The epididymal soluble prion protein forms a high-molecular-mass complex in association with hydrophobic proteins.
We have shown previously that a 'soluble' form of PrP (prion protein), not associated with membranous vesicles, exists in the male reproductive fluid [Ecroyd, Sarradin, Dacheux and Gatti (2004) Biol. Reprod. 71, 993-1001]. Attempts to purify this 'soluble' PrP indicated that it behaves like a high-molecular-mass complex of more than 350 kDa and always co-purified with the same set of proteins. The main associated proteins were sequenced by MS and were found to match to clusterin (apolipoprotein J), BPI (bacterial permeability-increasing protein), carboxylesterase-like urinary excreted protein (cauxin), beta-mannosidase and beta-galactosidase. Immunoblotting and enzymatic assay confirmed the presence of clusterin and a cauxin-like protein and showed that a 17 kDa hydrophobic epididymal protein was also associated with this complex. These associated proteins were not separated by a high ionic strength treatment but were by 2-mercaptoethanol, probably due to its action on reducing disulphide bonds that maintain the interaction of components of the complex. Our results suggest that the associated PrP retains its GPI (glycosylphosphatidylinositol) anchor, in contrast with brain-derived PrP, and that it is resistant to cleavage by phosphatidylinositol-specific phospholipase C. Based on these results, the identity of the associated proteins and the overall biochemical properties of this protein ensemble, we suggest that 'soluble' PrP can form protein complexes that are maintained by hydrophobic interactions, in a similar manner to lipoprotein vesicles or micellar complexes.
Ecroyd
Heath
H
Gamète Male et Fertilité, Institut National de la Recherche Agronomique, INRA-Nouzilly, 37380 Monnaie, France.
Belghazi
Maya
M
Dacheux
Jean-Louis
JL
Gatti
Jean-Luc
JL
D000595:Amino Acid Sequence; D000818:Animals; D051152:Clusterin / Q000378:metabolism; D004822:Epididymis / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism*; D011328:Prions / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D012756:Sheep; D012995:Solubility
D051152:Clusterin; D046912:Multiprotein Complexes; D011328:Prions