The non-glycosylated N-terminal domain of human thrombopoietin is a molten globule under native conditions

Shigeki Arai, Chie Shibazaki, Motoyasu Adachi, Yoshitake Maeda, Tomoyuki Tahara, Takashi Kato, Hiroshi Miyazaki, Ryota Kuroki

    Research output: Contribution to journalArticle

    Abstract

    Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1–163 residues of hTPO (hTPO163) including the N-terminal active site domain (1–153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO163 is higher than that of the full-length glycosylated hTPO (ghTPO332). In order to clarify the structural and physicochemical properties of hTPO163, circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO163 undergoes an induced-fit conformational change (+19.0% for helix and −16.7% for β-strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO163 is a multistate transition; hTPO163 is thermally stabilized upon receptor (c-Mpl) binding, as indicated with raising the midpoint (Tm) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO163 indicate that hTPO163 exists as a molten globule under native conditions, which may enable the induced-fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C-terminal domain (154–332 residues) and glycosylation assists the folding of the N-terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO163 might be reduced, if the conformational variability of hTPO163 is restricted by mutation and/or by the addition of C-terminal domain with glycosylation to keep its conformation suitable for the c-Mpl recognition.

    Original languageEnglish
    JournalFEBS Journal
    DOIs
    Publication statusPublished - 2019 Jan 1

    Fingerprint

    Thrombopoietin
    Circular Dichroism
    Glycosylation
    Molten materials
    Differential Scanning Calorimetry
    Differential scanning calorimetry
    Thrombopoiesis
    Antibody Affinity
    Antibodies
    Transition Temperature
    Platelets
    Neutralizing Antibodies
    Thrombocytopenia
    Conformations
    Catalytic Domain
    Intercellular Signaling Peptides and Proteins
    Blood Platelets
    Hot Temperature
    Ligands
    Mutation

    Keywords

    • CD
    • DSC
    • human thrombopoietin
    • molten globule

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

    Cite this

    The non-glycosylated N-terminal domain of human thrombopoietin is a molten globule under native conditions. / Arai, Shigeki; Shibazaki, Chie; Adachi, Motoyasu; Maeda, Yoshitake; Tahara, Tomoyuki; Kato, Takashi; Miyazaki, Hiroshi; Kuroki, Ryota.

    In: FEBS Journal, 01.01.2019.

    Research output: Contribution to journalArticle

    Arai, Shigeki ; Shibazaki, Chie ; Adachi, Motoyasu ; Maeda, Yoshitake ; Tahara, Tomoyuki ; Kato, Takashi ; Miyazaki, Hiroshi ; Kuroki, Ryota. / The non-glycosylated N-terminal domain of human thrombopoietin is a molten globule under native conditions. In: FEBS Journal. 2019.
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    abstract = "Human thrombopoietin (hTPO) is a primary hematopoietic growth factor that regulates megakaryocytopoiesis and platelet production. The non-glycosylated form of 1–163 residues of hTPO (hTPO163) including the N-terminal active site domain (1–153 residues) is a candidate for treating thrombocytopenia. However, the autoantigenicity level of hTPO163 is higher than that of the full-length glycosylated hTPO (ghTPO332). In order to clarify the structural and physicochemical properties of hTPO163, circular dichroism (CD) and differential scanning calorimetry (DSC) analyses were performed. CD analysis indicated that hTPO163 undergoes an induced-fit conformational change (+19.0{\%} for helix and −16.7{\%} for β-strand) upon binding to the neutralizing antibody TN1 in a manner similar to the coupled folding and binding mechanism. Moreover, DSC analysis showed that the thermal transition process of hTPO163 is a multistate transition; hTPO163 is thermally stabilized upon receptor (c-Mpl) binding, as indicated with raising the midpoint (Tm) temperature of the transition by at least +9.5 K. The conformational variability and stability of hTPO163 indicate that hTPO163 exists as a molten globule under native conditions, which may enable the induced-fit conformational change according to the type of ligands (antibodies and receptor). Additionally, CD and computational analyses indicated that the C-terminal domain (154–332 residues) and glycosylation assists the folding of the N-terminal domain. These observations suggest that the antibody affinity and autoantigenicity of hTPO163 might be reduced, if the conformational variability of hTPO163 is restricted by mutation and/or by the addition of C-terminal domain with glycosylation to keep its conformation suitable for the c-Mpl recognition.",
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    AU - Arai, Shigeki

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    AU - Maeda, Yoshitake

    AU - Tahara, Tomoyuki

    AU - Kato, Takashi

    AU - Miyazaki, Hiroshi

    AU - Kuroki, Ryota

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