1. A structural core within apolipoprotein C-II amyloid fibrils identified using hydrogen exchange and proteolysis.
- Author
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Wilson LM, Mok YF, Binger KJ, Griffin MD, Mertens HD, Lin F, Wade JD, Gooley PR, and Howlett GJ
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Amyloid ultrastructure, Apolipoprotein C-II isolation & purification, Apolipoprotein C-II ultrastructure, Benzothiazoles, Chromatography, High Pressure Liquid, Circular Dichroism, Deuterium Exchange Measurement, Dose-Response Relationship, Drug, Electrophoresis, Agar Gel, Endopeptidase K pharmacology, Fluorescent Dyes, Humans, Hydrolysis, Kinetics, Mass Spectrometry, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Folding, Protein Structure, Tertiary, Serine Endopeptidases pharmacology, Spectrometry, Fluorescence, Thiazoles, Time Factors, Trypsin pharmacology, Amyloid chemistry, Apolipoprotein C-II chemistry, Deuterium metabolism, Hydrogen metabolism
- Abstract
Plasma apolipoproteins show alpha-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19-37 and 57-74 with reduced amide proton exchange rates compared to monomeric apoC-II. The C-terminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II(56-76). Synthetic apoC-II(56-76) readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to full-length apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60-70. We postulate that the ability of apoC-II(60-70) to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo.
- Published
- 2007
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