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• W2024164061 abstract "In transmissible spongiform encephalopathies, the cellular prion protein (PrPC) undergoes a conformational change from a prevailing α-helical structure to a β-sheet-rich, protease-resistant isoform, termed PrPSc. PrPC has two characteristics: a high affinity for Cu2+ and a strong pH-dependent conformation. Lines of evidence indicate that PrPSc conformation is dependent on copper and that acidic conditions facilitate the conversion of PrPC → PrPSc. In each species, PrPSc exists in multiple conformations, which are associated with different prion strains. In sporadic Creutzfeldt-Jakob disease (sCJD), different biochemical types of PrPSc have been identified according to the size of the protease-resistant fragments, patterns of glycosylation, and the metal-ion occupancy. Based on the site of cleavage produced by proteinase K, we investigated the conformational stability of PrPSc under acidic, neutral, and basic conditions in 42 sCJD subjects. Our study shows that only one type of sCJD PrPSc, associated with the classical form, shows a pH-dependent conformation, whereas two other biochemical PrPSc types, detected in distinct sCJD phenotypes, are unaffected by pH variations. This novel approach demonstrates the presence of three types of PrPSc in sCJD. In transmissible spongiform encephalopathies, the cellular prion protein (PrPC) undergoes a conformational change from a prevailing α-helical structure to a β-sheet-rich, protease-resistant isoform, termed PrPSc. PrPC has two characteristics: a high affinity for Cu2+ and a strong pH-dependent conformation. Lines of evidence indicate that PrPSc conformation is dependent on copper and that acidic conditions facilitate the conversion of PrPC → PrPSc. In each species, PrPSc exists in multiple conformations, which are associated with different prion strains. In sporadic Creutzfeldt-Jakob disease (sCJD), different biochemical types of PrPSc have been identified according to the size of the protease-resistant fragments, patterns of glycosylation, and the metal-ion occupancy. Based on the site of cleavage produced by proteinase K, we investigated the conformational stability of PrPSc under acidic, neutral, and basic conditions in 42 sCJD subjects. Our study shows that only one type of sCJD PrPSc, associated with the classical form, shows a pH-dependent conformation, whereas two other biochemical PrPSc types, detected in distinct sCJD phenotypes, are unaffected by pH variations. This novel approach demonstrates the presence of three types of PrPSc in sCJD. proteinase K sporadic Creutzfeldt-Jakob disease N-glycosidase F polyacrylamide gel electrophoresis full-length truncated The mammalian prion diseases are a group of fatal neurodegenerative disorders that exhibit sporadic, inherited or infectious presentation (1Prusiner S.B. Science. 1991; 252: 1515-1522Crossref PubMed Scopus (1737) Google Scholar). These disorders are characterized by the accumulation in affected brains of a misfolded pathogenic isoform of host-encoded cellular prion protein (PrPC), named PrPSc, which is insoluble in nondenaturing detergents and partially resistant to proteolysis (2Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar). PrPSc and PrPC differ in their secondary and tertiary structures, but not in the amino acid sequence (3Stahl N. Baldwin M.A. Teplow D.B. Hood L. Gibson B.W. Burlingame A.L. Prusiner S.B. Biochemistry. 1993; 32: 1991-2002Crossref PubMed Scopus (533) Google Scholar), as an effect of increased β-sheet content and decreased α-helices in the pathological protein (4Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (742) Google Scholar,5Pan K.-M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2061) Google Scholar).PrPC is a glycosylphosphatidylinositol-anchored glycoprotein (6Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (894) Google Scholar) with two potential N-linked glycosylation sites, encoded by a single gene located on human chromosome 20 (7Kretzschmar H.A. Stowring L.E. Westaway D. Stubblebine W.H. Prusiner S.B. DeArmond S.J. DNA (N. Y.). 