Seminal vesicle secretory protein SVSVII binds to the sperm surface by interacting with lipid components of membrane rafts.

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

La Plata

Conferencia; 8th International Conference on Lipid Binding Proteins, LBP 2013; 2013

SP-06: MOUSE SEMINAL VESICLE SECRETORY PROTEIN SVS VII BINDS TO THE SPERM SURFACE BY INTERACTING WITH LIPID COMPONENTS OF MEMBRANE RAFTS Jorgelina Buschiazzo1,2*, Lucia Zalazar3*, Rosana de Castro3 and Andreina Cesari3 1Lab. Biotecnología de la Reproducción-Departamento de Producción Animal, Instituto Nacional de Tecnología Agropecuaria (INTA), EEA Balcarce, Balcarce, Argentina. 2Instituto de Investigaciones Bioquímicas de Bahía Blanca (UNS- CONICET), Bahía Blanca, Argentina. 3Laboratorio de Biología de microorganismos y gametas, Instituto de Investigaciones Biológicas, CCTCONICET, Universidad Nacional de Mar del Plata, Mar del Plata, Argentina. acesari@mdp.edu.ar 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 silico 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 CaCl . 2 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 CaCl and then 2 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. *Authors who equally contributed to the work. LBP 2013 La Plata - Argentina 35