Journal of Molecular Biology
A Highly Amyloidogenic Region of Hen Lysozyme
Introduction
The deposition of naturally soluble proteins as amyloid fibrils underlies a large family of clinical disorders including Alzheimer's disease, the spongiform encephalopathies and type II diabetes disease.1., 2. Because of the enormous importance of these diseases in the context of present-day human health and welfare, the molecular mechanism that leads to amyloid formation is under intensive investigation in many laboratories.3., 4. The results of a variety of recent studies have indicated that amyloid fibril formation from globular native proteins occurs via partially unfolded intermediates that subsequently associate, ultimately to form well-ordered mature fibrils.3., 5. Formation of such intermediates appears to be critical for the onset of fibril formation, since these species are capable of strong intermolecular interactions due to the exposure of at least part of the polypeptide main chain and hydrophobic side-chains that are otherwise buried in the overall fold of the native protein. This view has led to the proposal that all polypeptide chains can, in principle, form amyloid aggregates under the appropriate experimental conditions.5., 6.
A large proportion of physiologically relevant amyloid deposits in tissue are made up of protein fragments derived from relatively larger protein precursors.5., 7. For example, the fibrils associated with Alzheimer's disease are formed by peptide fragment(s) resulting from the limited proteolysis of the amyloid precursor protein (APP) by secretases. Other examples of amyloidogenic polypeptides for which fragmentation or the proteolytic processing of a protein precursor precedes amyloid deposition include serum amyloid A protein, gelsolin, apolipoprotein A1, prolactin, calcitonin, BriL, transthyretin, medin and fibrinogen among others.5., 7. Protein fragments derived by limited proteolysis of proteins are particularly vulnerable to aggregation because they can usually only adopt, at most, partially folded states, and cannot establish the long-range interactions present in the original intact native protein or precursor. In particular, a common property of many fragments is that they contain hydrophobic clusters of residues that can trigger protein aggregation.8 Protein fragmentation by limited proteolysis, therefore, appears to be an important phenomenon underlying a substantial proportion of amyloid diseases.
Human lysozyme has been shown to form amyloid fibrils in individuals suffering from non-neuropathic systemic amyloidosis, all of which have point mutations in the lysozyme gene.9., 10., 11. The individuals affected are heterozygous for the mutation and carry amyloid deposits in different tissues containing the variant but not wild-type lysozyme.12 The formation of amyloid deposits is usually slow, but is generally fatal by the fifth decade of life. The only effective treatment at present involves transplantation of damaged organs, such as kidneys, when amyloid deposition causes their malfunction.12 The properties of two amyloidogenic lysozyme mutants (I56T and D67H) have been studied in detail and, when compared to those of the wild-type protein, the mutants were found to have reduced structural stability and altered folding kinetics.13., 14., 15. Moreover, in a recent study, it has been reported that the amyloidogenic mutant D67H of human lysozyme shows a significant destabilization of the β-domain (residues 40–82) and the adjacent C-helix (residues 89–100), regions which have been suggested to be critical for the amyloid formation process.16., 17., 18., 19. It has been hypothesized that protein aggregation derives from a lower thermodynamic stability and structural cooperativity of the mutant proteins giving rise to an increased propensity to adopt a partially unfolded state.14
Wild-type human lysozyme was found to form amyloid fibrils that are similar to those extracted from pathological deposits when incubated in vitro at pH 2.0 for several days.18 Krebs et al.19 obtained amyloid aggregates also from hen egg-white lysozyme (HEWL) utilizing a variety of methods, such as incubation of the protein in acidic solution (pH 2.0–4.0) at elevated temperatures (37–65 °C) for several days. Amyloid aggregates of HEWL can also be obtained by incubating the protein at neutral pH in the presence of trifluoroethanol19 or ethanol,20 both of which destabilize the native protein structure and favor protein aggregation. In a recent study, it was shown that reduction of the four disulfide bonds in HEWL causes the protein to form amyloid fibrils more readily than does the fully native protein.21 It has also been shown that bovine α-lactalbumin, a member of the lysozyme/lactalbumin superfamily, with a native structure very similar to that of lysozyme despite its low level of amino acid sequence identity, forms fibrils much more easily if three of its four disulfide bridges are reduced.22 These results support the principle that the ready conversion of a protein into the amyloid structure requires at least partial unfolding prior to aggregation to fibrils. These findings are also consistent with the observation that the lower thermodynamic stability of the pathological mutants of human lysozyme allows the formation of a partially unfolded intermediate able to trigger amyloid formation under much milder experimental conditions than those required by the wild-type protein.14
In the present study, we have analyzed the formation of amyloid fibrils by HEWL, as this protein is readily available and represents an excellent experimental system through which to study the determinants of protein aggregation, being highly homologous to human lysozyme in structure, although only 40% identical in its sequence. Here, amyloid fibrils from HEWL were obtained under rather harsh solution conditions involving incubation of the protein for several days at pH 2.0 and 65 °C. Analysis of the fibrils formed in this way shows that they are composed mainly of fragments, corresponding primarily to peptides involving residues 49–101 of HEWL. In order to gain further insights into the aggregation properties of HEWL, the propensities of different fragments of the protein to aggregate were examined. In order to achieve this objective, protein fragments were produced by limited proteolysis of HEWL using pepsin in acidic solution.23., 24., 25., 26., 27. Two fragments present in high abundance, 57–107 and 1–38/108–129, were isolated and purified to homogeneity. Fragment 57–107 encompasses part of the β-domain and all the amino acid residues forming the C-helix of the native protein and contains two intramolecular disulfides Cys64–Cys80 and Cys76–Cys94. Fragment 1–38/108–129 consists of fragments 1–38 and 108–129 covalently linked by the two disulfide bridges Cys6–Cys127 and Cys30–Cys115; it contains α-helices A, B and D of native HEWL. The conformational and aggregation properties of these two fragment species were evaluated by circular dichroism (CD) measurements and electron microscopy (EM). It was found that only fragment 57–107 readily forms amyloid fibrils under the solution conditions used here and is therefore likely to represent a key region responsible for triggering the aggregation process of the entire protein.
