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

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. 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.