

Preparation of Anti-WGA16 —The purified WGA16 mixedwith Freund’s complete adjuvant (Wako, Osaka, Japan) wasintraperitoneally injected into Ddy mice (female, 8 weeks old;Nippon SLC). The mice were boosted twice with WGA16 (10�g/mouse) mixed with Freund’s incomplete adjuvant every 2weeks. Blood was collected 1 week after each boost, and theserum was collected. The anti-WGA16 IgG antibody (anti-WGA16) was purified from the obtained anti-WGA16 serumusing Protein G-Sepharose 4 Fast Flow (Amersham Biosci-ences) according to the manufacturer’s protocol.

Amino Acid Sequencing—Purified WGA16 was digested witheither trypsin (Promega) or endopeptidase Lys-C (Sigma), andpeptides were further purified by HPLC on an ODS column andsubmitted to N-terminal sequencing. For amino acid sequenceanalysis, WGA16 (5 �g) was applied onto a PVDF membraneusing a Pro SorbTM kit (PerkinElmer Life Sciences) and washedwith 0.1% trifluoroacetic acid (TFA). The WGA16-containingspot on the PVDF membrane was punched out and added with aBioBrene solution. It was washed with 0.1% TFA and then addedwith methanol. After air drying of methanol, it was subjected toamino acid sequencing by automated Edman degradation on aProcise HT analyzer (Applied Biosystems, Foster City, CA)

cDNA Cloning of WGA16 —Preparation of total RNA andoligo(dT)-derived cDNA from pig male reproductive organs(testis, epididymis, seminal vesicles, prostate, and bulboure-thral glands) and rapid amplification of cDNA ends (RACE)were performed as described previously (30). Based on a partialamino acid sequence that was identified as homologous toZG16 homolog B from S. scrofa, the cDNA fragment forWGA16 was amplified by reverse transcription polymerasechain reaction (RT-PCR) from prostate and bulbourethralglands using the following primers: ZG16-F-P (5�-CAGATGT-TCGGGAACGGAAAAGGCTCC-3�) and ZG16-R-P (5�-TATAATGTGCTCGCCGGGNTGCAGGAT-3�). Amplifiedfragment was subcloned into a pGEM Easy vector (Promega)for sequencing. An insert was amplified by M13 promoter andterminator and sequenced. Because we observed significant dif-ferences between the sequenced amplified fragment and ZG16mRNA sequence, we assessed the full WGA16 sequence by 5�-and 3�-RACE PCR using primers (3�-GSP1, 5�-CTAATCAAG-AGTATCCAGGTGAGGTAT-3�; 3�-GSP2, 5�-GGATCCTC-CTGGAGTGAAAAATACGGA-3�; 5�-GSP1, 5�-AGTGATT-CCCGTCTCATTGTCTTTACT-3�; 5�-GSP2, 5�-GGTGATG-AAATAGGAGCCTTTTCCGTT-3�). Finally, the full WGA16cDNA sequence was cloned using sense (5�-GAAGCCATGC-TGCTGTGGCTA-3�) and antisense (5�-CCCTCTCAGAAT-GGGTCGTC-3�) primers. The amplified full-length cDNA wascloned into pGEM plasmid (pGEM-WGA16). The sequence ofthe amplified fragment was investigated by DNA sequencing.The cDNA sequence corresponds to the expressed sequencetag FS704065 sequence (this sequence also corresponds toAK396685.1, AK396699.1, and AK400881.1 from other cDNA




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libraries; all of those expressed sequence tags were obtainedfrom prostate, confirming prostate specificity). For quality eval-uation of mRNA prepared from various organs, RT-PCR foractin was done using specific primers: 5�-CTGGAGAAGAGC-TACGAGCTGC-3� and 5�-CGTGGCACTTCATGATGGA-3�. Real-time PCR was performed as described previously (27),using the above described primers ZG16-F-P and ZG16-R-Pand Ssof FastTM EvaGreenSuper mix (Bio-Rad).

Preparation of Recombinant WGA16 —The DNA encodingWGA16 lacking a signal peptide was amplified by PCR withsense primers: 5�-CCTGCGATATCGGGCAGATGTTCGGG-3�(EcoRV site underlined) and antisense primer 5�-GAATTCG-AAGCTTCTTCAGAATGGGTC-3� (HindIII site underlined),using the pGEM-WGA16 as a template. The amplified productwas digested with EcoRV and HindIII and inserted into thecomplementary sites in pET32a(�) vector (Novagen) to pre-pare a plasmid, pET-WGA16, that encoded a fusion protein ofthioredoxin and His-tagged, signal peptide-deleted WGA16.Escherichia coli BL21 (DE3) pLysS (Stratagene) was trans-formed with pET-WGA16 and grown at 37 °C in 2-YT mediumto reach A600 � 0.6. After a 4-h induction at 37 °C with 0.5 mM

isopropyl �-D-thiogalactoside, cells were harvested and sus-pended in PBS. After sonication, thioredoxin-His-tagged WGA16fusion protein was purified using nickel-nitrilotriacetic acid-aga-rose affinity columns (Qiagen) according to the manufacturer’sprotocol. The purified fusion protein was subjected to digestionwith recombinant enterokinase (Novagen), followed by applica-tion on a second nickel-nitrilotriacetic acid-agarose column. Afterelution, fractions containing His-tagged WGA16 (WGA16-His)were concentrated on an Amicon Ultrafiltration cell and buffer-exchanged to PBS. WGA16-His was then biotinylated accordingto the manufacturer’s protocol. Ten �g of WGA16 (final volume500 �l) in PBS were transferred onto YM-3 membrane. Two �l of10 mM solution of biotinylation reagent (EZ-Link Sulfo-NHS-LC-Biotin) were added. The mixture was incubated at room temper-ature for 30 min, extensively washed on YM-3 membrane toremove residual biotinylation reagent, and used as biotinylatedrecombinant WGA16 (biotin-rWGA16). Biotinylation efficiencywas tested by dot blot on nitrocellulose membrane using Strepta-vidin and anti-WGA16.

To explore the role of some specific amino acid residues inthe carbohydrate binding activity of WGA16, point mutationsin pET-WGA16 construct were generated at Trp-138 or Tyr-140 using QuikChange II site-directed mutagenesis kits (Agi-lent Technologies). Use of the following primers resulted in theconstruction of pET-WGA16(W138A) (sense, 5�-TACGGGCA-GTTCGCGCTCTATGGCATC-3�; antisense, 5�-GATGCCAT-AGAGCGCGAACTGCCCGTA-3�) and pET-WGA16(Y140A)(sense, 5�-CAGTTCTGGCTCGCTGGCATCACGGGC-3�;antisense, 5�-GCCCGTGATGCCAGCGAGCCAGAACTG-3�). Recombinant W138A and Y140A mutants were preparedas described above. To evaluate roles of basic patches, BC1(Lys-53 and Lys-73) and BC2 (Lys-25, Arg-91, Arg-93, and Lys-117), DNA fragments encoding WGA16 with alanine mutantsof BC1 (K53A and K73A � AAG to GCG and AAA to GCA,respectively) and BC2 (K25A, R91A, R93A, and K117A � AAAto GCA, AGG to GCG, AGG to GCG, and AAG to GCG,respectively) flanked by HindIII and XbaI sites were synthe-

sized (Genewiz Inc., Suzhou, China) and cloned into pCold-His6-streptag(st)-PDI vector (31) with HindIII and XbaI sites.Using these plasmids and pCold-WGA16 (see below), recom-binant WGA16 and its BC1 and BC2 mutants were prepared asdescribed below.

