Final Report Summary - MCIBC (Molecular characterization of the interaction of β -2 microglobulin with collagen)
B-microglobulin (b2m) is one of the polypeptide chains forming the major histocompatibility complex class I (MHC I) that is expressed on the surface of all nucleated cells. b2m continuously dissociates from MHC I and, in healthy subjects, is normally removed from the plasma in the kidneys, where the majority of the protein is degraded. However, in the case of renal failure the removal of b2m is disrupted causing an increase in serum concentration of b2m up to 60 times higher than the normal concentration. This increase of concentration leads to the accumulation of b2m in joints and cartilage tissue, causing progressive bone and joint destruction and resulting in severe skeletal morbidity. This pathological condition, Dialysis-Related Amyloidosis (DRA), affects more than 700,000 patients word-wide. Although WT b2m does not aggregate in vitro at physiological pH, the analysis of ex vivo deposits of DRA patients showed that b2m forms fibrils and often, these b2m fibrils are associated with collagen fibrils, which are one of the main components of connective tissue. The specificity of b2m for joints, and the fact that joints are rich in collagen, suggest a key role of in the mechanism of b2m accumulation. The role of glycosaminoglycans (GAGs) could also be important in disease, as connective tissue is rich in GAGs, in addition, heparin, which is a GAG used as an anticoagulant during dialysis. So, the objective of this project was to characterise the aggregation process of b2m at relevant physiological conditions and determine the role of collagen and heparin in the aggregation process.
The aggregation of b2m into amyloid fibrils is a complex mechanism, composed of different steps. The nucleation phase involves the monomer misfolding and/or the formation of oligomers. The elongation phase involves extension of nuclei and fibril formation. This process has recently been described as containing multiple different events, which occur simultaneously. Elongation (addition of oligomers or monomers at the end of the fibrils), secondary nucleation (formation of fibrils on the surface of the preformed ones) and fragmentation (breaking of fibrils forming more fibril ends), that usually happens when the samples are agitated. Detailed kinetic analysis, therefore, can be used to reveal the role of different components in the local environment on different processes occurring during aggregation.
In this project we have measured the effect of heparin and collagen on spontaneous (non seeded) fibril growth to study the effect of these macromolecules on the nucleation phase as well as their effect on seeded fibril growth kinetics which reveal the effect on elongation and secondary nucleation. This kinetic study was complemented by other biochemical techniques, including NMR and coprecipitate experiments to determine which species (monomer, oligomer or fibrils) interact with collagen.
As shown in panels a,b and c of the figure attached, the presence of collagen inhibits primary nucleation of b2m at high collagen concentrations (higher than 0.47mg/mL) accelerates the kinetics at lower collagen concentrations (less than 0.12mg/mL) suggesting an interaction between misfolded monomers or oligomers and collagen. In the presence of fibril seeds fibril growth occurs via two steps, the elongation process (0-20h) and secondary nucleation. In the presence of collagen secondary nucleation is inhibited (d, e) and in the presence of heparin it is enhanced (g). Collagen only slightly inhibits secondary nucleation in the presence of heparin (h). The data show that elongation rate is enhanced by heparin, but collagen does not cause any additional change (f). The half time b2m, b2m in the presence of heparin and heparin and collagen are shown in panel (i), the half time of WT in the presence of collagen can not be represented as secondary nucleation does not take place in the time measured. Panel (i) shows that the fibril formation is clearly decreased by the presence of collagen and increased in the presence of heparin. In the presence of heparin the presence of collagen has a subtler effect on the half time.
Similar experiments were carried out in the presence of the b2m variant in which the 6 residues of the N terminal are lacking. No effect was observed in the kinetics by collagen and an enhancement was observed in the presence of heparin
The experiments showed have been carried out in the presence of collagen in its fibril form. Experiments in the presence of collagen in the single chain form (precursor of the fibril form) and collagen like peptides have no effect in the kinetics.
In brief, the data showed that collagen and heparin have opposite effects on b2m aggregation, while collagen inhibits both primary and secondary nucleation process, heparin enhances primary nucleation, secondary nucleation and elongation, and heparin competes for the effects of collagen.
The aggregation of b2m into amyloid fibrils is a complex mechanism, composed of different steps. The nucleation phase involves the monomer misfolding and/or the formation of oligomers. The elongation phase involves extension of nuclei and fibril formation. This process has recently been described as containing multiple different events, which occur simultaneously. Elongation (addition of oligomers or monomers at the end of the fibrils), secondary nucleation (formation of fibrils on the surface of the preformed ones) and fragmentation (breaking of fibrils forming more fibril ends), that usually happens when the samples are agitated. Detailed kinetic analysis, therefore, can be used to reveal the role of different components in the local environment on different processes occurring during aggregation.
In this project we have measured the effect of heparin and collagen on spontaneous (non seeded) fibril growth to study the effect of these macromolecules on the nucleation phase as well as their effect on seeded fibril growth kinetics which reveal the effect on elongation and secondary nucleation. This kinetic study was complemented by other biochemical techniques, including NMR and coprecipitate experiments to determine which species (monomer, oligomer or fibrils) interact with collagen.
As shown in panels a,b and c of the figure attached, the presence of collagen inhibits primary nucleation of b2m at high collagen concentrations (higher than 0.47mg/mL) accelerates the kinetics at lower collagen concentrations (less than 0.12mg/mL) suggesting an interaction between misfolded monomers or oligomers and collagen. In the presence of fibril seeds fibril growth occurs via two steps, the elongation process (0-20h) and secondary nucleation. In the presence of collagen secondary nucleation is inhibited (d, e) and in the presence of heparin it is enhanced (g). Collagen only slightly inhibits secondary nucleation in the presence of heparin (h). The data show that elongation rate is enhanced by heparin, but collagen does not cause any additional change (f). The half time b2m, b2m in the presence of heparin and heparin and collagen are shown in panel (i), the half time of WT in the presence of collagen can not be represented as secondary nucleation does not take place in the time measured. Panel (i) shows that the fibril formation is clearly decreased by the presence of collagen and increased in the presence of heparin. In the presence of heparin the presence of collagen has a subtler effect on the half time.
Similar experiments were carried out in the presence of the b2m variant in which the 6 residues of the N terminal are lacking. No effect was observed in the kinetics by collagen and an enhancement was observed in the presence of heparin
The experiments showed have been carried out in the presence of collagen in its fibril form. Experiments in the presence of collagen in the single chain form (precursor of the fibril form) and collagen like peptides have no effect in the kinetics.
In brief, the data showed that collagen and heparin have opposite effects on b2m aggregation, while collagen inhibits both primary and secondary nucleation process, heparin enhances primary nucleation, secondary nucleation and elongation, and heparin competes for the effects of collagen.
