Abstract
Elucidation of structure and biological properties of the prion protein scrapie (PrP(Sc)) is fundamental to an understanding of the mechanism of conformational transition of cellular (PrP(C)) into disease-specific isoforms and the pathogenesis of prion diseases. Unfortunately, the insolubility and heterogeneity of PrP(Sc) have limited these studies. The observation that a construct of 106 amino acids (termed PrP106 or miniprion), derived from mouse PrP and containing two deletions (Delta 23-88, Delta 141-176), becomes protease-resistant when expressed in scrapie-infected neuroblastoma cells and sustains prion replication when expressed in PrP(0/0) mice prompted us to generate a corresponding synthetic peptide (sPrP106) to be used for biochemical and cell culture studies. sPrP106 was obtained successfully with a straightforward procedure, which combines classical stepwise solid phase synthesis with a purification strategy based on transient labeling with a lipophilic chromatographic probe. sPrP106 readily adopted a beta-sheet structure, aggregated into branched filamentous structures without ultrastructural and tinctorial properties of amyloid, exhibited a proteinase K-resistant domain spanning residues 134-217, was highly toxic to primary neuronal cultures, and induced a remarkable increase in membrane microviscosity. These features are central properties of PrP(Sc) and make sPrP106 an excellent tool for investigating the molecular basis of the conformational conversion of PrP(C) into PrP(Sc) and prion disease pathogenesis.
Highlights
** Assistant Telethon Scientist (DTI, Fondazione Telethon). 1 The abbreviations used are: PrP, prion protein; PrP106, PrP-derived construct of 106 amino acids; PrPC, cellular isoform of PrP; PrPSc, scrapie isoform of PrP; dsPrP106, derivatized synthetic PrP106; Recombinant PrP106 (rPrP106), recombinant PrP106; sPrP106, synthetic PrP106; DCM, dichloromethane; Fmoc, N-(9-fluorenyl)methoxycarbonyl; FP, fluorescence polarization; GdnHCl, guanidine hydrochloride; HPLC, high performance liquid chromatography; MALDI MS, matrix-assisted laser desorption/ionization mass spectrometry; PA, phosphatidic acid; PC, phosphatidylcholine; PG, phosphatidylglycerol; PK, proteinase K; termed PrP scrapie (PrPSc), in the central nervous system
Purification, and Characterization of PrP106 — PrP106 was synthesized by Fmoc-based stepwise solid phase synthesis (SSPS) with some modifications
It should be recalled that the N terminus of sPrP106 is the only available site for the reaction because the N termini of truncated peptides are acetylated in the capping step after each coupling cycle
Summary
Peptide Synthesis, Derivatization, and Purification—PrP106 (GQGGGTHNQWNKPSKPKTNMKHMAGAAAAGAVVGGLGGYMLGSAMSRPMIHFDCVNITIKQHTVTTTTKGENFTETDVKMMERVVEQMCVTQYQKESQAYYDGRRS) was synthesized by SSPS on an automated Applied Biosystems synthesizer model 433A at 0.1 mM scale with p-hydroxymethylphenoxymethyl resin from Fmoc-protected Lamino acid derivatives. CD experiments were performed after the addition of liposome suspensions to sPrP106 solution in the same buffer, to obtain a final peptide concentration of 0.1 mg/ml and a protein:lipid molar ratio of 1:25. SPrP106 was dissolved in deionized water and added to the medium for the acute treatment (final concentration 5 and 10 M), on day 5 of culture, and the cell viability was determined 24 h later. Neuronal cells were detached mechanically, centrifuged gently at 550 ϫ g for 10 min, washed with saline, resuspended in 2.5 ml of 2 M 1,6-diphenyl-1,3,5-hexatriene in 5 mM sodium acetate, pH 5.5, and incubated for 30 min at room temperature. The FP value (expressed as arbitrary units) was determined at 25 °C, before and 10 min after the addition of an increasing concentration of sPrP106 to the cells or liposome suspensions placed in the cuvette. For prediction of membrane protein topology the TMHMM method, based on a hidden Markov model developed by Anders Krogh and Erik Sonnhammer, was applied (19) (www.cbs.dtu.dk/services/TMHMM/)
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