title: Molecular characterization of a disease associated conformational epitope on GAD65 recognised by a human monoclonal antibody b96.11.
abstract: Autoantibodies to the 65kDa isoform of glutamate decarboxylase (GAD65) are associated with type I diabetes and recognise highly conformational epitope(s) that remain to be defined. The human recombinant Fab from mAb b96.11 inhibits binding of most GAD65 antibody positive sera from patients and its epitope has previously been localized to the middle region of GAD65. Recent studies indicate that b96.11 antibody specificity predicts the risk of developing type 1 diabetes in prediabetic individuals. We describe the use homology modelling, protein-protein docking simulations and biopanning of random peptide phage displayed libraries with b96.11 to predict contact amino acids on the interface of GAD65/Fab b96.11 complex. Further analysis by in vitro mutagenesis of GAD65 followed by radioimmunoprecipitation refined the amino acids contributing to the b96.11 epitope. Our studies show an interface characterized by a protruding antibody-combining site centered on the long heavy chain CDR3 loop of Fab b96.11 establishing interactions with the critical residue Phe(344) in the core of the epitope on GAD65, surrounded by charged sites within (375)RK(376) and (305)DER(307). The epitope requires residues from both middle and the C-terminal domains, and is the first precise definition of an epitope on GAD65. The nature of the b96.11 epitope leads to considerations of potential structural variations for differences in antigenicity between the isoforms GAD65 and GAD67. The study shows the utility of using a combination of in silico techniques and experimental data for molecular characterization and localization of conformational epitopes for which crystal structures are lacking.
authors.affiliation: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia.
authors.forename: Gustavo
authors.initials: G
authors.lastname: Fenalti
authors.forename: Christiane S
authors.initials: CS
authors.lastname: Hampe
authors.forename: Karen
authors.initials: K
authors.lastname: O'connor
authors.forename: J Paul
authors.initials: JP
authors.lastname: Banga
authors.forename: Ian R
authors.initials: IR
authors.lastname: Mackay
authors.forename: Merrill J
authors.initials: MJ
authors.lastname: Rowley
authors.forename: Ossama
authors.initials: O
authors.lastname: El-Kabbani
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001327:Autoimmune Diseases / Q000201:enzymology / Q000276:immunology* / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology*; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal; D000939:Epitopes; D007140:Immunoglobulin Fab Fragments; D007527:Isoenzymes; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2