Methionine 129 Variant of Human Prion Protein Oligomerizes More Rapidly than the Valine 129 Variant
2004; Elsevier BV; Volume: 279; Issue: 30 Linguagem: Inglês
10.1074/jbc.m401754200
ISSN1083-351X
AutoresAbdessamad Tahiri‐Alaoui, Andrew C. Gill, Petra Disterer, William James,
Tópico(s)Neurological diseases and metabolism
ResumoThe human PrP gene (PRNP) has two common alleles that encode either methionine or valine at codon 129. This polymorphism modulates disease susceptibility and phenotype of human transmissible spongiform encyphalopathies, but the molecular mechanism by which these effects are mediated remains unclear. Here, we compared the misfolding pathway that leads to the formation of β-sheet-rich oligomeric isoforms of the methionine 129 variant of PrP to that of the valine 129 variant. We provide evidence for differences in the folding behavior between the two variants at the early stages of oligomer formation. We show that Met129 has a higher propensity to form β-sheet-rich oligomers, whereas Val129 has a higher tendency to fold into α-helical-rich monomers. An equimolar mixture of both variants displayed an intermidate folding behavior. We show that the oligomers of both variants are initially a mixture of α- and β-rich conformers that evolve with time to an increasingly homogeneous β-rich form. This maturation process, which involves no further change in proteinase K resistance, occurs more rapidly in the Met129 form than the Val129 form. Although the involvement of such β-rich oligomers in prion pathogenesis is speculative, the misfolding behavior could, in part, explain the higher susceptibility of individuals that are methionine homozygote to both sporadic and variant Creutzfeldt-Jakob disease. The human PrP gene (PRNP) has two common alleles that encode either methionine or valine at codon 129. This polymorphism modulates disease susceptibility and phenotype of human transmissible spongiform encyphalopathies, but the molecular mechanism by which these effects are mediated remains unclear. Here, we compared the misfolding pathway that leads to the formation of β-sheet-rich oligomeric isoforms of the methionine 129 variant of PrP to that of the valine 129 variant. We provide evidence for differences in the folding behavior between the two variants at the early stages of oligomer formation. We show that Met129 has a higher propensity to form β-sheet-rich oligomers, whereas Val129 has a higher tendency to fold into α-helical-rich monomers. An equimolar mixture of both variants displayed an intermidate folding behavior. We show that the oligomers of both variants are initially a mixture of α- and β-rich conformers that evolve with time to an increasingly homogeneous β-rich form. This maturation process, which involves no further change in proteinase K resistance, occurs more rapidly in the Met129 form than the Val129 form. Although the involvement of such β-rich oligomers in prion pathogenesis is speculative, the misfolding behavior could, in part, explain the higher susceptibility of individuals that are methionine homozygote to both sporadic and variant Creutzfeldt-Jakob disease. The transmissible spongiform encephalopathies are a group of fatal, neurodegenerative disorders that affect humans and animals and are believed to be caused by a novel class of infectious pathogen, the prion (1Prusiner S.B. Trends Biochem. Sci. 1996; 21: 482-487Google Scholar, 2Weissmann C. FEBS Lett. 1996; 389: 3-11Google Scholar). These diseases have attracted considerable interest not only because of their unique biology but also because of the appearance of new variant of Creutzfeldt-Jakob disease (vCJD) 1The abbreviations used are: vCJD, variant of CJD; CJD, Creutzfeldt-Jakob disease; HPLC, high performance liquid chromatography; SEC, size exclusion chromatography; RP, reversed-phase; PK, proteinase K. 1The abbreviations used are: vCJD, variant of CJD; CJD, Creutzfeldt-Jakob disease; HPLC, high performance liquid chromatography; SEC, size exclusion chromatography; RP, reversed-phase; PK, proteinase K. (3Will R.G. Ironside J.W. Zeidler M. Cousens S.N. Estibeiro K. Alperovitch A. Poser S. Pocchiari M. Hofman A. Smith P.G. Lancet. 