The conversion of recombinant human mast cell prochymase to enzymatically active chymase by dipeptidyl peptidase I is inhibited by heparin and histamine

Abstract

Chymase, a major product of mast cell activation, is secreted as a fully active enzyme. We have prepared recombinant human prochymase and have investigated the conditions under which it may be activated by dipeptidyl peptidase I (DPP I). The gene for human chymase was cloned in a baculovirus vector and expressed in High Five insect cells, and the recombinant protein purified by heparin-agarose and gel-filtration chromatography. The purified prochymase was homogeneous by SDS/PAGE with the same molecular mass as native human chymase, and its identity confirmed by N-terminal sequence analysis and Western blotting with chymase-specific antibodies. Treatment with DPP I to remove the Nterminal dipeptide prosequence resulted in enzymatically active chymase, with substrate and inhibitor profiles very similar to those of the native human enzyme. The activation of prochymase by DPP I was strongly inhibited by heparin (IC 50 ϭ 0.5 µg ml Ϫ1 ) and histamine (IC 50 ϭ 2 mM), though these mast cell products had little effect on the action of DPP I towards a low molecular-mass substrate. The pH optimum of DPP I was also higher and narrower with prochymase. The inhibitory action of heparin was lost at NaCl or KCl concentrations sufficient to elute prochymase from a heparin agarose column. Dextran sulphate was as inhibitory as heparin, whereas chondroitin sulphate C was more than 10-fold less effective. Our findings suggest that the activation of prochymase might be restricted to the early stages of vesicle maturation, when the pH is close to neutrality and the histamine and heparin concentrations are low.Keywords : chymase ; dipeptidyl peptidase I; heparin; histamine ; mast cell.Chymase is a serine protease with chymotrypsin-like sub-poprotein B-100, an early step in the aetiology of atherosclerotic strate specificity which is synthesised and stored in the secretory plaques [11]. granules of mast cells along with tryptase (a serine protease with Unlike the digestive proteases trypsin and chymotrypsin, trypsin-like substrate specificity), heparin and histamine [1]. chymase and tryptase, along with other granulocyte proteases, These are released in response to allergens or other stimuli. Al-are not stored as proenzymes, but in forms that are instantly though, in humans, chymase is most abundant in a mast cell active upon release from the secretory granule [1]. However, it subpopulation which predominates in connective tissue, it may is unlikely that proteases would be active in storage. An investialso be present in most, if not all, mast cells in mucosal tissue gation into the factors that might regulate the activity of chy- [2]. This protease has been implicated in defense against helmin-mase within the granule revealed that chymase is relatively inthic parasites, in allergic reactions, in cardiovascular disease, and active at pH 5.5 [12], the reported pH within the mast cell granin chronic inflammatory diseases [3]. Actions attributed to chy-ule [13]. Furthermore, heparin was found to accentuate this efmase include the conversion...

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