Identification of classical anaphylatoxin as the des-Arg form of the C5a molecule: evidence of a modulator role for the oligosaccharide unit in human des-Arg74-C5a.

A functionally active and potentially lethal fragment of the fifth component of complement (C5) is generated during complement activation in serum from animals of various species. This factor, termed the "classical" anaphylatoxin, was isolated from porcine serum and was identified chemically as the des-Arg derivative of the well-characterized C5a molecule. Unlike the C3a and C4a anaphylatoxins, porcine C5a does not require the COOH-terminal arginyl residue for spasmogenic activity. Further degradation of porcine des-Arg(74)-C5a by carboxypeptidase Y removed glycine-73 and leucine-72 and decreased the intrinsic spasmogenic activity by >90%. Hence, we conclude that, although the arginyl residue is not essential, the COOH-terminal sequence Leu-Gly-Arg contributes structural information that accounts for >90% of C5a activity. Human des-Arg(74)-C5a, like its porcine counterpart, has instrinsic anaphylatoxin activity; however, higher concentrations were needed to contract the guinea pig ileal tissue (i.e., 1 muM for human des-Arg(74)-C5a versus 1 nM for porcine des-Arg(74)-C5a). Furthermore, the des-Arg form of human C5a was only 0.1% as active as porcine des-Arg(74)-C5a for enhancing vascular permeability in guinea pig skin. In addition to these biological differences, numerous chemical differences exist between the human and porcine des-Arg(74)-C5a molecules, the most prominent feature being an oligosaccharide entity associated uniquely with the human C5a. When the oligosaccharide unit of human des-Arg(74)-C5a was removed by glycosidases, leaving a single glucosamine residue attached to the side chain of asparagine-64, activity was enhanced. The human des-Arg(74)-C5a molecule devoid of the complex oligosaccharide unit exhibited 10-fold stronger spasmogenic activity and 20- to 50-fold greater permeability-enhancing activity than did human des-Arg(74)-C5a containing the oligosaccharide. Consequently, the oligosaccharide associated with human C5a modulates or suppresses potentially harmful activities of this anaphylatoxin. The relatively high levels of spasmogenic activity associated with porcine des-Arg(74)-C5a indicate that this factor is poorly controlled by endogenous serum carboxypeptidase, whereas human C5a is virtually inactivated by the enzyme. Hence, the influence of this oligosaccharide in suppressing human des-Arg(74)-C5a activity is of major physiologic importance in protecting man from potentially toxic effects of this complement factor.