Authors
Maria Monti, Serena Principe, Sofia Giorgetti, Palma Mangione, Gianpaolo Merlini, Anne Clark, Vittorio Bellotti, Angela Amoresano, Piero Pucci
ARTICLETopological investigation of amyloid fibrils obtained from  2-microglobulin |
| Maria Monti 2 1, Serena Principe 1, Sofia Giorgetti 4 3, Palma Mangione 4 3, Gianpaolo Merlini 4 3, Anne Clark 5, Vittorio Bellotti 4 3, Angela Amoresano 1, Piero Pucci 2 1 * |
1Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, via Cinthia 6, 80126 Napoli, Italy
2CEINGE Biotecnologie Avanzate, scarl, Napoli, Italy
3Dipartimento di Biochimica, Centro Interdipartimentale di Biologia Applicata, Università di Pavia, Pavia, Italy.
4Laboratorio di Biotecnologie-Centro per lo studio delle Amiloidosi IRCCS, Policlinico S. Matteo, Pavia, Italy
5Diabetes Research Laboratories, Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Infirmary, Woodstock Road, Oxford, UK
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| email: Piero Pucci (pucci@unina.it) |
*Correspondence to Piero Pucci, Dipartimento di Chimica Organica e Biochimica, via Cinthia 6, I-80126 Napoli, Italy; fax: 39-081-674313.
Amyloidosis amyloid fibrils  2-microglobulin limited proteolysis mass spectrometry |
Amyloid fibrils of patients treated with regular hemodialysis essentially consists of 2-microglobulin (2-m) and its truncated species  N62-m lacking six residues at the amino terminus. The truncated fragment has a more flexible three-dimensional structure and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology of synthetic fibrils obtained from intact 2-m and truncated N62-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of 2-m within the fibrillar polymer. The distribution of prefential proteolytic sites observed in both fibrils revealed that the central region of the protein, which had been easily cleaved in the full-length globular 2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of 2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that 2-m molecules in the fibrils consist of an unaccessible core comprising residues 20-87 with the strands I and VIII being not constrained in the fibrillar polymer and exposed to the proteases. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to occur in vivo to generate the truncated forms of 2-m occuring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native 2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given. |
Received: 11 March 2002; Revised: 8 July 2002; Accepted: 9 July 2002
10.1110/ps.0206902
About DOI
10.1110/ps.0206902
About DOI
