In silico identification and wet lab validation of novel cryptic B cell epitopes in ZnT8 zinc transporter autoantigen

Zinc transporter 8 (ZnT8) is a novel immunodominant autoantigen, associated with Type-1 diabetes. A non-synonymous polymorphism (R325W) in its gene is associated with Type-2 diabetes. In this study, we performed an in silico B cell epitope prediction followed by wetlab validation of ZnT8. Apart from the previously characterized polymorphic epitope (BE-5 TAASR*DS), two novel epitopes BE-2 (N-terminus) and BE-6 (C-terminus) were identified. Wet lab validation of these epitopes was carried out by measuring ZnT8 specific isotypes (IgG, IgM and IgA) in the sera of Normal Glucose Tolerant (NGT), Type-1 diabetic (T1DM) and Type-2 diabetic (T2DM) patients by indirect ELISA. Unexpectedly, compared to NGT, significantly decreased levels of IgG and IgA isotypes was seen in T1DM subjects without complications. IgM levels were reduced in T1DM subjects with retinopathy. Newly diagnosed T1DM subjects initiated on insulin therapy showed an increase in IgA and decrease in IgM titre. Like T1DM, significantly reduced level of IgG, IgM and IgA was seen in T2DM subjects. For the first time, we have identified novel cryptic B cell epitopes in ZnT8 autoantigen against which the naturally occurring autoantibody levels were found to be reduced in diabetes.