Mechanism by which heparin proteoglycan modulates mast cell chymase activity.
Chymases are highly basic chymotrypsin-like serine proteases expressed exclusively by mast cells. Large amounts of chymases complexed with heparin proteoglycan (PG) are released in vivo during mast cell activation. The tight binding of chymase to heparin PG results in increased activity of the protease toward certain substrates, e.g., thrombin and MeO-Suc-Arg-Pro-Tyr-pNA (S-2586). In this study, the mechanism by which heparin PG modulates chymase activity was investigated, using thrombin and various chromogenic peptide substrates as model substrates. Incubation of thrombin with oligonucleotides that block the heparin-binding site of thrombin abolished the stimulatory effect of heparin PG on thrombin inactivation. Further, thrombin mutants with defects in their heparin-binding regions were less efficiently inactivated by chymase-heparin PG than wild type thrombin. These findings suggest a model for chymase stimulation where heparin PG may promote the chymase-catalyzed cleavage of heparin-binding substrates by simultaneously binding to both chymase and substrate. Experiments in which various chromogenic peptide substrates were utilized showed that heparin PG enhanced the activity of chymase toward positively charged peptide substrates such as S-2586, whereas the cleavage of uncharged substrates was not affected by the presence of heparin PG. On the basis of the latter findings, an alternative stimulation mechanism is discussed where heparin PG may stimulate chymase activity by blocking positively charged regions in chymase, thereby reducing the level of electrostatic repulsion between chymase and positively charged substrates.
Pejler
G
G
Department of Veterinary Medical Chemistry, The Biomedical Center, Swedish University of Agricultural Sciences, Uppsala. Gunnar.Pejler@vmk.slu.se
Sadler
J E
JE
D000818:Animals; D002384:Catalysis; D053818:Chymases; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D006493:Heparin / Q000031:analogs & derivatives* / Q000737:chemistry; D006868:Hydrolysis; D008407:Mast Cells / Q000201:enzymology*; D008956:Models, Chemical; D011509:Proteoglycans / Q000737:chemistry*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012697:Serine Endopeptidases / Q000378:metabolism*; D055672:Static Electricity; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D011509:Proteoglycans; C036090:heparin proteoglycan; D006493:Heparin; D012697:Serine Endopeptidases; D053818:Chymases; D013917:Thrombin