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.

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.