Role of the single disulphide bond of beta(2)-microglobulin in amyloidosis in vitro.
The aggregation of beta(2)-microglobulin (beta(2)m) into amyloid fibrils occurs in the condition known as dialysis-related amyloidosis (DRA). The protein has a beta-sandwich fold typical of the immunoglobulin family, which is stabilized by a highly conserved disulphide bond linking Cys25 and Cys80. Oxidized beta(2)m forms amyloid fibrils rapidly in vitro at acidic pH and high ionic strength. Here we investigate the role of the single disulphide bond of beta(2)m in amyloidosis in vitro. We show that reduction of the disulphide bond destabilizes the native protein such that non-native molecules are populated at neutral pH. These species are prone to oligomerization but do not form amyloid fibrils when incubated for up to 8 mo at pH 7.0 in 0.4 M NaCl. Over the pH range 4.0-1.5 in the presence of 0.4 M NaCl, however, amyloid fibrils of reduced beta(2)m are formed. These fibrils are approximately 10 nm wide, but are shorter and assemble more rapidly than those produced from the oxidized protein. These data show that population of non-native conformers of beta(2)m at neutral pH by reduction of its single disulphide bond is not sufficient for amyloid formation. Instead, association of one or more specific partially unfolded molecules formed at acid pH are necessary for the formation of beta(2)m amyloid in vitro. Further experiments will now be needed to determine the role of different oligomeric species of beta(2)m in the toxicity of the protein in vivo.
Smith
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DP
School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT UK.
Radford
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SE
D000682:Amyloid / Q000737:chemistry / Q000378:metabolism; D000686:Amyloidosis / Q000378:metabolism*; D052160:Benzothiazoles; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008958:Models, Molecular; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013844:Thiazoles / Q000378:metabolism; D001613:beta 2-Microglobulin / Q000737:chemistry* / Q000378:metabolism*
D000682:Amyloid; D052160:Benzothiazoles; D004220:Disulfides; D013844:Thiazoles; D001613:beta 2-Microglobulin; C009462:thioflavin T