Two distinct forms of human mast cell chymase

Abstract

Chymase, a chymotrypsin-like protease secreted by human mast cells, is generally considered to be a single enzyme. However, by heparin-agarose chromatography of high-salt extracts of human skin, we have consistently resolved three peaks of chymotryptic activity, eluting at 0.4 M NaCl (peak A), 1.0Ϫ 1.2 M NaCl (peak B) and 1.8Ϫ2.0 M NaCl (peak C), with peak B containing 75Ϫ90% of the recovered activity. Each peak retained its identity upon rechromatography. The three peaks of activity were similar in substrate specificity and inhibitor profile and distinctly different from other chymotryptic enzymes, including cathepsin G and the stratum corneum chymotryptic enzyme. Examination of different tissues revealed that peak C was virtually absent from synovial tissue, was present as a minor component in skin and heart, but constituted the predominant chymotryptic activity in lung. Peaks B and C from skin tissue were further purified by chromatography on Sephacryl S-200. Both had a molecular mass of 28Ϫ29 kDa, yielded the N-terminal sequence reported for chymase, and on western blots reacted with a panel of polyclonal, monoclonal and antipeptide antibodies against chymase. Chymase C required higher concentrations of NaCl to overcome the stimulatory effects of heparin than did chymase B, but had a similar pH profile. Thus, human chymase exists in at least two distinct but similar forms, and the differences in heparin binding and tissue distribution could have important consequences for enzyme function.

Is there a role for mast cells in psoriasis?

“…Thus, tryptase can diVuse through the extracellular matrix and reach more distant target sites in the skin whereas chymase functions rather locally. However, diVerent forms of chymase have been described in respect to the binding aYnity to heparinagarose column, possibly owing to diVerences in glycosylation, and therefore chymase form with low binding aYnity may diVuse more easily after release than the high-aYnity form [114]. Although chymase binds eYciently to heparin, the enzyme remains active also in the absence of heparin, a feature which diVers from that of tryptase.…”

“…However, heparin can modulate the interaction of chymase with cells, protein substrates and inhibitors [74,159,195]. A clearly distinct feature of chymase is that it can be inactivated by a range of endogenous protease inhibitors, including 1 -proteinase inhibitor ( 1 -PI), 1 -antichymotrypsin ( 1 -AC) [114,161] 2 -macroglobulin [196], secretory leukocyte proteinase inhibitor [195] and squamous cell carcinoma antigen-2 [165]. However, chymase can also eVectively degrade 1 -PI and 1 -AC and, in fact, this reaction proceeds faster than the inhibition of chymase by these inhibitors [161] suggesting a complex regulation between the proteinase and its inhibitors.…”

Selective Alterations in Mast Cell Subsets and Eosinophil Infiltration in Two Complementary Types of Intestinal Inflammation: Ascariasis and Crohn’s Disease

“…74 g mucosa and 63 g underlying non-mucosa were processed separately for chymase extraction as previously described [36]. In brief, frozen tissues were pulverised in a blender and extracted three times in low-salt buffer and then three times in high-salt buffer.…”

“…After washing with 10 column volumes of the equilibration buffer, the chymase isoforms were eluted with a 0.4-2.0 M NaCl gradient. Chymase enzymatic activity was monitored by the hydrolysis of N-succinylAla-Ala-Pro-Phe-p-nitro anilide (Sigma) in microtitre plates using the kinetics program of a Dynatech MR5000 plate reader [36]. …”

“…Samples were applied in 0.4 M NaCl, 10 mM MOPS, pH 6.8 and eluted with a 0.4-2.0 M NaCl gradient. 1 Fractions are defined as in McEuen et al [36]: peak A, eluted without binding to column; peak B, eluted at approximately 1.0 M NaCl; peak C, eluted at 1.8-2.0 M NaCl.…”