Labeling of ε-Lysine Crosslinking Sites in Proteins with Peptide Substrates of Factor XIIIa and Transglutaminase

• Published in: Proceedings of the National Academy of Sciences - PNAS 1990-11, Vol.87 (21), p.8472-8475
• Main Authors: Parameswaran, K. N., Velasco, P. T., Wilson, J., Lorand, L.
• Format: Article
• Language: English
• Subjects: Amines; Amino Acid Sequence; Amino acids; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biochemistry; Biological and medical sciences; Cadaverine - analogs & derivatives; Cross-Linking Reagents; Crosslinking; Crystallins; Crystallins - metabolism; Enzyme substrates; Enzymes; Esters; Fibrin - metabolism; Fundamental and applied biological sciences. Psychology; Gels; General aspects, investigation methods; Indicators and Reagents; Kinetics; Lens, Crystalline - metabolism; Lysine; Molecular Sequence Data; Oligopeptides - chemical synthesis; protein-glutamine gamma -glutamyltransferase; Proteins; Rabbits; Substrate Specificity; Transglutaminases - metabolism
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.87.21.8472

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.