Extracellular matrix components modulate different stages in β2-microglobulin amyloid formation

Núria Benseny-Cases,Theodoros K Karamanos,Cody L Hoop,Jean Baum,Sheena E Radford

doi:10.1074/jbc.ra119.008300

Abstract

Amyloid deposition of WT human β2-microglobulin (WT-hβ2m) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis. In vitro, WT-hβ2m does not form amyloid fibrils at physiological pH and temperature unless co-solvents or other reagents are added. Therefore, understanding how fibril formation is initiated and maintained in the joint space is important for elucidating WT-hβ2m aggregation and dialysis-related amyloidosis onset. Here, we investigated the roles of collagen I and the commonly administered anticoagulant, low-molecular-weight (LMW) heparin, in the initiation and subsequent aggregation phases of WT-hβ2m in physiologically relevant conditions. Using thioflavin T fluorescence to study the kinetics of amyloid formation, we analyzed how these two agents affect specific stages of WT-hβ2m assembly. Our results revealed that LMW-heparin strongly promotes WT-hβ2m fibrillogenesis during all stages of aggregation. However, collagen I affected WT-hβ2m amyloid formation in contrasting ways: decreasing the lag time of fibril formation in the presence of LMW-heparin and slowing the rate at higher concentrations. We found that in self-seeded reactions, interaction of collagen I with WT-hβ2m amyloid fibrils attenuates surface-mediated growth of WT-hβ2m fibrils, demonstrating a key role of secondary nucleation in WT-hβ2m amyloid formation. Interestingly, collagen I fibrils did not suppress surface-mediated assembly of WT-hβ2m monomers when cross-seeded with fibrils formed from the N-terminally truncated variant ΔN6-hβ2m. Together, these results provide detailed insights into how collagen I and LMW-heparin impact different stages in the aggregation of WT-hβ2m into amyloid, which lead to dramatic effects on the time course of assembly.

Highlights

Amyloid deposition of WT human ␤2-microglobulin (WTh␤2m) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis
These results provide detailed insights into how collagen I and LMW-heparin impact different stages in the aggregation of WT human ␤2-microglobulin (WT-h␤2m) into amyloid, which lead to dramatic effects on the time course of assembly
Because native WT-h␤2m does not form amyloid at neutral pH or at the slightly acidified pH in affected joints [18] unless co-solvents or copper ions are added (13, 16 –19, 28 –32) (Fig. 1A), we investigated how collagen I that is found in the extracellular matrix (ECM) and GAGs affect the kinetics of aggregation of WT-h␤2m

Summary

Edited by Ursula Jakob

Amyloid deposition of WT human ␤2-microglobulin (WTh␤2m) in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis. Assembly of WT-h␤2m monomers when cross-seeded with fibrils formed from the N-terminally truncated variant ⌬N6h␤2m Together, these results provide detailed insights into how collagen I and LMW-heparin impact different stages in the aggregation of WT-h␤2m into amyloid, which lead to dramatic effects on the time course of assembly. Amyloid fibrils have been found associated with collagen fibrils in ex vivo deposits from DRA patients [13], and monomers of both WT-h␤2m and its natural proteolytic product, ⌬N6-h␤2m, which lacks the N-terminal six amino acids, have been shown to have weak (KD: 4.1 ϫ 10Ϫ4 M and 4.9 ϫ 10Ϫ6 M, respectively [15]) affinities for collagen I at the pathophysiologic pH of 6.4 [12] Despite this evidence of the importance of LMW-heparin and collagen in amyloid formation, the mechanism(s) by which. They reveal how the local environment can have a dramatic effect in defining the rate and mechanisms of protein assembly into amyloid

Results

Collagen preparation

Protein preparation

Determination of the lag time of fibril growth