Heparin-Agarose Affinity Chromatography—We performedchromatography according to the manufacturer’s instruction.Briefly, a 1-ml heparin-agarose (Sigma) column was equili-brated in 10 mM Tris-HCl, pH 7.5, 150 mM NaCl. 1 ml of dilutedseminal plasma (1 mg/ml) was applied and mixed for 30 min at4 °C. Flow-through was collected, and the column was sequen-tially washed with 10 ml of equilibration buffer; washed with 5ml of 10 mM Tris-HCl, pH 7.5, 1 M NaCl; and finally eluted with5 ml of 10 mM Tris-HCl, pH 7.5, 2 M NaCl. The separationprofile was monitored by 15% SDS-PAGE followed by eithersilver staining or immunoblotting with anti-WGA16. For theheparin binding assay of recombinant BC1 and BC2 mutants ofWGA16, supernatants of the lysate of E. coli Rosetta2 express-ing BC1 and BC2 mutants of WGA16 were mixed with 20 �l ofheparin-agarose at 4 °C for 16 h. The pellet was washed with 10mM sodium phosphate, 150 mM NaCl, and the supernatant andpellet were obtained. The pellet was treated with 1 M NaCl,sodium phosphate, and the supernatant (1 M NaCl fraction) andthe pellet (gel) were analyzed for the recombinant WGA16molecules by anti-WGA antibody as described above.

Glycan Array—The glycan array was performed using the gly-can array Glycan-I (S-Bio Business Division, Sumitomo BakeliteCo., Tokyo, Japan). Biotin-rWGA16 was diluted at a concentra-tion of 10 �g/ml in reaction buffer TBS (50 mM Tris-HCl, pH 7.5,100 mM NaCl, 1 mM MnCl2, 1 mM MgCl2) containing 0.5% Tween20. Seventy �l of the solution were applied onto the glycan arrayslide surface and incubated at room temperature for 2 h. The slidewas washed in TBS for 1 min under gentle agitation and thenrinsed with pure water twice. After drying under air blowing, theslide was incubated with 2 �g/ml Cy3-streptavidin (GE Health-care) in 0.5% Tween 20-TBS at room temperature for 1 h. The slidewas washed with TBS and with pure water and dried under airblowing. The fluorescent intensity was immediately quantitatedby a Typhoon scanner (�ex � 532 nm, �em � 575 nm, PMT 600,medium sensitivity, resolution 50 �m).

N-Glycosylation Profiling—Delipidation of seminal plasmawas performed by sequential extraction of 500 �l of seminalplasma with 20 volumes of chloroform/methanol mixture (2:1,v/v) and then with the same volume of chloroform/methanol/water (2:1:0.8, v/v/v) solution. The resultant aqueous phase wasused as delipidated seminal plasma. Delipidated seminalplasma or purified WGA16 was suspended in a solution of 6 M

guanidinium chloride and 5 mM EDTA in 0.1 M Tris-HCl, pH 8,and agitated at 4 °C for 4 h. Dithiothreitol was added to a finalconcentration of 20 mM and incubated at 37 °C for 5 h, followedby the addition of iodoacetamide to a final concentration of 50mM and further incubation in the dark at room temperature forovernight. The reduced and alkylated samples were dialyzedagainst water at 4 °C and lyophilized. The recovered proteinsamples were then digested with tosylphenylalanyl chloro-methyl ketone-treated trypsin (Pierce) at 37 °C overnight in 50mM ammonium bicarbonate buffer (pH 8.4). Crude peptidesand glycopeptides were loaded onto a C18 Sep-Pak cartridge




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(Waters). Contaminants were washed off with 5% aqueous ace-tic acid, and the bound peptides and glycopeptides were elutedwith a stepwise gradient of 20, 40, and 60% 1-propanol in water.Eluted fractions were pooled, dried down, and incubated withpeptide:N-glycanase F from Flavobacterium meningosepticum(Takara Bio Co.) at 37 °C overnight in 50 mM ammonium bicar-bonate buffer, pH 8.4. Released N-glycans were separated frompeptides and glycopeptides using the same C18 Sep-Pak proce-dure and then further purified on a graphitized column. ForMALDI-TOF MS glycan profiling, native compounds in waterwere mixed 1:1 with 2,5-dihydroxybenzoic acid matrix (10mg/ml in acetonitrile/H2O, 0.1% TFA (50:50)), spotted on thetarget plate, and dried under vacuum. Data acquisition wasperformed manually on an Ultraflex III mass spectrometer(Bruker) operated in the reflectron mode. Laser shots wereaccumulated until a satisfactory signal/noise ratio was achievedwhen combined and smoothed.

Carbohydrate Analyses—The monosaccharide compositionswere determined by gas-liquid chromatography (GLC) asdescribed previously (32). To determine the sialic acid contentof WGA16, sialic acids were liberated directly by strong hydroly-sis in 0.1 N TFA at 80 °C for 2 h and then reacted with a volumeof 1,2-diamino-4,5-methylenedioxybenzene reagent at 50 °Cfor 2 h (33). The monomeric 1,2-diamino-4,5-methylenedioxy-benzene-sialic acid derivatives were analyzed by a JASCOLC-900 HPLC system equipped with a JASCO FP-920 fluores-cence detector (�ex � 373 nm, �em � 448 nm) operating iso-cratically at 1.0 ml/min on a TSK-gel ODS-120T (250 � 4.6mm) with a solvent mixture of acetonitrile/methanol/water(7:9:84, v/v/v) and identified by referring to the elution posi-tions of standard Neu5Ac and Neu5Gc derivatives. Digestionwith peptide:N-glycanase F, sialidase from Arthrobacter ure-afaciens (Nacalai), or N-acetylhexosaminidase from jack bean(Seikagaku Co.) was performed on purified glycoproteinaccording to the manufacturer’s instructions.

Expression and Purification of Recombinant WGA16 forCrystallization—DNA fragments encoding pig WGA16 (aminoacid residues 18 –166) were subcloned into pCold-His6-streptag(st)-PDI vector (31) for production of recombinantprotein (pCold-WGA16). E. coli Rosetta2 (DE3) (Novagen) wastransformed with the plasmid, and the cells were grown in LBmedium at 37 °C. After induction with 0.1 mM isopropyl �-D-thiogalactoside (Wako), the cells were cultured at 15 °C for12 h. The harvested cells were resuspended in a buffer contain-ing 50 mM Tris-HCl (pH 8.0) containing 50 mM NaCl andsonicated in the presence of Bugbuster (Novagen). After cen-trifugation, the supernatants were collected and applied to anickel-Sepharose column (GE Healthcare) equilibrated withPBS. After washing the column with PBS, the proteins wereeluted with PBS containing 500 mM imidazole. The His6-st-PDI-fused protein was then digested by TEV protease at 4 °Cfor 12 h. The digested protein was passed through a nickel-Sepharose column and applied to a HiLoad 16/60 Superdex 75pg column (GE Healthcare) equilibrated with PBS. The purifiedprotein was replaced with 8 mM Na2HPO4, 1 mM KH2PO4, 177mM NaCl, and 3 mM KCl, 30 mM arginine (pH 7.4) for crystalform A or 10 mM CAPS buffer (pH 9.0) for crystal form B andused for the crystallization.

Crystallization, X-ray Data Collection, and Structure Deter-mination—Crystal conditions were screened by the sitting dropvapor diffusion method, in which 0.5 �l of protein solution wasmixed with an equal volume of reservoir solution. Crystal wasobtained using the following reservoir solution: 0.5 M sodiumchloride, 0.1 M sodium citrate tribasic dehydrate, pH 5.6, 2%(v/v) ethyleneimine polymer (for crystal form A) and 0.1 M MES(pH 6.5), 1.6 M (NH4)2SO4, and 10% (v/v) 1,4-dioxane (for crys-tal form B). Data sets were collected from synchrotron radia-tion (1.0000 Å wavelength) at the NE3A and NW12A beamlinesof the Photon Factory, High Energy Accelerator ResearchOrganization (Tsukuba, Japan). The crystal was cryoprotectedwith a reservoir solution containing 25% (v/v) ethylene glycol(crystal form A) or 25% (v/v) glycerol (crystal form B). Thediffraction data were processed using HKL2000 (34). The struc-ture of WGA16 was solved by the molecular replacementmethod using the program Molrep (35), and human ZG16bstructure (Protein Data Bank code 3AQG) (36) was used as atemplate. Further model building was manually performedusing the program COOT (37). Refinement was carried outusing the program REFMAC5 (38). The stereochemical qualityof the final models was assessed by PROCHECK (39). Crystal-lographic parameters and refinement statistics are summarizedin Table 4. Coordinates and structure factors for WGA16 incrystal forms A and B have been deposited at the Protein DataBank with accession numbers 3WOB and 3WOC, respectively.Images of the three-dimensional structures were preparedusing PyMOL (Schroedinger, LLC, New York).

