Unfolding, Aggregation, and Seeded Amyloid Formation of Lysine-58-Cleaved β2-Microglobulin

• Published in: Biochemistry (Easton) Vol. 44; no. 11; pp. 4397 - 4407
• Main Authors: Heegaard, Niels H. H, Jørgensen, Thomas J. D, Rozlosnik, Noémi, Corlin, Dorthe B, Pedersen, Jesper S, Tempesta, Anna G, Roepstorff, Peter, Bauer, Rogert, Nissen, Mogens H
• Format: Journal Article
• Language: English
• Published: American Chemical Society 22-03-2005
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi047594t

β2-Microglobulin (β2m) is the amyloidogenic protein in dialysis-related amyloidosis, but the mechanisms underlying β2m fibrillogenesis in vivo are largely unknown. We study a structural variant of β2m that has been linked to cancer and inflammation and may be present in the circulation of dialysis patients. This β2m variant, ΔK58-β2m, is a disulfide-linked two-chain molecule consisting of amino acid residues 1−57 and 59−99 of intact β2m, and we here demonstrate and characterize its decreased conformational stability as compared to wild-type (wt) β2m. Using amide hydrogen/deuterium exchange monitored by mass spectrometry, we show that ΔK58-β2m has increased unfolding rates compared to wt-β2m and that unfolding is highly temperature dependent. The unfolding rate is 1 order of magnitude faster in ΔK58-β2m than in wt-β2m, and at 37 °C the half-time for unfolding is more than 170-fold faster than at 15 °C. Conformational changes are also reflected by a very prominent Congo red binding of ΔK58-β2m at 37 °C, by the evolution of thioflavin T fluorescence, and by changes in intrinsic fluorescence. After a few days at 37 °C, in contrast to wt-β2m, ΔK58-β2m forms well-defined high molecular weight aggregates that are detected by size-exclusion chromatography. Atomic force microscopy after seeding with amyloid-β2m fibrils under conditions that induce minimal fibrillation in wt-β2m shows extensive amyloid fibrillation in ΔK58-β2m samples. The results highlight the instability and amyloidogenicity under near physiological conditions of a slightly modified β2m variant generated by limited proteolysis and illustrate stages of amyloid formation from early conformational variants to overt fibrillation.