![]() ![]() The representative applications include substrate profiling of protein-modifying enzymes in proteomics research, such as caspase 22, viral protease and a kinase 23, metalloprotease 24, and enzymes involved in the modification of post-translationally modified peptides 25, 26, and demonstrate that substrate preference can be studied in great detail by mRNA/cDNA display. Utilization of mRNA/cDNA display for activity-based selection of enzyme substrates has been described to a much lower extent. Numerous reports exist on mRNA/cDNA display being applied to affinity-based screening and selection of peptides and antibody fragments 15, 16, 17, 18, 19, 20, 21. The convenience of complete control of expression/screening conditions, stable genotype–phenotype linkage, incorporation of unnatural amino acids and ability to handle large libraries (up to the 10 12 variants) makes mRNA/cDNA display a preferred screening method. However, to this date, these probes have not been optimized in terms of amino acid preference at Gln-surrounding positions or used to investigate TG substrate preference in detail.Īn in vitro screening method, mRNA/cDNA display, pioneered by two groups, Roberts and Szostak’s group 13 and Nemoto and Yanagawa’s group 14 relies on the formation of a covalent link between the genotype (mRNA or cDNA) and phenotype (protein of interest) via puromycin. These probes have been developed by selection from random peptide libraries using phage display technology, and are available for studies of TG isozymes. Artificial fluorescently labeled Gln-peptide probes (‘Hitomi peptides’) have greatly contributed to in vitro and in situ detection and measurement of TG activity 7, 8, 9, 10, 11, 12. Although widely studied, more work is needed to fully understand the biological function of TGs, for which sensitive and specific in situ detection of TG activity is desired. Aberrant expression and function of TGs cause serious effects on human health and cause conditions such as hemorrhage, celiac disease, cancer, fibrosis, Alzheimer's and Huntington’s disease, and lamellar ichthyosis 6. In mammals, eight different types of TGs have been identified (TG1, TG2, TG3, TG4, TG5, TG6, TG7, and factor XIII), with functions ranging from blood clotting, epidermis and hair follicle formation, wound healing, apoptosis, extracellular matrix formation and cell adhesion 5. Microbial TGs have been involved in a variety of biotechnological applications and industrial use 2, 3, 4. The presence of TGs is vast across kingdoms of plants, animals and microorganisms. Since transamidation proceeds via the formation of an acyl-enzyme intermediate, which is a rate-limiting step, TGs show high specificity towards acyl donors, while reacting on a variety of acyl acceptors such as lysine residues of proteins and small primary amines 1. Owing to their transamidation activity, TGs are known for their role in crosslinking of proteins and peptides. As a result, a stable isopeptide bond is formed, resistant to proteolytic degradation. Transglutaminases (TGs: EC 2.3.2.13) are enzymes catalyzing transamidation, a transfer reaction between an acyl donor (peptidyl glutamine) and an acyl acceptor (amino group of lysine). This platform will be further used for the substrate profiling of other TG isozymes, as well as for the selection and evolution of larger biomolecules. Furthermore, the obtained information on substrate profiling can be used to identify potential TG2 protein targets. The highly enriched peptides indeed contained the indicated sequence and showed a higher reactivity as TG2 substrates than the peptide previously selected by phage display, thus representing the novel candidate peptide probes for TG2 research. After screening and selection of the control peptide library randomized at the reactive glutamine, a combinatorial library of displayed peptides randomized at positions − 1, + 1, + 2, and + 3 from the reactive glutamine was screened followed by NGS and bioinformatic analysis, which indicated a strong preference of TG2 towards peptides with glutamine at position − 1 (Gln-Gln motif), and isoleucine or valine at position + 3. Here, we developed a platform using cDNA display and next-generation sequencing (NGS) for rapid and comprehensive substrate profiling of transglutaminase 2 (TG2), an enzyme crosslinking glutamine and lysine residues in proteins. CDNA display is an in vitro display technology based on a covalent linkage between a protein and its corresponding mRNA/cDNA, widely used for the selection of proteins and peptides from large libraries (10 12) in a high throughput manner, based on their binding affinity.
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