1996; 347: 921-925Google Scholar), which appears to be caused by dietary exposure to the causative agent of bovine spongiform encephalopathy (4Bruce M.E. Will R.G. Ironside J.W. McConnell I. Drummond D. Suttie A. McCardle L. Chree A. Hope J. Birkett C. Cousens S. Fraser H. Bostock C.J. Nature. 1997; 389: 498-501Google Scholar, 5Hill A.F. Desbruslais M. Joiner S. Sidle K.C. Gowland I. Collinge J. Doey L.J. Lantos P. Nature. 1997; 389 (and 526): 448-450Google Scholar). At the heart of disease pathogenesis lies a poorly understood structural rearrangement of PrP, a host-encoded glycoprotein of the nervous and lymphoid systems. The normal cellular form of the prion protein (PrPC) undergoes a conversion that leads to the accumulation of an abnormal, conformationally altered isoform (PrPSc). According to the "protein-only" hypothesis of prion propagation, PrPSc is the principal or sole component of transmissible prions (6Prusiner S.B. Science. 1991; 252: 1515-1522Google Scholar). PrPSc differs from PrPC by increased β-sheet content, increased resistance to proteinase K, and an oligomeric rather than a monomeric state (7Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Google Scholar). The human PrP gene (PRNP) exists in two major allelic forms that encode either methionine or valine at codon 129, with allele frequencies of 0.63 and 0.37 in western European and American populations, respectively (8Collinge J. Palmer M.S. Dryden A.J. Lancet. 1991; 337: 1441-1442Google Scholar, 9Windl O. Dempster M. Estibeiro J.P. Lathe R. de Silva R. Esmonde T. Will R. Springbett A. Campbell T.A. Sidle K.C. Palmer M.S. Collinge J. Hum. Genet. 1996; 98: 259-264Google Scholar). This polymorphism is a key determinant of susceptibility to sporadic (10Palmer M.S. Dryden A.J. Hughes J.T. Collinge J. Nature. 1991; 352: 340-342Google Scholar) and acquired (8Collinge J. Palmer M.S. Dryden A.J. Lancet. 1991; 337: 1441-1442Google Scholar, 11Brown P. Cervenakova L. Goldfarb L.G. McCombie W.R. Rubenstein R. Will R.G. Pocchiari M. Martinez-Lage J.F. Scalici C. Masullo C. Neurology. 1994; 44: 291-293Google Scholar) prion diseases and may affect age at onset (12Baker H.E. Poulter M. Crow T.J. Frith C.D. Lofthouse R. Ridley R.M. Lancet. 1991; 337: 1286Google Scholar, 13Cervenakova L. Goldfarb L.G. Garruto R. Lee H.S. Gajdusek D.C. Brown P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13239-13241Google Scholar, 14Parchi P. Giese A. Capellari S. Brown P. Schulz-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: 224-233Google Scholar). Based on the analysis of 300 sporadic CJD subjects, Parchi et al. (14Parchi P. Giese A. Capellari S. Brown P. Schulz-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: 224-233Google Scholar) identified six distinct clinicopathological variants of sCJD, which appeared to be specified largely by the genotype at codon 129 and the physiochemical properties of PrPSc. More recently, an extensive analysis, which included the metal ion-dependent conformation of PrPSc, of a large number of sCJD cases showed that PrPSc types are associated with the residue encoded at codon 129, the duration of illness, and with neuropathological phenotype (15Hill A.F. Joiner S. Wadsworth J.D. Sidle K.C. Bell J.E. Budka H. Ironside J.W. Collinge J. Brain. 2003; 126: 1333-1346Google Scholar). PrPSc types 1 and 4 have so far been detected only in Met129 homozygotes, type 4 being uniquely associated with vCJD, type 3 in cases containing a Val129 allele, and type 2 in any PRNP codon 129 genotype (16Collinge J. Sidle K.C. Meads J. Ironside J. Hill A.F. Nature. 1996; 383: 685-690Google Scholar). The codon 129 polymorphism seemed to modulate the pattern of neuropathology and the extent of lesions in sCJD, as shown in a study that involved 70 patients who died in France between 1994 and 1998 (17Hauw J.J. Sazdovitch V. Laplanche J.L. Peoc'h K. Kopp N. Kemeny J. Privat N. Delasnerie-Laupretre N. Brandel J.P. Deslys J.P. Dormont D. Alperovitch A. Neurology. 2000; 54: 1641-1646Google Scholar). Codon 129 polymorphism also has epistatic effects on the phenotypic effects of mutations elsewhere in the prion gene. For example, the Asp178 → Gln mutation combined with a methionine at position 129 results in fatal familial insomnia (18Goldfarb L.G. Petersen R.B. Tabaton M. Brown P. LeBlanc A.C. Montagna P. Cortelli P. Julien J. Vital C. Pendelbury W.W. Haltia M. Wills P.R. Hauw J.J. McKeever P.E. Monari L. Schrank B. Swergold G.D. Autilio-Gambetti L. Gajdusek D.C. Lugaresi E. Gambetti P. Science. 