In Situ Hybridization—Tissues (prostate, epididymis, andtestis) from S. scrofa were fixed using 4% paraformaldehydesolution and embedded in paraffin. Tissue sections (4 �m) wereadhered to glass slides and deparaffinized in xylene andhydrated in a graded series of alcohols.

For probe to WGA16 mRNA, a sequence with a size of 120nucleotides (5�-CTGGCGAGCACATCATAAGCATCTATG-GAAGGTACAGGACCTTCCTTCAGCACGTGACTCTGA-TCACCAACCAAGGGCGCTCAGCCTCATTCGGACTGG-AAACTGGCAAGGGCTTCTTCG-3�) was designed in theregion of WGA16 mRNA using the G-Probe software (Genetyx,Tokyo, Japan). As a control probe, a 120-nucleotide � phagesequence having no similarity to any of the mammalian sequenceswas used in all in situ hybridization systems. Digoxigenin-labeledcomplementary RNA probe was synthesized using the AmpliScribeT7-Flash transcription kit (Epicenter, Madison, WI).

The procedure for in situ hybridization was the same asreported earlier (40) with the following exception. The hybrid-ization was performed in a solution containing 50% formamide,2� standard saline citrate (300 mM NaCl, 30 mM sodium citrate,pH 7.0), 1.0 mg/ml transfer RNA, 1.0 mg/ml salmon sperm DNA,1.0 mg/ml BSA, 1.0% SDS, 1 mg/ml doxycycline, and 3.0 �g/mldigoxigenin-labeled complementary RNA probe at 37 °C for 16 h.Hybridization signals were detected with the nitro blue tetrazoli-um/5-bromo-4-chloro-3-indolyl phosphate system (RocheApplied Science). The resulting tissue sections on glass slides werephotographed with a slide scanner (MIRAX Desk, Carl Zeiss).

Immunohistochemistry—Tissue sections (4 �m thick) hydratedas described above were washed three times with PBS and thenwere treated with 3% H2O2 for 5 min, followed by dipping of




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tissue sections in boiling 10 mM sodium citrate buffer (pH 6.0)for 20 min. The resulting tissue sections were washed threetimes with PBS and incubated with 5% fetal bovine serum (FBS)in TB-S (25 mM Tris-HCl, 150 mM NaCl, pH 7.2) for 20 min, inorder to suppress nonspecific binding of antibody to tissue sec-tions (blocking). After blocking, the sections were treated withor without mouse anti-WGA16 serum (1:100 dilution) in 5%FBS-TBS at 4 °C overnight and washed three times with TB-S,followed by treatment with peroxidase-conjugated anti-mouseIgG (H�L) (Wako) (1:500 dilution) in 5% FBS-TBS for 60 minat room temperature. Then the resulting sections were washedthree times in TBST (25 mM Tris-HCl, 150 mM NaCl, 0.05%Tween 20, pH 7.2) and incubated in a 3,3�-diaminobenzidinetetrahydrochloride solution (Sigma-Aldrich) to develop perox-idase signals. For histological analysis, serial sections of thoseused for in situ hybridization and immunohistochemical anal-ysis were subjected to hematoxylin staining. The sections onglass slides thus processed were photographed using a MIRAXDesk scanner (Carl Zeiss).

Glycan/Heparin-mediated Removal of WGA16 from SpermSurface—Uncapacitated sperm were incubated with NCM orwith 10 mM monosaccharide (Gal, GlcNAc, or Neu5Ac), 10 mM

activated sugar (UDP-Gal, UDP-GlcNAc, or CMP-Neu5Ac), orwith increasing concentrations (0.5, 1, and 2 mg/ml) of heparin inNCM at 39 °C in 5% CO2 for 30 min. The sperm suspensions werecentrifuged (150 � g, 10 min) to pellet down intact sperm andwashed twice with NCM. Sperm lysates were quantified by a BCAassay and applied onto a 15% SDS-polyacrylamide gel at 15 �g ofprotein/lane. WGA16 was quantified by the intensity of the immu-nostaining band on Western blotting with anti-WGA16.

RESULTS

Discovery and Purification of WGA16 —To examine whetherglycosylation changes occur on the sperm surface during capac-itation, sperm lysate and the lipid raft that was prepared as lowdensity detergent-insoluble membranes from uncapacitatedand capacitated pig sperm were analyzed by lectin blotting.Sperm capacitation was monitored by antiphosphotyrosineblot analysis using 4G10 (Fig. 1A). Specific phosphorylationincrease on proteins at 32 and 42 kDa, together with character-istic sperm hyperactivity, was observed as described previously(10, 14). In parallel, we monitored the capacitation processusing several lectins (Fig. 1, B and C). No drastic changes inlectin staining profile could be observed using lectins such asconcanavalin A (�Man), GS-IB4 (�Gal), peanut agglutinin(Gal�1,3GalNAc), and M. amurensis agglutinin (Sia�2,3Gal�1,4GlcNAc/Glc) (Fig. 1B). We found that, after capacitation, lowmolecular weight, WGA lectin epitope-bearing proteins dramati-cally decreased at the sperm surface, especially at the 16–25-kDacomponent in the lipid raft (Fig. 1, C and D). This observation wasexclusively made using WGA lectin, specific toward terminal�-HexNAc and Neu5Ac residues. We thus focused on the lipidraft-associated 16–25-kDa protein that was removed from thesperm plasma membrane during capacitation.

This 16 –25-kDa protein was initially purified from spermlysate. Then a protein with similar properties was also detectedin seminal plasma (Fig. 2, A–D). Sperm lysate was applied to aCM-Toyopearl 650 M column (Fig. 2A). The 16 –25-kDa pro-

tein was eluted around 0.4 M NaCl, as revealed by WGA lectinblotting. The WGA lectin-positive fractions were then appliedonto a WGA-agarose column. The 16 –25-kDa protein waseluted with 100 mM or 1 M GlcNAc. These elution fractionswere separated on a Sephacryl S-100 column (Fig. 2C). Based onthe silver staining and WGA lectin blotting, the WGA lectin-pos-itive components were eluted at positions corresponding to theirmolecular weights, indicating that they are homogeneous in thatothercontaminantsareabsentandthattheyarepresentasamono-mer with a different glycosylation state. We named the 16–25-kDaprotein WGA16. Notably, other low molecular weight proteins,such as 16-kDa PSP-I/II, which were identified by proteomic anal-ysis, were eluted as oligomers prior to WGA16 (fractions 38–48;Fig. 2C). When contaminants were present, WGA16 was furtherpurified on a CM-Toyopearl 650 M column to finally obtain pureWGA16. Similar procedures were used in purification of the sem-inal plasma-derived WGA16 (Fig. 2B for the anion exchange chro-matography; Fig. 2D for Sephacryl S-100 gel filtration). From 200ml of semen, 5 ml of sperm cells and 195 ml of seminal plasma wereobtained. Purification yields were, respectively, 0.4 mg from 5 ml ofsperm and 22 mg from 195 ml of seminal plasma. Thus, we canconclude that about 2% of WGA16 in semen were present on thesperm cells.

