title: Two amino acids in glutamic acid decarboxylase act in concert for maintenance of conformational determinants recognised by Type I diabetic autoantibodies.
abstract: OBJECTIVE
  Glutamic acid decarboxylase 65 is a major autoantigen in Type I (insulin-dependent) diabetes mellitus, autoimmune polyendocrine syndrome and stiff-man syndrome. These disorders are characterised by the presence of multiple autoantibodies to the autoantigen which can be distinguished in a variety of different ways. We have investigated the role of single amino-acid mutations in glutamic acid decarboxylase 65 in distinguishing the binding of serum antibodies and a variety of patient-derived human IgG monoclonal antibodies directed to different determinants of the autoantigen.
  
  
  METHODS
  We identified a mutant of glutamic acid decarboxylase 65 that contained four single amino-acid mutations from the wild-type molecule. The role of these mutations was investigated by site-directed mutagenesis. We investigated the binding of patient-derived serum antibodies to glutamic acid decarboxylase 65 to a number of single and double amino-acid mutants using immunoprecipitation with labelled, recombinant antigen. To overcome the heterogeneity of different anti-glutamic acid decarboxylase 65 antibodies present in a patient's serum, the binding of a panel of eleven patient-derived human monoclonal antibodies recognising different determinants on the autoantigen was also studied.
  
  
  RESULTS
  Two replacements in glutamic acid decarboxylase 65 at Asn247Ser and Leu574Pro were identified that preferentially influence the anti-glutamic acid decarboxylase 65 serum antibodies of Type I diabetic patients, without statistically significantly effecting those recognised in other disorders. Single or double amino-acid replacements Asn247Ser and Leu574Pro in the autoantigen showed differential affects on expression of epitopes recognised by the human monoclonals. The double replacement of Asn247Ser and Leu574Pro in glutamic acid decarboxylase 65 resulted in the loss of binding of all eleven human monoclonal antibodies, irrespective of their epitope recognition. In contrast, single replacement of Leu574Pro statistically significantly reduced the binding of some carboxyl terminal-directed antibodies such as MICA 1, MICA 3 and DP-A without influencing the binding of other monoclonals. Replacement of Asn247Ser did not, however, influence the binding of any patients serum or human monoclonal antibodies.
  
  
  CONCLUSIONS
  Two distantly spaced amino acids, Asn247 and Leu574 in glutamic acid decarboxylase 65 were identified that act in concert to greatly influence the conformational structure of the autoantigen and statistically significantly influence the binding of antibodies present in Type I diabetic sera. The single or double amino-acid mutants can be used to distinguish some anti-glutamic acid decarboxylase-65 autoantibodies and could prove useful in distinguishing Type I diabetic from autoimmune polyendocrine syndrome and stiff-man syndrome patients' sera as well as to study changes in antibody patterns during disease progression.
authors.affiliation: Guy's, King's, St Thomas' School of Medicine, Division of Medicine, London, UK.
authors.forename: T I
authors.initials: TI
authors.lastname: Tree
authors.forename: N G
authors.initials: NG
authors.lastname: Morgenthaler
authors.forename: N
authors.initials: N
authors.lastname: Duhindan
authors.forename: K E
authors.initials: KE
authors.lastname: Hicks
authors.forename: A M
authors.initials: AM
authors.lastname: Madec
authors.forename: W A
authors.initials: WA
authors.lastname: Scherbaum
authors.forename: J P
authors.initials: JP
authors.lastname: Banga
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001323:Autoantibodies / Q000097:blood* / Q000737:chemistry; D001483:Base Sequence; D001666:Binding Sites, Antibody; D017931:DNA Primers; D003924:Diabetes Mellitus, Type 2 / Q000097:blood / Q000276:immunology*; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood / Q000737:chemistry; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D017931:DNA Primers; D007074:Immunoglobulin G; D007527:Isoenzymes; D011994:Recombinant Proteins; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2