Chaperone activity of α B-crystallin is responsible for its incorrect assignment as an autoantigen in multiple sclerosis

Abstract

For 15 y, α B-crystallin (heat shock protein [Hsp] B5) has been labeled an autoantigen in multiple sclerosis (MS) based on humoral and cellular responses found in humans and animal models. However, there have been several scientific inconsistencies with this assignment, ranging from studies demonstrating small differences in anticrystallin responses between patients and healthy individuals to the inability of crystallin-specific T cells to induce symptoms of experimental allergic encephalomyelitis in animal models. Experiments in this article demonstrate that the putative anti-HspB5 Abs from 23 MS patients cross-react with 7 other members of the human small Hsp family and were equally present in normal plasma. Biolayer interferometry demonstrates that the binding was temperature dependent, and that the calculated K(a) increased as the concentration of the sHsp decreased. These two patterns are characteristic of multiple binding sites with varying affinities, the composition of which changes with temperature, supporting the hypothesis that HspB5 bound the Ab and not the reverse. HspB5 also precipitated Ig heavy and L chains from sera from patients with MS. These results establish that small Hsps bind Igs with high affinity and refute much of the serological data used to assign α B-crystallin as an autoantigen.

Figure 1

Average binding of Igs in MS patients' and normal sera to each of the recombinant human sHsps. A, Differential binding of Igs to the family of human sHsps from sera of 23 MS patients; (B) comparison of the relative signals from sera from 13 healthy individuals with that of sera from an additional 4 MS patients. Error bars represent SD of both the variation between the individual samples and the small differences from four separate readings.

Figure 2

Binding kinetics of sHsps to polyclonal murine anti-human Fc Ab using biolayer interferometry. Schematic diagram displaying the design of the probe and how the rates of association and dissociation are measured by moving eight probes into different rows of a 96-well plate (A). Temperature dependence of HspB5 binding to anti-Fc Abs demonstrated by analyzing the rate of 10 μM HspB5 binding to the Ab-coated probes at 23, 37, and 40°C (B). The calculated dissociation constants of HspB5 (C) and HspB1-B8 (D) with the Ab-coated surface over a range of concentrations from 10 μM to 123 nM in PBS at 40°C decrease as the concentration of the Hsp decreased. Each assay was done a minimum of two times.