Semipreparative Chromatographic Method to Purify the Normal Cellular Isoform of the Prion Protein in Nondenatured Form

✍ Scribed by Paola Pergami; Howard Jaffe; Jiri Safar

• Publisher: Elsevier Science
• Year: 1996
• Tongue: English
• Weight: 248 KB
• Volume: 236
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1006/abio.1996.0132

✦ Synopsis

Recent developments in the genetics and molecular A fundamental step in the pathogenesis of spongibiology of spongiform encephalopathies (prion disform encephalopathies (prion diseases) is the convereases) confirm the key role of prion protein (PrP Sc ) 2 in sion of the cellular isoform of prion protein (PrP C ) into susceptibility, disease transmission, incubation time, the infectious form (scrapie isoform, PrP Sc ), apparand pathogenesis. PrP Sc and its normal cellular isoform ently by a conformational mechanism. Comparison of PrP C are encoded by the same gene (1), and have the the native secondary and tertiary structures of both same molecular weight (2). In spite of these similariproteins is essential to elucidate the molecular basis ties, PrP Sc and PrP C have distinctly different physicoof this transformation. To obtain sufficient quantities chemical behaviors: PrP Sc copurifies with infectivity; it of native-like PrP C , we have developed a semipreparais proteinase K-resistant; and it forms scrapie amyloid tive method to purify PrP C from hamster brains. PrP C (PrP27-30), whereas PrP C does not (3, 4). was solubilized from purified synaptosomal and micro-Since direct biochemical studies of PrP Sc have failed somal membranes by the nonionic detergent n-octylto reveal a difference in posttranslational modifications b-glucopyranoside; the soluble fraction was loaded at that could distinguish it from PrP C (5), it is possible pH 7.5 onto a semipreparative cation-exchange TSKthat the ability to replicate and form amyloid results SP-5PW (HPLC) column. The fractions eluted by linear from a conformational transition of the PrP molecule. NaCl gradient and enriched for PrP C were sequen-Such a process may be triggered by a point mutation tially purified using an immobilized ion-affinity HPLC in the PrP molecule or by an hypothetical ligand (6). column charged by Co 2/ , followed by wheat germ ag-To understand the molecular basis of this transition, glutinin (WGA)-affinity HPLC or size-exclusion HPLC it is essential to compare the native conformations of (SE-HPLC) using a TSK G3000SW column. More than PrP Sc and PrP C . 95% purity was achieved after SE-HPLC as estimated A recent work (7) using circular dichroism (CD) meaby quantitative densitometry of the silver-stained surement showed that PrP C has high a-helix and low SDS-PAGE gel; the recovery of total brain PrP C was b-sheets content, while PrP Sc (8) has more than a 40% §8%. The purified PrP C was a monomer with an intact b-sheet content, suggesting that the conversion of a-N-terminus, and with a Stoke's radius of 26 A ˚, corresponding to that expected from the molecular weight helices into b-sheets underlies formation of the profor a native protein. The presence of the native-like tein's infectious form. We have recently demonstrated conformation was further verified by peptide mapping that scrapie amyloid (prion) protein is an aggregated after limited trypsin proteolysis, and by the apparent unfolding in guanidine hydrochloride, as detected by 2 Abbreviations used: BSA, bovine serum albumin; Gdn HCl, gua-SE-HPLC.