Abstract
Using spectroscopic, calorimetric and microscopic methods, we demonstrate that calcium binds to beta-2-microglobulin (β2m) under physiological conditions of pH and ionic strength, in biological buffers, causing a conformational change associated with the binding of up to four calcium atoms per β2m molecule, with a marked transformation of some random coil structure into beta sheet structure, and culminating in the aggregation of the protein at physiological (serum) concentrations of calcium and β2m. We draw attention to the fact that the sequence of β2m contains several potential calcium-binding motifs of the DXD and DXDXD (or DXEXD) varieties. We establish (a) that the microscopic aggregation seen at physiological concentrations of β2m and calcium turns into actual turbidity and visible precipitation at higher concentrations of protein and β2m, (b) that this initial aggregation/precipitation leads to the formation of amorphous aggregates, (c) that the formation of the amorphous aggregates can be partially reversed through the addition of the divalent ion chelating agent, EDTA, and (d) that upon incubation for a few weeks, the amorphous aggregates appear to support the formation of amyloid aggregates that bind to the dye, thioflavin T (ThT), resulting in increase in the dye's fluorescence. We speculate that β2m exists in the form of microscopic aggregates in vivo and that these don't progress to form larger amyloid aggregates because protein concentrations remain low under normal conditions of kidney function and β2m degradation. However, when kidney function is compromised and especially when dialysis is performed, β2m concentrations probably transiently rise to yield large aggregates that deposit in bone joints and transform into amyloids during dialysis related amyloidosis.
Highlights
Human b2-microglobulin (b2m), known as the MHC-I light chain, is a small protein constituent of all Class-I major histocompatibility (MHC-I) complexes displayed on the surfaces of human cells [1]
We monitored calcium-induced aggregation of b2m at a physiological pH of 7.4, and a physiological temperature of 37uC, using a b2m concentration of,0.8 mM which is comparable to that seen in healthy individuals (1–3 mM), and various increasing concentrations of the calcium ion [up to 0.9 mM] well below those observed in the serum of healthy individuals (1–2 mM)
This increased scattering is evidence of the presence of micro-aggregated protein and hints at the possibility of b2m aggregation occurring in the serum, since comparable concentrations of b2m were used with lower-than-normal concentration of calcium
Summary
Human b2-microglobulin (b2m), known as the MHC-I light chain, is a small protein constituent of all Class-I major histocompatibility (MHC-I) complexes displayed on the surfaces of human cells [1]. In patients suffering from renal dysfunction, the degradation of b2m in the kidney becomes compromised [3], leading to elevated b2m concentrations in the serum Under such conditions, b2m levels can be as high as 25–60 times the concentrations seen in healthy humans [4]. An apparent consequence of these elevated concentrations is that b2m tends to aggregate and deposit as insoluble amyloid precipitates within the joints of patients receiving hemodialysis-based treatment. This leads to Dialysis Related Amyloidosis (DRA), a condition which includes carpal tunnel syndrome, amyloid arthopathy, and pathological bone disruption [5],[6]. There is much interest, in the aggregation and deposition of this small seven b-stranded (anti-parallel b-sandwich) protein
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