Acidic pH and Detergents Enhance in Vitro Conversion of Human Brain PrPC to a PrPSc-like Form (2002)

In the presence of a low concentration of denaturants or detergents, acidic pH triggers a conformational transition of α-helices into β-sheets in recombinant prion protein (PrP), likely mimicking some aspects of the transformation of host-encoded normal cellular PrP (PrPC) into its pathogenic isoform (PrPSc). Here we observed the effects of acidic pH and guanidine hydrochloride (GdnHCl) on the physicochemical and structural properties of PrPC derived from normal human brain and determined the ability of the acid/GdnHCl-treated PrP to form a proteinase K (PK)-resistant species in the absence and presence of PrPSc template. After treatment with 1.5 mGdnHCl at pH 3.5, PrPC from normal brain homogenates was converted into a detergent-insoluble form similar to PrPSc. Unlike PrPSc, however, the treated brain PrPC was protease-sensitive and retained epitope accessibility to monoclonal antibodies 3F4 and 6H4. Brain PrPC treated with acidic pH/GdnHCl acquired partial PK resistance upon further treatment with low concentrations of sodium dodecyl sulfate (SDS). Formation of this PrPSc-like isoform was greatly enhanced by incubation with trace quantities of PrPSc from Creutzfeldt-Jakob disease brain. Acid/GdnHCl-treated brain PrP may constitute a “recruitable intermediate” in PrPSc formation. Further structural rearrangement seems essential for this species to acquire PK resistance, which can be promoted by the presence of a PrPSc template. In the presence of a low concentration of denaturants or detergents, acidic pH triggers a conformational transition of α-helices into β-sheets in recombinant prion protein (PrP), likely mimicking some aspects of the transformation of host-encoded normal cellular PrP (PrPC) into its pathogenic isoform (PrPSc). Here we observed the effects of acidic pH and guanidine hydrochloride (GdnHCl) on the physicochemical and structural properties of PrPC derived from normal human brain and determined the ability of the acid/GdnHCl-treated PrP to form a proteinase K (PK)-resistant species in the absence and presence of PrPSc template. After treatment with 1.5 mGdnHCl at pH 3.5, PrPC from normal brain homogenates was converted into a detergent-insoluble form similar to PrPSc. Unlike PrPSc, however, the treated brain PrPC was protease-sensitive and retained epitope accessibility to monoclonal antibodies 3F4 and 6H4. Brain PrPC treated with acidic pH/GdnHCl acquired partial PK resistance upon further treatment with low concentrations of sodium dodecyl sulfate (SDS). Formation of this PrPSc-like isoform was greatly enhanced by incubation with trace quantities of PrPSc from Creutzfeldt-Jakob disease brain. Acid/GdnHCl-treated brain PrP may constitute a “recruitable intermediate” in PrPSc formation. Further structural rearrangement seems essential for this species to acquire PK resistance, which can be promoted by the presence of a PrPSc template. An insoluble, β-sheet-rich isoform of the prion protein (herein generically designated PrPSc) 1The abbreviations used are: PrPSc, pathogenic prion protein; PrPC, cellular prion protein; PK, proteinase K; rPrP, recombinant prion protein; hurPrP, recombinant human PrP; GdnHCl, guanidine hydrochloride; CJD, Creutzfeldt-Jakob disease; PMCA, protein misfolding cyclic amplification; PBS, phosphate-buffered saline; BSA, bovine serum albumin is the only known component of the infectious prion particle associated with the prion diseases, which are a group of transmissible, fatal neurodegenerative diseases in humans and animals. PrPSc is derived from its normal cellular isoform (PrPC), which is rich in α-helical structure, by a posttranslational process involving a conformational transition (1Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar). While the primary structure of PrPC is identical to that of PrPSc, secondary and tertiary structural changes are responsible for the distinct physicochemical properties of the two isoforms. PrPC exists as a detergent-soluble monomer and is readily degraded by protease K (PK), whereas the infectious isoform PrPSc forms detergent-insoluble aggregates and shows high resistance to PK digestion and to phosphatidylinositol-specific phospholipase C-mediated release (2Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google Scholar). According to the “protein only” theory of prion infectivity, PrPC is converted to PrPSc by a template-directed process catalyzed by PrPSc. This disease-related in vivo transition has been modeled in vitro, in which PrPC can be converted to a protease-resistant form by contact with PrPSc (3Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (792) Google Scholar, 4Saborio G.P. Soto C. Kascsak R.J. Levy E. Kascsak R. Harris D.A. Frangione B. Biochem. Biophys. Res. Commun. 