| β2-Microglobulin (β2M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). Although glycated P2M is found as a major component of the amyloid deposits in HAA, the effect of glycation on the misfolding and aggregation of β2M has not been studied so far. D-ribose, present in all living cells and blood, is not only an efficient glycating agent, but also an essential component for energy production in the human body. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β2M in vitro. We find that glycating agent D-ribose interacts with human β2M to generate AGEs which form aggregates in a time-dependent manner. The molecular mass of ribosylated β2M monomer incubated for3days increased up to12,694Da. The extra803Da indicated that about6ribose groups were bound to β2M on average, compared with that of the native P2M. Ribosylated β2M molecules are highly oligomerized compared with unglycated β2M, and have a granular morphology. Furthermore, we demonstrated that ribosylated β2M aggregates could not be digested by trypsin and showed increased resistance to PK. Therefore, once glycated β2M aggregates have formed, they are difficult to be degraded by proteases and can persist in human tissues for a long period.Next, we employed MTT reduction assay to measure the cytotoxicity of ribosylated β2M to human SH-SY5Y neuroblastoma cells and human foreskin fibroblast FS2cells. Because glycated β2M is the major component of the amyloid deposits in HAA, we tried to define the cytotoxic role of ribosylated β2M aggregates. Our data demonstrate that ribosylated P2M aggregates are highly toxic to both SH-SY5Y cells and FS2cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2cells, indicating that the cytotoxicity of ribosylated β2M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, D-ribose reacts with β2M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. In the light of the higher cytotoxicity of ribosylated β2M aggregates than unglycated β2M aggregates, once glycated β2M aggregates have formed, they can persist in human tissues for a long time period and can be more harmful to cells than unglycated β2M aggregates, emphasizing the need for lowering circulating glycated β2M levels in dialysis patients. These findings suggest that ribosylated β2M aggregates could contribute to the dysfunction and death of cells and could play an important role in pathogenesis of β2M-associated diseases such as HAA.Finally, we investigated the effects of macromolecular crowding agents, Ficoll70and dextran70, on the depolymerization of amyloid fibrils at pH7.5, as well as on fibril formation by P2M at an acidic pH, using thioflavin T binding assays, transmission electron microscopy and circular dichroism. On the one hand, depolymerization of β2M amyloid fibrils formed by β2M was inhibited by Ficoll70and dextran70in a dose-dependent way. On the other hand, macromolecular crowding dramatically accelerated amyloid formation by monomeric β2M at an acidic pH. A sigmoidal equation has been used to fit these kinetic data, yielding lag times and apparent rate constants for the growth of fibrils of β2M. These biochemical data indicate that macromolecular crowding significantly accelerated the nucleation step of P2M fibril formation. The above results suggest that crowded physiological environment could enhance the deposition of β2M amyloid fibrils in vivo, possibly by accelerating the formation of β2M amyloid fibrils and inhibiting depolymerization of β2M amyloid fibrils at the same time. |
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