Topological investigation of amyloid fibrils obtained from beta2-microglobulin

Abstract

Amyloid fibrils of patients treated with regular hemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-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 beta2-m and truncated DeltaN6beta2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of beta2-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 beta2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of beta2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that beta2-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 beta2-m occurring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native beta2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.

Fig. 1.

Electron microscopy analysis. EM analysis of the fibrils obtained from the insoluble precipitate of ΔN6β2-m at pH 4. Space bar, 100 nm.

Fig. 2.

Thioflavin assay. The persistence of fibrillar structure in the proteolysis experimental conditions (pH 7.5 ▴), in β2-m fibrillogenesis conditions (pH 4 O), and in the buffer used to solubilize the proteolysed fibrils (40% CH3CN 0.4% TFA ▪ ) was monitored by thioflavine assay.

Fig. 3.

Limited proteolysis experiment with trypsin. HPLC analysis of the aliquot withdrawn following 15 min of trypsin incubation of the fibrils from β2-m (A) and ΔN6β2-m (B). The indicated fractions were manually collected and the eluted peptides were identified by ESMS.

Fig. 4.

Limited proteolysis experiment with chymotrypsin. HPLC analysis of the aliquot withdrawn following 15 min of chymotrypsin incubation of the fibrils from β2-m (A) and ΔN6β2-m (B). The indicated fractions were collected manually, and the eluted peptides were identified by ESMS.

Fig. 5.

Schematic representation of the results obtained from limited proteolysis experiments. The preferential cleavage sites common to the soluble ΔN6β2-m (A) and to the fibrils originated from both intact β2-m (B) and ΔN6β2-m (C) are highlighted in light gray; those observed on intact β2-m (B) and ΔN6β2-m (C) at the fibrillar state only are highlighted in dark gray.

Fig. 6.

Possible fibrillogenic pathway of β2-m. Native β2-m undergoes conformational changes to generate the partly unfolded intermediate (A) characterized by an amyloidogenic conformation whose overall topologic structure resembles that of the truncated ΔN6β2-m form. The transient component (A) originates the accumulation of soluble aggregates (B), which, in turn, drives the entire equilibrium toward the formation of fibrils (C). When the extracellular proteases attempt to digest the insoluble material, the tightly packed structure of the fibril core prevents proteolysis and addresses protease action toward the amino-and carboxy-terminal ends (D).