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.
Experimental conditions required for the expression of maximum C5 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 degrees C for 30 min. Employing 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 (molecular weight, 90,000) and C5 alpha 5 (molecular weight, 25,000) fragments that remained disulfide bonded to the modified C5 molecule (C5'try). Detailed structural-functional analyses clearly indicated the 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 further demonstrated that at least 90% of the total recoverable C5a-like biological activity was mediated by the 210,000 molecular weight 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.
D002634:Chemotaxis, Leukocyte; D002850:Chromatography, Gel; D003182:Complement C5 / Q000378:metabolism*; D015936:Complement C5a; D005341:Fibrinolysin / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D009504:Neutrophils / Q000502:physiology; D010196:Pancreatic Elastase / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D010447:Peptide Hydrolases / Q000494:pharmacology*; D013917:Thrombin / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D003182:Complement C5; D010446:Peptide Fragments; D015936:Complement C5a; D010447:Peptide Hydrolases; D010196:Pancreatic Elastase; D014357:Trypsin; D013917:Thrombin; D005341:Fibrinolysin