Glycosaminoglycans Enhance the Trifluoroethanol-Induced Extension of β2-Microglobulin–Related Amyloid Fibrils at a Neutral pH

Abstract

Abstract. ␤ 2 -Microglobulin-related (A␤2M) amyloidosis is a frequent and serious complication in patients on long-term dialysis, and ␤ 2 -microglobulin is a major structural component of A␤2M amyloid fibrils. Several biologic molecules inhibiting the depolymerization of A␤2M amyloid fibrils at a neutral pH were found recently. The effect of trifluoroethanol and glycosaminoglycans (GAG) on the extension of the fibrils at a neutral pH was investigated with the use of fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy. Trifluoroethanol at concentrations of up to 20% (vol/vol) caused fibril extension of heparin-stabilized seeds, inducing a subtle change in the tertiary structure of ␤ 2 -microglobulin and stabilizing the fibrils at a neutral pH. This extension reaction followed a first-order kinetic model. In addition, some GAG, especially heparin, dose-dependently enhanced the fibril extension. These results suggest that some GAG, especially heparin, may bind to the fibrils and enhance their deposition in vivo. Thus, the experimental system described here should be useful to search for the factors that accelerate A␤2M amyloid deposition in vivo. In addition, the interference of the binding of GAG to A␤2M amyloid fibrils may be an attractive therapeutic modality.␤ 2 -Microglobulin-related (A␤2M) amyloidosis is a frequent and serious complication in patients on long-term dialysis (1). Carpal tunnel syndrome and destructive arthropathy associated with cystic bone lesions are the major clinical manifestations of A␤2M amyloidosis (2,3). Several biochemical and cell biologic studies have revealed that intact ␤ 2 -microglobulin (␤2-m) is a major structural component of amyloid fibrils deposited in the synovial membrane of the carpal tunnel (4 -7), but the mechanism of the deposition of these amyloid fibrils is still incompletely understood. Although the retention of ␤2-m in the plasma seems to be prerequisite (8), other factors, such as the age of the patient, the duration of dialysis, and the type of dialysis membrane used, may also be involved (9 -11).We and other groups have proposed that a nucleationdependent polymerization model could explain the general mechanism of amyloid fibril formation in vitro, in various types of amyloidosis (12)(13)(14)(15)(16)(17). This model consists of two phases: a nucleation phase and an extension phase. Nucleus formation requires a series of association steps of monomers, which are thermodynamically unfavorable, representing the rate-limiting step in amyloid fibril formation in vitro. Once the nucleus (n-mer) has been formed, further addition of monomers becomes thermodynamically favorable, resulting in rapid extension of amyloid fibrils according to a first-order kinetic model (12,14,16).The extension of A␤2M amyloid fibrils, as well as the formation of the fibrils from ␤2-m, is greatly dependent on the pH of the reaction mixture, with the optimum pH at approximately 2.0 to 3.0 (16,17). However, they readily depolymerize into monomeri...

Polymorphism of β2-Microglobulin Amyloid Fibrils Manifested by Ultrasonication-enhanced Fibril Formation in Trifluoroethanol

“…The main effect of TFE is expected to be a weakening of hydrophobic interactions with a strengthening of electrostatic interactions (including polar and charge-charge interactions) that leads to denaturation of the rigid native structure of proteins (43,44) and stabilization of the ␣-helical structure (45,46). Especially at higher concentrations of TFE, the ␣-helical structure is dominant with intramolecularly stabilized hydrogen bond networks, whereas little intermolecular association or aggregation.…”

Metal binding sheds light on mechanisms of amyloid assembly

“…These include limited proteolysis, 11 incubation at acidic pH, 12 and exposure to detergents or organic solvents. 13,14 In 2001, we discovered that β2m is a Cu 2+ -binding protein. 4 Whereas β2m apo is not amyloidogenic, β2m holo is aggregation-prone.…”

Monitoring the Interaction between β2-Microglobulin and the Molecular Chaperone αB-crystallin by NMR and Mass Spectrometry

“…In vitro, however, the transition does not occur spontaneously at neutral pH but requires moderately acidic conditions and proper ionic strength or the presence of chaotropes (20 -22). The formation of ␤2m fibrils at neutral pH occurs upon the addition of seeds and 2,2,2-trifluoroethanol, surfactants, or glycosaminoglycans (23,24). Furthermore, temperature and pH conditions similar to those occurring upon inflammation in periarticular compartments enhance aggregation (25) and trigger fibril formation in the presence of collagen fibers (26) and heparin (27).…”