title: Evaluating the conformation of recombinant domain I of β(2)-glycoprotein I and its interaction with human monoclonal antibodies.
abstract: Pathogenic antiphospholipid antibodies (aPL) cause the antiphospholipid syndrome (APS) by interacting with domain I (DI) of beta-2-glycoprotein I (β(2)GPI). The aPL/β(2)GPI complex then exerts pathogenic effects on target cells. We previously described periplasmic bacterial expression of native and mutated variants of DI, and reported the presence of immunodominant epitopes at positions 8-9 (D8/D9) and position 39 (R39). Mutations at these positions strongly influenced the ability of recombinant DI to bind patient-derived IgG aPL and to inhibit pathogenic effects of these aPL in a mouse model of APS. We now describe an improved cytoplasmic bacterial expression system allowing higher yield of DI. We demonstrate that the nuclear magnetic resonance (NMR) spectra of a (15)N,(13)C-isotope-labelled sample of the recombinant DI protein exhibit properties consistent with the structure of DI in crystal structure of intact β(2)GPI. Mutations at D8/D9 and R39 had limited impact on the NMR spectrum of DI indicating maintenance of the overall fold of the DI domain. We investigated interactions between five variants of DI and ten monoclonal human IgG antibodies, all derived from the IgG aPL antibody IS4 by sequence manipulation and in vitro expression. Arginine residues at positions 100 and 100g in IS4V(H) CDR3 play a particularly important role in binding to DI, but this is unlikely to be due to electrostatic interactions with negatively charged amino acids on DI. Both the strength of binding to DI and the ability to discriminate different DI variants varies between the different IgG antibodies tested. There was no simple relationship between these binding properties and antibody pathogenicity.
authors: Centre for Rheumatology, Division of Medicine, University College London, London WC1E 6JF, UK. c.pericleous@ucl.ac.uk
authors: Charis
authors: C
authors: Pericleous
authors: Jennifer
authors: J
authors: Miles
authors: Diego
authors: D
authors: Esposito
authors: Acely
authors: A
authors: Garza-Garcia
authors: Paul C
authors: PC
authors: Driscoll
authors: Anastasia
authors: A
authors: Lambrianides
authors: David
authors: D
authors: Latchman
authors: David
authors: D
authors: Isenberg
authors: Anisur
authors: A
authors: Rahman
authors: Yiannis
authors: Y
authors: Ioannou
authors: Ian
authors: I
authors: Giles
mesh_terms: D017152:Antibodies, Antiphospholipid / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D016736:Antiphospholipid Syndrome / Q000276:immunology; D001666:Binding Sites, Antibody / Q000276:immunology; D006801:Humans; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D016133:Polymerase Chain Reaction; D011485:Protein Binding / Q000276:immunology; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology; D053482:beta 2-Glycoprotein I / Q000737:chemistry* / Q000276:immunology*
chemical_list: D017152:Antibodies, Antiphospholipid; D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D053482:beta 2-Glycoprotein I