TYROSINE IS A POTENTIAL SITE FOR COVALENT ATTACHMENT OF ACTIVATED COMPLEMENT COMPONENT C3

Activation of C3 results in the generation of metastable C3b, which ha s been shown to preferentially react with the hydroxyl groups of carbo hydrates and with specific serine and threonine residues in proteins. In this study we have examined the reactivity of metastable C3b with t he third type of hydroxyl group present in proteins, tyrosine (Tyr). T he results demonstrated that Tyr reacts with the thioester of metastab le C3b and that this reactivity was 11-fold better than that of threon ine, 47-fold better than serine and 50-fold better than the reactivity of carbohydrates. Model peptides containing Tyr showed even higher re activity than free Tyr, demonstrating that incorporation into peptide structures does not block C3b attachment. The site of attachment was f ound to be in the alpha'-chain of C3b and the bond was hydroxylamine s ensitive, indicating an ester linkage. The stability of the C3b-Tyr co mplex was measured under physiological conditions (pH 7.4, 37 degrees C) and compared to the stability of other C3b complexes. C3b-Tyr decay ed 50% in 19 hr at 37 degrees C, but C3b bound to a Tyr-containing pep tide was more stable, exhibiting a t(1/2) of 53 hr. The ester linked c omplexes C3b-IgG and C3b-glycerol were less stable each exhibiting a t in of approximately 8 hr. As yet, only two specific C3b attachment sit es on proteins have been identified, Ser(12/17) in C4b and Thr(144) in IgG(1). The present evidence demonstrates that Tyr residues are highl y reactive and that the C3b-Tyr linkage is stable. The findings sugges t that complexes involving tyrosine residues as the site of attachment will also be found.