Ultrasonication-induced Amyloid Fibril Formation of β2-Microglobulin

Abstract

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on ␤ 2 -microglobulin (␤2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded ␤2-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 ␤2-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 ␤2-m, producing fibrils adapted to the respective pH.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 (1-4). Among various amyloidogenic proteins,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 (5-12). Dialysis-related amyloidosis is a common and serious complication in patients receiving hemodialysis for more than 10 years (5, 6). ␤2-m, a typical immunoglobulin domain made of 99 residues, is present as the non-polymorphic light chain of the class I major histocompatibility complex (13). As part of its normal catabolic cycle, ␤2-m dissociates from the class I major histocompatibility complex and is transported in serum to the kidneys where the majority (95%) of it is degraded (6). Renal failure disrupts the clearance of ␤2-m from the serum, and moreover the ␤2-m does not pass through the dialysis membrane, resulting in an increase in the ␤2-m concentration by up to 50-fold in the blood circulation (6). By a mechanism that is currently not well understood, ␤2-m then self-associates to form amyloid fibrils under physiological conditions.The incubation of ␤2-m in vitro under acidic conditions in the presence or absence of seed fibrils results in the formation of high yields of amyloid fibrils with a range of different morphologies (7-11). In contrast, the generation of a substantial amount of amyloid fibrils at neutral pH has be...

Ultrasonication: An Efficient Agitation for Accelerating the Supersaturation-Limited Amyloid Fibrillation of Proteins

“…35) The ThT fluorescence increased dramatically without a lag-phase. AFM images revealed the formation of long and straight fibrils with a diameter of 3 nm.…”

“…Ohhashi et al 35) studied in detail the effects of ultrasonication on monomeric 2-m. First, the same conditions as used for the standard seed-dependent fibrillation experiments (i.e., 0.3 mg/mL 2-m monomer at pH 2.5 containing 0.1 mol/L NaCl) were used to examine the effects of ultrasonication. After the reaction mixture was prepared in an Eppendorf tube, ultrasonic treatment was started with the tube placed in a water bath-type ultrasonicator (Elekon ELESTEIN 070-GOT) at 37 C. The effects of ultrasonication were monitored by fluorometric analysis with thioflavin T (ThT), a specific dye for amyloid fibrils.…”

“…37) However, even in the presence of 0.5 mmol/L SDS, the fibril formation took several days at pH 7.0. Here, Ohhashi et al 35) examined the effects of ultrasonication at pH 7.0 in the presence of 0.5 mmol/L SDS.…”

“…All these results indicated that ultrasonication is also useful for the formation of fibrils even at pH 7.0. 35) 3. Ultrasonication-Induced Formation of the Minimum Sized Fibrils 3.1 Opposing effects of the ultrasonication-dependent breakage and induction Repetitive ultrasonication pulses of 1 min with a quiescent incubation period of 9 min resulted in the formation of relatively long fibrils, followed by the breakdown into short fibrils [ Fig.…”

Interplay Between Mechanochemistry and Sonochemistry

“…1 min), cause the formation of subsequent fibrils (self-seeding). The process is pH-dependent and AFM images indicate that fibrils with diameters of more than 7 nm are obtained at pH 7.0, which are thicker than those formed at pH 2.5 [112]. Fibril breakage occurs at longer irradiation times, although ultrasound also produces a uniform fibril length distribution.…”