Identification of the Core Structure of Lysozyme Amyloid Fibrils by Proteolysis

Abstract

Human lysozyme variants form amyloid fibrils in individuals suffering from a familial non-neuropathic systemic amyloidosis. In vitro, wild-type human and hen lysozyme, and the amyloidogenic mutants can be induced to form amyloid fibrils when incubated under appropriate conditions. In this study, fibrils of wild-type human lysozyme formed at low pH have been analyzed by a combination of limited proteolysis and Fouriertransform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates. After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a significant minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive β-sheet structure and a substantial element of non β-sheet or random structure that is reduced significantly in the fibrils after digestion. The sequence 32-108 includes the β-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme. The present proteolytic data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain. The majority of amyloid fibrils formed from lysozyme under the conditions used here contain a core structure involving some 50% of the polypeptide chain that is flanked by proteolytically accessible N and C-terminal regions.

The Mechanism of Fibril Formation of a Non-inhibitory Serpin Ovalbumin Revealed by the Identification of Amyloidogenic Core Regions

“…Proteolytic Mapping of the Core Region of the OVA FibrilThe role of the hB region in the heat-induced fibril formation of OVA was further assessed by limited proteolysis, which has been exploited successfully to analyze aspects of fibril formation (55). Proteolytic cleavage generally occurs at flexible regions of a polypeptide chain, and the structured regions of the amyloid fibril correspond to the most highly protease-resistant region.…”

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

“…In a previous study, it was shown by a combined approach of limited proteolysis and Fourier transform infrared spectroscopy (FTIR) that the core region of mature fibrils from human lysozyme produced in vitro at low pH is primarily formed by residues 32-108 in the 130-polypeptide chain of the protein and that the rest of the chain is relatively unstructured. 14 The segment 32-108 corresponds to the region found to undergo cooperative local unfolding in the amyloidogenic variants I56T and D67H of human lysozyme 15,16 and to be amyloidogenic in the homologous hen lysozyme. 17,18 Given the high stability of wild-type human lysozyme, [19][20][21] conditions such as low pH and high temperature or ethanol have been previously used to populate a partially unfolded state that is prone to form fibrils.…”

Multisite aggregation of p53 and implications for drug rescue

“…Protein aggregation occurs in many diseases (7)(8)(9). The best mechanistically characterized examples involve the polymerization of aggregationprone peptides (10,11) or small proteins to give well-defined fibrils based on a regular repeat structure (12)(13)(14)(15)(16)(17)(18). The fibrillar aggregates have a characteristic cross-β X-ray diffraction pattern and bind such dyes as Congo Red and Thioflavine T (ThT) (16).…”