Genomics and Proteomics of the Reproductive System
(M535) THE EPIDIYMAL SOLUBLE PRION PROTEIN IS ASSOCIATED WITH HYDROPHOBIC PROTEINS IN A HIGH MOLECULAR WEIGHT STRUCTURE.
Gatti, Jean-Luc1, Ecroyd, Heath2, Belghazi, Maya1, Dacheux, Jean-Louis1, 1 Station de Physiologie de la Reproduction et des Comportements, INRA, Nouzilly, France2 The University of Adelaide, Adelaide, Australia
ABSTRACT- The cellular prion protein (PrpC) is a glycoprotein of unknown function found in almost all tissues where it is usually attached to the external face of cell membranes by a glycosylphosphatidylinositol (GPI) anchor. The conformational conversion of PrpC to its pathological isoform (PrpSC) and subsequent spread of PrpSC to other tissues is thought to be responsible for the progression of spongiform encephalopathies (TSEs), such as bovine spongiform encephalopathy (BSE), scrapie in sheep and Creutzfeldt-Jakob disease in human. It has been suggested that the biochemical conversion of PrpC to PrpSC is an autocatalytic process, however, its interactions with an unidentified factor (named protein X) has also been proposed. We have previously reported that a soluble form of the prion protein, not associated with membraneous vesicles, exist in the male reproductive fluid (Ecroyd H, Sarradin P, Dacheux J-L and Gatti J-L. 2004. Biol. Reprod. 71; 993-1001). During attempts to purify this soluble prion protein we found that it behaves like a high molecular weight protein of more than 400 kDa and always co-purified with the same set of proteins. The main proteins associated were identified by mass spectrometry as clusterin (apolipoprotein-J), bacterial permeability increasing-protein (BPI), beta-mannosidase, and beta-galactosidase. Immunoblotting confirmed the presence of clusterin and show that a hydrophobic 17 kDa epididymal protein was also associated. These proteins were not separated by a high ionic strength treatment but were by beta-mercaptoethanol, certainly due to its action on reducing disulfide bonds that maintain multimers of clusterin alpha and beta chains. Surprinsingly, the associated-Prp retained its GPI-anchor that was resistant to cleavage by PI-PLC. Based on these results, the identity of the associated proteins, and the overall biochemical properties of this protein ensemble, we suggest that soluble Prp is part of a high molecular weight structure maintained mainly by hydrophobic interactions, in a micelle- or lipoprotein-like complex. The existence of such prion-associated structure in other body fluids, as well as their possible role in the transport of PrpSC, remains to be elucidated.
KEY WORDS: epididymis, prion, mass spectrometry, sheep