title: Topological investigation of amyloid fibrils obtained from beta2-microglobulin.
abstract: Amyloid fibrils of patients treated with regular hemodialysis essentially consists of beta2-microglobulin (beta2-m) and its truncated species DeltaN6beta2-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 beta2-m and truncated DeltaN6beta2-m was investigated by the limited proteolysis/mass spectrometry approach that appeared particularly suited to gain insights into the structure of beta2-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 beta2-m, was fully protected in the fibrillar form. In addition, the amino- and carboxy-terminal regions of beta2-m became exposed to the solvent in the fibrils, whereas they were masked completely in the native protein. These data indicate that beta2-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 beta2-m occurring in natural fibrils. On the basis of these data, a possible mechanism for fibril formation from native beta2-m is discussed and an explanation for the occurrence of truncated protein species in natural fibrils is given.
authors.affiliation: Dipartimento di Chimica Organica e Biochimica, Università di Napoli Federico II, via Cinthia 6, Italy.
authors.forename: Maria
authors.initials: M
authors.lastname: Monti
authors.forename: Serena
authors.initials: S
authors.lastname: Principe
authors.forename: Sofia
authors.initials: S
authors.lastname: Giorgetti
authors.forename: Palma
authors.initials: P
authors.lastname: Mangione
authors.forename: Gianpaolo
authors.initials: G
authors.lastname: Merlini
authors.forename: Anne
authors.initials: A
authors.lastname: Clark
authors.forename: Vittorio
authors.initials: V
authors.lastname: Bellotti
authors.forename: Angela
authors.initials: A
authors.lastname: Amoresano
authors.forename: Piero
authors.initials: P
authors.lastname: Pucci
mesh_terms: D000682:Amyloid / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008666:Metalloendopeptidases; D008854:Microscopy, Electron; D001613:beta 2-Microglobulin / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D000682:Amyloid; D001613:beta 2-Microglobulin; D010450:Endopeptidases; D002918:Chymotrypsin; D008666:Metalloendopeptidases; C058921:endoproteinase Asp-N