1992; 258: 806-808Google Scholar). In contrast, the same mutation with a valine encoded at position 129 results in familial CJD (19Goldfarb L.G. Haltia M. Brown P. Nieto A. Kovanen J. McCombie W.R. Trapp S. Gajdusek D.C. Lancet. 1991; 337: 425Google Scholar). Similarly, Val129 homozygotes in association with the Phe198 → Ser mutation predispose patients to the Indiana kindred variant of Gerstmann-Straussler-Scheinker disease (20Dlouhy S.R. Hsiao K. Farlow M.R. Foroud T. Conneally P.M. Johnson P. Prusiner S.B. Hodes M.E. Ghetti B. Nat. Genet. 1992; 1: 64-67Google Scholar). Furthermore, an increased prevalence of genotype Val/Val at the polymorphic site 129 has been described in patients with early onset Alzheimer's disease (21Dermaut B. Croes E.A. Rademakers R. Van den Broeck M. Cruts M. Hofman A. van Duijn C.M. Van Broeckhoven C. Ann. Neurol. 2003; 53: 409-412Google Scholar) and a shift in cognitive decline toward early age in carriers of PRNP129 Val/Val has been reported recently (22Croes E.A. Dermaut B. Houwing-Duistermaat J.J. Van den Broeck M. Cruts M. Breteler M.M. Hofman A. van Broeckhoven C. van Duijn C.M. Ann. Neurol. 2003; 54: 275-276Google Scholar). Despite the clear importance of the polymorphism at codon 129 in the PRNP gene and its link to disease susceptibility and pathogenesis, the molecular mechanisms by which these effects are mediated remain unclear. The in vitro, thermodynamic stability of recombinant PrP is not affected by the Met129 → Val mutation or by other substitutions related to inherited human prion diseases (23Liemann S. Glockshuber R. Biochemistry. 1999; 38: 3258-3267Google Scholar). Structural studies, however, have shown evidence for hydrogen bonding between residues 128 and 178, which might provide a structural basis for the highly specific influence of polymorphism in position 129 on disease phenotype that segregates with Asp178 → Gln (24Riek R. Wider G. Billeter M. Hornemann S. Glockshuber R. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11667-11672Google Scholar). Molecular dynamic simulations of low pH-induced conformational conversion of PrP has provided further clues as to the role of residue 129 (25Alonso D.O. DeArmond S.J. Cohen F.E. Daggett V. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2985-2989Google Scholar). In these simulations, Met129 seems to interact with Val122 leading to the recruitment of more N-proximal residues into an expanding β-sheet. As a complementary approach, we have chosen to study the folding properties of the two PrP allelomorphs experimentally by examining the misfolding pathway that leads to the formation of β-sheet-rich oligomeric isoforms (26Baskakov I.V. Legname G. Baldwin M.A. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; 277: 21140-21148Google Scholar). We show that PrP-Met129 has a higher propensity to form β-sheet-rich oligomers, whereas PrP-Val129 has a higher tendency to fold into α-helical-rich monomers. We also provide evidence that the dynamics of maturation of the oligomers differ between the two variants. The maturation process occurs over time at the expense of the α-helical-like monomers at a faster rate for PrP-Met129 than for PrP-Val129. Once the oligomers from either allelomorph have been formed they show similar proteinase K resistance that does not change throughout the oligomer maturation process. The observed differences in the misfolding of PrP-Met129 and PrP-Val129 could explain the high susceptibility of individuals that are methionine homozygote to sporadic as well as variant CJD. Cloning of PrP Genes and Protein Purification—Genomic DNA encoding methionine/valine at codon 129 of PRNP gene (18Goldfarb L.G. Petersen R.B. Tabaton M. Brown P. LeBlanc A.C. Montagna P. Cortelli P. Julien J. Vital C. Pendelbury W.W. Haltia M. Wills P.R. Hauw J.J. McKeever P.E. Monari L. Schrank B. Swergold G.D. Autilio-Gambetti L. Gajdusek D.C. Lugaresi E. Gambetti P. Science. 