1999; 258: 470-475Crossref PubMed Scopus (61) Google Scholar). Recently, it has been reported that this conversion can be promoted by protein misfolding cyclic amplification (PMCA), a process analogous to the polymerase chain reaction for nucleic acids (5Saborio G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1022) Google Scholar). Studies using recombinant PrP (rPrP) have indicated that the structural transition of α-helix to β-sheet and concomitant self-association can be triggered by acidic pH combined with detergents in vitro (6Swietnicki 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, 7Jackson G.S. Hill A.F. Joseph C. Hosszu L. Power A. Waltho J.P. Clarke A.R. Collinge J. Biochim. Biophys. Acta. 1999; 1431: 1-13Crossref PubMed Scopus (113) Google Scholar, 8Hornemann S. Glockshuber R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6010-6014Crossref PubMed Scopus (240) Google Scholar). We have also demonstrated that acid-induced conformational transition and aggregation may be associated with protonation of the acidic amino acids aspartate (Asp) and glutamate (Glu) using a peptide (195–213) corresponding to the C-terminal region of PrP (9Zou W.Q. Yang D.S. Fraser P.E. Cashman N.R. Chakrabartty A. Eur. J. Biochem. 2001; 268: 4885-4891Crossref PubMed Scopus (7) Google Scholar). These data suggest that PrP in acidic organelles may participate in specific folding pathways, forming molecular species structurally and physicochemically distinct from PrPC. Although studies using rPrP have provided important information about PrP structural transition, three-dimensional structure, thermodynamic stability, and folding pathways, the absence of co-factor molecules and posttranslational modifications, such as N-linked glycans and the C-terminal glycosylphosphatidylinositol anchor, may confound attempts to model faithfully in vivo PrP conversion events. In the present study, we have observed the effect of acidic pH and guanidine hydrochloride (GdnHCl) on the physicochemical and structural properties of cellular PrP derived from normal human brain. Importantly, we find that acid/GdnHCl-treated human brain PrP is a superior substrate for in vitro conversion than untreated PrP. Phenylmethylsulfonyl fluoride and proteinase K were purchased from Sigma. Magnetic beads (Dynabeads M-280 Tosylactivated) were from Dynal Co. (Dynal AS, Oslo, Norway). Mouse monoclonal antibody 6H4 from Prionics Co. (Zürich, Switzerland) recognizes the sequence DYEDRYYRE in the prion protein (human PrP residues 144–152). Mouse monoclonal antibody 3F4 from Signet Laboratories, Inc. (Dedham, MA) recognizes an epitope of human PrP residues 109–112 including residues MKHV. Horseradish peroxidase-conjugated sheep anti-mouse antibody was purchased fromAmersham Biosciences. Necropsied human brain tissue was collected within 24 h of death. The normal human brain was obtained from an individual determined by histology to be free of neurological disorders, and a prion-infected brain was from an individual with Creutzfeldt-Jakob disease (CJD) confirmed by histological examination and Western blot analysis to show the presence of PrPSc. Both samples were homozygous for Met/Met at codon 129. Mouse brains were from wild-type and PrP−/− mice. All tissues were frozen immediately after collection and stored at −80 °C. 10% (w/v) brain homogenates were prepared in lysis buffer (100 mm NaCl, 10 mm EDTA, 0.5% Nonidet P 40 (Nonidet P-40), 0.5% sodium deoxycholate, 10 mm Tris-HCl, pH 7.5). Samples were mixed with an equal volume of 2× electrophoresis loading buffer (6% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, 4 mm EDTA, 20% glycerol, 125 mm Tris-HCl, pH 6.8) and boiled for 10 min. Proteins were separated by 12% SDS-PAGE and electrotransferred onto polyvinylidene difluoride membranes at 125 mA for 2 h. The membranes were blocked with 5% nonfat milk in TBST (10 mm Tris-HCl, pH 7.6, 150 mm NaCl, 0.05% Tween 20) overnight at 4 °C or 1 h at 37 °C prior to incubation with antibodies. Membrane-bound proteins were probed with 6H4 antibody at 1:5,000 or with 3F4 antibody at 1:50,000. Washing with TBST, the blot was incubated with horseradish peroxidase-conjugated sheep anti-mouse antibody at 1:3,000. After washing with TBST, the proteins were visualized by enhanced chemiluminescence + plus (ECL + Plus, Amersham Biosciences). 