Expression of C5a-like biological activities by the fifth component of human complement (C5) upon limited digestion with noncomplement enzymes without release of polypeptide fragments.

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Abstract
Experimental conditions required for the expression of maximum C5 [complement component 5] activation upon limited trypsin hydrolysis were determined to be 0.008 mol of trypsin/mol C5 in a reaction mixture containing 1 mg C5/ml veronal-buffered saline incubated at 37.degree. C for 30 min. Using these optimal incubation conditions, the primary or preferred site of trypsin hydrolysis of the C5.alpha.-chain resulted in the production of C5.alpha.1 (MW, 90,000 [daltons]) and C5.alpha.5 (MW, 25,000) fragments that remained disulfide bonded to the modified C5 molecule (C5''try). Detailed structural-functional analyses clearly indicated that trypsin-mediated conversion of the C5.alpha.-chain to C5.alpha.1 and C5.alpha.5 was responsible for the acquisition of neutrophil lysosomal enzyme-releasing and chemotactic activities. Gel filtration column chromatography under physiological ionic strength, pH 7.4, or in the presence of 0.2% SDS [sodium dodecyl sulfate] further demonstrated that at least 90% of the total recoverable C5a-like biological activity was mediated by the 210,000 MW forms of trypsin-modified C5. Other physiologically relevant, noncomplement protease enzymes (.alpha.-thrombin, plasmin and elastase) also activated C5 to express C5a-like reactivities. Analysis of .alpha.-thrombin-induced, C5.alpha.-chain cleavage events by SDS-polyacrylamide slab gel electrophoresis indicated that the mechanism of .alpha.-thrombin-activation of C5 is similar to that described for trypsin. Reconciliation of this novel mechanism of C5 activation by trypsin with previously published results, and a discussion of the biological significance of noncomplement enzyme-mediated activation of C5 as it might relate to inflammatory processes in vivo, was presented.