1992; 258: 806-808Google Scholar) was extracted from the blood of a heterozygote individual using standard phenolchloroform methods. The fragment of the PRNP gene spanning codon 90 through 231 was amplified with the oligonucleotide primers 5′-cgggatcccatgcaaggaggtggcacccacagtcagtggaacaagccg-3 (and 5′-cccaagcttcatgctcgatcctctctggtaataggcctgag-3′ in 100 μl of PCR buffer that contained 300 ng of each DNA, 2 units of Taq DNA polymerase, 200 μm concentration of each dNTP, 0.2 μm concentration each primer, 3.5 μm MgCl2, 10 mm Tris-HCl, pH 9.0, 0.1% Triton X-100, 50 mm KCl. The following PCR conditions were used: an initial denaturation (95 °C, 3 min) followed by 25 cycles of denaturation (95 °C, 1 min), annealing (55 °C, 1 min), extension (72 °C, 1 min), and a final elongation (72 °C, 8 min). Restriction sites BamHI and HindIII, including extra nucleotides for efficient cleavage close to the ends, were introduced in the primers. The PCR fragment was cloned into the pTrcHis2B vector that incorporated a C-terminal His tag (Invitrogen, Paisley, UK) according to the manufacturer's instructions. The identity of human PrP clones was confirmed by sequencing with the BigDye Terminator v3.0 on ABI-Prism 3100 Genetic Analyzer (Applied Biosystems). The clones corresponding to PrP90–231 with Met129 or Val129 were identified by comparison with human PrP clones available in the databases (GenBank™ accession numbers: M13667 and P04156). Escherchia coli expression and purification of recombinant human PrP was performed as described previously (27Jackson G.S. Hill A.F. Joseph C. Hosszu L. Power A. Waltho J.P. Clarke A.R. Collinge J. Biochim. Biophys. Acta. 1999; 1431: 1-13Google Scholar) except that the buffer used to solubilize inclusion bodies did not contain dithiothreitol. Stocks of highly purified proteins were stored in 6 m guanidine hydrochloride, 50 mm Tris-HCl, pH 7.2. Oligomer Formation by HPLC Gel Filtration and Dialysis Methods— Rapid refolding of proteins into oligomeric isoforms was carried out at concentrations of 10 and 30 mg/ml. PrP-Met129 and PrP-Val129 were denatured in 6 m guanidine hydrochloride, 50 mm Tris-HCl, pH 7.2, and were injected (100 μl) onto a size exclusion chromatography (SEC) column (TSK®-Gel SWXL G3000 HPLC column, 7.8 × 300 mm, Phenomenex, Macclesfield, UK), equilibrated in 20 mm sodium acetate, 0.2 m NaCl, pH 3.7, 1 m urea, and 0.02% sodium azide (26Baskakov I.V. Legname G. Baldwin M.A. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; 277: 21140-21148Google Scholar). This rapid oligomer formation on SEC-HPLC corresponded to time 0 as opposed to time course analysis of oligomer formation during dialysis. This time course analysis of oligomer formation was carried out as follows. 1 ml of 6 m guanidine hydrochloride-denatured proteins (10 or 30 mg/ml) was dialyzed at room temperature against 2 liters of 20 mm sodium acetate, 0.2 m NaCl, pH 3.7, 2 m urea by use of a Slide-A-Lyser dialysis cassette (Perbio Science UK Ltd., Tattenhall, UK) with a 10-kDa cutoff. Aliquots were withdrawn, after carefully shaking the cassette to ensure homogeneity, after 30 min and 2, 4, and 24 h and analyzed by SEC as described above. Protein peaks were manually collected for subsequent analysis. Circular dichroism was carried as described previously (28Baskakov I.V. Legname G. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2001; 276: 19687-19690Google Scholar) to assess the secondary structure of the protein. Analytical Reversed-phase HPLC—Protein fractions collected during SEC analysis were kept in SEC elution buffer at room temperature and analyzed after 2, 12, 30, 110, 140, 200, and 300 days by analytical reversed-phase (RP) HPLC (Sephasil® C4, 5 μm, 4.6 × 250 mm) (Amersham Biosciences, Little Chalfont, UK). Proteins were eluted with a linear gradient of H2O + 0.1% trifluoroacetic acid to 95% acetonitrile + 0.09% trifluoroacetic acid over 25 min. All HPLC separations were performed at room temperature with a flow rate of 1 ml/min by means of a PerkinElmer Life Sciences HPLC system composed of a Binary LC pump 250 and a diode array detector 235C controlled by Total Chrome software version 6.2 (PerkinElmer Life Sciences, Seer Green, UK) through a PE Nelson 600 series link. The eluent was monitored by UV absorption at 280 nm. Proteinase K Digestion—Recombinant human PrP collected from SEC-HPLC was subjected to proteinase K digestion after 2, 12, and 140 days maturation of the oligomers. The solution was incubated at 37 °C with a ratio of 1:24 PK