title: Human single-chain variable fragment antibody inhibits macrophage migration inhibitory factor tautomerase activity.
abstract: Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine, secreted from a variety of immune cells, that regulates innate and adaptive immune responses. Elevation of MIF levels in plasma correlates with the severity of inflammatory diseases in humans. Inhibition of MIF or its tautomerase activity ameliorates disease severity by reducing inflammatory responses. In this study, the human single-chain variable fragment (HuScFv) antibody specific to MIF was selected from the human antibody phage display library by using purified recombinant full-length human MIF (rMIF) as the target antigen. Monoclonal HuScFv was produced from phage-transformed bacteria and tested for their binding activities to rMIF by indirect enzyme-linked immunosorbent assay as well as to native MIF by western blot analysis and immunofluorescence assay. The HuScFv with highest binding signal to rMIF also inhibited the tautomerase activities of both rMIF and native MIF in human monoblastic leukemia (U937) cells in a dose-dependent manner. Mimotope searching and molecular docking concordantly demonstrated that the HuScFv interacted with Lys32 and Ile64 in the MIF tautomerase active site. To the best of our knowledge, this is the first study to focus on MIF-specific fully-human antibody fragment with a tautomerase-inhibitory effect that has potential to be developed as anti-inflammatory biomolecules for human use.
authors.affiliation: Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Mayuri
authors.initials: M
authors.lastname: Tarasuk
authors.affiliation: Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Ornnuthchar
authors.initials: O
authors.lastname: Poungpair
authors.affiliation: Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Duangporn
authors.initials: D
authors.lastname: Ungsupravate
authors.affiliation: Department of Biochemistry, Faculty of Sciences, Kasetsart University, Bangkok, Thailand.
authors.forename: Kunan
authors.initials: K
authors.lastname: Bangphoomi
authors.affiliation: Laboratory for Research and Technology Development, Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
authors.affiliation: Division of Molecular Medicine, Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Pa-Thai
authors.initials: PT
authors.lastname: Yenchitsomanus
mesh_terms: D000893:Anti-Inflammatory Agents / Q000008:administration & dosage*; D020134:Catalytic Domain; D045744:Cell Line, Tumor; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007113:Immunity, Innate; D019746:Intramolecular Oxidoreductases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D007938:Leukemia / Q000188:drug therapy* / Q000276:immunology; D008263:Macrophage Migration-Inhibitory Factors / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism*; D011485:Protein Binding / Q000276:immunology; D057127:Single-Chain Antibodies / Q000008:administration & dosage* / Q000378:metabolism
chemical_list: D000893:Anti-Inflammatory Agents; D008263:Macrophage Migration-Inhibitory Factors; D057127:Single-Chain Antibodies; D019746:Intramolecular Oxidoreductases; C515598:MIF protein, human