Unfolding, aggregation, and seeded amyloid formation of lysine-58-cleaved beta 2-microglobulin.
Beta(2)-microglobulin (beta(2)m) is the amyloidogenic protein in dialysis-related amyloidosis, but the mechanisms underlying beta(2)m fibrillogenesis in vivo are largely unknown. We study a structural variant of beta(2)m that has been linked to cancer and inflammation and may be present in the circulation of dialysis patients. This beta(2)m variant, DeltaK58-beta(2)m, is a disulfide-linked two-chain molecule consisting of amino acid residues 1-57 and 59-99 of intact beta(2)m, and we here demonstrate and characterize its decreased conformational stability as compared to wild-type (wt) beta(2)m. Using amide hydrogen/deuterium exchange monitored by mass spectrometry, we show that DeltaK58-beta(2)m has increased unfolding rates compared to wt-beta(2)m and that unfolding is highly temperature dependent. The unfolding rate is 1 order of magnitude faster in DeltaK58-beta(2)m than in wt-beta(2)m, and at 37 degrees C the half-time for unfolding is more than 170-fold faster than at 15 degrees C. Conformational changes are also reflected by a very prominent Congo red binding of DeltaK58-beta(2)m at 37 degrees C, by the evolution of thioflavin T fluorescence, and by changes in intrinsic fluorescence. After a few days at 37 degrees C, in contrast to wt-beta(2)m, DeltaK58-beta(2)m forms well-defined high molecular weight aggregates that are detected by size-exclusion chromatography. Atomic force microscopy after seeding with amyloid-beta(2)m fibrils under conditions that induce minimal fibrillation in wt-beta(2)m shows extensive amyloid fibrillation in DeltaK58-beta(2)m samples. The results highlight the instability and amyloidogenicity under near physiological conditions of a slightly modified beta(2)m variant generated by limited proteolysis and illustrate stages of amyloid formation from early conformational variants to overt fibrillation.
Heegaard
Niels H H
NH
Department of Autoimmunology, Statens Serum Institut, DK-2300 Copenhagen S, Denmark. nhe@ssi.dk
Jørgensen
Thomas J D
TJ
Rozlosnik
Noémi
N
Corlin
Dorthe B
DB
Pedersen
Jesper S
JS
Tempesta
Anna G
AG
Roepstorff
Peter
P
Bauer
Rogert
R
Nissen
Mogens H
MH
D000682:Amyloid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D052160:Benzothiazoles; D001665:Binding Sites; D002850:Chromatography, Gel; D003224:Congo Red / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008239:Lysine / Q000235:genetics* / Q000378:metabolism*; D018625:Microscopy, Atomic Force; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013696:Temperature; D013844:Thiazoles / Q000378:metabolism; D013997:Time Factors; D001613:beta 2-Microglobulin / Q000235:genetics* / Q000378:metabolism* / Q000648:ultrastructure
D000682:Amyloid; D052160:Benzothiazoles; D005456:Fluorescent Dyes; D013844:Thiazoles; D001613:beta 2-Microglobulin; C009462:thioflavin T; D003224:Congo Red; D008239:Lysine