Scrapie PrP 27-30 is a sialoglycoprotein

Abstract

The major scrapie prion protein, designated PrP 27-30, exhibited both charge and size heterogeneity after purification from infected hamster brains. Eight or more discrete charge isomers of PrP 27-30 with isoelectric points ranging from approximately pH 4.6 to 7.9 were found by using non-equilibrium pH gradient electrophoresis in the first dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. The charge isomers were detected by silver staining as well as by radioiodination. The procedures used to disaggregate PrP 27-30 before electrophoresis in the first dimension do not appear to be responsible for the charge heterogeneity. However, heating PrP 27-30 to 100°C for 15 min in 0.1 N NaOH or 0.1 N HCI resulted in modification of the protein and alteration of its electrophoretic pattern. A PrP 27-30 fragment (molecular weight, 17,100 to 21,900) obtained by cyanogen bromide cleavage also exhibited charge and size heterogeneity. Periodic acid-Schiff staining of PrP 27-30 electrophoresed into sodium dodecyl sulfate-polyacrylamide gels demonstrated that carbohydrate residues are attached to the protein. Digestion of PrP 27-30 with neuraminidase and endo-,l-N-acetylglucosaminidase H resulted in significant changes in the isoelectric pH of PrP 27-30 isomers, whereas digestion with alkaline phosphatase had no effect. Our results demonstrate that PrP 27-30 is a sialoglycoprotein; this is consistent with several properties of this protein and of the scrapie prion.

Prions Adhere to Soil Minerals and Remain Infectious

“…Changes in pH often alter interactions between clay surfaces and sorbed proteins [27,28]. Incubation of Mte-bound PrP Sc in 100 mM phosphate buffer at pH 2.5 or 11.5, proton activities substantially higher and lower than the reported isoelectric points for PrP Sc [29], failed to release the protein ( Figure 3B). Likewise, increases in ionic strength (0.1 M or 1 M NaCl) failed to remove detectable PrP Sc from Mte ( Figure 3C).…”

Regional and phenotype heterogeneity of cellular prion proteins in the human brain

“…After translation, PrP C is modified at the amino and carboxyl terminus (Hope et al, 1986;Stahl et al, 1990) and by attachment of a glycan moiety and the formation of a disulphide bond (Kretzschmar et al, 1986). Fully processed PrP C then attaches to the cell membrane by means of the glycosylphosphatidyl-inositol (GPI) anchor (Bolton et al, 1985;Endo et al, 1989;Stahl et al, 1992).…”

The major polypeptide of scrapie-associated fibrils (SAF) has the same size, charge distribution and N-terminal protein sequence as predicted for the normal brain protein (PrP).

“…A major component of hamster fibrils has been described as a protease-resistant glycoprotein with an apparent mol. wt of 27 000 -30 000 (PrP27 -30) (Diringer et al, 1983;Bolton et al, 1982Bolton et al, , 1984Bolton et al, , 1985McKinley et al, 1983;Prusiner et al, 1982Prusiner et al, , 1983. The partial amino acid sequence of this fibril protein (PrP) has been determined by protein microsequencing Multhaup et al, 1985;Oesch et al, 1985) and a possible complete primary structure inferred from the cDNA sequence of its mRNA (Oesch et al, 1985;Chesebro et al, 1985).…”

“…This finding has led to the inclusion of a proteinase K digestion step m all recent methods that have been used in attempts to purify scrapie infectivity (Diringer et al, 1983;Hilmert and Diringer, 1984; Bolton et al, 1982;Prusiner et al, 1983). SAF and infectivity co-purify, at least in part, during the subcellular fractionation of scrapie-infected brain (Diringer et al, 1983;Somerville et al, 1986) and these fibrils, and their major protein component (PrP27 -30) (Diringer et al, 1983;McKinley et al, 1983;Prusiner et al, 1982Prusiner et al, , 1983, have also been described as protease resistant (Diringer et al, 1983;Bolton et al, 1982Bolton et al, , 1984Bolton et al, , 1985Prusiner et al, 1984). The association of PrP27 -30, SAF and infectivity has led to the speculation that SAF are a form of the scrapie agent (Merz et al, 1983a;Diringer et al, 1983;Somerville et al, 1986), and as a scrapie-specific nucleic acid has yet to be found in SAF, that PrP27 -30 is, in itself, the infectious entity (Bolton et al, , 1985McKinley et al, 1983;Prusiner et al, 1982Prusiner et al, , 1983.…”

“…SAF and infectivity co-purify, at least in part, during the subcellular fractionation of scrapie-infected brain (Diringer et al, 1983;Somerville et al, 1986) and these fibrils, and their major protein component (PrP27 -30) (Diringer et al, 1983;McKinley et al, 1983;Prusiner et al, 1982Prusiner et al, , 1983, have also been described as protease resistant (Diringer et al, 1983;Bolton et al, 1982Bolton et al, , 1984Bolton et al, , 1985Prusiner et al, 1984). The association of PrP27 -30, SAF and infectivity has led to the speculation that SAF are a form of the scrapie agent (Merz et al, 1983a;Diringer et al, 1983;Somerville et al, 1986), and as a scrapie-specific nucleic acid has yet to be found in SAF, that PrP27 -30 is, in itself, the infectious entity (Bolton et al, , 1985McKinley et al, 1983;Prusiner et al, 1982Prusiner et al, , 1983. This latter notion appears to be incompatible both with the occurrence of > 15 distinct strains of scrapie (Dickinson et al, 1984) which indicates it has a host-independent genome (Kimberlin, 1986), and with the evidence that the PrP sequence is encoded by a single gene in the host genome (Oesch et al, 1985).…”