Autoantibody reactivity to tetraspanin-7 is directed to the cytoplasmic domains of the protein: implications for immunoassay design and immunotherapy

Aims: Knowledge of regions of autoantigens recognised by the immune system in Type 1 diabetes is crucial for the development of antigen-specific immune intervention to prevent Type 1 diabetes and for efficient disease prediction and diagnosis. We identified tetraspanin-7 (Tspan7), a multi-pass secretory granule transmembrane protein, as an important autoantigen in Type 1 diabetes. The aim of this study was to localise regions of the molecule recognised by Tspan7 autoantibodies in disease.

Methods: For epitope localisation, chimeric constructs of the cytoplasmic, transmembrane and ectodomains of Tspan7 expressed on the backbone of a similar, but non-immunoreactive family member, Tspan6, were constructed as fusions with a luciferase tag. Chimeric proteins were expressed in mammalian cells, extracted and tested against type 1 diabetes autoantibodies using a luminescent immunoprecipitation system. Site-directed mutagenesis was performed to identify amino acids important for autoantibody binding.

Results: Antibodies in Type 1 diabetes bound constructs containing the cytoplasmic and transmembrane domains of Tspan7, but failed to bind those containing only Tspan7 ectodomains. All three cytoplasmic domains of Tspan7 were required for optimal autoantibody binding. However, immunoreactivity was also observed to constructs containing the C-terminal tail of Tspan7. Mutagenesis of residues that differ between Tspan7 and Tspan6 identified F240 and A243 as important residues in the C-terminal cytoplasmic tail required for antibody binding.

Conclusions: Immunoreactivity to Tspan7 was directed to the relatively short cytoplasmic domains, particularly to the 15-residue long cytoplasmic tail. Transmembrane domains may also be required to maintain structural integrity of the cytoplasmic domains for antibody recognition. Autoantibody epitopes to all three secretory granule membrane autoantigens in Type 1 diabetes (Tspan7, IA-2 and ZnT8) are localised to the cytoplasmic domains of the proteins. The short lengths of the Tspan7 cytoplasmic domains will facilitate design of immunoassays and of interventions to block immune responses to the protein.