Labeling of epsilon-lysine crosslinking 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 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.
Parameswaran
K N
KN
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208.
Velasco
P T
PT
Wilson
J
J
Lorand
L
L
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002103:Cadaverine / Q000031:analogs & derivatives; D003432:Cross-Linking Reagents; D003459:Crystallins / Q000378:metabolism; D005337:Fibrin / Q000378:metabolism; D007202:Indicators and Reagents; D007700:Kinetics; D007908:Lens, Crystalline / Q000378:metabolism; D008239:Lysine; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis*; D011817:Rabbits; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*
D003432:Cross-Linking Reagents; D003459:Crystallins; D007202:Indicators and Reagents; D009842:Oligopeptides; D005337:Fibrin; D011503:Transglutaminases; C008542:monodansylcadaverine; D008239:Lysine; D002103:Cadaverine