Lysine 58-cleaved beta2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis

TY - JOUR

T1 - Lysine 58-cleaved beta2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis

AU - Giorgetti, Sofia

AU - Stoppini, Monica

AU - Tennent, Glenys A

AU - Relini, Annalisa

AU - Marchese, Loredana

AU - Raimondi, Sara

AU - Monti, Maria

AU - Marini, Sara

AU - Østergaard, Ole

AU - Heegaard, Niels H H

AU - Pucci, Piero

AU - Esposito, Gennaro

AU - Merlini, Giampaolo

AU - Bellotti, Vittorio

PY - 2007/2

Y1 - 2007/2

N2 - The lysine 58 cleaved and truncated variant of beta(2)-microglobulin (DeltaK58-beta2m) is conformationally unstable and present in the circulation of a large percentage of patients on chronic hemodialysis, suggesting that it could play a role in the beta2-microglobulin (beta2m) amyloid fibrillogenesis associated with dialysis-related amyloidosis (DRA). However, it has yet to be detected in the amyloid deposits of such patients. Here, we extracted amyloid fibrils, without denaturation or additional purification, from different amyloidotic tissues of two unrelated individuals suffering from DRA, and characterized them by high-sensitivity bidimensional gel electrophoresis (2D-PAGE), immunoblotting, MALDI time-of-flight mass spectrometry, and protein sequencing. To confirm whether or not this species could be identified by our proteomic approaches, we mapped its location in 2D-PAGE, in mixtures of pure DeltaK58-beta2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of beta2m. Using this approach, the two known principal isoforms found in beta2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (DeltaN6-beta2m). In contrast, we found no evidence for the presence of DeltaK58-beta2m.

AB - The lysine 58 cleaved and truncated variant of beta(2)-microglobulin (DeltaK58-beta2m) is conformationally unstable and present in the circulation of a large percentage of patients on chronic hemodialysis, suggesting that it could play a role in the beta2-microglobulin (beta2m) amyloid fibrillogenesis associated with dialysis-related amyloidosis (DRA). However, it has yet to be detected in the amyloid deposits of such patients. Here, we extracted amyloid fibrils, without denaturation or additional purification, from different amyloidotic tissues of two unrelated individuals suffering from DRA, and characterized them by high-sensitivity bidimensional gel electrophoresis (2D-PAGE), immunoblotting, MALDI time-of-flight mass spectrometry, and protein sequencing. To confirm whether or not this species could be identified by our proteomic approaches, we mapped its location in 2D-PAGE, in mixtures of pure DeltaK58-beta2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of beta2m. Using this approach, the two known principal isoforms found in beta2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (DeltaN6-beta2m). In contrast, we found no evidence for the presence of DeltaK58-beta2m.

KW - Amyloid/chemistry

KW - Amyloidosis/metabolism

KW - Electrophoresis, Gel, Two-Dimensional

KW - Humans

KW - Immunoblotting

KW - Lysine/chemistry

KW - Microscopy, Atomic Force

KW - Renal Dialysis

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - beta 2-Microglobulin/chemistry

U2 - 10.1110/ps.062563507

DO - 10.1110/ps.062563507

M3 - Journal article

C2 - 17242436

SN - 0961-8368

VL - 16

SP - 343

EP - 349

JO - Protein Science

JF - Protein Science

IS - 2

ER -