Purification of N-Acetyllactosamine-Binding

Journal of Reproduction and Development, Vol. 58, No 1, 2012 —Original Article — Purification of N-acetyllactosamine-binding Activity from the Porcine Sperm Membrane: Possible Involvement of an ADAM Complex in the Carbohydrate-binding Activity of Sperm Etsuko MORI1), Hiroyuki FUKUDA2), Shinobu ImaJOH-Ohmi2), Tsuneatsu MORI1) and Seiichi TAKasaKI1) 1)Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan 2)Medical Proteomics Laboratory, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan Abstract. Although the importance of carbohydrate recognition by sperm during egg zona pellucida binding has been widely reported, the sperm molecular species that recognize the carbohydrates are poorly characterized. Our previous cytochemical study indicated that two kinds of carbohydrate-binding proteins are expressed on porcine sperm heads—one recognizes N-acetyllactosamine (Galβ1-4GlcNAc-), and the other recognizes the Lewis X structure (Galβ1-4(Fucα1-3) GlcNAc-). For this report, we used proteomic techniques to characterize the sperm proteins that bind N-acetyllactosamine. Porcine sperm plasma membrane was solubilized with a detergent solution and subjected to sequential chromatography with dextran sulfate agarose, affinity, and hydroxyapatite, and the binding activities in the eluates were monitored by a solid-phase binding assay. The tryptic peptides of two proteins most likely associated with the binding activities were subjected to tandem mass spectrometry sequencing. A subsequent database search identified one of the two proteins as predicted disintegrin and metalloprotease domain-containing protein 20-like (XP_003128672). The other protein was identified as disintegrin and metalloprotease domain-containing protein 5 (AB613817) by database searches for homologous amino acid sequences, cDNA cloning, nucleotide sequencing and nucleotide database searches. Furthermore, two-dimensional blue native/SDS-PAGE demonstrated that they formed a variety of non-covalent complexes. Therefore, these ADAM complexes probably are responsible for the N-acetyllactosamine-binding activity. An affinity-purified fraction containing these ADAM complexes showed zona pellucida-binding activity, though the activity was relatively weak, and the presence of another zona pellucida-binding protein that probably works in concert with these ADAM complexes was suggested. Immunofluorescence testing suggested that ADAM20-like was localized on the anterior part of the sperm plasma membrane. Key words: ADAM, Carbohydrate-binding, Sperm, Zona pellucida (J. Reprod. Dev. 58: 117–125, 2012) n mammals, sperm interact with specific carbohydrate chains glycodelin-A as well as to the ZP [18]; none of these, however, is Ifound on the zona pellucida (ZP) of eggs, and these interac- widely accepted as a sperm carbohydrate-binding protein involved tions seem to be species-selective [1]. Extensive analyses of ZP in ZP binding. We previously characterized porcine ZP oligosac- carbohydrate chains have been performed in pigs [2–5], mice [6, charides [2, 3, 5]. Using those results, we then determined which 7] and cattle [8], and the involvement of N-acetyllactosamine and oligosaccharides were recognized by intact porcine sperm and an Lewis X moieties on the ZP in sperm binding was reported in isolated sperm plasma membrane [9, 19]. We found that porcine pigs [9, 10] and mice [11, 12]. For cattle, the α-mannosyl residues sperm recognizes N-acetyllactosamine-bearing oligosaccharides of ZP N-glycans are involved in binding [13]. Conversely, little is with or without sialic acid present at the non-reducing ends of known about which sperm proteins recognize ZP carbohydrates. N-glycans. After removal of the galactose residues, the sperm no Murine sperm galactosyltransferase has been the most extensively longer recognized the oligosaccharides. The binding of sperm to studied carbohydrate-binding protein [14], but it appears unlikely the ZP was inhibited by asialo-N-glycans from fetuin. Additionally, that it participates in the initial gamete adhesion process [15]. porcine sperm recognized a Lewis X-bearing oligosaccharide. The Other carbohydrate-binding proteins have been implicated, i.e., sperm protein(s) that recognizes N-acetyllactosamine is distinct rat galactosyl receptor, which is identical to the rat hepatic lectin from the one(s) that recognizes the Lewis X moiety because the receptor 2/3 [16], porcine AWN-1, which interacts with core 1 binding of the sperm membrane to an N-acetyllactosamine-bearing O-glycans [17], and human fucosyltransferase, which binds to probe was not inhibited by a Lewis X-bearing probe and vice versa. The binding of sperm to these carbohydrates was calcium ion-independent, which indicated that the carbohydrate-binding