LABELING OF EPSILON-LYSINE CROSS-LINKING SITES IN PROTEINS WITH PEPTIDE-SUBSTRATES OF FACTOR-XIIIA AND TRANSGLUTAMINASE

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 γ-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 >> Boc-AQQIV > Boc-QQIV, and for transglutaminase, Boc-QQIV > Boc-AQQIV > 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 α-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.