1986; 5: 315-324Crossref PubMed Scopus (294) Google Scholar). In its mature form the human protein spans residues 23–231 of the polypeptide predicted from cDNA sequence and exists under diglycosylated, monoglycosylated, and unglycosylated species. PrPC is composed of an N-terminal unstructured domain, which contains four copies of an octapeptide sequence (PHGGGWGQ) between residues 60 and 91, and a C-terminal region with two short β-sheet structures of four residues each and three α-helical regions (5Pan K.-M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2061) Google Scholar). Previous studies have identified the octarepeat sequence as a Cu2+ binding motif and have assessed that, in the presence of copper, each octapeptide forms an α-helix and acts as an α-helical formation promoter on the region comprising residues 84–103 (8Miura T. Hori-i A. Takeuchi H. FEBS Lett. 1996; 396: 248-252Crossref PubMed Scopus (141) Google Scholar). Copper binding stoichiometry is pH-dependent: four octapeptides chelate four Cu2+ at pH 7.4, but bind only two Cu2+ at pH 6. Under acidic conditions, Cu2+ is bound by two histidine residues of different octapeptides, whereas the His-triglycine segment of a single octapeptide represents the binding site at pH 7.4 (9Miura T. Hori-i A. Mototani H. Takeuchi H. Biochemistry. 1999; 38: 11560-11569Crossref PubMed Scopus (247) Google Scholar). The effect of pH on PrPC conformation is not, however, limited to its influence on Cu2+. At low pH, recombinant forms of human90–231 (10Swietnicki W. Petersen R. Gambetti P. Surewicz W.K. J. Biol. Chem. 1997; 272: 27517-27520Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and murine121–231 (11Hornemann S. Glockshuber R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6010-6014Crossref PubMed Scopus (240) Google Scholar) PrPC undergo conformational changes, with an increase in β-sheet content and loss of α-helical structures.Within a single mammalian species, PrPSc exists under multiple conformations that are associated with distinct disease phenotypes. Upon experimental transmission, prion strains are distinguished according to differences in incubation time, regional distribution of cerebral lesions, and the pattern of proteinase K (PK)1-induced proteolytic cleavage of PrPSc (12Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar). Biochemical evidence for the notion that PK cleaves the N-terminal region of PrPSc at different sites in different strains has been obtained so far both in animal (13Bessen R.A. Marsh R.F. J. Virol. 1994; 68: 7859-7868Crossref PubMed Google Scholar) and human (14Collinge J. Sidle K.C.L. Ironside J. Hill A.F. Nature. 1996; 383: 685-690Crossref PubMed Scopus (1581) Google Scholar, 15Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A.F. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (719) Google Scholar, 16Telling G.C. Parchi P. DeArmond S.J. Cortelli P. Montagna P. Gabizon R. Mastrianni J. Lugaresi E. Gambetti P. Prusiner S.B. Science. 1996; 274: 2079-2082Crossref PubMed Scopus (746) Google Scholar) prion diseases. In addition, based on the occupancy of the N-glycosylation sites or the ratio of the three PK-resistant PrPSc glycoforms (diglycosylated, monoglycosylated, and un-glycosylated), further biochemical types of PrPSc have been classified (14Collinge J. Sidle K.C.L. Ironside J. Hill A.F. Nature. 1996; 383: 685-690Crossref PubMed Scopus (1581) Google Scholar). Decoding the biochemical signature of individual prion strains may allow for a more precise molecular classification and may reveal environmental risk factors into human disorders with unknown etiology, such as sporadic Creutzfeldt-Jakob disease (sCJD).In earlier large scale studies, conflicting results have been obtained on the types of PrPSc in sCJD. Parchi et al.(15Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A.F. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (719) Google Scholar, 17Parchi P. Giese A. Capellari S. Brown P. Schultz-Schaeffer W. Windl O. Zerr I. Budka H. Kopp N. Piccardo P. Poser S. Rojiani A. Streichemberger N. Julien J. Vital C. Ghetti B. Gambetti P. Kretzschmar H. Ann. Neurol. 