Equatorin: Identification and Characterization of the Epitope of the MN9 Antibody in the Mouse1

Abstract

Equatorin (MN9 antigenic molecule) is a widely distributed acrosomal protein in mammalian sperm. During the acrosome reaction, some amount of equatorin translocates to the plasma membrane, covering the equatorial region. From the results of studies of both in vitro and in vivo fertilization inhibition using the MN9 antibody, equatorin has been suggested to be involved in fusion with the oolemma. In the present study, we cloned equatorin and, using mass spectrometry and carbohydrate staining, found it to be a highly glycosylated protein. Equatorin is a sperm-specific type 1 transmembrane protein, and glycosidase treatment and recombinant protein assays verified that it is an N,O-sialoglycoprotein. In addition, the gamete interaction-related domain recognized by the MN9 antibody is posttranslationally modified. The modified domain was identified near threonine 138, which was most likely to be O-glycosylated when analyzed by amino acid substitution, dephosphorylation, and O-glycosylation inhibitor assays. Immunogold electron microscopy localized the equatorin N-terminus, where the MN9 epitope is present, on the acrosomal membrane facing the acrosomal lumen. These biochemical properties and the localization of equatorin are important for further analysis of the translocation mechanism leading to gamete interaction.

Membrane Fusions During Mammalian Fertilization

“…SPESP1 (sperm equatorial segment protein 1; [132]) is a candidate, based on the finding that Spesp1 -/-males produce slightly smaller litters than wild type controls (22%), and have sperm with reduced (although not completely deficient) ability to undergo sperm-oocyte fusion [77]. Other proteins associated with sperm-oocyte fusion and exposure or rearrangement after acrosome exocytosis include equatorin [133,134], Sperm Acrosomal Membrane-Associated the phenotype of the knockout [82]. Numerous other molecules have also been suggested to participate in mammalian gamete membrane interaction (sperm binding and/or sperm-oocyte membrane fusion; Fig.…”

The mechanism of sperm–egg interaction and the involvement of IZUMO1 in fusion

“…Equatorin on plasma membrane is considered to function in sperm-egg fusion, because MN9 antibody inhibits both in vitro 33 and in vivo fertilization systems 34 without affecting zona-penetrating ability of sperm. The equatorin gene is cloned 35 and has been identified as a type 1 transmembrane protein of a 40-60 kDa and N,O-sialoglycoprotein.…”

“…35,[56][57][58] SPESP1 was one of the candidates functioning for fusion, because anti-SPESP1 antibody was known to inhibit human sperm from fusing with hamster eggs as well as inhibiting sperm-egg fusion in mouse. 59 A mouse line lacking Spesp1 was produced by homologous recombination and the fertilizing ability of male mice was examined.…”

Equatorin-Related Subcellular and Molecular Events During Sperm Priming for Fertilization in Mice

“…When examined by immunogold electron microscopy, the immunogold Yamatoya et al 2009 ) particles are abundant on the inner acrosomal membrane in the principal region and equatorial segment (Ito et al 2013 ). However, on the outer acrosomal membrane, the gold particles are depleted in the principal region but abundant at the equatorial segment, whereas gold particles are not detected on the outer acrosomal membrane in the apical region, suggesting that equatorin is absent or very poor on the outer acrosomal membrane in the apical region.…”

“…Equatorin was purifi ed by immunoprecipitation using the MN9 antibody, and purifi ed equatorin was identifi ed by LC-MS/MS analysis ( Fig. 7.1 ) (Yamatoya et al 2009 ). A Mascot search revealed a single signifi cant candidate, 4930579C15Rik, the human orthologue of which is reported to be frequently deleted in cancer (Ruiz et al 2000 ).…”