Covalent binding and hemolytic activity of complement proteins.

Abstract

We report the inactivation of the third component of complement (C3) In this study, we treated C3 with primary amines and compared the rate of decay of C3 hemolytic activity with the rate of loss of surface-binding activity. We find both rates to be identical, indicating that both activities are causally related. We also present data to support our hypothesis (7) that there is an internal ester bond within C3 and that C3b binds to RS by the transfer of the acyl group of the internal ester to a hydroxyl group on the RS. C4 and C5 were also treated with hydroxylamine and methylamine. C4, which binds covalently to membrane surfaces (8), behaves like C3 in these experiments. C5, which does not bind covalently to cell surfaces (8), is not inactivated by hydroxylamine and methylamine.

Function of the Factor I Modules (FIMS) of Human Complement Component C6

“…This is accomplished readily for Alternative Pathway activators. These activators include rabbit erythrocytes, yeast, and bacterial cells walls, all of which provide a large number of nucleophilic acceptors for transacylation with nascent C3b* (30,31). Furthermore, relative to cells that are activators of the Classical Pathway, those that engage the Alternative Pathway are better able to shield C3b from degradation by plasma control proteins (32).…”

Formation of the initial C3 convertase of the alternative complement pathway. Acquisition of C3b-like activities by spontaneous hydrolysis of the putative thioester in native C3.

“…It has previously been shown (4,6,8) that the reaction of C3 with methylamine results in the simultaneous loss of hemolytic activity and the incorporation of 1 tool of methylamine per mol of C3. This reaction has been shown to expose a titratable SH group.…”

“…It has also been known that treatment of C3 with simple amines such as hydrazine abolished its hemolytic activity (3). Recently, evidence has accumulated that strongly supports the existence of an intramolecular thioester bond in native C3 (4)(5)(6)(7)(8). Modification of the thioester by incorporation of 1 mol of methylamine destroyed the potential of C3 to bind to biological targets of complement, induced conformational changes in the molecule (9) and led to the expression of functional binding sites for complement proteins resembling those of enzymatically generated C3b (4,10).…”

Sequence determination of the thiolester site of the fourth component of human complement.

“…This thiol is also released on activation of C3 to C3b (13). These data, and the observed stoichiometric relationship between uptake of methylamine into a glutamyl residue and titration of a thiol (10), led to the hypothesis that an internal thiolester exists in C3 and that covalent bond formation involves transfer ofthe acyl group from the thiol to a hydroxyl contained on the acceptor molecule (10,11,14). These studies of C3 have now been extended to C4.…”

“…These studies of C3 have now been extended to C4. Treatment with nitrogen nucleophiles such as methylamine again results in the titration of a single reactive group, the release of a thiol (which is also contained in C4b), and the loss of covalent bondforming potential (14)(15)(16)(17)(18)(19). Recently, in addition to ester bonds between these molecules and binding surfaces, amide (hydroxylamine-resistant) bonds have also been proposed (20,21).…”