Section snippets
Characterization of amyloid fibrils formed from degradation of HEWL at low pH
Amyloid fibrils from HEWL were obtained by incubating concentrated solutions of the protein (1 mM) in 10 mM HCl (pH 2.0) at 65 °C for up to ten days. Following this procedure, HEWL forms protein aggregates with typical amyloid features.19 In particular, long, straight and unbranched protein fibrils with diameters of ∼10 nm are clearly evident in EM images (Figure 1A). In addition, some fibrils show a clear twisting of protofilaments, as often observed with amyloid fibrils. HEWL fibril samples
Discussion
Wild-type human and hen lysozymes can be induced to aggregate into amyloid fibrils when incubated at pH 2.0 or at neutral pH in the presence of trifluoroethanol, ethanol or moderate concentrations of protein denaturants.17., 18., 19., 20. Here, we have shown that incubation of HEWL for ten days at pH 2.0 and 65 °C, results in fibrils that are largely composed of fragments deriving from the partial acid hydrolysis of the protein at Asp-X (and X-Asp) peptide bonds. This protein fragmentation is
Materials
Hen egg-white lysozyme (HEWL), porcine pepsin and thioflavin-T (ThT) were purchased from the Sigma Chemical Company (St. Louis, MO). All other chemicals were of analytical reagent grade and were obtained from Sigma or Fluka (Buchs, Switzerland).
Preparation and characterization of amyloid fibrils
HEWL amyloid fibrils were prepared by incubating protein samples (1 mM) in 10 mM HCl at pH 2.0 and 65 °C for up to ten days. Aggregation of HEWL fragments 57–107 and 1–38/108–129 was monitored by incubating the fragments (0.55 mM) in 10 mM HCl at pH 2.0
Acknowledgements
We thank Mr Giuseppe Tognon for assistance with electron microscopy measurements and Dr Maria Francesca Mossuto for having conducted some of the experiments reported here. The critical reading of the manuscript by Dr Mark Krebs is also gratefully acknowledged. This work was supported by the Italian Ministry of University and Research (FIRB Project on Protein Folding and PRIN 2003), the Wellcome and Leverhulme Trusts (to J.Z. and C.M.D.) and a BBSRC grant (to C.M.D.).
References (68)
Protein misfolding, evolution and disease
Trends Biochem. Sci.
(1999)Protein aggregation: folding aggregates, inclusion bodies and amyloid
Fold. Des.
(1998)- et al.
Hereditary renal amyloidosis caused by a new variant lysozyme W64R in a French family
Kidney Int.
(2002) - et al.
A novel lysozyme mutation Phe57Ile associated with hereditary renal amyloidosis
Kidney Int.
(2003) - et al.
Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants
J. Struct. Biol.
(2000) - et al.
Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the beta-domain
J. Mol. Biol.
(2000) - et al.
Probing the partly folded states of proteins by limited proteolysis
Fold. Des.
(1997) - et al.
Probing the conformational state of apomyoglobin by limited proteolysis
J. Mol. Biol.
(1997) Cleavage at aspartic acid
Methods Enzymol.
(1967)Cleavage at aspartic acid
Methods Enzymol.
(1983)
Circular dichroism
Methods Enzymol.
The application of circular dichroism to study of protein folding and unfolding
Biochim. Biophys. Acta
The structure of amyloid fibrils by electron microscopy and X-ray diffraction
Advan. Protein Chem.
Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration
J. Mol. Biol.
Understanding how proteins fold: the lysozyme story so far
Trends Biochem. Sci.
Trifluoroethanol-assisted protein folding: fragment 53–103 of bovine alpha-lactalbumin
Biochim. Biophys. Acta
Identification of the peptide region that folds into a native conformation in the early stage of the renaturation of reduced lysozyme
Biochem. Biophys. Res. Commun.
Conformational properties of four peptides spanning the sequence of hen lysozyme
J. Mol. Biol.
Prediction of amyloid fibril-forming proteins
J. Biol. Chem.
Structural and folding dynamic properties of the T70N variant of human lysozyme
J. Biol. Chem.
Age-dependent deamidation of asparagine residues in proteins
Expt. Gerontol.
Structural alterations in the peptide backbone of beta-amyloid core protein may account for its deposition and stability in Alzheimer's disease
J. Biol. Chem.
Measurement of altered aspartyl residues in the scrapie associated form of prion protein
Biochem. Biophys. Res. Commun.
Calculation of protein extinction coefficients from amino acid sequence data
Anal. Biochem.
The amino acid sequence of egg-white lysozyme
J. Biol. Chem.
Folding proteins in fatal ways
Nature
Therapeutic approaches to protein-misfolding diseases
Nature
Protein aggregation and aggregate toxicity: new insights into protein folding, misfolding diseases and biological evolution
J. Mol. Med.
Protein folding and disease: a view from the first Horizon symposium
Nature Rev. Drug Discov.
Therapeutic strategies for human amyloid diseases
Nature Rev. Drug Discov.
Human lysozyme gene mutations cause hereditary systemic amyloidosis
Nature
Hereditary systemic amyloidosis with renal involvement
J. Nephrol.
The structure, stability, and folding process of amyloidogenic mutant human lysozyme
J. Biochem.
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