Characterisation of an autoreactive conformational epitope on GAD65 recognised by the human monoclonal antibody b78 using a combination of phage display, in vitro mutagenesis and molecular modelling.
Autoantibodies to the diabetes autoantigen, the 65kDa isoform of glutamic acid decarboxylase (GAD65), react with conformational epitopes defined according to linear sequences but not according to structural information, or contact sites with the antibody paratope. To ascertain such information for an exemplary human monoclonal antibody (mAb) to GAD65, b78, we combined antibody screening of phage-displayed peptide libraries, alanine mutagenesis of selected motifs, homology modelling of the PLP and C-terminal regions of GAD65, and molecular dynamics to examine for structural effects of mutagenesis. By phage display, mAb b78 selected phagotopes containing acidic residues (D, E), hydrophobic residues (Y, F or W) and LRS that localised to a possible surface-exposed conformational epitope on the combined homology model. Alanine mutants of GAD65 based on deduced contact residues were examined for binding with b78 and control sera. Mutation of (524)SRL(526), (572)DF(573) and (498)KPQ(500) reduced reactivity of b78 with mutant GAD65 > 50%. Molecular dynamics indicated that mutation of (498)KPQ(500) caused structural changes that could account for effects of this mutation. Thus phage display in combination with molecular modelling identified contact residues within a highly conformational epitope for mAb b78 in the C-terminus of GAD65. These techniques should have broad applicability to definition of epitope structure.
O'Connor
Karen H
KH
Department of Biochemistry and Molecular Biology, Monash University, Wellington Road, Clayton, Victoria 3800, Australia.
Banga
J Paul
JP
Darmanin
Connie
C
El-Kabbani
Ossama
O
Mackay
Ian R
IR
Rowley
Merrill J
MJ
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015551:Autoimmunity / Q000276:immunology; D018604:Epitope Mapping; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007074:Immunoglobulin G / Q000276:immunology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019151:Peptide Library; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007074:Immunoglobulin G; D007527:Isoenzymes; D019151:Peptide Library; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2