title: Epitope mapping of monoclonal antibody to integrin alphaL beta2 hybrid domain suggests different requirements of affinity states for intercellular adhesion molecules (ICAM)-1 and ICAM-3 binding.
abstract: Integrin undergoes different activation states by changing its quaternary conformation. The integrin beta hybrid domain acts as a lever for the transmission of activation signal. The displacement of the hybrid domain can serve to report different integrin activation states. The monoclonal antibody (mAb) MEM148 is a reporter antibody that recognizes Mg/EGTA-activated but not resting integrin alpha(L) beta2. Herein, we mapped its epitope to the critical residue Pro374 located on the inner face of the beta2 hybrid domain. Integrin alpha(L) beta2 binds to its ligands ICAM-1 and ICAM-3 with different affinities. Integrin is proposed to have at least three affinity states, and the position of the hybrid domain differs in each. We made use of the property of mAb MEM148 to analyze and correlate these affinity states in regard to alpha(L) beta2/intercellular adhesion molecule (ICAM) binding. Our study showed that Mg/EGTA-activated alpha(L)beta2 can adopt a different conformation from that activated by activating mAbs KIM185 or MEM48. Unlike ICAM-1 binding, which required only one activating agent, alpha(L) beta2/ICAM-3 binding required both Mg/EGTA and an activating mAb. This suggests that alpha(L)beta2 with intermediate affinity is sufficient to bind ICAM-1 but not ICAM-3, which requires a high affinity state. Furthermore, we showed that the conformation adopted by alpha(L)beta2 in the presence of Mg/EGTA, depicting an intermediate activation state, could be reverted to its resting conformation.
authors.affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551 and Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
authors.forename: Ren-Hong
authors.initials: RH
authors.lastname: Tang
authors.forename: Emilia
authors.initials: E
authors.lastname: Tng
authors.forename: S K Alex
authors.initials: SK
authors.lastname: Law
authors.forename: Suet-Mien
authors.initials: SM
authors.lastname: Tan
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D015703:Antigens, CD / Q000737:chemistry*; D015815:Cell Adhesion Molecules; D045744:Cell Line, Tumor; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000737:chemistry; D018604:Epitope Mapping / Q000379:methods; D000939:Epitopes / Q000737:chemistry; D006801:Humans; D047468:Immunoprecipitation; D018799:Intercellular Adhesion Molecule-1 / Q000737:chemistry*; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D000939:Epitopes; C483616:ICAM3 protein, human; D008024:Ligands; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1; D004533:Egtazic Acid; D011392:Proline