Ultrasonication-induced Amyloid Fibril Formation of β2-Microglobulin

Yumiko Ohhashi,Miho Kihara,Hironobu Naiki,Yuji Goto

doi:10.1074/jbc.m506501200

Abstract

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on beta2-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded beta2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native beta2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mm sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of beta2-m, producing fibrils adapted to the respective pH.

Highlights

Amyloidosis results from the deposition of normally soluble proteins into insoluble amyloid fibrils: long, unbranched, and often twisted fibrillar structures a few nanometers in diameter and predominantly composed of cross ␤-sheets [1,2,3,4]
Among various amyloidogenic proteins, ␤2-microglobulin (␤2-m)3 is a target of extensive study because of its clinical importance and suitable size for examining the relation between protein folding and amyloid fibril formation [5,6,7,8,9,10,11,12]
The lag time varied slightly depending on the experiment and on the volume of the mixture in the tube, suggesting that the formation of fibrils critically depends on the sonication energy

Summary

Introduction

Amyloidosis results from the deposition of normally soluble proteins into insoluble amyloid fibrils: long, unbranched, and often twisted fibrillar structures a few nanometers in diameter and predominantly composed of cross ␤-sheets [1,2,3,4]. They proposed that protein unfolding and aggregation are caused by the ultrasonication, leading to the formation of amyloid fibrils. In the seeding experiments with the sonication-induced fibrils, sonicated ␤2-m fibrils were added at a final monomer concentration of 0.42 and 2.5 ␮M to the acidic and neutral pH reaction mixtures containing 25 ␮M ␤2-m, respectively, and the solutions were incubated at 37 °C without agitation.

Results

Conclusion