Fibrillogenic Oligomers of Human Cystatin C Are Formed by Propagated Domain Swapping

Maria Wahlbom,Xin Wang,Veronica Lindström,Eric Carlemalm,Mariusz Jaskolski,Anders Grubb

doi:10.1074/jbc.m611368200

Abstract

Cystatin C and the prion protein have been shown to form dimers via three-dimensional domain swapping, and this process has also been hypothesized to be involved in amyloidogenesis. Production of oligomers of other amyloidogenic proteins has been reported to precede fibril formation, suggesting oligomers as intermediates in fibrillogenesis. A variant of cystatin C, with a Leu68-->Gln substitution, is highly amyloidogenic, and carriers of this mutation suffer from massive cerebral amyloidosis leading to brain hemorrhage and death in early adulthood. This work describes doughnut-shaped oligomers formed by wild type and L68Q cystatin C upon incubation of the monomeric proteins. Purified oligomers of cystatin C are shown to fibrillize faster and at a lower concentration than the monomeric protein, indicating a role of the oligomers as fibril-assembly intermediates. Moreover, the present work demonstrates that three-dimensional domain swapping is involved in the formation of the oligomers, because variants of monomeric cystatin C, stabilized against three-dimensional domain swapping by engineered disulfide bonds, do not produce oligomers upon incubation under non-reducing conditions. Redox experiments using wild type and stabilized cystatin C strongly suggest that the oligomers, and thus probably the fibrils as well, are formed by propagated domain swapping rather than by assembly of domain-swapped cystatin C dimers.

Highlights

Insoluble amyloid fibrils are found as deposits in patients with a range of diseases, e.g. Alzheimer disease, Parkinson disease, reactive amyloidosis, cystatin C amyloidosis, and the prionoses [1,2,3]
Patients suffering from Hereditary Cystatin C Amyloid Angiopathy (HCCAA) have L68Q cystatin C deposited as amyloid fibrils in the cerebral arteries, resulting in cerebral hemorrhage and death in early adulthood [7]
In Vitro Fibril Formation of wt and L68Q Cystatin C and wt Cystatin C Stabilized against Domain Swapping—We have previously shown that recombinant wt cystatin C forms amyloid fibrils in vitro in solutions at pH 2.0 [28]

Summary

Introduction

Insoluble amyloid fibrils are found as deposits in patients with a range of diseases, e.g. Alzheimer disease, Parkinson disease, reactive amyloidosis, cystatin C amyloidosis, and the prionoses [1,2,3]. The present work demonstrates that three-dimensional domain swapping is involved in the formation of the oligomers, because variants of monomeric cystatin C, stabilized against three-dimensional domain swapping by engineered disulfide bonds, do not produce oligomers upon incubation under non-reducing conditions.

Results

Conclusion