Characterization of amine donor and acceptor sites for tissue type transglutaminase using a sequence from the C-terminus of human fibrillin-1 and the N-terminus of osteonectin

Transglutaminase (TGase)-modified proteins are commonly observed in a wide range of biological systems. Therefore, the identification of TGase substrates and respective consensus sites may contribute to a better understanding of the physiological role of TGase. In this study, we identified enzyme-specific properties of two peptide sequences, EDGFFKI, derived from human fibrillin-1, and the previously characterized APQQEA, derived from human osteonectin. EDGFFKI was identified in a previous publication as an amine donor substrate for tissue TGase; APQ3Q4EA is an amine acceptor for this enzyme. A widely-used lysine donor mimic, monodansylcadaverine (MDC), was used as a control. EDGFFKI crosslinked specifically only to Q3 of the acceptor probe. The EDGFFKI sequence also showed enzyme specificity for tissue TGase while no reaction was observed with plasma TGase (Factor XIIIa), consistent with its natural occurrence in vivo. Using this substrate in biotinylated form we demonstrate its value as a tracer probe to detect endogenous TGase activity in human tissues as well as to target potential amine acceptor substrates via an enzyme-directed site-specific labeling. The results of this study show natively derived EDGFFKI and APQQEA are better and more specific indicators of endogenous tissue TGase activity as compared to a small molecule probe; this may be important in diagnostic applications. The specificity with which matrix sequences APQQEA and EDGFFKI interact with tissue TGase but not plasma TGase may also be crucial for understanding and controlling the function of these TGases in vivo and in tissue engineering.