WGA16 Is Related to the Jacalin-related Lectin Family—Forpurified WGA16 from sperm lysate and seminal plasma, N-ter-minal amino acid sequence on the native protein as well asinternal sequences on tryptic or Lys-C-digested peptides weredetermined. The results for seminal plasma WGA16 are shownin Table 1, A–D. Further analysis established that sperm surfaceand seminal plasma WGA16 correspond to the same proteinbecause they share the same proteolytic profiles and amino acidsequence. A computer-assisted search indicated that some pep-tide fragments match the deduced amino acid sequences frommRNA sequence XM_003124749.1, corresponding to S. scrofazymogen granule protein 16 (ZG16) homolog-B like. In PCRanalysis using primers after this nucleotide sequence, a 204-bpproduct was amplified for prostate, although no product wasobtained for testis, epididymis, seminal vesicles, or bulboure-thral gland. This partial cDNA sequence coded for a 68-aminoacid peptide (underlined in Table 1D). The sequence ofWGA16 full mRNA was determined by a 3�- and 5�-RACEapproach. Information obtained by RACE PCR was registeredas GenBankTM AB851481, which matched the sequence ataccession number FS704065: full-length enriched swine cDNAlibrary, adult prostate S. scrofa cDNA clone PST010091G03 5�mRNA sequence. The corresponding deduced amino acidsequence is shown in Table 1D. This sequence matches pep-tides obtained after tryptic or proteinase-Lys-C treatment(Table 1, A–C). WGA16 open reading frame consists of 498 bp(nucleotides 69 –566 in the FS704065 sequence), encoding aprotein of 166 amino acids with a predicted 17-amino acid sig-nal peptide (Table 1D). The deduced mature WGA16 corre-sponds to a 149-amino acid polypeptide chain with a molecularweight of 16,200 with a calculated pI of 8.94, consistent with thebiochemical characteristics of WGA16. N-terminal sequencingon native protein confirmed that first 17 amino acids corre-spond to signal peptide because native WGA16 starts at Gly-18.The nucleotide sequence of WGA16 shows 70 and 43% identi-




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ties with the predicted ZG16 homolog B-like from S. scrofa andwith human protein ZG16 homolog b (hZG16b), respectively.Those proteins belong to a large family of plant and animallectins, grouped as Jacalin-related lectins (JRLs). In animals, thefunction of JRLs such as ZG16 remains unclear. So far, thoseproteins have been exclusively found in tissues with high secre-tory activity (pancreas, submaxillary glands, liver, and intestine)(41, 42). This is the first case of the existence of a JRL member insperm and reproductive organs.

WGA16 Shows a Unique Glycosylation Profile—Althoughmajor seminal plasma glycoproteins can be revealed usingeither WGA or GS-IB4 lectin blotting, WGA16 was exclusivelyobserved using WGA (Fig. 3, A and B), suggesting a specificglycosylation pattern. The glycosylation profile of PSP-I/PSP-IIheterodimer, the major seminal plasma glycoproteins, showsdiantennary complex-type N-glycans bearing �1,3-fucosylated

or �2,6-sialylated N-acetyllactosamine (LacNAc) as well as di-N-acetyllactosamine (LacdiNAc) structure. The �-GlcNAc res-idues of non-sialylated antennae are usually substituted by�1,3-Fuc residues, and a majority of the non-sialylated LacNAcantennae are terminated by �1,3-Gal residues (43). In the caseof WGA16, the lack of reactivity toward GS-IB4 lectin (Fig. 3B)indicates that WGA16 glycans are devoid of terminal �-Galresidues.

N-glycan profiling of WGA16 and seminal plasma proteinsby mass spectrometry confirmed observations made by lectinblotting. Glycan structures were assigned by combining datafrom MALDI-MS with monosaccharide composition obtainedby gas chromatography as well as information from the liter-ature (43). Seminal plasma glycoproteins were confirmed tocontain N-glycans bearing terminal �-Gal residues but alsoterminal LacNAc or LacdiNAc units that can be substituted

FIGURE 1. Increased sperm tyrosine phosphorylation and decreased WGA lectin epitope in capacitated sperm. A, sperm tyrosine phosphorylation incontrol and capacitated sperm. An equal amount of control and capacitated sperm was used for immunoblotting (IB) with 4G10 IgG. Arrowheads mark anincrease in reactivity of the 42 and 32 kDa bands in capacitated sperm. B, concanavalin A (ConA), GS-IB4, peanut agglutinin (PAA), and M. amurensis agglutinin(MAA) lectin staining of lipid rafts in control and capacitated sperm. Shown are WGA lectin staining (C) and silver staining (D) of sperm lysate and lipid raftsin control and capacitated sperm. In D, samples were overloaded to clearly show the higher molecular weight region.




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by Fuc (Fig. 3C and Table 2). On the other hand, MS profilingof purified WGA16 exhibited a complex N-glycosylationpattern, with more than 10 different N-glycan structures(Fig. 3D). Assignment of WGA16 N-glycans confirmed theabsence of terminal �-Gal residues but a rather high contentof LacdiNAc antennae, with and without fucosylation. Theprominent peak at m/z 1891.9 corresponds to core-fucosy-lated diantennary LacdiNAc N-glycan (Fig. 3D and Table 2).Finally, both lectin blotting and MS analysis indicate thatWGA16 presents a very unique glycosylation pattern amongseminal plasma proteins, remarkably characterized by theabsence of terminal �1,3-Gal.

Carbohydrate compositions of WGA16 and seminal plasmaglycoproteins are shown in Table 3. The content of HexNAc(GalNAc and GlcNAc) is higher for WGA16 than for seminalplasma glycoproteins. The WGA epitope was lost afterN-acetylhexosaminidase treatment but not after sialidase treat-ment (Fig. 3E), suggesting that terminal HexNAc but notNeu5Ac residues are responsible for the WGA epitope of

WGA16. peptide:N-glycanase F treatment gave a sharp band at16 kDa on SDS-PAGE/anti-WGA blotting, whereas WGAepitope was lost on the 16-kDa product (Fig. 3E). These resultsindicate that the WGA epitope exclusively resides on N-gly-cans, which mainly contributes to the heterogeneity in molec-ular mass of WGA16. Based on the deduced amino acidsequence (Table 1D), there are two potential N-glycosylationsites at Asn-35 and Asn-124. At least Asn-35 is glycosylated,because this residue could not be assigned on the N-terminaland internal sequence analyses (Table 1, A–C). It should benoted that WGA epitope remained at 20 –23 kDa, in addition tothe 16-kDa product, after peptide:N-glycanase treatment, sug-gesting the presence of O-glycans with the WGA epitope.Because the mannose amount decreased but it was still detectedin WGA16 after peptide:N-glycanase treatment (data notshown), some N-glycans may be peptide:N-glycanase-resistant.Therefore, it remained unclear whether O-glycans also containthe WGA epitope. Structural analysis of the O-glycans shouldbe performed in detail in the future.

FIGURE 2. Purification of WGA16 from sperm lysate and seminal plasma. Cation exchange chromatography on Toyopearl 650 M monitored by silverstaining (top) and WGA-lectin blotting (bottom) of sperm lysate (A) and seminal plasma (B). In A, samples were overloaded to clearly show the higher molecularweight region by silver staining. Shown is gel filtration on Sephacryl S-100 of the sperm lysate-derived WGA16 fraction obtained by WGA-lectin chromatog-raphy (C) and the seminal plasma-derived WGA16 fraction obtained by WGA-lectin chromatography (D). Arrows, WGA16-containing fractions.




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Crystal Structure of WGA16 —We determined the crystalstructures of WGA16 (residues 18 –166) in two different crystalforms, A (Protein Data Bank code 3WOB) and B (Protein DataBank code 3WOC) at 2.6 and 2.4 Å resolution, respectively(Table 4, Fig. 4 (A–D), and Fig. 5 (A–C)). In crystal form A, oneWGA16 molecule is included in the asymmetric unit, whereasin the crystal form B, six WGA16 molecules (chains A, B, C, D,E, and F) were included in the asymmetric unit (Fig. 5C).WGA16 was found as a monomer in crystal form A, whereasdimer formation was observed in crystal form B. Both struc-tures are similar except for the N-terminal region, which isinvolved in dimerization in crystal form B (Fig. 5B). The dimerformation in the crystal form B is likely to be an artifact, becauseWGA16 eluted as a monomer on size exclusion chromatogra-phy (data not shown). Hereafter, we describe crystal form Aunless otherwise stated.

