Oxidation of Helix-3 methionines precedes the formation of PK resistant PrP

Abstract

While elucidating the peculiar epitope of the alpha-PrP mAb IPC2, we found that PrPSc exhibits the sulfoxidation of residue M213 as a covalent signature. Subsequent computational analysis predicted that the presence of sulfoxide groups at both Met residues 206 and 213 destabilize the alpha-fold, suggesting oxidation may facilitate the conversion of PrPC into PrPSc. To further study the effect of oxidation on prion formation, we generated pAbs to linear PrP peptides encompassing the Helix-3 region, as opposed to the non-linear complexed epitope of IPC2. We now show that pAbs, whose epitopes comprise Met residues, readily detected PrPC, but could not recognize most PrPSc bands unless they were vigorously reduced. Next, we showed that the alpha-Met pAbs did not recognize newly formed PrPSc, as is the case for the PK resistant PrP present in lines of prion infected cells. In addition, these reagents did not detect intermediate forms such as PK sensitive and partially aggregated PrPs present in infected brains. Finally, we show that PrP molecules harboring the pathogenic mutation E200K, which is linked to the most common form of familial CJD, may be spontaneously oxidized. We conclude that the oxidation of methionine residues in Helix-3 represents an early and important event in the conversion of PrPC to PrPSc. We believe that further investigation into the mechanism and role of PrP oxidation will be central in finally elucidating the mechanism by which a normal cell protein converts into a pathogenic entity that causes fatal brain degeneration.

Figure 1. Helix-3 sequences.

(A) Helix-3 sequences in different PrP species. The 206–214 region, comprising M206 and M213, is completely conserved in all species. (B) PrP peptides used for the generation of α Helix-3 antibodies. (C) PrP peptides used for the inhibition of α Helix 3 antibodies.

Figure 2. Testing for the activity of anti-Helix-3 antibodies: pAb RVC does not recognize PrP^Sc generated in prion infected brains.

(A) Activity of pAb RVC and RTC, as compared to the established mAb 6H4, against normal brain homogenates from bovine, mouse, humans and hamster. (B) Human and mouse normal brain homogenates were immunoblotted with pAb RVC and RTC alone or in the presence of diverse Helix-3 PrP peptides (see Figure 1C for the peptide sequences) (C) Brain homogenates from mice, hamster and humans (normal, prion-infected and prion-infected digested with proteinase K), were immunoblotted with mAb 6H4, with pAb RTC or RVC, or with rec Ab R1. D) Mouse scrapie (RML) and human CJD brains (E200K) were digested with PK, processed for MMA reduction as described in the methods and immunoblotted with α PrP mAb 6H4, IPC2, or pAb RVC.

Figure 3. pAb RVC does not recognize PrP^Sc generated in prion infected cells.

(A) Brain (normal, scrapie infected, as well as scrapie infected digested with PK) as well as normal and prion-infected cells, (N2a and GT1 infected either with the RML or the 22L prion strains) were extracted and immunoblotted with mAb IPC1 as compared to pAb RVC. (B): Effect of the MMA chemical reduction of proteinase K digested ScGT1 and Sc N2a cells on the PrP recognition by mAb IPC1, mAb IPC2 and pAb RVC.

Figure 4. Intermediate PrP forms are oxidized as PrP^Sc.

Sarkosyl extracted brain samples from normal and prion infected mice and humans were subjected to sucrose gradient centrifugation. Fractions from these gradients were digested in the presence or absence of proteinase K and immunoblotted with both mAb 6H4 and pAb RVC.

Figure 5. HuPrP E200K is spontaneously oxidized.

(A) Brain samples from scrapie infected mice and from humans suffering from familial E200K or sporadic CJD, were digested in the presence or absence of proteinase K and subsequently immunoblotted with mAb 6H4 or pAb RGM. The last 2 lanes of each gel comprise normal GT1 and proteinase K digested ScGT1 cells expressing a chimera Mo/Ha PrP form. (B) Human and mouse normal brain homogenates were immunoblotted with the RGM antibody alone or preincubated with several PrP peptides in the Helix-3 Met area. (C) Immunoblots of HuPrP(23–230) wt and E200K with 3F4 (recognizing the 109–112 region), DZS18 (recognizing oxidized Met residues in different proteins), IPC2 (recognizing non-oxidized M213) and RGM (recognizing non-oxidized M206). Blots were prepared in the absence of β-mercaptoethanol. (D) Thermal stability of HuPrP(23–230) wt and E200K probed by the relative change in the ellipticity at 220 nm as a function of temperature. Insert: Far-UV CD spectrum of HuPrP(23–230) wt and E200K.

Figure 6. Scheme of Helix 2 and 3 of PrP including epitopes of α Helix 3 antibodies.