PARENT SESSION

(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 (Prp^C) 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 Prp^C to its pathological isoform (Prp^SC) and subsequent spread of Prp^SC 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 Prp^C to Prp^SC 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 Prp^SC, remains to be elucidated.

KEY WORDS: epididymis, prion, mass spectrometry, sheep