100 μl of 10% brain homogenate was mixed with an equal volume of 3.0m GdnHCl (final concentration 1.5 m) in PBS at pH 7.4 or pH 3.5 adjusted with 1 n HCl, followed by incubation at room temperature with shaking. After 5 h, samples were mixed with 5 volumes of prechilled methanol and incubated at −20 °C for 2 h to precipitate the proteins. The samples were subjected to centrifugation at 16,000 × g for 20 at 4 °C to the acidic buffer and GdnHCl, and were in 100 μl of lysis buffer or in 100 μl of 0.05% 0.5% in PBS, pH to the The samples treated at pH 7.4 were designated as 100 μl of the treated or in lysis buffer was at × g at 4 °C for 1 h. detergent-soluble were to and detergent-insoluble were in an equal volume of lysis The of detergent-soluble PrP or detergent-insoluble PrP was determined by the of the treated 20 μl of was incubated with PK at 100 for 1 h at 37 and the digestion reaction was by of fluoride to 2 mm The was mixed with equal volumes of loading boiled for 10 and subjected to SDS-PAGE and monoclonal antibodies and at were to in PBS at 37 °C for 20 h and with washing buffer The beads were incubated with BSA, Tris-HCl, pH at 37 °C for 4 h to and with The beads were stored in PBS at 4 °C. of μl of beads was incubated with μl of lysis buffer in the presence of 5 μl of 10% (w/v) brain homogenate acidic or at room temperature for h. The beads were with washing buffer Nonidet Tween PBS, pH After the were and μl of loading buffer was such as and to antibody from the The samples were at °C for 5 and at for min. The were subjected to SDS-PAGE and in vitro conversion of proteins by prechilled methanol were in equal volumes of 0.05% 0.5% in PBS, pH than in lysis This buffer has been used in of PrPSc (5Saborio G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1022) Google Scholar). in vitro was in an volume of the substrate μl of and μl of 10% brain homogenate for the template-directed and the was incubated in a at 37 °C for h with shaking. After PK digestion at 37 °C for 1 h and in loading the samples were subjected to SDS-PAGE and with the of (6Swietnicki 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, 7Jackson G.S. Hill A.F. Joseph C. Hosszu L. Power A. Waltho J.P. Clarke A.R. Collinge J. Biochim. Biophys. Acta. 1999; 1431: 1-13Crossref PubMed Scopus (113) Google Scholar, 8Hornemann S. Glockshuber R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6010-6014Crossref PubMed Scopus (240) Google we have that recombinant an α-helix to β-sheet conformational transition upon treatment with 1.5 GdnHCl in PBS at pH 3.5, as indicated by and R. We the effect of on the physicochemical properties of cellular PrP derived from normal human brain After of acidic buffer and from normal human brain the detergent-soluble and of the samples in lysis buffer were separated by The of PrP in the and in the was determined by a of PrPC in the samples was in the detergent-soluble PrP in the treated brain homogenates was in the detergent-insoluble acidic pH and GdnHCl only a conformational transition of the recombinant can also a conversion from detergent-soluble PrPC into detergent-insoluble PrPSc-like species in PrP from brain treated protein retained the of to pH for at 1 a and further the effect of pH and GdnHCl on the of PrP in detergents, we of two on human brain of the of PrP in the presence of low concentrations of pH than or equal to 3.5, PrP from human brain insoluble, at pH equal to or than PrP was This pH to the a of the of the and that the in of PrP be associated with the protonation of acidic the effect of GdnHCl on the of PrP at low brain homogenates were incubated with concentrations of GdnHCl at pH in of the concentration of GdnHCl was to or of the brain PrP PrP at the concentration of GdnHCl than and equal to a concentration of GdnHCl at which brain proteins were on analysis acidic PrP may a structure at GdnHCl, the physicochemical properties of which may be by brain proteins. protease resistance is a of the PrPSc from structural conversion of the brain PrP treated with this samples were incubated with PK at concentrations at 37 °C for 1 h. in and acidic PrP an PK resistance at low concentrations of PK to or than 1 which is with the of A. W. Biochem. Biophys. Res. Commun. 