Mutant PrPSc conformers induced by a synthetic peptide and several prion strains

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrP^Sc(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4°C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrP^Sc(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a β-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrP^Sc(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrP^Sc in the brains of Tg196 mice. Our results argue that MoPrP^Sc(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrP^C(P101L) as well as in Tg196 mice expressing low levels of MoPrP^C(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a β-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.The discovery that brain fractions enriched for prion infectivity contain a protein (rPrP^Sc) that is resistant to limited proteolytic digestion advanced prion research (8, 37). N-terminal truncation of rPrP^Sc produced a protease-resistant fragment, denoted PrP 27-30, that is readily measured by Western blotting, enzyme-linked immunosorbent assay, or immunohistochemistry. The measurement of PrP^Sc was dramatically changed with the development of the conformation-dependent immunoassay (CDI), which permitted detection of full-length rPrP^Sc as well as previously unrecognized protease-sensitive forms of PrP^Sc (39).The CDI depends on using anti-PrP antibodies that react with an epitope exposed in native PrP^C but that do not bind to native PrP^Sc. Upon denaturation, the buried epitope in PrP^Sc becomes exposed and readily reacts with anti-PrP antibodies. Using the CDI, we discovered that most PrP^Sc is protease sensitive, which we designate sPrP^Sc. Whether sPrP^Sc is an intermediate in the formation of rPrP^Sc remains to be determined. In Syrian hamsters inoculated with eight different strains of prions, the ratio of rPrP^Sc to sPrP^Sc was different for each strain and the concentration of sPrP^Sc was proportional to the length of the incubation time (39).In earlier studies, transgenic (Tg) mice, denoted Tg2866, expressing high levels of PrP(P101L) were used to model Gerstmann-Sträussler-Scheinker (GSS) disease caused by the P102L point mutation. In the brains of several lines of mice expressing high levels of PrP(P101L), no rPrP^Sc(P101L) was detectable (26, 27, 47). This was particularly perplexing since these Tg mice expressing high levels of PrP(P101L) developed all facets of prion-induced neurodegeneration, including multicentric PrP amyloid plaques. Moreover, brain extracts from ill Tg2866 mice transmitted disease to Tg196 mice expressing low levels of PrP(P101L) that infrequently developed spontaneous neurodegeneration (29).In humans with GSS, several different mutations of the PrP gene (PRNP) resulting in nonconservative amino acid substitutions have been identified (23). In these patients, the clinical presentation, disease course, and amounts of rPrP^Sc in the brain are variable. Brain extracts from humans who died of GSS were inoculated into apes and monkeys, but the transmission rates were not correlated with the levels of PrP^Sc in the inoculum (1, 2, 9, 32). In a limited study, GSS(P102L) was transmitted to Tg mice expressing a chimeric mouse-human (MHu2 M) PrP transgene carrying the P102L mutation but not to Tg mice expressing MHu2M PrP without the mutation (47). In another study, GSS(P102L) human prions were transmitted to Tg mice expressing MoPrP(P101L) in which the transgene was incorporated through gene replacement (31). The use of gene replacement permits all of the regulatory elements that control the wild-type (wt) MoPrP gene to modulate the expression of MoPrP(P101L). In these mice, the expression level of MoPrP(P101L) in brain is likely to be similar to that in Tg196 mice.When we synthesized a 55-mer MoPrP peptide composed of residues 89 to 143 containing the P101L mutation and folded it under conditions favoring a β-structure, it induced neurodegeneration in Tg196 mice (29). When the peptide was not folded into a β-structure, it did not produce disease in Tg196 mice. We report here that the peptide-initiated disease in Tg196 mice could be serially transmitted to other Tg196 mice using brain extracts from the peptide-inoculated Tg196 mice. Using procedures derived from the CDI, brain extracts from inoculated Tg196 mice were found to contain sPrP^Sc(P101L), from which a 22- to 24-kDa PrP fragment was generated by limited digestion with proteinase K (PK) at 4°C and selective precipitation with phosphotungstate (PTA) (25, 39). In the interest of clarity, we have designated digestion at 4°C as “cold PK” and simply refer to standard digestion at 37°C as “PK.” To aid in distinguishing rPrP^Sc(P101L) from sPrP^Sc(P101L), their properties based on the work reported here and in other previously published papers are listed in Table ?Table11 (39, 40).

TABLE 1.

Characteristics of PrP(P101L) isoforms

Characteristic	Isoforma
	PrPc(P101L)	sPrPSc(P101L)	rPrPSc(P101L)
PrP epitopes (residues 90-125) in native state	Exposed	Buried	Buried
Precipitatable by PTA	?	+	+
Digestion with PK at 37°C (“PK”)	Dipeptides, tripeptides	Dipeptides, tripeptides	PrP 27-30
Digestion with PK at 4°C (“cold PK”)	Dipeptides, tripeptides	PrP 22-24	PrP 27-30
Infectious	?	?	+

Open in a separate window^a?, unknown; +, positive; ?, negative.In addition to inoculating Tg196 mice with brain extracts containing sPrP^Sc(P101L) or with the MoPrP(89-143,P101L) peptide, we inoculated Tg196 with several strains of prions carrying wt MoPrP^Sc-A or MoPrP^Sc-B. The 301V strain carrying wt MoPrP^Sc-B (22) exhibited similar abbreviated incubation times in both Tg196 mice and Prnp^b/b mice. In contrast, the RML and 139A strains carrying wt MoPrP^Sc-A showed prolonged incubation times in both Tg196 and Prnp^b/b mice (12, 33). Regardless of the host mouse strain, the 301V, RML, and 139A prion strains produced large amounts of rPrP^Sc in the brains of inoculated mice. Thus, the discovery of sPrP^Sc has for the first time provided a molecular signature for GSS prions that either arise spontaneously in mice or are induced by a synthetic peptide carrying the GSS mutation.