Antigen presentation of detergent-free glutamate decarboxylase (GAD65) is affected by human serum albumin as carrier protein

Abstract

The smaller isoform of glutamate decarboxylase (GAD65) is a major autoantigen in type 1 diabetes (TID). Its hydrophobic character requires detergent to keep the protein in solution, which complicates studies of antigen processing and presentation. In this study an attempt was made to replace detergent with human serum albumin (HSA) for in vitro antigen presentation. Different preparations of recombinant human GAD65 solubilized by HSA were incubated with Priess B cells (HLA DRB1⁎0401) and antigen presentation was tested with HLA DRB1⁎0401-restricted and epitope-specific T33.1 (GAD65 epitope 274–286) and T35 (GAD65 epitope 115–127) T-cell hybridomas. Specific epitope recognition by T33.1 (274–286) and T35 (115–127) cells varied between the different GAD65/HSA preparations, and a reverse pattern of antigen presentation was detected by the two hybridoma. The HSA-specific T-cell hybridoma 17.9 response to the different GAD65/HSA preparations followed the same pattern as that observed for the T33.1 cells. The content of immunoreactive GAD65 measured with four GAD65 antibodies indicated that the lowest GAD65 concentration resulted in the highest 274–286, but the lowest 115–127 presentation. This suggests that HSA–GAD65 interactions qualitatively affect the epitope specificity of GAD65 presentation. HSA may enhance the 274–286 epitope presentation, while suppressing the 115–127 epitope.