Mechanisms For β₂-microglobulin Amyloid Formation Modulated By Macromolecular Crowding And N-terminal Hexapeptide

Amyloid fibrils formed from proteins and peptides are aberrant aggregates which cause a wide range of lethal protein misfolding diseases, such as Alzheimer disease, Parkinson disease, Type II diabetes, and hemodialysis-associated amyloidosis (HAA). β2-Microglobulin (β2M) is the light chain of major histocompatibility complex class I. In normal metabolic cycles, it is regularly dissociated from the complex and degraded in the proximal tubulesof kidney. As a consequence of renal failure and renalmalfunction, the serum concentration of β2M in patient bearing long-term dialysisis increased up to 60-fold compared to healthy individuals, and forms amyloid deposition in osteoarticular tissues, leading to bone damage and destruction. The diseases are known as hemodialysis-associated amyloidosis (HAA), including carpal tunnel syndrome and bone cysts. Although other factors involved, HAA is mainly formed of wild-type human β2M and its N-terminal hexapeptide truncated mutant △N6. Therefore, the uncovering of the mechanism of β2M amyloid fibrils formation is of center importance for the reveal of pathogenesis of HAA and the development of therapeutic strategy.High concentration of proteins, polysaccharides and their complexes exist in cells and tissues of body and exert profound influence on biochemical reaction such as protein folding and misfolding. HAA involves the fibrillization of P2M and occurs in crowded physiological environments. However, how macromolecular crowding affects amyloid formation of β2M remains elusive. Here we study the effects of macromolecular crowding on amyloid formation and fibril disassembly of wild-type human β2M and its pathogenic mutant △N6. At pH 2.5, the presence of crowding agent (Ficoll 70 or dextran 70) not only dramatically accelerates the fibrillization of both wild-type P2M and its AN6 variant by stabilizing the nucleation phase, but also remarkably increases the amount of β2M fibrils. At pH 6.2, such an enhancing effect of macromolecular crowding on fibril formation is only observed for pathogenic mutant △N6, but not for wild-type P2M which does not form amyloid fibrils in the absence and presenceof a crowding agent. Thus, we propose that the monomers of β2M form the nuclei, which is enhanced by macromolecular crowding, followed by the step of fibril elongation. Furthermore, at physiological pH, macromolecular crowding remarkably inhibits β2M fibril disassembly by decreasing rate constants corresponding to fast and slow stages of fibril disaggregation. Our data demonstrates that macromolecular crowding favors the fibrillization of β2M by accelerating the nucleation step and inhibiting fibril disassembly. Our findings provide clear evidence for the pathology of HAA that macromolecular crowding should be taken into account.We also explore the role of N-terminal hexapeptide truncation in β2M amyloid formation. By analyzing and comparing the fibrillization kinetics of wild-type β2M and its △N6 variant, as well as fibrils’hydrophobicity and morphology, we found that wild-type β2M and its △N6 variant form distinct fibrils, named as’W-type’for wild-type β2M fibrils and ’N-type’ for △N6-β2M fibrils. The lag time of fibrillization kinetics of N-type fibril is longer than W-type fibril. The ANS fluorescence intensity of N-type fibril is much smaller than W-type fibril, indicating difference in hydrophobic amino acid exposure in fibrils. Furthermore, the morphology of two types of fibrils observed by TEM is very distinct. As revealed by seeded-propagation experiments, both wild-type β2M and its △N6 variant can be seeded by N-type fibril seeds, leading to the formation of N-type amyloid fibrils; however, only wild-type β2M can be seeded by W-type fibril seeds, leading to the formation of W-type fibrils, △N6-β2M can not be seeded by W-type fibril seeds. Our results suggest that N-terminal hexapeptide truncation of β2M alters fibrillization kinetics and morphology of β2M fibrils and N-terminal hexapeptide could play an important role in’cross-β’structure constitution of W-type fibrils.