The overall structure is similar to JRL family proteins, such asBanlec (44), human ZG16p (37), and human ZG16b (37).WGA16 assumes a �-prism fold consisting of three �-sheets(Fig. 4A). Each �-sheet is made up of 3– 4 �-strands (sheet I: �2,�11, and �12; sheet II: �3, �4, and �6; sheet III: �7–�10), form-ing three Greek key motifs. The N-glycosylation site, Asn-35, islocated at the loop between �2 and �3 strands and solvent-exposed. In Jacalin-related mannose-binding lectins, the sugar-binding site is composed of three loops (GG loop, binding loop,and recognition loop) and a key aspartic acid residue (Asp-151in human ZG16p) (Fig. 4B, bottom). Asp-151 in human ZG16pis replaced with Thr-143 in WGA16 (Fig. 4B, top). Character-istically, hydrophobic residues (Trp-138, Leu-139, and Tyr-140) are clustered in the region corresponding to the canonicalsugar-binding site of Jacalin-related mannose-binding lectins.It is therefore suggested that WGA16 does not bind to man-nose-type sugars.

Intriguingly, the crystal structure of WGA16 in crystal formB contains a sulfate ion at the �3-�4 loop region (Fig. 4C, top).

The bound sulfate was observed at the same site in chains A, B,C, D, and F, and the sulfate-binding site is close to the secondsugar binding site in Banlec (45). The sulfate ion interacts withN� of Lys-53 and main chain nitrogens of Pro-48 and Val-49.Similarly, the crystal structure of ZG16b contains phosphate atthe same site consisting of Leu-83, Leu-84, Leu-85, and Lys-87(Fig. 4C, bottom). These negatively charged ions may mimic thesulfate group of glycosaminoglycan (GAG). In fact, WGA16possesses a positively charged surface, as seen in human ZG16pand ZG16b (Fig. 4D), suggesting that the region is involved inbinding to GAGs, such as heparin, and is apart from the N-gly-cosylation site, Asn-35 (see “Discussion”).

WGA16 Has the Heparin-binding but Not Jacalin-relatedLectinic Activities—During the course of purification ofWGA16, we first noticed that WGA16 came to the heparin-binding fraction of seminal plasma proteins. On the heparin-agarose chromatography of seminal plasma (Fig. 6A), the elu-tion profiles of the 75- and 90-kDa proteins confirmed that theseparation procedure worked efficiently and indicated thatheparin-binding proteins started being eluted at 1 M NaCl.Anti-WGA16 immunoblotting showed that highly glycosylatedforms of WGA16 were rapidly eluted, whereas less glycosylatedforms were slowly eluted in 1 M and 2 M NaCl-eluted fractions,establishing WGA16 affinity to heparin. To further exploreWGA16 lectinic activity, recombinant WGA16 with an N-ter-minal His6 tag (rWGA16-His) was expressed in E. coli and puri-fied as a single band at 17 kDa on SDS-PAGE. Glycan bindingspecificity of WGA16 was analyzed using a glycoconjugatemicroarray comprising 29 immobilized compounds, includingseveral GAGs, as well as N- and O-glycans. WGA16 unambig-uously exhibits a selective binding to heparin (Fig. 6B). Addi-tionally, when the exposure time was extended, we couldobserve the weak binding to partially desulfated heparin D2Sand D6S but not to non-sulfated GAGs.

In the region corresponding to the canonical sugar-bindingsite of the JRL family, WGA16 is devoid of the typical GXXXDmotif. Instead, WGA16 contains hydrophobic amino acid res-idues Trp-138 and Tyr-140 in this region (Table 1D). As pre-dicted by the crystal structure of this region (Fig. 4B), WGA16had no binding activity to high mannnose type glycans (Fig. 6B).To investigate whether those residues were involved in the sug-ar-binding domain of WGA16, we performed point mutationsand explored heparin-binding properties of W138A and Y140Amutants. Because no changes in heparin affinity were observedfor either W138A or Y140A, we concluded that those residueswere not involved in heparin binding in WGA16.

The crystal structure of WGA16 exhibits two basic aminoacid clusters BC1 and BC2 that are presumably responsible forheparin-binding; BC1 contains Lys-53 and Lys-73 that are closeto the site corresponding to the second sugar binding site inBanlec (45), and BC2 contains Lys-25, Arg-91, Arg-93, and Lys-117 (Fig. 6C). The two alanine mutants for BC1 and BC2 werebacterially expressed and tested for binding ability to heparin-agarose resins (Fig. 6D). BC2 mutant bound to heparin-agaroselike WGA16, whereas BC1 mutant did not. These results indi-cate that BC1 is involved in the heparin-binding activity, con-sistent with the observation that Lys-53 interacts with sulfateion in the WGA16 crystal structure (Fig. 4C).

TABLE 1Determination of amino acid sequence of WGA16Native WGA16 was directly sequenced (A) or digested with either trypsin (B) orLys-C (C). Peptides were separated on an ODS column and sequenced by Edmandegradation. X represents a modified amino acid that could not be identified. D, thededuced amino acid sequence from the cDNA sequence (AB851481) is shown.Boxed sequence corresponds to a signal peptide, determined by bioinformaticsprediction and confirmed by sequencing of native WGA16. Underlined sequencesmatch the results from amino acid sequencing. Sequences corresponding to primersused for initial RT-PCR are double-underlined.




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Distribution of WGA16 in Male Reproductive Organs—Spe-cific polyclonal anti-WGA16 antibody showed the occurrenceof WGA16 protein in tissues of the prostate and bulbourethralglands. Tissue homogenates from boar reproductive organs weretested for the presence of WGA16 using both anti-WGA16 andWGA lectin (Fig. 7, A and B). The strongest immunostaining was

detected in prostate. The high molecular weight and intense WGAlectin signal indicate that it corresponds to heavily glycosylatedforms of WGA16. A weaker signal was detected in the bulboure-thral gland, corresponding to underglycosylated WGA16. Real-time PCR results showed that the WGA16 transcript was exclu-sively detected in prostate (Fig. 7C).

FIGURE 3. Comparative analysis of glycosylation profile from seminal plasma proteins and WGA16. Comparative lectin staining of seminal plasmaproteins separated on cation exchange column. Shown is lectin blotting with GS-IB4 (A) and WGA (B). Arrowheads, fractions containing WGA16. Shown areMALDI-MS profiles of N-glycans from seminal plasma proteins (C) and from seminal plasma-derived WGA16 (D). E, SDS-PAGE of WGA16 after treatment with�-hexosaminidase, sialidase, endo-�-galactosidase, and peptide:N-glycanase F (PNGaseF). Left, WGA lectin blotting; right, silver staining.




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In situ hybridization and immunohistochemistry using anti-WGA16 were performed on prostate, epididymis, and testis(Fig. 8). Whereas expression of WGA16 mRNA was detected intestis, cell layer of epididymis, and epithelial cells of prostate,

the protein-specific signal was exclusively detected in epithelialcells of prostate. Based on the results of Western blotting andimmunohistochemistry, it appears that WGA16 is a proteincharacteristic of the prostate gland. Taken together, the resultsindicate that WGA16 secretion mainly takes place in the pros-tate to be deposited on the sperm surface.

