Mouse seminal vesicle secretory protein svs vii binds to the sperm surface by interacting with lipid components of membrane rafts.

BUSCHIAZZO, J; ZALAZAR, L; DE CASTRO, R; CESARI, A

La Plata

Conferencia; 8th international Conference on LIPID BINDING PROTEINS; 2013

INIBIOLP- CONICET, CCT La Plata

Secretory proteins from seminal vesicle are known to participate in sperm physiology by interaction with the cell membrane during the transit along the male duct in mammals. Seminal vesicle secretion from mouse contains seven well defined proteins designated as SVS I?VII. SVS VII is a major protein described to bind the sperm surface, however its role is far from being elucidated. It is known that many sperm binding proteins are decapacitating molecules as they stabilize specialized domains for cellular signaling at the plasma membrane (membrane rafts) by binding to membrane phospholipids or cholesterol (Chol). In this work we produced two versions of recombinant SVS VII (GST-SVS VII and SVS VII-His6) and evaluated their ability to interact with the sperm membrane. Binding to the sperm surface was confirmed by immunocytochemistry. It was previously reported that purified native SVS VII binds to phosphatidylserine (PS) but not to phosphatidylcholine (PC). In sillico search for CRAC domains (Chol recognition aminoacid consensus) in SVS VII protein sequence revealed two binding motifs. In order to experimentally demonstrate whether SVS VII could bind Chol, SVS VII was allowed to interact with different liposomes (PS, PC and PC:Chol) in the presence or absence of 1.8 mM Ca2Cl. After centrifugation pellet and supernatant fractions were analyzed by SDS-PAGE. SVS VII bound to liposomes made of PS and PC:Chol when no Ca+2 was included. These interactions were confirmed through a fat western blot assay by spotting lipid standards (PS, PC and Chol) on a nitrocellulose membrane that was further incubated with either GST-SVS VII or SVS VII-His6 in the presence or absence of 1.8 mM Ca2Cl and then developed by their specific antibodies. These results suggest that interaction of SVS VII with sperm surface might be through plasma membrane domains containing PS and Chol making SVS VII a potential important candidate as a decapacitating protein likely acting via membrane rafts.