Genetic influence on the structural variations of the abnormal prion protein

Abstract

Prion diseases are characterized by the presence of the abnormal prion protein PrP(Sc), which is believed to be generated by the conversion of the alpha-helical structure that predominates in the normal PrP isoform into a beta-sheet structure resistant to proteinase K (PK). In human prion diseases, two major types of PrP(Sc), type 1 and 2, can be distinguished based on the difference in electrophoretic migration of the PK-resistant core fragment. In this study, protein sequencing was used to identify the PK cleavage sites of PrP(Sc) in 36 cases of prion diseases. We demonstrated two primary cleavage sites at residue 82 and residue 97 for type 1 and type 2 PrP(Sc), respectively, and numerous secondary cleavages distributed along the region spanning residues 74-102. Accordingly, we identify three regions in PrP(Sc): one N-terminal (residues 23-73) that is invariably PK-sensitive, one C-terminal (residues 103-231) that is invariably PK-resistant, and a third variable region (residues 74-102) where the site of the PK cleavage, likely reflecting the extent of the beta-sheet structure, varies mostly as a function of the PrP genotype at codon 129.

Figure 1

Presence of type 1 and type 2 PrP^Sc in human prion diseases. Brain homogenate was treated with PK, followed by incubation in the absence (−) or presence (+) of PNGase F. PK-resistant PrP^Sc was detected on immunoblots by using 3F4 mAb. (Top) Sporadic cases with codon 129 genotypes on both alleles. (Middle) Familial cases with a point mutation at indicated codons or an insert mutation, coupled with the codon 129 on the mutant allele. (Bottom) Acquired cases including those of iatrogenic CJD (iCJD), kuru, and the new variant CJD (nvCJD). Their codon 129 on both alleles is also indicated. Molecular size markers are shown in kilodaltons on the left.

Figure 2

Analysis of purified type 1 and type 2 PrP^Sc. (A) Silver staining of SDS/PAGE. (B) Coomassie blue staining of the sequencing membrane used for automated Edman degradation. Samples were either untreated (−) or treated (+) with PNGase F. Molecular size markers are shown in kilodaltons on the left.

Figure 3

Diagram of the structural variations of human PrP^Sc related to the PK cleavage patterns. Three distinct regions are represented along the full-length uncleaved PrP^Sc (residues 23–231): (i) a region that is PK-sensitive (wavy line); (ii) the “variable” region (broken line), which carries the multiple PK cleavage sites [residues 74–102; the primary (underlined) and secondary cleavages are indicated]; and (iii) a region that is invariably PK-resistant (residues 103–231, zigzagged line). The PK cleavage site, reflecting the size of the PK-resistant region and thus the conformation of PrP^Sc, is a function of PrP^Sc type and codon 129 genotype (indicated on the top and the right side of each line).