WGA16 Is Associated with Sperm through Sperm SurfaceGalactosyltransferase—As described above, WGA16 is secretedfrom prostate and deposited on the sperm surface, specificallyon the lipid rafts, during passage through genital tracts untilcapacitation (Fig. 1). One striking structural feature of WGA16lies in its very particular glycosylation profile, characterized bythe absence of terminal �-Gal residues, and the presence ofterminal GalNAc/GlcNAc residues, contrasting with otherseminal plasma glycoproteins (Fig. 3). To explore whether thesurface association of WGA16 is mediated through a specificinteraction with a sperm membrane lectin, uncapacitatedsperm were incubated with NCM containing 10 mM Gal,GlcNAc, or Neu5Ac or their activated forms (UDP-Gal,UDP-GlcNAc, and CMP-Neu5Ac). After incubation andextensive washing by NCM, sperm lysate was analyzed by SDS-PAGE and Western blotting with anti-WGA16 (Fig. 9A). Sur-prisingly, sperm surface WG16 was efficiently removed withUDP-Gal, a donor substrate for galactosyltransferase (GalT).This is of particular interest because the sperm surface �-galac-tosyltransferase (�-GalT) has been reported in various species,including pigs (46, 47). To confirm the presence of an activeGalT at the sperm surface and to validate that this GalT inter-acts with WGA16, sperm and the supernatant at the washingstep were analyzed by the SDS-PAGE/lectin blotting usingeither R. communis agglutinin (Gal�1,4GlcNAc-specific) orGS-IB4 lectin (�-Gal/GalNAc-specific) as well as by Westernblotting with anti-WGA16 (Fig. 9B). After incubation withUDP-Gal, sperm surface WGA16 decreased, whereas WGA16in the supernatant increased, suggesting that the presence ofUDP-Gal induces WGA16 transfer from the sperm surface tothe supernatant. In addition, the lectin blotting revealed thatupon UDP-Gal incubation, supernatant WGA16 becameR. communis agglutinin-, but not GS-IB4-reactive (i.e. the�-Gal, but not �-Gal, residues were newly formed). These

TABLE 2Comparison of PNGase F-released N-glycans from pig seminal plasmaand WGA16The molecular masses detected by MALDI/TOF-MS R� (M � Na)� of nativeoligosaccharides are listed. Relative abundance of species is roughly estimated fromthe respective signal intensities: �, 2–5%; �, 5–20%; ��, 21– 40%; ��, 41– 60%;��, 61– 80%; ��, 81–100%.

m/zOligosaccharide

compositionSeminal plasma

glycoproteins WGA16

1282.1 Hex3HexNAc3Fuc1 ��1444.2 Hex4HexNAc3Fuc1 ��1485.2 Hex3HexNAc4Fuc1 ��1606.3 Hex5HexNAc3Fuc1 ��1631.3 Hex3HexNAc4Fuc2 ��1647.3 Hex4HexNAc4Fuc1 �1688.3 Hex3HexNAc5Fuc1 � ��1809.6 Hex5HexNAc4Fuc1 ��1891.3 Hex3HexNAc6Fuc1 � ��1955.7 NeuAcHex5HexNAc4 � (�)1997.7 Hex4HexNAc5Fuc2 � �2038.7 Hex3HexNAc6Fuc2 �� 2054.7 Hex4HexNAc6Fuc1 �2133.3 Hex7HexNAc4Fuc1 ��2158.3 Hex5HexNAc5Fuc2 �2184.4 NeuAcHex3HexNAc6Fuc � �2257.7 Hex4HexNAc7Fuc1 �2337.4 Hex7HexNAc5Fuc1 �2378.4 Hex6HexNAc6Fuc1 (�)2402.7 Hex4HexNAc7Fuc2 �2499.4 Hex8HexNAc5Fuc1 �2539.8 Hex7HexNAc6Fuc1 �2661.4 Hex9HexNAc5Fuc1 �2701.4 Hex8HexNAc6Fuc1 (�)2743.5 Hex7HexNAc7Fuc1 (�)2946.7 Hex7HexNAc8Fuc1 (�)

TABLE 3Carbohydrate composition of WGA16 and seminal plasma glycoproteinsValues are given as molar ratios.

Monosaccharide Seminal plasma glycoproteins WGA16

% �mol % �molFuc 10.2 3.4Man 39.9 24.9Gal 16.1 17.8GalNAc 4.0 5.0GlcNAc 27.4 36.3Neu5Ac 2.3 12.5

TABLE 4Data collection and refinement statistics of WGA16 structures

Crystal form A Crystal form B

Data collectionsProtein Data Bank code 3WOB 3WOCSpace group P21 C2Unit cell parameters a, b, c (Å), � (degrees) 20.88, 52.33, 45.30, 103.18 134.48, 77.85, 87.19, 91.30Resolution (last shell data in parentheses) (Å) 50.0-2.60 (2.64-2.60) 50.0-2.40 (2.44-2.40)Rmerge (last shell) 16.2 (51.0) 6.9 (61.2)Mean ((I)/�(I)) (last shell data in parentheses) 7.3 (2.1) 22.4 (2.2)Completeness (last shell data in parentheses) (%) 99.8 (100.0) 99.7 (99.3)No. of unique reflections 3,847 35,861Average multiplicity (last shell data in parentheses) 3.7 (3.6) 5.7 (5.7)

Crystallographic refinement statisticsNo. of reflections used for refinement 3,654 33,943Reflections marked for RfreeRfree 28.3 32.0Rwork 21.4 25.2Average B-factors (Å2) 24.7 39.5Root mean square deviation of bond length (Å) 0.006 0.006Root mean square deviation of bond angle (degrees) 0.935 1.015Ramachandran plot: most favored/additional allowed/generously allowed/disallowed (%) 88.5/9.6/1.9/0.0 89.2/9.0/1.9/0.0




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results suggest that sperm surface GalT is present and specifi-cally interacts with WGA16 through its terminal HexNAc res-idues. It is unknown whether UDP-Gal is extracellularly pres-ent in genital tracts. The UDP-Gal-dependent WGA16 releasethat we observed requires high concentrations of UDP-Gal (e.g.1–10 mM) (Fig. 9A).4 Because UDP-Gal is not present extracel-lularly in vivo, we point out that UDP-Gal-dependent WGA16release does not occur under physiological conditions andtherefore is of no physiological relevance. This experimentonly allows confirmation that WGA16 retention occursthrough the �-GalT under physiological conditions.Another mechanism, probably involving female tract com-ponents, may explain WGA16 release from the sperm sur-face during in vivo capacitation.

Removal of WGA16 from Sperm Surface Is Mediated byHeparin—In vitro observations established that WGA16 wasremoved from the lipid rafts during capacitation (Fig. 1). How-ever, the molecular basis of in vivo capacitation remains

unclear. In the case of bovines, it has been clearly demonstratedthat heparin induces capacitation (48), and heparin-bindingproteins have been shown to modulate capacitation (49). Thepresence of heparin, heparan sulfate, and other sulfated glycos-aminoglycans has been demonstrated in follicular fluid of sows(50). Unlike in the case of bovines, heparin does not inducecapacitation in sows but rather enhances in vitro capacitation ofporcine sperm only under capacitating conditions (51). Ourresults demonstrated the strong affinity of WGA16 towardheparin (Fig. 6, A and B). We thus hypothesized that in vivoheparin or sulfated glycosaminoglycans could be involved inthe removal of WGA16 from the sperm surface. To test thishypothesis, uncapacitated pig sperm was incubated with NCMcontaining increasing concentrations of heparin, and both spermand supernatant obtained from the washing step were analyzed forthe amount of WGA16 by Western blotting with anti-WGA16(Fig. 10). Whereas it decreased in sperm lysate, the WGA16 signalincreased in supernatant in a heparin-dependent manner, indicat-ing that heparin can be responsible for WGA16 removal, probablythrough a direct WGA16-heparin interaction.4 E. Garenaux, C. Sato, and K. Kitajima, unpublished results.

FIGURE 4. WGA16 is a �-prism-fold protein with a positively charged surface. A, overall structure of pig WGA16 (residues 18–166) in crystal form A shown witha ribbon model. The �-sheet structures are highlighted in yellow (�-sheet I; �1-�2 and �11-�12), cyan (�-sheet II; �3–�6), and green (�-sheet III; �7–�10). Loops arecolored in gray. B, comparison of pig WGA16 and human ZG16p structures. Close-up views are shown with ribbon models for the WGA16 structure (crystalform A) (top) and human ZG16p structure in complex with O-methyl-mannose (Protein Data Bank entry 3VZF) (bottom). The residues of WGA16corresponding to ZG16p residues interacting with mannose are shown with stick models. Bound mannose in ZG16p is also shown with a ball and stickmodel. Potential hydrogen bonds are indicated as dotted lines. C, comparison of pig WGA16 and human ZG16b structures. Close-up views are shown forthe sulfate-binding site of WGA16 (crystal form B, chain B) (top) and the phosphate-binding site of human ZG16b (bottom) (36). Potential hydrogenbonds are indicated as dotted lines. D, positively charged patches of WGA16, ZG16b, and ZG16p. The surface models of WGA16 (left), ZG16p (middle), andZG16b (right) are colored according to electrostatic surface potential (blue, positive; red, negative; scale from �10 to �10 kT/e). The residues possiblyinvolved in heparin binding (Lys-36, Arg-37, and Arg-55) are indicated in the ZG16p structure (36, 68).