1998; PubMed Scopus Google however, was in PK the that PrPSc-like the species of PrP is identical to PrPSc. antibodies to of PrP have been used to conformational rearrangement in PrP structural C. R. Cohen F.E. S.B. J. Biol. 1997; PubMed Scopus Google Scholar, R. W. Gambetti P. Surewicz W.K. J. Biol. PubMed Scopus Google Scholar, S.B. R.J. Cohen F.E. J. Biol. 1999; PubMed Scopus Google Scholar, S. A. S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, E. A. S.B. J. 2001; PubMed Scopus Google Scholar, W. S. and A. of Scholar). We used with 3F4 antibody residues or 6H4 antibody residues to in epitope accessibility and acid/GdnHCl-treated brain proteins. In lysis buffer low concentrations of detergents Nonidet and 0.5% sodium 6H4 and 3F4 antibodies PrP from and acid/GdnHCl-treated brain homogenates that two are in the structural changes by the treatment In the of PrP from brain homogenates by the two antibodies was than that from normal brain homogenates 6H4 recognizes only PrPC and PrPSc C. P. B. 1999; PubMed Scopus Google and 3F4 is in PrPSc C. R. Cohen F.E. S.B. J. Biol. 1997; PubMed Scopus Google the of PrP in the brain homogenates be PrPC. These data that residues 109–112 and are in PrPSc molecules C. R. Cohen F.E. S.B. J. Biol. 1997; PubMed Scopus Google Scholar, C. P. B. 1999; PubMed Scopus Google and suggest that the of PrPC may be in the prion brain S. A. S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). acidic human brain PrP the epitope properties of PrPSc. Recently, has been to structural conversion of α-helices to β-sheets and aggregation of rPrP Raymond Raymond G.J. Caughey B. J. 2001; PubMed Scopus Google Scholar, E. S.B. Biol. Chem. 2001; PubMed Scopus Google Scholar). a low concentration of is present in the by G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1022) Google Scholar). acid/GdnHCl-treated protein can further structural rearrangement upon treatment with treated and brain samples were incubated in 0.05% 0.5% in PBS, pH 7.4 at 37 °C for h with shaking. in of a were in the acid/GdnHCl-treated PrP 4 was observed in 4 that may conformational in treated brain PrP. Although has been to aggregation and structural transition of recombinant acquire PK at pH Raymond Raymond G.J. Caughey B. J. 2001; PubMed Scopus Google which is with data with brain the of PrP from the treated PrP was greatly enhanced by incubation with trace quantities of PrPSc from brain homogenate 4 PrP a similar to that by brain conversion of treated human brain PrP in the presence of a PrPSc was confirmed in or PrP was derived from similar with brain PrP in 1 of a are protease-resistant of the PrPSc amplification of PrPSc was observed human was incubated with brain homogenates from treated wild-type or PrP−/− 4 an of human PrPSc in the wild-type brain and in PrP−/− brain or PBS These that the incubation the and of the PrPSc, are with amplification of protease-resistant PrP in the a of with the 6H4 antibody the of conversion of PrP by human species observed in prion protein conversion in vivo (1Prusiner S.B. Scott M.R. DeArmond S.J. Cohen F.E. Cell. 1998; 93: 337-348Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar, S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5168) Google and in vitro (3Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (792) Google Scholar, Raymond Raymond G.J. Caughey B. J. 2001; PubMed Scopus Google Scholar). the of PrPSc in wild-type PrP brain or PBS is of the that vivo of in brain is upon of PrP R. Chesebro B. Nature. 1998; PubMed Scopus Google Scholar, R. A. Raymond G.J. Caughey B. Chesebro B. J. 2001; PubMed Scopus Google Scholar). the conversion reaction only μl of 10% brain homogenate in μl of 10% normal brain and 1 and 2 of 4 a only of brain a with treated brain by According to the of and (5Saborio G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1022) Google of the PrP in human brain was derived from substrate in treated brain with in brain (5Saborio G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1022) Google Scholar). In this study, we have demonstrated that acidic pH and GdnHCl a transition of cellular PrP derived from normal human brain PrP is similar to PrPSc, protease and epitope of acidic human brain PK resistance upon further treatment with a low concentration of quantities of PrPSc greatly conversion of acidic human brain PrP to a PrPSc-like data suggest that conversion of PrPC to PrPSc may two which be in pH and denaturants may the of structural in which treated PrP than PrPC. The is upon further rearrangement that is by a PrPSc which is in vitro by low concentrations of a pH and denaturants can β-sheet conformational and self-association in recombinant PrP from species (6Swietnicki 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, 7Jackson G.S. Hill A.F. Joseph C. Hosszu L. Power A. Waltho J.P. Clarke A.R. Collinge J. Biochim. Biophys. 