A protein microarray-based in vitro transglutaminase assay platform for epitope mapping and peptide discovery

Transglutaminases (TGs) are a family of crosslinking enzymes catalyzing the formation of intra- and inter-molecular glutamine-lysine isopeptide bonds in a calcium dependent manner. Activation of transglutaminases is pathogenically associated with severe human diseases including neurodegenerations, cardiovascular diseases, and autoimmune diseases. Although continuous efforts determining the enzymes substrate preference have been witnessed, a high-throughput assay platform with the "omic" efficiency is still missing for the global identification of substrate-specific TG modification sites. In this study we report a protein microarray-based in vitro TG assay platform for rapid and large-scale (up to 30000 reactions per chip) determination of the glutamine (Q)-bearing TG modification motifs. With this platform we identified the Q16 in superoxide dismutase 1 and Q109 in alpha-synuclein as the primary modification sites for tissue transglutaminase (TG2), the most ubiquitous member of the enzyme family. Of particular interest, we found a close match between the modification motifs and published vaccine epitope sequences in alpha-synuclein, implying an essential and intrinsic role transglutaminase might play in the determination of immunodominant epitopes. Our data collectively suggest the glutamine and its follow-up five residues on the C terminal of a protein compose a minimal determinant motif for TG2 modification and, more importantly, the TG2 modification motifs determined by our platform could finally correspond to the substrates immunodominant epitope sequences in antigen processing. To establish an efficient approach to optimize the enzyme modification motifs and screen for site-specific interfering peptides, we scanned the known TG2 modification motifs with onchip amino-acid swapping and glutamine repeat addition, and obtained multiple variants with significantly upregulated TG2 reactivity. This approach could also be employed to improve the target peptides immunogenicity. Taken together, our synthetic transglutaminase assay platform might be able to deliver a precise epitope blueprint for immunotherapeutic targeting and provide pilot and directional studies for TG-based peptide discovery and immunogen design.