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DISCUSSION

If their implication in fertilization is clearly established, thefunction of seminal plasma proteins during capacitation, acro-some reaction, and sperm-egg interaction remains largelyunclear. Especially, no functional significance of their glycanchains has ever been elucidated. Because glycan chains on gly-colipids and glycosylphosphatidylinositol-anchored glycopro-teins are enriched in lipid rafts (52), particular glycan structuresmay need to be concentrated in the lipid rafts to be functional.Thus, our hypothesis is that lipid rafts must give a platform forglycan-involved interactions (18). Sperm capacitation is char-acterized by a drastic remodeling of sperm plasma membrane(9 –14). In our approach, we focused on lipid raft-localized gly-coproteins whose localization changed upon capacitation inorder to link the sperm lipid rafts to their involvement in spermcapacitation and in the function of seminal plasma glycopro-teins. In this study, we discovered a particular glycoprotein thatdisappeared from the sperm surface during capacitation. Wenamed this protein WGA16 because of its enrichment in theWGA lectin epitope. WGA16 is shown to be a prostate-derivedseminal plasma glycoprotein that is deposited on the spermsurface in the male genital tract at the moment of ejaculation.

We show that this deposition process is glycan-dependent andinvolves the binding of sperm surface �-GalT to the N-glycan ofWGA16. WGA16 is the only WGA-positive seminal plasmaglycoprotein that undergoes capacitation-dependent redistri-bution on the sperm surface, whereas other WGA-positive gly-coproteins, such as spermadhesins PSP-I/II and AQN-1,remain at the sperm surface (Fig. 1). Isolation and functionalcharacterization of WGA16 evidenced common features withthe spermadhesin family (PSP-I/II, AQN-3, AWN). The mainseminal plasma proteins, 18/20-kDa spermadhesins PSP-I andPSP-II, form heterodimers and are described as decapacitationfactors. They may be involved in several sequential steps offertilization through a multifunctional ability to bind to car-bohydrates, sulfated glycosaminoglycans, or phospholipids(53, 54). We established, based on comparison of both pri-mary and crystal structure, that WGA16 was not a sperma-dhesin, although it shares common features, particularlyaffinity toward heparin or sulfated GAGs. WGA16 belongsto the JRL family; however, WGA16 does not form a dimer,confirming that, unlike JRLs from plants that form oligomersby non-covalent interaction (55), animal JRLs, such ashZG16 or WGA16, consist of monomers (33).

FIGURE 5. Crystal structure of WGA16 dimer in crystal form B. A, a dimer structure of WGA16 (chain F and chain C) is shown. The sulfate ion is shown with a ball andstick model. B, superposition of WGA16 structures from crystal form A (cyan) and crystal form B, chain F (gray). C, superposed structures of WGA16 in crystal form Bhighlighting the sulfate-binding site. WGA16-bound sulfate ions were observed in five molecules corresponding to chains A, B, C, D, and F in the asymmetric unit.




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One of the striking features of WGA16 is the exclusiveabsence of �-Gal residues and abundance of terminal GlcNAcand GalNAc residues, which enables an interaction of WGA16with the sperm surface �1,4-GalT (Fig. 11). For the past 30years, several studies have been dedicated to defining the func-tion of �1,4-GalT in sperm-egg interaction (54). Resultsobtained on knock-out mice suggest that the most importantrole of �1,4-GalT in fertilization is to induce the acrosome reac-tion (56). Before the acrosome reaction occurs, sperm has tobind to ZP3 through the surface receptors, which induce exo-cytosis of acrosomal contents. Several studies to understand thenature of sperm receptors for the murine zona pellucida have

been conducted (57–59). The most compelling supporting evi-dence is in favor of the binding of �1,4-GalT to terminalGlcNAc residues on ZP3 O-glycans (60).

Sperm surface �1,4-GalT is known to occur in various spe-cies, including pigs (46, 47). Mouse ZP orthologues have beenidentified: ZPA, ZPB, and ZPC (61). It has been demonstratedthat �1,4-GalT interacts with ZPs via GlcNAc residues but that�1,4-GalT does not have a required function in porcine zonaadhesion (47, 62). Our results suggest that porcine �1,4-GalTwould instead be responsible for modulation of capacitation byretaining seminal plasma-derived stabilizing (decapacitating)factors like WGA16 on the sperm surface. By incubating sperm

FIGURE 6. WGA16 specifically binds to heparin through the basic patch 1 region. A, fractionation of seminal plasma proteins on a heparin-agarose column.Seminal plasma proteins were applied onto a 1-ml heparin-agarose column, sequentially washed, and eluted using increasing NaCl concentration. Chromato-graphic fractions were analyzed by 15% SDS-PAGE and visualized by silver staining or anti-WGA16 antibody. Samples were overloaded to clearly show thehigher molecular weight region by silver staining. B, WGA16 lectinic activity as determined by the glycan microarray. Biotinylated recombinant WGA16 wasdiluted at a concentration of 10 �g/ml in reaction buffer, applied onto the glycan array slide surface, and incubated at room temperature for 2 h. After washing,the slide was incubated with 2 �g/ml Cy3-Streptavidin at room temperature for 1 h. The slide was washed and dried. The fluorescent intensity was immediatelyquantitated on a Typhoon scanner (�ex � 532 nm, �em � 575 nm). The grid key is shown on the right. The white narrow and wide squares show heparin and De2Sand De6S Hep, respectively. The yellow square shows high mannose N-glycan. C, location of basic patches of WGA16. Two possible positively charged patchesthat may be involved in heparin binding activity are indicated as BC1 (Lys-53 and Lys-73) and BC2 (Lys-25, Arg-91, Arg-93, and Lys-117), respectively. The surfacemodel and its 180° turn of WGA16 are colored for lysine (blue) and arginine (light blue). D, site-directed mutagenesis experiments to localize the heparin-bindingsite of WGA16. The supernatant fraction (input) of E. coli transformed with mock, wild type, and its BC1 and BC2 mutants of WGA plasmids were mixed withheparin-Sepharose resin. After washing, the resin was treated with high salt solution to elute the bound molecules (1 M NaCl). Together with the remaining gel(Gel, strongly bound fraction), input and 1 M NaCl fractions were analyzed by Western blotting (IB) using anti-WGA16 antibody. The arrowheads show theelution position of the recombinant WGA16 proteins.




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with UDP-Gal, we could observe the modification of theWGA16 glycosylation profile, with a great increase of R. com-munis agglutinin lectin signal, as well as the specific release ofWGA16 from the sperm surface. We thus hereby identify anddescribe for the first time a binding partner for boar sperm�1,4-GalT and address the molecular basis of one particularaspect of boar sperm capacitation.

Apart from its unique glycosylation profile, another strikingaspect of WGA16 is its high homology with ZG16 family pro-teins, which belong to the JRL family. In humans, ZG16p hasbeen found in the liver, small intestine, and pancreas (42).

ZG16b, also called PAUF, has been described in the pancreasand in saliva (63, 64). For the first time, we report the presenceof a ZG16-like protein in reproductive tissues. WGA16 is highlyglycosylated, whereas human, mouse, and rat ZG16p sequencesdo not contain any potential N-glycosylation sites. Althoughnumerous serine and threonine residues are present in theZG16p sequence, rZG16p was purified from the pancreas as aclear sharp band, suggesting the absence of glycosylation (65).Sequence homology indicates that WGA16 is closer to humanand pig ZG16b than human ZG16p. A recent study (66)reported that ZG16b facilitates tumor growth and metastasisthrough enhancing cell adhesiveness by regulating interactionsbetween tumor cells and extracellular matrix molecules.

