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Both the cellular and scrapie isoforms of the prion protein (PrP) designated PrP c and PrP s" are encoded by a single-copy chromosomal gene and appear to be translated from the same 2.1-kb mRNA. PrP c can be distinguished from PrP s~ by limited proteolysis under conditions where PrP c is hydrolyzed and PrP s~ is resistant. We report here that PrP c can be released from the surface of both normal-control and scrapie-infected murine neuroblastoma (N2a) cells by phosphatidylinositol-specific phospholipase C (PIPLC) digestion and it can be selectively labeled with sulfo-NHS-biotin, a membrane impermeant reagent. In contrast, PrP s~ was neither released by PIPLC nor labeled with sulfo-NHS-biotin. Pulse-chase experiments showed that [35S]methionine was incorporated almost immediately into PrP c while incorporation into PrP s~

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Prion proteins (PrP) were localized in the brains of normal and scrapie-infected hamsters by immunohistochemistry and Western blotting. PrP monoclonal antibodies and monospecific anti-PrP peptide sera, which react with both the cellular (PrPC) and scrapie (PrPSc) isoforms of the prion protein, were used to locate PrP in tissue sections. In normal hamsters, PrPC was located primarily in nerve cell bodies throughout the CNS; whereas, in the terminal stages of scrapie, PrP immunoreactivity was shifted to the neuropil and was absent from most nerve cell bodies. Prion proteins were not uniformly dispersed throughout the gray matter of scrapie-infected hamster brains; rather, they were concentrated in those regions that exhibited spongiform degeneration and reactive astrogliosis. Since earlier studies showed that the level of PrPC remains constant during scrapie infection as measured in whole brain homogenates and no antibodies are presently available that can distinguish PrPC from PrPSc, we analyzed individual brain regions by Western blotting. Analysis of proteinase K-digested homogenates of dissected brain regions showed that most of the regional changes in PrP immunoreactivity that are seen during scrapie infection are due to the accumulation of PrPSc. These observations indicate that the tissue pathology of scrapie can be directly correlated with the accumulation of PrPSc in the neuropil, and they suggest that the synthesis and distribution of the prion protein has a central role in the pathogenesis of this disorder.

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.

The scrapie agent causes a degenerative nervous system disorder of sheep and goats. Considerable evidence indicates that the scrapie agent contains a protein that is necessary for infectivity [Prusiner, S. B., Groth, D. F., Cochran, S. P., Masiarz, F. R., McKinley, M. P. & Martinez, H. M. (1980) Biochemistry 19, 4883-4891], but direct demonstration of a protein moiety has been hampered by lack ofsufficiently purified preparations. Employing preparations of the scrapie agent enriched 100-to 1000-fold with respect to protein, we found that digestion by proteinase K destroyed more than 99.9% of the infectivity. Diethylpyrocarbonate, which chemically modifies amino acid residues in proteins with high efficiency, also inactivated the scrapie agent in these purified preparations. Reductions of infectivity by proteinase K and diethylpyrocarbonate were not observed with less purified preparations. The agent bound to phenyl-Sepharose could not be eluted with 8.5 M ethylene glycol; however, a combination of ethylene glycol and detergents did release the agent. These observations provide good evidence for a protein and for hydrophobic domains within the scrapie agent. Whether the protein required for infectivity is the same protein responsible for the hydrophobic properties of the scrapie agent remains to be established.The scrapie agent causes a degenerative nervous system disorder in sheep and goats and is considered a prototype for two similar disorders of humans: kuru and Creutzfeldt-Jakob disease (1). Many months or even years pass from the time of exposure or inoculation until onset of neurological dysfunction; hence the term "slow virus" disease. Because the unusual properties of the scrapie agent readily distinguish it from conventional viruses, we prefer to use the term "unusual slow viruslike agent". Studies on the structure of unusual slow virus-like agents are of great interest because they may provide new approaches to the investigation of many common degenerative diseases of unknown etiology.In this communication we show that the scrapie agent possesses a protein that is required for infectivity. The titer of the scrapie agent in a purified fraction was reduced by proteolytic digestion and by chemical modification. Hydrophobic chromatography provides further evidence for the hydrophobicity of the agent (2).Our findings that the scrapie agent contains a protein, to our knowledge, represent the most convincing identification so far of a macromolecule within the agent. The requirement. for a protein to maintain infectivity clearly separates the scrapie agent from infectious "naked" nucleic acids such as plant viroids (3). To date we have been unable to identify a nucleic acid within the agent. These results raise the possibility that the genome coding for the scrapie agent protein may not reside within the infectious particle itself. MATERIALS AND METHODSChemicals and Reagents. Crystalline proteinase K was purchased from Merck; micrococcal nuclease, phenylmethylsulfonyl fluoride (PhMeSO2F), and diethyl...

Considerable evidence suggests that the scrapie prion protein (PrP) is a component of the infectious particle. We studied the biogenesis and transmembrane orientation of an integral-membrane form of PrP in a cell-free transcription-linked translation-coupled translocation system programmed with a full-length PrP cDNA cloned behind the SP6 promoter. Translation of SP6 transcripts of the cDNA or of native mRNA from either normal or infected hamster brain in the absence of dog pancreas membranes resulted in the synthesis of a single PrP immunoreactive polypeptide (each polypeptide was the same size; Mr, 28,000), as predicted from the known sequence of the coding region. In the cotranslational presence of membranes, two additional forms were observed. Using peptide antisera specific to sequences from the amino-or the carboxy-tertninal domain of PrP together with proteinase K or endoglycosidase H digestion or both, we showed that one of these forms included an integrated and glycosylated form of PrP (Mr = 33,000) which spans the bilayer twice, with domains of both the amino and carboxy termini in the extracytoplasmic space. By these criteria, the other form appeared to be an unglycosylated intermediate of similar transmembrane orientation. The PrP cell-free translation products did not display resistance to proteinase K digestion in the presence of nondenaturing detergents. These results suggest that the PrP cell-free translation products most closely resemble the normal cellular isoform of the protein, since its homolog from infected brain was proteinase K resistant. The implications of these findings for PrP structure and function are discussed.