to protein in 20 mm sodium acetate, 0.2 m NaCl, pH 5.5. Aliquots of the digests were taken after 15, 30 and 60 min and snap-frozen for analysis by SDS-PAGE and mass spectrometry. On-line Capillary HPLC Nanospray Mass Spectrometry—Mass spectrometry was carried out on a Quattro II tandem quadrupole mass spectrometer (Micromass UK Ltd., Altrincham, UK) equipped with on-line capillary HPLC as detailed in Ref. 29Gill A.C. Ritchie M.A. Hunt L.G. Steane S.E. Davies K.G. Bocking S.P. Rhie A.G. Bennett A.D. Hope J. EMBO J. 2000; 19: 5324-5331Google Scholar. Briefly, the capillary HPLC was 180 μm inner diameter and was packed with 3.5-μm Jupiter C18 resin (Phenomenex, Macclesfield, UK). A flow rate of 1 μl/min was used, and proteins were eluted with a gradient from 0–70% solvent A to B, where solvent A was 95:5 water:acetonitrile with 0.05% trifluoroacetic acid, and solvent B was 95:5 water:acetonitrile with 0.05% trifluoroacetic acid. The eluent was passed directly to the mass spectrometer, which was operated in continuous flow nanospray mode. Full scan mass spectra (m/z 300–2100) were acquired every 5 s. Characterization of Recombinant Human PrP-Met129 and PrP-Val129—Recombinant human prion protein variants, HuPrP90–231 Met129 and HuPrP90–231 Val129, were purified to homogeneity by immobilized metal affinity chromatography (see supplementary Fig. 1) followed by RP-HPLC. Purified proteins were analyzed by mass spectrometry to confirm purity and identity. Omitting dithiothreitol from the buffer used to solubilize the inclusion bodies and allowing disulfide bond to form in the oxidizing environment of the immobilized metal affinity chromatography was found to yield a fully oxidized protein (see supplementary Fig. 1) that has characteristics of PrPC, such as a monomeric state, high α-helical content, and proteinase K sensitivity. All the studies presented in this paper were performed on proteins that included a C-terminal His tag. PrP-Met129 Has an Intrinsically Higher Propensity to Oligomerize than PrP-Val129—To assess the effect of the codon 129 polymorphic residue on the formation of non-native isoforms, both protein variants were denatured in 6 m guanidine hydrochloride, 50 mm Tris-HCl, pH 7.2, and allowed to fold under conditions favoring the formation of β-oligomer species (26Baskakov I.V. Legname G. Baldwin M.A. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; 277: 21140-21148Google Scholar). To dissect the in vitro folding pathway of prion protein misfolding we used SEC, dialysis, and CD. The denatured proteins (30 mg/ml) were injected onto a SEC column that had previously been equilibrated in 20 mm sodium acetate, 0.2 m NaCl, pH 3.7, 1 m urea, and 0.02% sodium azide and eluted with the same buffer. The elution profile of the Met129 variant shows two major peaks and one minor peak (Fig. 1A). Peak I (terminology as given by Baskakov et al. (26Baskakov I.V. Legname G. Baldwin M.A. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; 277: 21140-21148Google Scholar)) eluted at 5.68 min and corresponded to high molecular weight aggregates (Fig. 1A). Peak II eluted at 6.54 min and corresponded to the oligomeric isoform, while peak III, which eluted at 9.08 min, had a similar retention time to monomeric protein (Fig. 1A). The elution profile of Val129 variant shows the same three peaks but in different proportions, with peak II eluting as a shoulder to peak I (Fig. 1B). To mimic a heterozygote situation of an individual that carries both alleles, we have analyzed the folding behavior in a 1:1 mixture of Met129 and Val129 (Fig. 1C). The elution profile of the equimolar mixture showed three peaks as seen before (Fig. 1C). Integration of the peaks in Fig. 1, A and B, revealed that less than 2% of the Val129 variant had formed oligomers under these rapid refolding conditions (within the first 10 min of the SEC elution) as compared with more than 70% in the case of Met129 (Fig. 1D). In addition, the percentage of monomeric population that was formed within 10 min was calculated to be about 66% for the Val129 variant but only 24% for the Met129 variant (Fig. 1D). Integration of the peaks in the equimolar mixture showed that the percentage of oligomeric form was reduced to about 44%, corresponding to ∼30% reduction in the amount of