Amyloid Fibril Formation in the Context of Full-length Protein: EFFECTS OF PROLINE MUTATIONS ON THE AMYLOID FIBRIL FORMATION OF β2-MICROGLOBULIN

Takeshi Chiba,Yoshihisa Hagihara,Takashi Higurashi,Kazuhiro Hasegawa,Hironobu Naiki,Yuji Goto

doi:10.1074/jbc.m304473200

Abstract

Beta2-microglobulin (beta2-m), a typical immunoglobulin domain made of seven beta-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis. To understand the mechanism of amyloid fibril formation in the context of full-length protein, we prepared various mutants in which proline (Pro) was introduced to each of the seven beta-strands of beta2-m. The mutations affected the amyloidogenic potential of beta2-m to various degrees. In particular, the L23P, H51P, and V82P mutations significantly retarded fibril extension at pH 2.5. Among these, only L23P is included in the known "minimal" peptide sequence, which can form amyloid fibrils when isolated as a short peptide. This indicates that the residues in regions other than the minimal sequence, such as H51P and V82P, determine the amyloidogenic potential in the full-length protein. To further clarify the mutational effects, we measured their stability against guanidine hydrochloride of the native state at pH 8.0 and the amyloid fibrils at pH 2.5. The amyloidogenicity of mutants showed a significant correlation with the stability of the amyloid fibrils, and little correlation was observed with that of the native state. It has been proposed that the stability of the native state and the unfolding rate to the amyloidogenic precursor as well as the conformational preference of the denatured state determine the amyloidogenicity of the proteins. The present results reveal that, in addition, stability of the amyloid fibrils is a key factor determining the amyloidogenic potential of the proteins.

Highlights

␤2-Microglobulin (␤2-m), a typical immunoglobulin domain made of seven ␤-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis
Amyloid Fibril Formation Monitored by thioflavin T (ThT) Fluorescence—To analyze the effects of Pro mutations on the amyloidogenic potential, the seed-dependent amyloid fibril extensions of wild-type and mutant ␤2-ms were examined with wild-type seeds
The kinetics of fibril extension was affected to varying degrees by the mutations, and significant retardation of the extension reaction was observed for several mutants (i.e. L23P, L39P, H51P, L65P, and V82P)

Summary

Introduction

␤2-Microglobulin (␤2-m), a typical immunoglobulin domain made of seven ␤-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis. Only L23P is included in the known “minimal” peptide sequence, which can form amyloid fibrils when isolated as a short peptide This indicates that the residues in regions other than the minimal sequence, such as H51P and V82P, determine the amyloidogenic potential in the full-length protein. In other types of amyloidosis, such as familial amyloid polyneuropathy and dialysis-related amyloidosis, amyloid fibrils are made of the entire part of the originally globular proteins For these proteins, it has been shown that short peptides, called minimal (or essential) sequences, can form amyloid fibrils (4 –7). Dialysis-related amyloidosis is a common and serious complication in patients receiving long term hemodialysis for more than 10 years (16 –18) In this disease, full-length ␤2-microglobulin (␤2-m) is a major structural component of amyloid fibrils [16]. In amyloid fibrils of ␤2-m, more than 50% of amide protons, mostly located at the central regions of the protein, are highly protected from hydrogen/deuterium exchange (Fig. 1A), which is distinct from the protected amide protons of the native state

Results

Discussion

Conclusion