A Partially Structured Species of β2-Microglobulin Is Significantly Populated under Physiological Conditions and Involved in Fibrillogenesis

Abstract

The folding of ␤ 2 -microglobulin (␤ 2 -m), the protein forming amyloid deposits in dialysis-related amyloidosis, involves formation of a partially folded conformation named I 2 , which slowly converts into the native fold, N. Here we show that the partially folded species I 2 can be separated from N by capillary electrophoresis. Data obtained with this technique and analysis of kinetic data obtained with intrinsic fluorescence indicate that the I 2 conformation is populated to ϳ14 ؎ 8% at equilibrium under conditions of pH and temperature close to physiological. In the presence of fibrils extracted from patients, the I 2 conformer has a 5-fold higher propensity to aggregate than N, as indicated by the thioflavine T test and light scattering measurements. A mechanism of aggregation of ␤ 2 -m in vivo involving the association of the preformed fibrils with the fraction of I 2 existing at equilibrium is proposed from these results. The possibility of isolating and quantifying a partially folded conformer of ␤ 2 -m involved in the amyloidogenesis process provides new opportunities to monitor hemodialytic procedures aimed at the reduction of such species from the pool of circulating ␤ 2 -m but also to design new pharmaceutical approaches that consider such species as a putative molecular target.Dialysis-related amyloidosis represents an inevitable and severe complication of long term hemodialysis (1-4). Under this pathological condition, protein aggregates known as amyloid fibrils, accumulate in essential tissues, such as the skeletal muscle, interfering with their normal functions. A major constituent of the amyloid fibrils related to this pathological condition is ␤ 2 -microglobulin (␤ 2 -m).1 In its native form, this 99-residue protein is constituted by two ␤-sheets packed against each other to form a fold typical of the immunoglobulin superfamily (5). The two ␤-sheets, constituted by three and four strands, respectively, interact by means of hydrophobic interactions and a disulfide bridge that stabilizes further the ␤-sandwich structure.␤ 2 -m constitutes the light chain of the major histocompatibility complex class I (MHCI). A significant pool of ␤ 2 -m is also normally present in the plasma as a consequence of the constant release from the MHCI to allow the process of catabolic degradation in the kidney (1, 2). In chronic dialysis patients, the artificial membrane induces an inflammatory reaction, which causes the production and release of ␤ 2 -m to increase significantly (6). In addition, ␤ 2 -m cannot be filtered efficiently through the artificial membrane, resulting in an increase of the levels of soluble ␤ 2 -m from 0.3 to 30 g/ml, the range of concentrations observed within healthy individuals, to ϳ40 g/ml (7). The increase of free circulating ␤ 2 -m, the preferential substrate for amyloid deposition by this protein, is responsible, at least in part, for this form of amyloidosis in these patients (1, 2). Big efforts have been expended to improve the biocompatibility and performance of dialysis approaches. ...

Co-populated Conformational Ensembles of β2-Microglobulin Uncovered Quantitatively by Electrospray Ionization Mass Spectrometry

“…The data show that only the native protein is populated above pH 5.0. The spectra showed no evidence of a small population (ϳ15%) of a slowly converting nonnative species at physiological pH previously detected by stopped flow fluorescence and capillary electrophoresis (35,36). This could reflect differences in the buffer conditions used or would result if the partially folded and the nonnative states have similar charge state distributions.…”

Supramolecular Amyloid-like Assembly of the Polypeptide Sequence Coded by Exon 30 of Human Tropoelastin

“…8). The formation of viscous material is indicative of the formation of amyloid-like fibrils (48,49). The interaction of this material with Congo Red dye is shown in Fig.…”

Both the cis - trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly

“…In the case of β2m, such species are obtained when Pro32, normally in a cis conformation, slowly isomerizes to trans (13,33,35). Cis-to-trans isomerization of this residue exerts a destabilizing effect on protein structure (13,23), and ensuing conformational changes lead to exposure of hydrophobic residues and intermolecular aggregation via the D strand of the β-sandwich structure (6).…”