Molecular cloning and characterization of rat sperm surface antigen 2B1, a glycoprotein implicated in sperm-zona binding

Cloning and molecular characterization of the first aquatic hyaluronidase, SFHYA1, from the venom of stonefish (Synanceja horrida)

“…For instance, reported their failure to express enzymatically active human PH-20 hyaluronidase in E. coli. Hou et al (1996) reported epitope mapping of rat sperm surface antigen 2B1, a protein highly homologous to PH-20 hyaluronidases, using E. coli produced recombinant fragments of 2B1. The hyaluronidase activities of 2B1 was proven indirectly by showing that in both Western blot (using monoclonal antibody that recognizes 2B1) and zymogram of hyaluronidase activity of rat spermatozoa extracts, a 56-kDa protein was identified.…”

Rat Sperm 2B1 Glycoprotein (PH20) Contains a C-Terminal Sequence Motif for Attachment of a Glycosyl Phosphatidylinositol Anchor. Effects of Endoproteolytic Cleavage on Hyaluronidase Activity1

“…Guinea pig PH20 glycoprotein (gpPH20) is unusual in that it migrates from one type of membrane, the postacrosomal plasma membrane, onto a different type of membrane, the inner acrosomal membrane, after exocytosis of the acrosomal vesicle [5]. The rat orthologue of PH20 (original designation, 2B1, retained throughout this paper for the sake of consistency in the literature; [10,11]) is initially restricted to the sperm tail; but as part of the general membrane changes that accompany capacitation, it is transported across putative diffusion barriers in the neck and equatorial segment regions and accumulates on the acrosomal plasma membrane [9]. Sequence analyses of gpPH20 and its species orthologues have revealed significant homology to bee venom hyaluronidase, and, as a consequence, they have been found to degrade hyaluronic acid [11][12][13][14][15].…”

“…However, attempts to demonstrate a lipid anchor on 2B1 antigen by phosphatidylinositol phospholipase C (PI-PLC) digestion of whole spermatozoa, or of plasma membrane vesicles derived therefrom, have been unsuccessful [19]. Sequence analysis of 2B1 revealed a hydrophobic domain of approximately 23 amino acid residues toward the C-terminus suggestive of a membrane spanning region [11]. Like gpPH20 [21], 2B1 is endoproteolytically cleaved into a disulfidebridged heterodimer during sperm passage through the epididymis [19], but it not known whether cleavage is obligatory for its ability to migrate during capacitation or how it affects hyaluronidase activity.…”

Secreted Epididymal Glycoprotein 2D6 That Binds to the Sperm’s Plasma Membrane Is a Member of the β-Defensin Superfamily of Pore-Forming Glycopeptides1

“…9). As an additional control, parallel blots were probed with 2B1 McAb, which has been shown to recognize a peptide epitope [28]. Alkali treatment had no detectable effect on the ability of 2B1 McAb to recognize its antigen (Fig.…”

“…Western blots containing reduced cauda sperm proteins, previously separated by SDS-PAGE, were immersed in 55 mM NaOH, pH 12.0, at 4ЊC for various times up to 1 h, washed three times in PBS, and probed with 2D6 McAb and 2D6 polyclonal antibodies as described previously. As a positive control, parallel blots were probed with 2B1 McAb, which recognizes a peptide-based epitope [28].…”