Rat ZG16p has been demonstrated to bind to heparin,whereas human ZG16p binds to mannose. Classically, the sug-ar-binding site of mannose-binding type JRL consists of threeexposed regions (the GG loop, recognition loop, and bindingloop) at the top of the �-prism fold (31). In human ZG16p,mannose binding is mediated by the binding loop with a con-served motif, GXXXD. Rat ZG16p interaction with heparin hasbeen associated with a secondary sugar binding site between�-sheets 3 and 4 (N-terminal) but not with the classical bindingloop. In WGA16, as in human ZG16b, most of the conservedmotifs responsible for lectinic activity are absent or replaced.Particularly, in human ZG16b, the mannose-binding loopbetween �-sheets 11 and 12 containing the GXXXD motif ismodified (36). In WGA16, the binding loop between �-sheets11 and 12 is absent (i.e. there are no basic residues susceptible tobind to heparin). However, the site-directed mutagenesis stud-ies could clearly define the heparin-binding site, showing thatbasic cluster of Lys-53 and Lys-73 is involved. This binding siteis different from conventional mannose-binding sites but closeto the secondary binding site of Banlec or the correspondingsite of human ZG16b (Fig. 4D). It is striking that as for sperma-

FIGURE 7. Expression and occurrence of WGA16 in boar reproductiveorgans. A, Western blotting using anti-WGA16. B, WGA lectin blotting. Boarreproductive organs (testis, epididymis, seminal vesicle, prostate, and bul-bourethral gland, 10 �g/lane) were homogenized and subjected to 15% SDS-PAGE followed by immunostaining and lectin staining. C, real-time PCR formRNA expression of WGA16. The expression level in each organ was normal-ized by the expression of actin mRNA as described (27). The relative expres-sion is shown relative to that of testis set to 1.0. More than 1,000 times higherexpression was observed in prostate than in testis. pAb, polyclonal antibody.Error bars, S.E.

FIGURE 8. In situ hybridization (ISH) and immunohistochemistry (IHC) ofprostate (top), epididymis (middle), and testis (lower) for WGA16. Hema-toxylin and eosin staining (HE) is shown. In situ hybridization using comple-mentary RNA probes for negative control and WGA16 is shown. Also shown isimmunohistochemistry using anti-WGA16 and negative control for tissuesections of prostate, epididymis, and testis. The bars with numerals representscales. PS, prostate secretion; EP, epithelial cells of prostate; Sp, sperm; CE, celllayer of epididymis.




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dhesins, the modality of their interaction with sulfated GAGshas not yet been completely deciphered. Data suggest that, as itwas already observed for PSP-I (67), glycosylation of WGA16might influence its affinity toward heparin. Complex typeN-glycan-bearing PSP-I was recovered in the non-heparin-binding fraction, whereas highly mannose-substituted PSP-Iwas eluted in the heparin-binding fraction. However, mecha-nisms explaining the influence of glycosylation on heparinbinding are still unclear, and a future comparative study toestablish the molecular basis of heparin binding of seminalplasma proteins will be of interest.

Finally, from a functional point of view, it would be interest-ing to point out that N-glycosylation sites and a potential lec-tinic site (sulfate binding site) in WGA16 were present at twoopposite sides of the protein. As summarized in Fig. 11, wehypothesize that this functional asymmetry illustrates the func-tional duality of WGA16. Indeed, WGA16 is deposited at thesperm surface upon ejaculation via recognition of its N-glycanby the �1,4-GalT. Seminal plasma contains numerous factorsthat modulate the capacitation process in vivo during spermresidence in the female’s genital tract (4). We hereby provide forthe first time a model explaining the molecular mechanism ofdeposit and release of a particular seminal plasma protein. Wehypothesize that WGA16 blocks and protects the substrate-binding site of �1,4-GalT until capacitation occurs and thatthose phenomena are regulated by GAG content (i.e. heparin,chondroitin sulfate, and so on) of the female tract environ-ments. Once in the high GAG concentration region of the ovi-duct, WGA16 is removed from the sperm surface throughinteraction with sulfated GAGs; only then can the substrate-binding site of �1,4-GalT be exposed to interact with the acro-

FIGURE 9. Galactosyltransferase-dependent binding of WGA16 on thesperm surface. A, Western blotting (IB) of sperm treated with various sugarsand nucleotide sugars. Uncapacitated sperm were incubated with NCM (Con-trol) or 10 mM monosaccharide (Gal, GlcNAc, or Neu5Ac) or 10 mM activatedsugar (UDP-Gal, UDP-GlcNAc, or CMP-Neu5Ac) in NCM at 39 °C in 5% CO2 for 30min. The sperm suspensions were centrifuged, and sperm pellets werewashed twice with NCM. Sperm lysates were subjected to Western blottingwith anti-WGA16 antibody. B, Western blotting of UDP-Gal-treated spermand its washing supernatant. Uncapacitated sperm were treated with NCM(Control) or 10 mM UDP-Gal at 39 °C in 5% CO2 for 30 min and centrifuged toobtain pellet (Sperm) and supernatant (Wash). Those samples were sub-jected to Western blotting using anti-WGA16 (left) and lectin blotting withR. communis agglutinin (RCA) (middle) and GS-IB4 (right), recognizingGal�1,4GlcNAc and Gal� residues, respectively. Samples for lysate lanes wereslightly overloaded to show the signals in the supernatant that were causedby low sensitivity of the lectin staining.

FIGURE 10. Heparin-mediated removal of WGA16 from sperm surface.Uncapacitated sperm were incubated with NCM or with increasing concen-trations (0.5, 1, and 2 mg/ml) of heparin in NCM at 39 °C in 5% CO2 for 30 min.Sperm suspensions were centrifuged to pellet down intact sperm andwashed twice with NCM. Sperm lysates were applied onto a 15% SDS-PAGE at15 �g of protein/lane. WGA16 was quantified by the intensity of the immu-nostaining band on Western blotting with anti-WGA16 antibody. CBB, Coo-massie Brilliant Blue.

FIGURE 11. Schematic model for glycan-mediated adhesion and removalof sperm surface WGA16. Mainly through its specific N-glycans, WGA16 isthe privileged binding partner for GalT, resulting in the specific attachment ofWGA16 to lipid rafts at the sperm surface. The attachment takes place in malegenital tract (GT) after WGA16 is secreted from prostate gland. Later in thefemale genital tract, the presence of sulfated glycosaminoglycans in the ovi-duct induces removal of WGA16 from the sperm surface, facilitating capaci-tation. At the same time, sperm surface GalT is exposed for commitment tobind to ZP glycans, leading to acrosome reaction. Therefore, WGA16 mayplay a role in suppressing ZP-GalT binding-mediated signaling beforecapacitation.




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some reaction induced by ZP glycoproteins. It has been deter-mined that in mice, �1,4-GalT seems to directly interact withZP components, implying a direct role in fertilization. So far,based on sequence homology, we have not been able to identifythe WGA16 equivalent in mice. Deeper studies on murine sem-inal plasma content, particularly WGA lectin-positive, heparin-binding proteins, might allow us to further study the role of�1,4-GalT in fertilization. Deciphering and comparing themolecular mechanisms underlying capacitation in several spe-cies may provide answers about the cascade of events that musttake place before fertilization occurs.

Acknowledgments—We thank Masahiko Tanizawa, Kozoh Ishikawa,and Yasutoshi Ishikawa (Ishikawa Pig Farm Co., Ltd., Aichi, Japan)for kind and constant provision of fresh ejaculated sperm. We aregrateful to Drs. Satoshi Ohkura and Tsukasa Matsuda (Nagoya Uni-versity) and Dr. Nongnuj Tanphaichitr (Ottawa Hospital ResearchInstitute) for valuable discussions. We also thank Drs. Kay-Hooi Khooand Chia-Wei Lin (Adademia Sinica, Taiwan) for technical adviceand critical reading of the manuscript.

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Yamaguchi, Chihiro Sato and Ken KitajimaKanazawa, Ami Goshima, Mitsuru Chiba, Hiroshi Yasue, Akemi Ikeda, Yoshiki

Estelle Garénaux, Mayumi Kanagawa, Tomoyuki Tsuchiyama, Kazuki Hori, Takerua Prostate-derived Heparin-binding Protein WGA16 from Boar Sperm

Discovery, Primary, and Crystal Structures and Capacitation-related Properties of

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