Tyrosine is a potential site for covalent attachment of activated complement component C3.
Activation of C3 results in the generation of metastable C3b, which has been shown to preferentially react with the hydroxyl groups of carbohydrates and with specific serine and threonine residues in proteins. In this study we have examined the reactivity of metastable C3b with the third type of hydroxyl group present in proteins, tyrosine (Tyr). The results demonstrated that Tyr reacts with the thioester of metastable C3b and that this reactivity was 11-fold better than that of threonine, 47-fold better than serine and 50-fold better than the reactivity of carbohydrates. Model peptides containing Tyr showed even higher reactivity than free Tyr, demonstrating that incorporation into peptide structures does not block C3b attachment. The site of attachment was found to be in the alpha'-chain of C3b and the bond was hydroxylamine sensitive, indicating an ester linkage. The stability of the C3b-Tyr complex was measured under physiological conditions (pH 7.4, 37 degrees C) and compared to the stability of other C3b complexes. C3b-Tyr decayed 50% in 19 hr at 37 degrees C, but C3b bound to a Tyr-containing peptide was more stable, exhibiting a t1/2 of 53 hr. The ester linked complexes C3b-IgG and C3b-glycerol were less stable each exhibiting a t1/2 of approximately 8 hr. As yet, only two specific C3b attachment sites on proteins have been identified, Ser1217 in C4b and Thr144 in IgG1. The present evidence demonstrates that Tyr residues are highly reactive and that the C3b-Tyr linkage is stable. The findings suggest that complexes involving tyrosine residues as the site of attachment will also be found.
Sahu
A
A
Department of Biochemistry, University of Texas Health Science Center, Tyler 75710, USA.
Pangburn
M K
MK
D000595:Amino Acid Sequence; D001665:Binding Sites; D003167:Complement Activation; D003176:Complement C3 / Q000737:chemistry*; D003179:Complement C3b / Q000737:chemistry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
D003176:Complement C3; D010455:Peptides; D014443:Tyrosine; D003179:Complement C3b