Topological investigation of amyloid fibrils obtained from beta2-microglobulin.

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of β2-microglobulin (β2-m) and its truncated species ΔN6β2-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact β2-m and truncated ΔN6β2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of β2-m within the fibrillar polymer. The distribution of prefential proteolytic sites observed in both fibrils revealed that the central region of the protein, which had been easily cleaved in the full-length globular β2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of β2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that β2-m molecules in the fibrils consist of an unaccessible core comprising residues 20-87 with the strands I and VIII being not constrained in the fibrillar polymer and exposed to the proteases. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to occur in vivo to generate the truncated forms of β2-m occuring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native β2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.