Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Mapping the epitope for a monoclonal antibody derived from an autoimmune response against glutamic acid decarboxylase 65 (#179)

David E Hoke , Liah Clark , James Green , Kasper D Rand , Susanne Stander , John Jimma , Tam TTN Nguyen , Nathan P Cowieson , Ashley M Buckle 1
  1. Monash University, Clayton, VIC, Australia

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is produced by two distinct genetically encoded proteins named glutamic acid decarboxylase (GAD) 65 kDa (GAD65) and GAD67. While these enzymes are important for neurotransmitter homeostasis, they have also been shown to be differentially recognised by autoantibodies during the pathology of type I, autoimmune diabetes.  Since these proteins share 70% sequence identity, determining the aetiology of this differential immune response is important in identifying the underlying cause of autoimmune disorders.  Our lab previously determined the crystal structures of GAD65 and GAD67 and also showed increased flexibility in the GAD65 isoform which is a target of antibody responses in type I diabetes.  It is hypothesised that subtle structural differences between the two proteins are responsible for the ability of antibodies to recognise GAD65 but not GAD67.  Previous studies aimed at determining the epitope for a patient-derived monoclonal antibody (b96.11) were made before the structures of the two GAD isoforms were known and before an appreciation of the different dynamics of the two proteins.  These pre-structural experiments led to mutagenesis studies that are predicted to alter the structure and/or dynamics of GAD65 in ways that would confound the results of epitope mapping studies.  In this work, we have used two unbiased approaches to determine the epitope that b96.11 binds within GAD65.  SAXS data on a complex between GAD65 and b96.11 yields a set of distance restraints for the complex.  Secondly, hydrogen-deuterium exchange mass spectrometry was used to find protected regions on GAD65 when incubated with b96.11.  These data are used to provide a detailed molecular model of the GAD65 / b96.11 interaction.