Received: July 27, 2011 protein(s) is not related to the hepatic asialoglycoprotein receptor Accepted: October 3, 2011 or to selectins. For the study reported herein, we identified sperm Published online in J-STAGE: November 4, 2011 ©2012 by the Society for Reproduction and Development membrane proteins that probably have N-acetyllactosamine-binding Correspondence: E Mori (e-mail: [email protected]) activity as disintegrin and metalloprotease domain-containing 118 MORI et al. protein 20-like (ADAM20-like) and disintegrin and metallopro- 7.4, containing 1.6 M NaCl, 30 mM MgCl2 and 50 mM KCl) was tease domain-containing protein 5 (ADAM5) by a combination of added to the suspension to restore isotonicity. The homogenized column chromatography, a solid-phase binding assay and proteomic cell suspension was centrifuged at 3000 × g (10 min, 4 C), and then techniques. ZP-binding activity and localization of these proteins the supernatant was centrifuged at 5,000 × g (10 min, 4 C). The was elucidated. obtained supernatant was ultracentrifuged at 100,000 × g (30 min, 4 C), and the sperm plasma membrane pellet was collected. The Materials and Methods plasma membrane was resuspended in Hepes-buffered saline containing 0.8 mM PMSF and 10 mM EDTA and reultracentrifuged Preparation of glycoprotein probes and glycoprotein-coupled at 230,000 × g (30 min, 4 C). This washing step was repeated Sepharose 4B three times. The membrane thus obtained (50 mg wet weight) was Asialo-α1-acid glycoprotein (As-α1-AGP) was prepared by mild incubated in 10 mM Hepes-buffered saline, pH 7.8, containing 1% acid hydrolysis of human α1-acid glycoprotein (Sigma-Aldrich, St. (w/v) Triton X-100, 0.5% (w/v) n-octyl-β-D-thioglucoside, 10 mM Louis, MO, USA) in ~20 mM HCl (pH 2) at 100 C for ~30 min while EDTA and protease inhibitor cocktail (Complete, Mini, Roche, monitoring the release of sialic acid by thin layer chromatography. Indianapolis, IN, USA) on ice for 60 min and then centrifuged at As-α1-AGP was biotinylated, and an aliquot was subjected to 100,000 × g for 60 min. The supernatant was subjected to dextran β-galactosidase digestion [11] to obtain the asialo-agalacto-α1-acid sulfate agarose column chromatography (5 K, 3 ml, GIBCO BRL, glycoprotein (AsAg-α1-AGP) probe. For affinity chromatography Karlsruhe, Germany). The resin was equilibrated with buffer A matrixes, As-α1-AGP was conjugated to CNBr-activated Sepharose (10 mM Hepes, pH 7.8, 10 mM EDTA, 0.1% (w/v) Triton X-100) 4B (GE Healthcare, Buckinghamshire, UK) as described by the that contained 0.15 M NaCl and, after loading the sample, was manufacturer, and an aliquot was subjected to β-galactosidase washed with 20 ml of this buffer. Bound protein was eluted with digestion to prepare AsAg-α1-AGP-coupled Sepharose. buffer A containing 0.4 M NaCl and then with buffer A containing 1.2 M NaCl. Fractions of 2 ml were collected. The fraction Solid-phase binding assays that had the greatest N-acetyllactosamine-binding activity was N-acetyllactosamine-binding activity was monitored through- concentrated by a Nanosep ultracentrifugation device (10 K, Pall out the purification process by the following assay. Samples of Life Sciences, Port Washington, NY, USA) into 100 µl, and NaCl chromatographic eluates were incubated with Bio-Beads SM-2 was adjusted to 0.5 M. Half of the sample was chromatographed for detergent removal (100-fold excess with respect to detergent, through a column of As-α1-AGP-coupled Sepharose, and the other w/w, Bio-Rad Laboratories, Hercules, CA, USA) for 2 h at room half was chromatographed through a column of AsAg-α1-AGP- temperature with vigorous shaking. Each recovered supernatant coupled Sepharose. Each column had a volume of 1 ml. The resins was added into a well of an EIA plate. N-acetyllactosamine-binding were initially equilibrated with buffer A containing 0.5 M NaCl. activity was detected using a biotin-labeled As-α1-AGP probe (10 µg/ Fractions (0.5 ml) were collected at a flow rate of 0.1 ml/min first ml). A biotin-labeled AsAg-α1-AGP probe (10 µg/ml) served as with 5 ml of buffer A containing 0.5 M NaCl and then with 2 ml the control. A sample in a well was incubated with each probe for of 0.1 M glycine-Cl, pH 2.5, 0.1% (w/v) Triton-X 100. Fractions 45 min and then incubated for 30 min with horseradish peroxidase from the As-α1-AGP-coupled Sepharose chromatography that had conjugated avidin, both at 4 C. For blocking and dilution, 10 mM N-acetyllactosamine-binding activity were pooled, concentrated, Tris-buffered saline containing 2% casein and 0.05% (w/v) Tween and buffer exchanged by a Nanosep ultracentrifugation device 20 was used. The intensity of horseradish peroxidase reaction was (10 K) with 10 mM sodium phosphate,

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