1999; 46: 224-233Crossref PubMed Scopus (1186) Google Scholar) have proposed a molecular classification of sCJD based on the analysis of PrP codon 129, encoding either methionine (M) or valine (V), and the physicochemical properties of two types of the PK-resistant PrPSc, as resolved after an SDS 13% polyacrylamide gels electrophoresis (SDS-PAGE) separation: type 1, with a core fragment (the PK-resistant PrPSc polypeptide backbone after removal of sugar chains) of 21 kDa, and type 2, migrating 2 kDa faster. On the contrary, Wadsworth et al.(18Wadsworth J.D.F. Hill A. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (277) Google Scholar) were able to characterize three PrPSc types after separation on 16% Tris-glycine gels, with type 1 found in subjects of the MM genotype, type 2 in all genotypes, and type 3 only in MV or VV genotypes. These three types of PrPSc are further distinguished based on their metal-ion occupancy. Type 1 and type 2 PrPSc upon metal-ion chelation undergo conformational changes that expose an apparently common PK cleavage site, whereas type 3 is unaffected by metal chelators (18Wadsworth J.D.F. Hill A. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (277) Google Scholar). Given the well known influence of pH on PrPC conformation and the lack of reports addressing the influence of pH on the conformational stability of PrPSc, we undertook a biochemical study on sCJD PrPSc in an attempt to assess pH-dependent strain-specific conformations.RESULTSWe first used conventional Western blot methods, with lysis buffer at pH 7.4, to analyze protease-resistant PrPSc in all cases. Based upon the banding pattern of the three PK-resistant PrPSc species and the apparent molecular mass of the core fragment, two types of PrPSc were observed. In 24 subjects (MM and VV genotypes), PK-cleavage products of the three PrPSc glycoforms migrated at ∼30, 27, and 21 kDa (Fig.1, lane 1), all of them shifting to a 21-kDa zone upon deglycosylation (Fig. 1, lane 3), whereas in 18 subjects (MM, MV, VV genotypes) PK-resistant PrPSc species were resolved at ∼28, 26, and 19 kDa (Fig.1, lane 2), with enzymatic deglycosylation yielding a band at ∼19 kDa (Fig. 1, lane 4). When we investigated the physicochemical properties of these two types of PrPSc at pH values other than 7.4, three patterns could be distinguished, based on the size of PK cleavage products.In 15 subjects (MM and VV) with the 21-kDa PrPSc, hereafter designated as group 1, the cleavage products of the three PrPSc bands, corresponding to the di-, mono-, and unglycosylated PK-resistant PrPSc, as obtained at pH 4 and 6, had a molecular mass value ∼1.5 kDa higher than digestion fragments generated at pH 7.4; conversely, when the samples were solubilized in lysis buffer at pH 8, the three PK-resistant PrPSc glycoforms showed a faster migration, corresponding to an additional loss of ∼1 kDa as compared with species generated at pH 7.4 (Fig. 2, A andB). The possibility of a direct pH influence on the electrophoretic mobility of PrPSc was ruled by exposing PK-treated and untreated samples to low and high pH before electrophoretic separation. Subjects of group 1 had a distinct clinical course with early dementia, myoclonus, and periodic sharp-wave electroencephalographic activity, overlapping the typical myoclonic sCJD variant, or cortical visual disturbances, consistent with the Heidenhain variant. These two sCJD variants represent clinical phenotypes of the classical form (17Parchi P. Giese A. Capellari S. Brown P. Schultz-Schaeffer W. Windl O. Zerr I. Budka H. Kopp N. Piccardo P. Poser S. Rojiani A. Streichemberger N. Julien J. Vital C. Ghetti B. Gambetti P. Kretzschmar H. Ann. Neurol. 