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Biol. 1997; PubMed Scopus Google reported in this region to were in in PrP The in the 3F4 epitope is with this the accessibility to the 6H4 epitope that the region C-terminal to residues to PrPSc is upon structural changes in structural changes in of the are responsible for the and enhanced of acidic human brain PrP. for structural residues an acidic transition in a C-terminal of S. Glockshuber R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6010-6014Crossref PubMed Scopus (240) Google and the of a for in treated at pH A. Glockshuber R. S. Biophys. J. 2001; Full Text Full Text PDF PubMed Scopus Google of which are of the human brain PrP some protease resistance of PrPSc in the presence of low concentration and The may the of treated rPrP molecules Raymond Raymond G.J. Caughey B. J. 2001; PubMed Scopus Google and and further structural The conversion of human brain PrP was greatly enhanced by the presence trace of PrPSc. These data suggest that acidic PrP an substrate for the PrPC to PrPSc the of PrPSc-like properties by acidic pH/GdnHCl brain PrP is similar to the PrP conversion in prion PrPC is in the and the to the it is to the by a glycosylphosphatidylinositol in or S.B. Cell. Full Text PDF PubMed Scopus Google Scholar, B. Chesebro B. J. PubMed Google Scholar). PrPC is into from which of the molecules are to the D.A. 1999; PubMed Google Scholar, Harris D.A. J. Biol. Chem. 268: Full Text PDF PubMed Google Scholar). of the molecules are followed by of the Harris D.A. J. Biol. Chem. 268: Full Text PDF PubMed Google Scholar, A. Scott A. L. S.B. J. Biol. PubMed Scopus Google Scholar). PrPC is degraded in the Harris D.A. J. Biol. Chem. 268: Full Text PDF PubMed Google Scholar, R.J. C. L. J. J. 1994; PubMed Scopus Google a of acidic that PrP is an enhanced substrate for PrP conversion an acidic for conversion in acidic in of W. PubMed Scopus Google chain M.R. L. W. R. Proc. Natl. Acad. Sci. U. S. 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The data in this after PrP properties distinct from the which are retained the protein is to with the of and of PrPC is the that only PrPC has been to be converted into A. S.B. Biol. Cell. PubMed Scopus (240) Google Scholar, A. S.B. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In prion diseases, it is that PrPC is to PrPSc by a template-directed process that can be in D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (792) Google Scholar, 4Saborio G.P. Soto C. Kascsak R.J. Levy E. Kascsak R. Harris D.A. Frangione B. Biochem. Biophys. Res. Commun. 1999; 258: 470-475Crossref PubMed Scopus (61) Google Scholar, G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1022) Google Scholar). that acidic human brain PrP a superior substrate for this reaction be to PrPSc in tissues and in which the is present in low is also that the conversion of PrP into a form may PrPSc in with two in vitro PrP conversion reported (3Kocisko D.A. Come J.H. Priola S.A. Chesebro B. Raymond G.J. Lansbury P.T. Caughey B. Nature. 1994; 370: 471-474Crossref PubMed Scopus (792) Google Scholar, 4Saborio G.P. Soto C. Kascsak R.J. Levy E. Kascsak R. Harris D.A. Frangione B. Biochem. Biophys. Res. Commun. 1999; 258: 470-475Crossref PubMed Scopus (61) Google in which a or a of PrPSc are for the conversion to (5Saborio G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1022) Google have also reported an in in which trace concentrations of brain PrPSc can be by a process to the brain by (5Saborio G.P. Permanne B. Soto C. Nature. 2001; 411: 810-813Crossref PubMed Scopus (1022) Google to amplification of human PrPSc. data that PrPC is a superior substrate to untreated amplification of human PrPSc and also the for is that acidic human brain PrP may be to the conformational of prion protein conversion in the molecular and posttranslational in and that PrPSc in vitro We and for the human brain and for on the and for We and Gambetti for of brain.