Lysine 58-cleaved β2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis

Sofia Giorgetti, Monica Stoppini, Glenys A. Tennent, Annalisa Relini, Loredana Marchese, Sara Raimondi, Maria Monti, Sara Marini, Ole Østergaard, Niels H.H. Heegaard, Piero Pucci, Gennaro Esposito, Giampaolo Merlini, Vittorio Bellotti

    Research output: Contribution to journalArticle

    Abstract

    The lysine 58 cleaved and truncated variant of β2-microglobulin (ΔK58-β2m) 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 β2-microglobulin (β2m) 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 ΔK58-β2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of β2m. Using this approach, the two known principal isoforms found in β2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (ΔN6-β2m). In contrast, we found no evidence for the presence of ΔK58-β2m.

    Original languageEnglish (US)
    Pages (from-to)343-349
    Number of pages7
    JournalProtein Science
    Volume16
    Issue number2
    DOIs
    StatePublished - Feb 1 2007

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    Dialysis
    Amyloidosis
    Electrophoresis
    Amyloid
    Lysine
    Electrophoresis, Gel, Two-Dimensional
    Protein Isoforms
    Gels
    Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
    Protein Sequence Analysis
    Amyloid Plaques
    Immunoblotting
    Denaturation
    Proteomics
    Renal Dialysis
    Mass Spectrometry
    Purification
    Mass spectrometry
    Amino Acids
    Proteins

    Keywords

    • Amyloid fibrils
    • Dialysis related amyloidosis
    • Proteolized variants of β2-microglobulin
    • Proteomics

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology

    Cite this

    Lysine 58-cleaved β2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis. / Giorgetti, Sofia; Stoppini, Monica; Tennent, Glenys A.; Relini, Annalisa; Marchese, Loredana; Raimondi, Sara; Monti, Maria; Marini, Sara; Østergaard, Ole; Heegaard, Niels H.H.; Pucci, Piero; Esposito, Gennaro; Merlini, Giampaolo; Bellotti, Vittorio.

    In: Protein Science, Vol. 16, No. 2, 01.02.2007, p. 343-349.

    Research output: Contribution to journalArticle

    Giorgetti, S, Stoppini, M, Tennent, GA, Relini, A, Marchese, L, Raimondi, S, Monti, M, Marini, S, Østergaard, O, Heegaard, NHH, Pucci, P, Esposito, G, Merlini, G & Bellotti, V 2007, 'Lysine 58-cleaved β2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis', Protein Science, vol. 16, no. 2, pp. 343-349. https://doi.org/10.1110/ps.062563507
    Giorgetti, Sofia ; Stoppini, Monica ; Tennent, Glenys A. ; Relini, Annalisa ; Marchese, Loredana ; Raimondi, Sara ; Monti, Maria ; Marini, Sara ; Østergaard, Ole ; Heegaard, Niels H.H. ; Pucci, Piero ; Esposito, Gennaro ; Merlini, Giampaolo ; Bellotti, Vittorio. / Lysine 58-cleaved β2-microglobulin is not detectable by 2D electrophoresis in ex vivo amyloid fibrils of two patients affected by dialysis-related amyloidosis. In: Protein Science. 2007 ; Vol. 16, No. 2. pp. 343-349.
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    abstract = "The lysine 58 cleaved and truncated variant of β2-microglobulin (ΔK58-β2m) 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 β2-microglobulin (β2m) 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 ΔK58-β2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of β2m. Using this approach, the two known principal isoforms found in β2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (ΔN6-β2m). In contrast, we found no evidence for the presence of ΔK58-β2m.",
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    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

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    N2 - The lysine 58 cleaved and truncated variant of β2-microglobulin (ΔK58-β2m) 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 β2-microglobulin (β2m) 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 ΔK58-β2m, and extracts of amyloid fibrils from patients, to a discrete region of the gel distinct from other isoforms of β2m. Using this approach, the two known principal isoforms found in β2m amyloid were identified, namely, the full-length protein and the truncated species lacking six N-terminal amino acid residues (ΔN6-β2m). In contrast, we found no evidence for the presence of ΔK58-β2m.

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