Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors

Powers, James C.; Tanaka, Takumi; Harper, J. Wade; Minematsu, Yoshihiro; Barker, L; Lincoln, David W.; Crumley, Katherine V.; Fräki, Jorma E.; Schechter, Norman M.; Lazarus, Geraldine Genevive

doi:10.1021/bi00329a037

Cited by 166 publications

(98 citation statements)

References 39 publications

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“…A previous study in which a benzyl thioester substrate was compared with its p-nitroanilide counterpart (SucVal-Pro-Phe-S-Bzl versus Suc-Val-Pro-Phe-NH-Np) [23] found that the thioester was 10-times more reactive than the p-nitroanilide with human skin chymase and 171-times more reactive with cathepsin G, a result broadly in accord with the findings presented here. As reported previously [24], cathepsin G was found to be more sensitive than chymase to inhibition by aprotinin.…”

Section: Discussionsupporting

confidence: 90%

Two distinct forms of human mast cell chymase

McEuen

Gaça

Buckley

et al. 1998

European Journal of Biochemistry

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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.

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Section: Discussionsupporting

confidence: 90%

Two distinct forms of human mast cell chymase

McEuen

Gaça

Buckley

et al. 1998

European Journal of Biochemistry

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“…Previous studies, utilizing peptide 4-nitroanilide substrates, indicated that substrates with a Phe residue at the P, position are preferentially cleaved by RMCP-1 (Powers et al, 1985). Our results, identifying the PhelG-GlylF bond in thrombin as a target for RMCP-1, are thus in agreement with these observations.…”

Section: Discussionsupporting

confidence: 92%

Identification of the Proteolytic Thrombin Fragments Formed after Cleavage with Rat Mast Cell Protease 1

Pejler

Karlström²,

Berg

1995

European Journal of Biochemistry

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We have previously identified rat mast cell protease 1 (RMCP-I), a chymotrypsin-like secretory granule serine protease, as a potent inactivator of thrombin. The present study outlines the cleavage pattern obtained after degradation of thrombin by RMCP-1. The cleavage sites in thrombin were identified by N-terminal amino acid sequence analysis of recovered thrombin fragments. Incubation of thrombin with RMCP-1 resulted in the rapid formation of a 37-kDa fragment, due to cleavage of the PhelG-GlylF bond in the thrombin A chain (numbering of amino acid residues according to topological equivalencies with chymotrypsinogen). Further incubation resulted in cleavage of the Trp148-Thr149 bond in the B chain, along with the formation of fragments of 27 kDa and 15 kDa. When the RMCP-lhhrombin mixtures were incubated further, successive degradation of the 37-kDa, 27-kDa and 15-kDa fragments was observed, along with cleavage of the Tyrl17-Ile118 bond in the B chain and the formation of fragments of 12, 9 and 6 kDa. No residual thrombin activity was detected after the degradation process had proceeded to this stage. Heparin was shown to markedly enhance the rate of thrombin degradation by RMCP-1.

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“…The corresponding peptide was synthesized, and this substrate was hydrolyzed 40 times more efficiently than S-2586 in the presence of heparin. When comparing the results in the phage display analysis of rMCP-4 with the substrate specificity analysis for rMCP-2 performed by Powers et al (8), several features of these enzymes were shown to be shared. The most preferred P3 amino acid for rMCP-2 was shown to be Val, followed by Leu, Met, Thr, and Phe, a result that correlates well with the phage display result for rMCP-4.…”

Section: Expression Purification and Analysis Of Recombinant Rmcp-4 -mentioning

confidence: 96%

“…rMCP-5 (initially designated as rMCP-3) is the rat ␣-chymase and the homologue to the single human chymase found so far. The rat mast cell ␤-chymases rMCP-1 and rMCP-2, expressed by connective tissue mast cells and MMC, respectively, have been extensively investigated with regard to tissue distribution, charge, heparin binding, substrate specificity, inhibitor susceptibility, and structure (7)(8)(9)(10). Recent data suggest that MMC mediators, in particular rMCP-2, directly induce physiological changes in the gastrointestinal tract, such as an increase in epithelial permeability (11).…”

mentioning

confidence: 99%

Rat Mast Cell Protease 4 Is a β-Chymase with Unusually Stringent Substrate Recognition Profile

Karlson

Pejler

Fröman

et al. 2002

Journal of Biological Chemistry

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Activated mast cells release a variety of potent inflammatory mediators including histamine, cytokines, proteoglycans, and serine proteases. The serine proteases belong to either the chymase (chymotrypsin-like substrate specificity) or tryptase (trypsin-like specificity) family. In this report we have investigated the substrate specificity of a recently identified mast cell protease, rat mast cell protease-4 (rMCP-4). Based on structural homology, rMCP-4 is predicted to belong to the chymase family, although rMCP-4 has previously not been characterized at the protein level. rMCP-4 was expressed with an N-terminal His tag followed by an enterokinase site substituting for the native activation peptide. The enterokinase-cleaved fusion protein was labeled by diisopropyl fluorophosphate, demonstrating that it is an active serine protease. Moreover, rMCP-4 hydrolyzed MeO-Suc-Arg-Ala-Tyr-pNA, thus verifying that this protease belongs to the chymase family. rMCP-4 bound to heparin, and the enzymatic activity toward MeO-SucArg-Ala-Tyr-pNA was strongly enhanced in the presence of heparin. Detailed analysis of the substrate specificity was performed using peptide phage display technique. After six rounds of amplification a consensus sequence, Leu-Val-Trp-Phe-Arg-Gly, was obtained. The corresponding peptide was synthesized, and rMCP-4 was shown to cleave only the Phe-Arg bond in this peptide. This demonstrates that rMCP-4 displays a striking preference for bulky/aromatic amino acid residues in both the P1 and P2 positions.Mast cells are potent inflammatory cells with well known harmful effects, e.g. during allergic reactions. However, their primary beneficial function in vivo is most likely their involvement in the expulsion of nematode parasites and in the defense against bacterial infections (1-3). Two main types of mast cells have been identified in rodents, the connective tissue mast cells, which mainly reside in connective tissue and the peritoneum, and the mucosal mast cells (MMC), 1 which reside mainly in the mucosa of the respiratory and intestinal tracts.Both types of rodent mast cells are activated by antigen cross-linking of surface-bound IgE and respond by rapidly releasing mediators stored in their granules. The inflammatory mediators include histamine, proteoglycans, prostaglandins, cytokines, and neutral proteases (4). Mast cells are a particularly rich source of serine proteases belonging to the tryptase family, the chymase family, and the recently identified mMCP-8 family. Proteases belonging to the mMCP-8 family have as yet unknown substrate specificity. The tryptase family of proteases cleaves their substrate at the C-terminal side of basic amino acids (Arg and Lys), whereas the chymase family has chymotrypsin-like substrate specificity, i.e. they cleave peptides after aromatic amino acids (Phe, Tyr, and Trp). Based on phylogenetic relationships, the chymase family can be further subdivided into two groups, the ␣-and the ␤-chymases (5). In nonrodent mammals only a single ␣-chymase can be found, whereas in...

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Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors

Cited by 166 publications

References 39 publications

Two distinct forms of human mast cell chymase

Two distinct forms of human mast cell chymase

Identification of the Proteolytic Thrombin Fragments Formed after Cleavage with Rat Mast Cell Protease 1

Rat Mast Cell Protease 4 Is a β-Chymase with Unusually Stringent Substrate Recognition Profile

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