Novel purification method for mammalian seminal plasma phospholipid‐binding proteins reveals the presence of a novel member of this family of protein in stallion seminal fluid

Abstract

A family of bull seminal plasma (BSP) phospholipid-binding proteins (BSP proteins), potentiate heparin- and HDL-induced capacitation. The homologous proteins have been purified from stallion and boar seminal plasma, and detected in low concentrations in other mammalian seminal plasma. In this study, we developed a new isolation method for mammalian seminal plasma choline phospholipid-binding proteins wherein they are present in low concentrations. The method is based on the interaction of this family of proteins with egg yolk low-density lipoprotein fraction (LDF). In order to demonstrate the feasibility of the method, we incubated LDF with alcohol precipitates of bull, boar, and stallion seminal plasma. LDF were re-isolated by ultracentrifugation along with bound proteins. LDF with associated proteins were dialyzed, lyophilized, and delipidated. BSP homologous proteins were finally purified by p-aminophenyl phosphorylcholine (PPC)-agarose and/or gelatin-agarose chromatographies, and analyzed by SDS-PAGE. With this new protocol, phospholipid-binding proteins of bull, boar, and stallion seminal plasma were recovered almost 100%. A new 12 kDa stallion seminal plasma protein of the same family was also isolated and partially sequenced. The radio-immunoassay (RIA) data showed that 10 mg of LDF can bind all BSP proteins present in 120 mg of alcohol precipitated BSP proteins. These results confirm the efficiency of the method and that the LDF step could be used for the isolation of all BSP proteins homologs from different mammalian species.

Proteomic analysis of the reproductive tract fluids from tropically-adapted Santa Ines rams

“…This might also be the case for RSVP 22, that would explain the fact that we found high molecular weight spots (60 kDa) corresponding to this protein in our seminal plasma maps. Members of the BSP family are expressed in the reproductive fluids of several species, including bovine [16,22], equine [35], swine [36] and goats [30]. BSPs interact with sperm phospholipids and stimulate both cholesterol efflux and permeabilization of the membrane, events typically linked to sperm capacitation [37].…”

Murine Binder of SPerm Homolog 2 (BSPH2): The Black Sheep of the BSP Superfamily1

“…It is also the case for BSP homologs found in other ungulates, such as stallion, boar, goat, bison, and ram [27][28][29][30][31][32]. All BSP proteins have a common structure.…”

Murine binder of sperm protein homolog 1: a new player in HDL-induced capacitation

“…Other members of the BSP superfamily have been identified in species such as boar, ram, goat, stallion, and bison and more recently in human and mice (Calvete et al 1995, Menard et al 2003, Villemure et al 2003, Boisvert et al 2004, Fan et al 2006. The BSP proteins are all structurally similar as they are composed of a variable N-terminal domain followed by two fibronectin type II (Fn2) domains arranged in tandem (Manjunath et al 2009).…”