oligomers as compared with the situation of the Met129 alone (Fig. 1D). Also the amount of the monomeric population in the mixture 1:1 increased by about 17% when compared with that of the Met129 alone (Fig. 1D). The monomeric forms of Met129 and Val129 variants yielded CD spectra with two minima at 208 and 222 nm, indicative of a predominantly α-helical conformation (data not shown). The CD spectra of protein from peak II of PrP-Met129 and peaks I and II of PrP-Val129 did not give clear information about the protein conformations but suggested that these fractions were composed of a mixture of α-helical and β-sheet forms. To investigate the formation and evolution of the oligomers that eluted in peak II in more detail, we used a slower dialysis method of refolding that allowed analysis at different times after initiation of the refolding. Two protein concentrations, 30 and 10 mg/ml, were analyzed in parallel. The composition of the dialysis buffer was the same as that used in SEC elution buffer, except that, to maximize the formation of oligomers, the urea concentration was increased to 2 m (28Baskakov I.V. Legname G. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2001; 276: 19687-19690Google Scholar). After 30-min refolding, and at 30 mg/ml, both allelomorphs adopted predominately oligomeric forms (peak II) with virtually no monomeric protein present (peak III); this elution profile did not change over the course of the experiment (Fig. 2A). Similar results were obtained with a mixture (1:1) of Met129 and Val129 (data not shown). The CD spectra of oligomers collected at 30 min and 1 h showed that the population was not dominated by β-sheet-rich structures but contained some α-helical-rich protein (Fig. 2, C and D). However, by 4 h the CD spectra of the protein contained in peak II from both allelomorphs were typical of proteins possessing high amounts of β-sheet, and the spectra remained constant thereafter. In contrast, at 10 mg/ml (Fig. 2B) the kinetics of oligomerization of PrP-Met129 were significantly different from those of the PrP-Val129. The disappearance of aggregated proteins from peak I occurred more rapidly in the Met129 variant than in the Val129 variant, which suggests that, under oligomer-promoting conditions, the aggregated material from PrP-Met129 has a higher propensity to convert into oligomer than aggregated protein from PrP-Val129. To test this, we took aggregated proteins from peak I of PrP-Met129 and PP-Val129 from SEC at time 0 and monitored their evolution over time by re-injecting them onto the same SEC. Time course comparison between the two allelomorphes clearly demonstrated the higher propensity of the Met129 variant to oligomerize than the Val129 variant (see supplementary Fig. 2). Virtually all aggregated PrP-Met129 converted into oligomeric isoforms over a period of 12 days, whereas a substantial amount of PrP-Val129 remained as aggregates. To investigate the behavior of monomeric species in peak III under the same oligomeric-promoting conditions, we compared the rate of oligomerization of the Met129 variant to that of the Val129 variant collected from SEC at time 0. Time course comparison between the two allelomorphs clearly revealed that monomeric Met129 has a higher propensity to oligomerize than monomeric Val129. This difference in oligomerization rates was apparent after 2 days of incubation at room temperature (see supplementary Fig. 2). From these data we conclude that, under these conditions, the Met129 variant has an intrinsically higher propensity to form oligomer isoforms than the Val129. The high oligomerization propensity of the methionine variant becomes even more apparent under the highest protein concentration and rapid refolding conditions. Since this is true for denatured, aggregated, or natively folded monomeric protein, the higher propensity of oligomerization is clearly independent of the starting state of the protein. The Oligomers of Both Met129 and Val129 Variants Are Conformationally Heterogeneous and Show Different Kinetics of Maturation—It has been shown that the oligomeric isoforms of mouse and Syrian hamster PrP are formed of populations of structurally heterogeneous proteins (26Baskakov I.V. Legname G. Baldwin M.A. Prusiner S.B. Cohen F.E. J. Biol. Chem. 2002; 277: 21140-21148Google Scholar). RP-HPLC analysis showed that reduced forms of recombinant mouse PrP existed in multiple β-sheet-rich isoforms with distinct retention times (30Lu B.Y. Beck P.J. Chang J.Y. Eur. J. Biochem. 