1999; 46: 224-233Crossref PubMed Scopus (1186) Google Scholar).Figure 2Western immunoblots with 3F4 antibody of PrPSc followingPKtreatment in the region of pH 4–8.The frontal cortex (A, C, E) and cerebellar cortex (B, D, F) from representative sCJD subjects of group 1 (A, B), group 2 (C, D), and group 3 (E,F) are shown. On the top, pH values of lysis buffer are indicated; molecular masses are shown in kilodaltons on both sides. The size of PK-resistant PrPSc products is affected by acidic and basic pH only in group 1 subjects, but not in groups 2 and 3. G–I show that the molecular mass of the PK-resistant deglycosylated PrPSc is sensitive to pH changes only in group 1; these results suggest that the conformation of both glycosylated and unglycosylated PrPSc in group 1 is pH-dependent.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In marked contrast, in 9 subjects, MM genotypes and termed group 2, with the PK-resistant fragment of 21 kDa, the migration of digestion products was insensitive to variations in pH (Fig. 2, C andD). In these cases the three glycoforms migrated to zones of ∼30, 27, and 21 kDa either at low or high pH. At variance with group 1, subjects of this group had an ataxic-dementing sCJD variant of longer duration. Our findings suggest that the physicochemical properties of PrPSc in group 1 and group 2, although apparently similar when examined at pH 7.4, may be further distinguished upon conformational changes induced by acidic and basic pH values.In sCJD subjects (MM, MV, and VV genotypes, group 3) with the 19-kDa core fragment, treatment at different pH values did not affect the molecular masses of PrPSc species generated by PK digestion (Fig. 2, E and F).The results obtained in each single subject were highly reproducible in at least three repetitions for a given sample and were detected in all investigated brain regions.To confirm that changes in migration similarly affected glycosylated and unglycosylated PrPSc species, we examined PK digestion products after removal of sugar chains. As expected, changes in pH affected only the migration of the PrPSc core fragment of group 1 subjects (Fig. 2G), whereas the size of the PrPSc core fragment of group 2 (Fig. 2H) and 3 (Fig. 2I) subjects was unchanged.To gain insight into whether naturally occurring species of the PrPSc types detected in the three groups differed in their polypeptide backbone, detergent-insoluble PrPSc-enriched fractions were prepared from the frontal cortex of each case and analyzed by immunoblots. In group 1 subjects, the insoluble PrPSc had an intact N-terminal region, as suggested by comparable signal intensities with 3F4 and 5B2 antibodies (Fig.3, lanes 1 and 2). Conversely, the remaining cases of groups 2 and 3 were all characterized by prevailing amounts of truncated PrPScfragments, as suggested by a more intense signal with 3F4 (Fig. 3,lanes 4 and 6) as compared with anti-N antibody (Fig. 3, lanes 3 and 5), reflecting the presence of additional bands which lack the N-terminal region. These results suggest that the PrPSc is mostly present under full-length isoforms in group 1, whereas it is mostly recovered under N-terminally truncated fragments in groups 2 and 3. Accordingly, we designated the three types of PrPSc as type-1full-length (FL), type-2truncated (T)and type-3T.Figure 3Detergent insolubility of PrPScinsCJDbrains. PrPSc was sedimented by high speed centrifugation and immunoblotted, after SDS-PAGE, with 5B2 (lanes 1, 3, and 5) and 3F4 (lanes 2,4, and 6) antibodies. In sCJD brains from group 2 (lane 4) and group 3 (lane 6), but not from group 1 (lane 2), there are significant amounts of truncated PrPSc fragments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In summary, the PrPSc bands of group 1, named type-1FL, naturally occur as full-length species, change in conformation over the pH range 4–8, and migrate to a 21-kDa zone after PK treatment at pH 7.4 and enzymatic deglycosylation. Conversely, type-2T of group 2 and type-3T detected in group 3 mostly exist as N-terminally truncated fragments, show no response to pH, and generate PK-resistant PrPSc core fragments of 21 and 19 kDa, respectively.Based on physicochemical properties of our three PrPSctypes under the same experimental conditions of Parchi et al. (15Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A.F. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (719) Google Scholar, 17Parchi P. Giese A. Capellari S. Brown P. Schultz-Schaeffer W. Windl O. Zerr I. Budka H. Kopp N. Piccardo P. Poser S. Rojiani A. Streichemberger N. Julien J. Vital C. Ghetti B. Gambetti P. Kretzschmar H. Ann. Neurol. 1999; 46: 224-233Crossref PubMed Scopus (1186) Google Scholar) (see all lanes at pH 7.4 of Fig. 2, A–I), it is evident that both type-1FL and type-2Tcorrespond to their type 1, whereas the type-3T matches their type 2.Regarding the classification of Wadsworth et al. (18Wadsworth J.D.F. Hill A. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (277) Google Scholar) no appropriate biochemical relation can be defined due to different experimental conditions.However, according to the electrophoretic migration of PK-resistant PrPSc types, disease phenotype and codon 129 genotype, it may be argued that our type-1FL corresponds to their type-1, our type2T to part of their type 2, and our type-3T to their type 3.DISCUSSIONThe results of the present study show that the conformation of the PrPSc associated with the classical form of sCJD, encompassing the myoclonic and Heidenhain variant, is dependent on pH and clearly distinguishable from the pathological isoforms detected in two other sCJD phenotypes. Interestingly, this distinctive physicochemical property clearly differentiates two PrPSctypes, namely type-1FL and type-2T, found in classical sCJD and in the ataxic-dementing variant, respectively, which, upon conventional Western blot analyses, share the migration of the three glycoforms and the size of the PrPSc core fragment. Although having in common the codon 129 genotype, subjects with type-2T PrPSc show different clinical presentation and disease duration significantly longer than patients with type-1FL PrPSc (data not shown), thus supporting the biochemical detection of two PrPScstrains.Recent sequencing studies have shown that the major PK cleavage site of the 21-kDa isoform associated with sCJD occurs at Gly-82 (22Parchi P. Zou W. Wang W. Brown P. Capellari S. Ghetti B. Kopp N. Schultz-Schaeffer W.J. Kretzschmar H.A. Head M.W. Ironside J.W. Gambetti P. Chen S.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10168-10172Crossref PubMed Scopus (266) Google Scholar), although different secondary cleavages are observed depending on the genotype. Accordingly, in MM and MV genotypes a secondary cleavage is observed at Gly-78, whereas in VV subjects additional N-terminal species, starting at Gly-86, are detected (22Parchi P. Zou W. Wang W. Brown P. Capellari S. Ghetti B. Kopp N. Schultz-Schaeffer W.J. Kretzschmar H.A. Head M.W. Ironside J.W. Gambetti P. Chen S.G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 10168-10172Crossref PubMed Scopus (266) Google Scholar). Interestingly, all these cleavage sites occur in the region of the third and fourth octapeptide, a region that under normal and pathological conditions may bind Cu2+. On the contrary, N-terminal species of the 19-kDa isoform start at Gly-97, in a Cu2+-free post-octarepeat domain.Two physicochemical properties of the PrPSc associated with classical sCJD are: the acquisition of its conformation in the presence of copper (18Wadsworth J.D.F. Hill A. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (277) Google Scholar) and a pH-dependent conformational stability (present study). Upon metal-ion chelation (in a lysis buffer at pH 7.4 and with Cu2+-chelators titrated to pH 8) (18Wadsworth J.D.F. Hill A. Joiner S. Jackson G.S. Clarke A.R. Collinge J. Nat. Cell Biol. 1999; 1: 55-59Crossref PubMed Scopus (277) Google Scholar), PK digestion products have molecular masses lower than PK-resistant PrPSc species obtained in the absence of metal chelators, thus suggesting that removal of Cu2+ induces a loss of β-sheet conformation in the octapeptide region. Our results, showing an increase in molecular masses of PK-resistant species at low pH and a decrease at pH 8, speak in favor of an increase in β-sheet conformation under acidic conditions and a loss of β-sheets under basic conditions. Therefore, the expected loss of two Cu2+ions per PrPSc molecule at low pH is not in keeping with the present findings, unless, as demonstrated at pH 6 for the normal octapeptide motif (9Miura T. Hori-i A. Mototani H. Takeuchi H. Biochemistry. 