Oxidation of Helix-3 methionines precedes the formation of PK resistant PrP.
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.
Canello
Tamar
T
Department of Neurology, The Agnes Ginges Center for Human Neurogenetics, Hadassah University Hospital, and Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem, Israel.
Frid
Kati
K
Gabizon
Ronen
R
Lisa
Silvia
S
Friedler
Assaf
A
Moskovitz
Jackob
J
Gasset
María
M
Gabizon
Ruth
R
D000906:Antibodies; D000937:Antigen-Antibody Reactions; D001923:Brain Chemistry; D000939:Epitopes; D008715:Methionine / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000276:immunology; D018621:PrPC Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D018620:PrPSc Proteins / Q000096:biosynthesis* / Q000737:chemistry; D011494:Protein Kinases / Q000378:metabolism; D055550:Protein Stability; D017433:Protein Structure, Secondary
D000906:Antibodies; D000939:Epitopes; D010446:Peptide Fragments; D018621:PrPC Proteins; D018620:PrPSc Proteins; D008715:Methionine; D011494:Protein Kinases