Substrate profiling of human Transglutaminase 1 using cDNA display and next-generation sequencing

Abstract

Human transglutaminase 1 regulates skin development and is linked with several disease conditions with yet fully unknown mechanisms. To uncover all of its roles in health and disease, the understanding of protein substrates and their reactivity with transglutaminase 1 is necessary. To gain insight into the substrate profile of human transglutaminase 1, this study uses an in vitro selection system based on the cDNA display technology to screen two displayed peptide libraries differing in the number and position of mutated sites relative to the reactive glutamine, in terms of their reactivity to transglutaminase 1. Analysis of the selected DNA pools of by next-generation sequencing and in-house bioinformatics methods revealed a detailed transglutaminase 1 substrate profile indicating preferred and non-preferred amino acid sequences. We have identified a peptide with sequence AEQHKLPSKWPF showing high reactivity to human transglutaminase 1 and low reactivity to transglutaminase 2 and transglutaminase 3. The position weight matrix consisting of per residue amino acid enrichment factors of all selected peptides was used to search human proteins by our in-house search algorithm and identify highly scoring sequence motifs in their primary structure. The search identified six already known transglutaminase 1 substrate proteins as highly scored hits as well as a list of candidate substrates that are under investigation.