1999; 38: 11560-11569Crossref PubMed Scopus (247) Google Scholar), residual Cu2+ ions are shared between two histidine residues of different octapeptide chains. However, the presumed effect of pH on Cu2+ binding does not fully explain results obtained at pH 4 and 8. Although it remains to be determined whether either the stoichiometry or the type of Cu2+binding are maintained after the conversion of PrPC into PrPSc, our result may suggest a direct effect of pH on PrPSc conformation.Why Is PrPSc conformation pH-sensitive only in one type of the sCJD-associated proteins?Although caution is dictated, our present results point to differences in the polypeptide backbone of the naturally occurring PrPSc, which contains the N terminus in group 1, whereas is mostly N-terminally truncated in the other two groups. This suggests that the effect of pH on the different types of PrPSc is strictly dependent on the presence of the N-terminal portion of the protein. Based on the present observations, it can also be suggested that, at variance with the N-terminal region, the conformation of the C-terminal portion of PrPSc is apparently insensitive to changes in pH. This property further differentiates PrPScfrom PrPC (10Swietnicki W. Petersen R. Gambetti P. Surewicz W.K. J. Biol. Chem. 1997; 272: 27517-27520Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar).As an alternative explanation, both Cu2+ binding at the N-terminal region could be simultaneously responsible for changes observed in type-1FL PrPSc; it is of interest to note that the pattern of progressive fragmentation in going from pH 4 to pH 8 (see G) follows the titration curve of the imidazole ring in His. Assuming a pK = 6.5, the latter would be fully protonated at pH 4 and extensively protonated (70%) at pH 6.0, but would only be 11% protonated at 7.4 and would bear only 5% positive charge at pH 8.0. The extent of His protonation, together with possible differences in the polypeptide backbone, might be responsible for the conformational transitions occurring in the type-1FL PrPSc reported here.Taken together, our data show evidence of three PrPSc types in our unselected consecutive series of sCJD patients and provide a novel reproducible method to differentiate PrPSc types in prion diseases. The mammalian prion diseases are a group of fatal neurodegenerative disorders that exhibit sporadic, inherited or infectious presentation (1Prusiner S.B. Science. 1991; 252: 1515-1522Crossref PubMed Scopus (1737) Google Scholar). These disorders are characterized by the accumulation in affected brains of a misfolded pathogenic isoform of host-encoded cellular prion protein (PrPC), named PrPSc, which is insoluble in nondenaturing detergents and partially resistant to proteolysis (2Prusiner S.B. Science. 1997; 278: 245-251Crossref PubMed Scopus (852) Google Scholar). PrPSc and PrPC differ in their secondary and tertiary structures, but not in the amino acid sequence (3Stahl N. Baldwin M.A. Teplow D.B. Hood L. Gibson B.W. Burlingame A.L. Prusiner S.B. Biochemistry. 1993; 32: 1991-2002Crossref PubMed Scopus (533) Google Scholar), as an effect of increased β-sheet content and decreased α-helices in the pathological protein (4Caughey B.W. Dong A. Bhat K.S. Ernst D. Hayes S.F. Caughey W.S. Biochemistry. 1991; 30: 7672-7680Crossref PubMed Scopus (742) Google Scholar,5Pan K.-M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2061) Google Scholar). PrPC is a glycosylphosphatidylinositol-anchored glycoprotein (6Stahl N. Borchelt D.R. Hsiao K. Prusiner S.B. Cell. 1987; 51: 229-240Abstract Full Text PDF PubMed Scopus (894) Google Scholar) with two potential N-linked glycosylation sites, encoded by a single gene located on human chromosome 20 (7Kretzschmar H.A. Stowring L.E. Westaway D. Stubblebine W.H. Prusiner S.B. DeArmond S.J. DNA (N. Y.). 