RESUMEN
Transglutaminases are a family of enzymes that catalyze cross-linking reactions between proteins. Among the members, there is currently no information regarding the substrate preferences of transglutaminase 7 (TG7), that would clarify its physiological significance. We previously obtained several highly reactive substrate peptide sequences of transglutaminases from a random peptide library. In this study, we screened for preferred substrate sequences for TG7 from a phage-displayed 12-mer peptide library. The most preferred sequence was selected based on reactivity and isozyme specificity. We firstly exhibited the tendency for the preference of substrate sequence for TG7. Then, using the most efficient peptide, Z3S, we established an in vitro assay system to assess enzymatic activity of TG7.
Asunto(s)
Biblioteca de Péptidos , Péptidos/química , Péptidos/metabolismo , Transglutaminasas/química , Secuencia de Aminoácidos , Sitios de Unión , Activación Enzimática , Datos de Secuencia Molecular , Unión Proteica , Especificidad por SustratoRESUMEN
Mammalian transglutaminases (TGs) are a family of enzymes that catalyze the formation of covalent crosslinks between glutamine and lysine residues in proteins. These catalytic reactions play roles in several essential biological processes, including blood coagulation, skin formation, and stabilization of the extracellular matrix. Among the members of this family, factor XIII and TGs 1-5 have been characterized well, but very little is known about the novel members TG6 and TG7. Recently, however, autoantibodies against TG6 were found in a patient with gluten ataxia, a disease caused by enzymatically modified gluten-derived peptides in neuronal cells. To characterize the possible physiological functions of TG6, in this study we screened a phage-displayed random peptide library to find highly reactive glutamine donor substrate peptides. From several candidate peptides, one sequence, designated Y25, appeared to have the highest reactivity. The Y25 sequence also has apparent isozyme specificity when evaluated by incorporation of the labeled glutamine acceptor substrate as a fusion protein with glutathione-S-transferase. Also, the sequence retained high reactivity as well as the isozyme specificity in the peptide form. Analyses with the biotin-labeled and fluorescence-labeled peptides showed TG6 to be an active enzyme and react to specific substrates in the skin, which is consistent with the results of the expression pattern of its transcripts.