Purification of N-acetyllactosamine-binding Activity from the Porcine Sperm Membrane: Possible Involvement of an ADAM Complex in the Carbohydrate-binding Activity of Sperm

Abstract

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][118][119][120][121][122][123][124][125] 2012) I n mammals, sperm interact with specific carbohydrate chains found on the zona pellucida (ZP) of eggs, and these interactions seem to be species-selective [1]. Extensive analyses of ZP carbohydrate chains have been performed in pigs [2][3][4][5], mice [6,7] and cattle [8], and the involvement of N-acetyllactosamine and Lewis X moieties on the ZP in sperm binding was reported in pigs [9,10] and mice [11,12]. For cattle, the α-mannosyl residues of ZP N-glycans are involved in binding [13]. Conversely, little is known about which sperm proteins recognize ZP carbohydrates. Murine sperm galactosyltransferase has been the most extensively studied carbohydrate-binding protein [14], but it appears unlikely that it participates in the initial gamete adhesion process [15]. Other carbohydrate-binding proteins have been implicated, i.e., rat galactosyl receptor, which is identical to the rat hepatic lectin receptor 2/3 [16], por...

Lactadherin is a candidate oviduct Lewis X trisaccharide receptor on porcine spermatozoa

“…There are few reports of glycan affinity of ADAMs proteins. Authors of one previous publication using asialo-α1-acid and asialo-aga-lacto- α1-acid affinity chromatography, reported that N -acetyllactosamine but not Le X had affinity for ADAM5 from epididymal boar spermatozoa (Mori et al ., 2012). On the contrary, we found that porcine epididymal and ejaculated spermatozoa recognize N -acetyllactosamine but bind much less N -acetyllactosamine than suLe X (Silva et al ., 2013).…”