Molecular mimicries of linear epitopes between pancreatic glutamic acid decarboxylase (GAD65) and proteins from Escherichia coli

Type1 diabetes (T1D) is characterised by autoimmunity tob-cellauto-antigens such as GAD65 and insulin. GAD auto-antibodiestarget epitopes whose isoforms may present in microorganisms, andcontribute as an environmental factor in development of diabeticautoimmune responses. This study searched a range of micro-organisms for GAD 65 mimicries. Purified GAD protein was pro-duced using Halo-Tag technology in Chinese Hamster Ovary (CHO)cells, and used to stimulate production of GAD antiserum in mice.The GAD antiserum was titrated and used to screen total proteinsamples from 40 bacteria and five yeasts, using a dot blot technique.Interestingly, strong immunological reaction was seen with bacteriaof the Enterobacteriaceae family. The positive samples were sub-jected to western blotting which produced immune active proteinbands. ForE. colifour bands were detected (40, 36, 22, 18 kDa).These were identified using mass spectrometry as an outer mem-brane protein A, formate dehydrogenase, superoxide dismutase andDNA starvation protein, respectively. These proteins expressed sig-nificant epitopes 1, 4, 4, 2, respectively, with strong homology (upto 70%) with the GAD epitopes. It was found that one epitope fromE. coliwas highly similar to several epitopes on GAD, whereas otherepitopes fromE. coliwere similar to a single and different epitopeon GAD. All these epitopes occur at the C-terminal region of GAD(residues 419565), a region previously reported to be targeted byauto-antibodies. This suggests that those epitopes may be inducers for autoimmunity particularly in individuals who are immune-compromised or genetically predisposed for T1D.