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

Abstract

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.