1986; 5: 315-324Crossref PubMed Scopus (294) Google Scholar). In its mature form the human protein spans residues 23–231 of the polypeptide predicted from cDNA sequence and exists under diglycosylated, monoglycosylated, and unglycosylated species. PrPC is composed of an N-terminal unstructured domain, which contains four copies of an octapeptide sequence (PHGGGWGQ) between residues 60 and 91, and a C-terminal region with two short β-sheet structures of four residues each and three α-helical regions (5Pan K.-M. Baldwin M. Nguyen J. Gasset M. Serban A. Groth D. Mehlhorn I. Huang Z. Fletterick R.J. Cohen F.E. Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10962-10966Crossref PubMed Scopus (2061) Google Scholar). Previous studies have identified the octarepeat sequence as a Cu2+ binding motif and have assessed that, in the presence of copper, each octapeptide forms an α-helix and acts as an α-helical formation promoter on the region comprising residues 84–103 (8Miura T. Hori-i A. Takeuchi H. FEBS Lett. 1996; 396: 248-252Crossref PubMed Scopus (141) Google Scholar). Copper binding stoichiometry is pH-dependent: four octapeptides chelate four Cu2+ at pH 7.4, but bind only two Cu2+ at pH 6. Under acidic conditions, Cu2+ is bound by two histidine residues of different octapeptides, whereas the His-triglycine segment of a single octapeptide represents the binding site at pH 7.4 (9Miura T. Hori-i A. Mototani H. Takeuchi H. Biochemistry. 1999; 38: 11560-11569Crossref PubMed Scopus (247) Google Scholar). The effect of pH on PrPC conformation is not, however, limited to its influence on Cu2+. At low pH, recombinant forms of human90–231 (10Swietnicki W. Petersen R. Gambetti P. Surewicz W.K. J. Biol. Chem. 1997; 272: 27517-27520Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and murine121–231 (11Hornemann S. Glockshuber R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6010-6014Crossref PubMed Scopus (240) Google Scholar) PrPC undergo conformational changes, with an increase in β-sheet content and loss of α-helical structures. Within a single mammalian species, PrPSc exists under multiple conformations that are associated with distinct disease phenotypes. Upon experimental transmission, prion strains are distinguished according to differences in incubation time, regional distribution of cerebral lesions, and the pattern of proteinase K (PK)1-induced proteolytic cleavage of PrPSc (12Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (819) Google Scholar). Biochemical evidence for the notion that PK cleaves the N-terminal region of PrPSc at different sites in different strains has been obtained so far both in animal (13Bessen R.A. Marsh R.F. J. Virol. 1994; 68: 7859-7868Crossref PubMed Google Scholar) and human (14Collinge J. Sidle K.C.L. Ironside J. Hill A.F. Nature. 1996; 383: 685-690Crossref PubMed Scopus (1581) Google Scholar, 15Parchi P. Castellani R. Capellari S. Ghetti B. Young K. Chen S.G. Farlow M. Dickson D.W. Sima A.A.F. Trojanowski J.Q. Petersen R.B. Gambetti P. Ann. Neurol. 1996; 39: 767-778Crossref PubMed Scopus (719) Google Scholar, 16Telling G.C. Parchi P. DeArmond S.J. Cortelli P. Montagna P. Gabizon R. Mastrianni J. Lugaresi E. Gambetti P. Prusiner S.B. Science. 1996; 274: 2079-2082Crossref PubMed Scopus (746) Google Scholar) prion diseases. In addition, based on the occupancy of the N-glycosylation sites or the ratio of the three PK-resistant PrPSc glycoforms (diglycosylated, monoglycosylated, and un-glycosylated), further biochemical types of PrPSc have been classified (14Collinge J. Sidle K.C.L. Ironside J. Hill A.F. Nature. 1996; 383: 685-690Crossref PubMed Scopus (1581) Google Scholar). Decoding the biochemical signature of individual prion strains may allow for a more precise molecular classification and may reveal environmental risk factors into human disorders with unknown etiology, such as sporadic Creutzfeldt-Jakob disease (sCJD). In earlier large scale studies, conflicting results ha" @default.
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