ALys amyloidosis caused by compound heterozygosity in Exon 2 (Thr70Asn) and Exon 4 (Trp112Arg) of the lysozyme gene

The aggregation process of wild-type human lysozyme at pH 3.0 and 60°C has been analyzed by characterizing a series of distinct species formed on the aggregation pathway, specifically the amyloidogenic monomeric precursor protein, the oligomeric soluble prefibrillar aggregates, and the mature fibrils. Particular attention has been focused on the analysis of the structural properties of the oligomeric species, since recent studies have shown that the oligomers formed by lysozyme prior to the appearance of mature amyloid fibrils are toxic to cells. Here, soluble oligomers of human lysozyme have been analyzed by a range of techniques including binding to fluorescent probes such as thioflavin T and 1-anilino-naphthalene-8-sulfonate, Fourier transform infrared spectroscopy, and controlled proteolysis. Oligomers were isolated after 5 days of incubation of the protein and appear as spherical particles with a diameter of 8-17 nm when observed by transmission electron microscopy. Unlike the monomeric protein, oligomers have solventexposed hydrophobic patches able to bind the fluorescent probe 1-anilinonaphthalene-8-sulfonate. Fourier transform infrared spectroscopy spectra of oligomers are indicative of misfolded species when compared to monomeric lysozyme, with a prevalence of random structure but with significant elements of the β-sheet structure that is characteristic of the mature fibrils. Moreover, the oligomeric lysozyme aggregates were found to be more susceptible to proteolysis with pepsin than both the monomeric protein and the mature fibrils, indicating further their less organized structure. In summary, this study shows that the soluble lysozyme oligomers are locally unfolded species that are present at low concentration during the initial phases of aggregation. The nonnative conformational features of the lysozyme molecules of which they are composed are likely to be the factors that confer on them the ability to interact inappropriately with a variety of cellular components including membranes.

Section: Introductionmentioning

confidence: 99%

Characterization of Oligomeric Species on the Aggregation Pathway of Human Lysozyme

Frare

Mossuto

Laureto

et al. 2009

“…1 Since 1993, six naturally occurring lysozyme variants associated with amyloid disease (I56T, F57I, F57I/T70N, W64R, D67H and T70N/W112R), [2][3][4][5] and one non-amyloidogenic variant (T70N), 6 have been discovered. In-depth characterization of two of the disease-associated variants, I56T and D67H, as well as the non-diseaserelated T70N variant, has been performed using a variety of biophysical techniques.…”

Section: Introductionmentioning

confidence: 99%

The Extracellular Chaperone Clusterin Potently Inhibits Human Lysozyme Amyloid Formation by Interacting with Prefibrillar Species

Kumita

Poon

Caddy

et al. 2007

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid disease. The aggregation of the amyloidogenic variant I56T is inhibited significantly at clusterin to lysozyme ratios as low as 1:80 (i.e. one clusterin molecule per 80 lysozyme molecules). Experiments indicate that under the conditions where inhibition of aggregation occurs, clusterin does not bind detectably to the native or fibrillar states of lysozyme, or to the monomeric transient intermediate known to be a key species in the aggregation reaction. Rather, it seems to interact with oligomeric species that are present at low concentrations during the lag (nucleation) phase of the aggregation reaction. This behavior suggests that clusterin, and perhaps other extracellular chaperones, could have a key role in curtailing the potentially pathogenic effects of the misfolding and aggregation of proteins that, like lysozyme, are secreted into the extracellular environment.

“…On this basis, the ability to form amyloid fibrils has been suggested to be a general property of the polypeptide chain. 5,[11][12][13] The single-point mutants of lysozyme I56T, F57I, W64R and D67H, [14][15][16][17] and the two double mutants F57I/T70N and W112R/T70N, 16,18 have been found to form amyloid plaques in individuals suffering from non-neuropathic hereditary amyloidosis. Another natural variant, T70N, has been found in 5% of the British population and 12% of the Caucasian Canadian population but has not so far been associated with disease.…”

Section: Introductionmentioning

confidence: 99%

Identification of the Core Structure of Lysozyme Amyloid Fibrils by Proteolysis

Frare

Mossuto

Laureto

et al. 2006

143

115

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.