Labeling of epsilon-lysine crosslinking sites in proteins with peptide substrates of factor XIIIa and transglutaminase.

Abstract

Peptides patterned on the N-terminal sequence of fibronectin were synthesized and tested for amine acceptor qualities in reactions with dansylcadaverine catalyzed either by coagulation factor XIIIa or intracellular transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13). On the basis of inverse half-saturations of the enzymes, the order of acceptor substrate affinity for factor XIIIa was pEAQQIV much greater than Boc-AQQIV greater than Boc-QQIV, and for transglutaminase, Boc-QQIV greater than Boc-AQQIV greater than pEAQQIV (amino acid residues are shown in one-letter code; pE, pyroglutamic acid; Boc, tert-butyloxycarbonyl). Sequence analysis of dansylcadaverine-substituted pEAQQIV indicated that the first of the two adjacent glutamine residues was the target of enzymatic modification. Boc-QIV showed no substrate activity with either enzyme. Crosslinking of crystallins in Ca2(+)-treated rabbit lens homogenate was readily inhibited by Boc-QQIV, Boc-AQQIV, and pEAQQIV, as was the formation of alpha-chain polymers in human fibrin by pEAQQIV in the presence of human factor XIIIa. SDS/PAGE analysis suggested that the inhibitory peptides selectively blocked the electron donor functionalities in these enzymatic crosslinking reactions.