title: Epitope mapping for monoclonal antibody reveals the activation mechanism for αVβ3 integrin.
abstract: Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs) were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.
authors.affiliation: Department of Anatomy, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan. kamata@z7.keio.jp
authors.forename: Tetsuji
authors.initials: T
authors.lastname: Kamata
authors.forename: Makoto
authors.initials: M
authors.lastname: Handa
authors.forename: Sonomi
authors.initials: S
authors.lastname: Takakuwa
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Sato
authors.forename: Yohko
authors.initials: Y
authors.lastname: Kawai
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Ikeda
authors.forename: Sadakazu
authors.initials: S
authors.lastname: Aiso
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D018604:Epitope Mapping; D005340:Fibrinogen / Q000378:metabolism; D006801:Humans; D039302:Integrin alphaVbeta3 / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D039302:Integrin alphaVbeta3; D005340:Fibrinogen