Pathogenic D76N Variant of β2-Microglobulin: Synergy of Diverse Effects in Both the Native and Amyloid States.

Abstract

Simple SummaryElevated β2-microglobulin (β2m) serum levels cause serious complications in patients on long-term kidney dialysis by depositing in the form of amyloid fibrils in the osteoarticular system. Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m mutant exhibiting normal serum levels and a distinct, visceral deposition pattern. D76N β2m showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Despite the extensive research, the molecular bases of the aberrant aggregation of β2m in vivo remains elusive. Here, using a variety of biophysical techniques, we investigated the role of the pathogenic D76N mutation in the amyloid formation of β2m by point mutations affecting the stabilizing ion-pairs of β2m. We found that, relative to WT β2m, the exceptional amyloidogenicity of the pathogenic D76N β2m variant is realized by the synergy of diverse effects of destabilized native structure, higher sensitivity to negatively charged amphiphilic molecules and polyphosphate, more effective fibril nucleation, higher conformational stability of fibrils, and elevated affinity for extracellular matrix proteins. Understanding the underlying molecular mechanisms might help to find target points for effective treatments against diseases associated with the deleterious aggregation of proteins.β2-microglobulin (β2m), the light chain of the MHC-I complex, is associated with dialysis-related amyloidosis (DRA). Recently, a hereditary systemic amyloidosis was discovered, caused by a naturally occurring D76N β2m variant, which showed a structure remarkably similar to the wild-type (WT) protein, albeit with decreased thermodynamic stability and increased amyloidogenicity. Here, we investigated the role of the D76N mutation in the amyloid formation of β2m by point mutations affecting the Asp76-Lys41 ion-pair of WT β2m and the charge cluster on Asp38. Using a variety of biophysical techniques, we investigated the conformational stability and partial unfolding of the native state of the variants, as well as their amyloidogenic propensity and the stability of amyloid fibrils under various conditions. Furthermore, we studied the intermolecular interactions of WT and mutant proteins with various binding partners that might have in vivo relevance. We found that, relative to WT β2m, the exceptional amyloidogenicity of the pathogenic D76N β2m variant is realized by the deleterious synergy of diverse effects of destabilized native structure, higher sensitivity to negatively charged amphiphilic molecules (e.g., lipids) and polyphosphate, more effective fibril nucleation, higher conformational stability of fibrils, and elevated affinity for extracellular components, including extracellular matrix proteins.