Major cleavage sites by PK are indicated by arrows; GLPglycolipid; CHO- complex N-glycosylation chains. rPrPSc from different cortical areas of the same sCJD Type 1 (lanes I-III in the left panel) and Type 2 (lanes I-IV in the right panel) case before and after deglycosylation. Asterisk (*) and double dagger (#) point to the bands of PK and PNGase F, respectively. (b) Western blot analysis of the purified human MM1 and MM2 sCJD prions before and after deglycosylation. The lower panels are from the same WB taken after longer exposure to detect less abundant low mass fragments of rPrPSc. The molecular weights of the marker proteins are in kD.(PDF) ppat.1004832.s002.pdf (2.8M) GUID:?0999EB95-F985-4275-8984-14AA804B9806 Data 4-hydroxyephedrine hydrochloride Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract The infectious pathogen responsible for prion diseases is the misfolded, aggregated form of the prion protein, PrPSc. In contrast to recent progress in studies of laboratory rodent-adapted prions, current understanding of the molecular basis of human prion diseases and, especially, their vast phenotypic diversity is very limited. Here, we have purified proteinase resistant PrPSc aggregates from two major phenotypes of sporadic Creutzfeldt-Jakob disease (sCJD), decided their conformational stability and replication tempo gene, there are two major subtypes of sCJD: MM1 and MM2. These types differ with regard to the progression rate of the disease, pattern of proteinase K (PK)-resistant fragments of infectious prion protein aggregates PrPSc, (Fig 1a), neuropathological characteristics of brain lesions, and transmissibility properties in transgenic mice [4C10]. Open in a separate window Fig 1 Schematic representation of PK-resistant fragments in rPrPSc corresponding to Type 1 (MM1) and Type 2 (MM2) sCJD prions and molecular characteristics of purified human rPrPSc used in structural studies.(a) Outline 4-hydroxyephedrine hydrochloride of classification of Type 1 and Type 2 human prions based on proteolytic fragmentation of PrPSc [5,52]. Major cleavage sites by PK are indicated by arrows; GLPglycolipid; CHO- complex N-glycosylation chains. The codes above light blue brackets represent monoclonal antibodies used PSTPIP1 in differentiation of various domains of human prions, and the numbers below these brackets indicate linear epitopes recognized by these antibodies. (b) Distinct glycosylation patterns and electrophoretic mobilities of purified human Type 1 (MM1) and Type 2 (MM2) sCJD rPrPSc (homozygous for methionine (M) in codon 129) used in structural studies. To differentiate Type 1, Type 2 prions, and their C-terminal fragments, Western blots of purified rPrPSc (fraction 8; F8) from the brain homogenate (BH) of type MM1 and MM2 sCJD were developed with mAb 12B2 (epitope residues 89C93) [53], mAb 3F4 (epitope residues 107C112) [54], and rabbit polyclonal antibody 2301 (epitope residues 220C231) [55]. 4-hydroxyephedrine hydrochloride The lower panels correspond to prolonged exposure of the same WB to detect less abundant low molecular weight fragments of 4-hydroxyephedrine hydrochloride rPrPSc. (c) Distinct fragmentation patterns of purified MM1 and MM2 sCJD prions 4-hydroxyephedrine hydrochloride in silver stained SDS-PAGE before and after deglycosylation with PNGase F. The symbols (*) and (#) indicate bands corresponding to PK and PNGase F, respectively. The molecular weights of marker proteins are in kDa. A substantial progress has been made in recent years in prion research using laboratory rodent-adapted, cloned prion strains. These studies revealed, among others, that phenotypic variability of these model prions is usually directly linked to (and likely encoded in) structural differences of PrPSc, and suggested that prion replication rates are inversely proportional to conformational stability of rodent PrPSc (as defined by the concentration of denaturant needed to dissociate/unfold PrPSc aggregates) [11,12]. By contrast, our understanding of the molecular basis of human prions such as those causing sCJD is far less advanced. These prions are.