Characterization of an Amine-Reactive, Heat-Labile Acyl Residue in the Fourth Component of Human Complement, C41

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Abstract
We have investigated the nature of the reactive site in human C4, which shows covalent binding with alkylamines, heat-induced proteolysis, and hemolytic activity. Incubation of C4 with [14C]methylamine resulted in the irreversible incorporation of 1 mol of methylamine into the α chain of C4. The amine incorporation was a peculiar feature of native C4 and was not observed with hemolytically inactive C4b. Incorporation of methylamine interfered with the activation of C4 by Cls. [14C]Methylamine-binding C4, like C4b, was susceptible to proteolytic digestion by a serum protease, C3b/C4b inactivator (C3b/C4bINA) and yielded large C4c (Mr = 150,000) and small radioactive C4d (Mr=45,000) fragments. As observed with C3 and α2-macroglobulin, heat-induced proteolysis of the α chain of C4 proceeded to yield two α chain fragments of 60,000 and 40,000 dalton. The N-termini of the 60,000 and 40,000 dalton fragments were identified as pyro-glutamic acid and aspartic acid (asparagine), respectively. Since the N-terminus of the α chain was aspartic acid, the 40,000 dalton fragment appeared to be derived from the N-terminal end of the α chain. Thus, it appeared likely that an active acyl residue, probably an activated glutamyl residue, was located at 40,000 dalton from the N-terminal end of the α chain. Native C4 contained a single thiol residue and did not yield any additional thiol residue upon incorporation of methylamine. This result excludes the possibility that, by analogy with C3 and α2-macroglobulin, a putative internal thiolester linkage might also be responsible for activation of the acyl residue in C4. The cleavage of C4 with 2-nitro-5-thiocyanobenzoic acid (NTCB) and the determination of the N-termini of two α chain fragments revealed that the thiol residue was located at 40,000 daltons from the N-terminal end of the α chain. This result suggested that the thiol residue might be located close to the activated acyl residue in the a chain. Treatment of C4 with thiol reagents did not result in loss of the hemolytic activity, indicating that the thiol residue was not responsible for the hemolytic activity of C4.