2001; 268: 3767-3773Google Scholar, 31Lu B.Y. Chang J.Y. Biochemistry. 2001; 40: 13390-13396Google Scholar). Accordingly, we used RP-HPLC to investigate the effect of the residue encoded at codon 129 on the composition of the oligomer population that eluted from SEC in peak II. Aliquots of this fraction were incubated at room temperature in SEC elution buffer (20 mm sodium acetate, 0.2 m NaCl, pH 3.7, 1 m urea, and 0.02% sodium azide) and analyzed at various times (Fig. 3). After 2 days of incubation, the RP chromatogram of PrP-Met129 showed two peaks with distinct retention times (Fig. 3A); however, for PrP-Val129 only one major peak was observed (Fig. 3B). A second peak could be seen only after 12 days of incubation. The two peaks were designated IIa and IIb, with shorter and longer retention times, respectively. To rule out the possibility that the two peaks represented unexpected modification to the proteins, we analyzed them by mass spectrometry (Fig. 4). The deconvoluted mass spectra of PrP-Met129 IIa and PrP-Met129 IIb demonstrate that they represent proteins of the same molecular mass (Fig. 4, A and B). Similarly, the deconvoluted mass spectra of PrP-Val129 IIa and PrP-Val129 IIb showed that they also represented proteins with similar molecular masses (Fig. 4, C and D). From these analyses we infer that no covalent differences are evident and that the two peaks observed by RP-HPLC represent different conformations, with different amounts of hydrophobic residues exposed at the protein surface.Fig. 4Nanospray mass spectrometry analysis of peaks IIa and IIb after 12 days of oligomerization. Peaks IIa and IIb that were resolved by reversed-phase HPLC during the oligomerization of Met129 and Val129 were manually collected and immediately subjected to mass spectrometry analysis. A and B are electrospray mass spectra and deconvoluted (see insets) mass spectra of peaks IIa and IIb, respectively, of the Met129 oligomer. C and D are electrospray mass spectra and deconvoluted (see insets) mass spectra of peaks IIa and IIb, respectively, of Val129 oligomer.View Large Image Figure ViewerDownload (PPT) Two important observations can be clearly made from reversed-phase chromatograms. First, the proportions of the two forms of PrP-Met129 and PrP-Val129 were different; the proportion of protein eluting in peak IIa was higher for PrP-Met129 after 12 days of incubation (Fig. 3, A versus B), while for PrP-Val129 there was more protein that eluted in peak IIb. Second, the proportions of the two peaks changed over time at different rates for Met129 and Val129 variants. In particular, the proportion of protein eluting in peak IIb decreased at a higher rate in the oligomer of PrP-Met129 (Fig. 3A) than in the oligomer of PrP-Val129 (Fig. 3B). Peak IIb of the oligomer from both allelomorphs seemed to have a retention time similar to that of the corresponding α-helical monomers. Because RP-HPLC uses differences in the hydrophobic properties to achieve separation between bio-molecules, it can be concluded that the protein population present in the highly retained peak IIb has more surface-exposed hydrophobic residues than the protein population present in peak IIa. Furthermore, the protein population present in peak IIb appears to undergo conversion to become recruited into the population that eluted in peak IIa. Over time, this conversion seemed to occur much quicker with PrP-Met129 than with PrP-Val129 (Fig. 3, A versus B). Because of the long storage time of both allelomorphs in SEC elution buffer at room temperature, we have used mass spectrometry to assess the integrity of the samples (see supplementary Fig. 3). After 2 and 12 days of oligomerization, both allelomorphs had molecular masses that matched those predicted from the amino acid sequences. However, after 30 days of oligomerization, the deconvoluted mass spectra of both variants showed an additional species with a molecular mass that corresponded to the cleavage of the N-terminal two residues from the prot
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