Degradation of human anaphylatoxin C3a by rat peritoneal mast cells: a role for the secretory granule enzyme chymase and heparin proteoglycan.
Purified human C3a was iodinated (125I-C3a) and used to study the interaction of labeled peptide with rat peritoneal mast cells (RMC). Cellular binding of 125I-C3a occurred within 30 sec, followed by a rapid dissociation from the cell. Both the binding of 125I-C3a and the rate of dissociation from the cell were temperature dependent. At 0 degrees C, the binding of 125I-C3a was increased and the rate of dissociation reduced, as compared with 37 degrees C. Once 125I-C3a was exposed to RMC, it lost the ability to rebind to a second batch of RMC. Analysis of the supernatants by trichloroacetic acid (TCA) precipitation and electrophoresis in sodium dodecyl sulfate polyacrylamide gels (SDS PAGE) revealed a decrease in the fraction of 125I precipitable by TCA and the appearance of 125I-C3a cleavage fragments. Pretreatment of RMC with enzyme inhibitors specific for chymotrypsin, but not trypsin, abrogated the degradation of 125I-C3a. Treatment of RMC bearing 125I-C3a with bis (sulfosuccinimidyl) suberate (BS3) covalently cross-linked the 125I-C3a to chymase, the predominant enzyme found in the secretory granules. Antiserum directed against chymase precipitated 125I-C3a from extracts of RMC treated with BS3. Indirect immunofluorescence of RMC by using the IgG fraction of goat anti-rat chymase showed that chymase is present on the surface of unstimulated cells. Neither purified chymase nor heparin proteoglycan alone had any appreciable effect on 125I-C3a, but together they resulted in prompt degradation of the 125I-C3a. Immunoabsorption of RMC sonicates with specific antibody for chymase completely abrogated the ability of these sonicates to degrade 125I-C3a. The results indicate that 125I-C3a binds to RMC and is promptly degraded by chymase in the presence of heparin proteoglycan.
D000706:Anaphylatoxins / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D053818:Chymases; D003176:Complement C3 / Q000378:metabolism*; D015926:Complement C3a; D003432:Cross-Linking Reagents; D003594:Cytoplasmic Granules / Q000201:enzymology* / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000276:immunology / Q000378:metabolism / Q000502:physiology*; D005260:Female; D005455:Fluorescent Antibody Technique; D006493:Heparin / Q000031:analogs & derivatives* / Q000502:physiology; D006801:Humans; D007163:Immunosorbent Techniques; D008407:Mast Cells / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D010455:Peptides / Q000378:metabolism*; D010529:Peritoneal Cavity / Q000166:cytology; D011480:Protease Inhibitors / Q000494:pharmacology; D011509:Proteoglycans / Q000502:physiology*; D051381:Rats; D011917:Rats, Inbred Lew; D012697:Serine Endopeptidases; D012995:Solubility; D013388:Succinimides
D000706:Anaphylatoxins; D003176:Complement C3; D003432:Cross-Linking Reagents; D010455:Peptides; D011480:Protease Inhibitors; D011509:Proteoglycans; D013388:Succinimides; C036090:heparin proteoglycan; D015926:Complement C3a; D006493:Heparin; C035760:bis(sulfosuccinimidyl)suberate; D010450:Endopeptidases; D012697:Serine Endopeptidases; D053818:Chymases