Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity

Chymases are mast cell serine proteases with chymotrypsin-like primary substrate specificity. Amino acid sequence comparisons of alpha-chymases from different species indicated that certain rodent alpha-chymases have a restricted S1 pocket that could only accommodate small amino acids, i.e. they may, despite being classified as chymases, in fact display elastase-like substrate specificity. To explore this possibility, the alpha-chymase, rat mast cell protease 5 (rMCP-5), was produced as a proenzyme with a His6 purification tag and an enterokinase-susceptible peptide replacing the natural propeptide. After removal of the purification tag/enterokinase site by enterokinase digestion, rMCP-5 bound the serine-protease-specific inhibitor diisopropyl fluorophosphate, showing that rMCP-5 was catalytically active. The primary specificity was investigated with chromogenic substrates of the general sequence succinyl-Ala-Ala-Pro-X-p-nitroanilide, where the X was Ile, Val, Ala, Phe or Leu. The activity was highest toward substrates with Val or Ala in the P1 position, whereas low activity toward the peptide with a P1 Phe was observed, indicating that the substrate specificity of rMCP-5 indeed is elastase-like. The extended substrate specificity was examined utilizing a phage-displayed random nonapeptide library. The preferred cleavage sequence was resolved as P4-(Gly/Pro/Val), P3-(Leu/Val/Glu), P2-(Leu/Val/Thr), P1-(Val/Ala/Ile), P1'-(Xaa), and P2'-(Glu/Leu/Asp). Hence, the extended substrate specificity is similar to human chymase in most positions except for the P1 position. We conclude that the rat alpha-chymase has converted to elastase-like substrate specificity, perhaps associated with an adoption of new biological targets, separate from those of human alpha-chymase.     

Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile

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-Suc-Arg-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.     

Guinea pig chymase is leucine-specific: a novel example of functional plasticity in the chymase/granzyme family of serine peptidases

To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the alpha clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig alpha-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by alpha1-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster alpha-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase.     

Structural basis for elastolytic substrate specificity in rodent alpha-chymases

Divergence of substrate specificity within the context of a common structural framework represents an important mechanism by which new enzyme activity naturally evolves. We present enzymological and x-ray structural data for hamster chymase-2 (HAM2) that provides a detailed explanation for the unusual hydrolytic specificity of this rodent alpha-chymase. In enzymatic characterization, hamster chymase-1 (HAM1) showed typical chymase proteolytic activity. In contrast, HAM2 exhibited atypical substrate specificity, cleaving on the carboxyl side of the P1 substrate residues Ala and Val, characteristic of elastolytic rather than chymotryptic specificity. The 2.5-A resolution crystal structure of HAM2 complexed to the peptidyl inhibitor MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone revealed a narrow and shallow S1 substrate binding pocket that accommodated only a small hydrophobic residue (e.g. Ala or Val). The different substrate specificities of HAM2 and HAM1 are explained by changes in four S1 substrate site residues (positions 189, 190, 216, and 226). Of these, Asn(189), Val(190), and Val(216) form an easily identifiable triplet in all known rodent alpha-chymases that can be used to predict elastolytic specificity for novel chymase-like sequences. Phylogenetic comparison defines guinea pig and rabbit chymases as the closest orthologs to rodent alpha-chymases.     

Angiotensin II generation by mast cell alpha- and beta-chymases

Mast cells secrete alpha- and beta-chymases. Primate alpha-chymases generate angiotensin (AT) II by selectively hydrolyzing AT I's Phe(8)-His(9) bond. This is distinct from the AT converting enzyme (ACE) pathway. In humans, alpha-chymase is the major non-ACE AT II-generator. In rats, beta-chymases destroy AT II by cleaving at Tyr(4)-Ile(5). Past studies predicted that AT II production versus destruction discriminates alpha- from beta-chymases and that Lys(40) in the substrate-binding pocket determines alpha-chymase Phe(8) specificity. This study examines these hypotheses by comparing AT II generation by human alpha-chymase (containing Lys(40)), dog alpha-chymase (lacking Lys(40)), and mouse mMCP-4 (a beta-chymase lacking Lys(40); orthologous to AT II-destroying rat chymase rMCP-1). The results suggest that human and dog alpha-chymase generate AT II exclusively and with comparable efficiency, although dog chymase contains Ala(40) rather than Lys(40). Furthermore, AT II is the major product generated by degranulation supernatants from cultured dog mast cells, which release tryptases and dipeptidylpeptidase as well as alpha-chymase. In contrast to rMCP-1, mMCP-4 beta-chymase readily generates AT II. Although there is competing AT I hydrolysis at Tyr(4), mMCP-4 does not destroy AT II quickly once it is formed. We conclude (1) that chymases are the dominant AT I-hydrolyzing mast cell peptidases, (2) that residues other than Lys(40) are key determinants of alpha-chymase AT I Phe(8) specificity, (3) that beta-chymases can generate AT II, and (4) that alpha- and beta-chymases are not strictly dichotomous regarding AT I cleavage specificity.     

Characterization of the gene encoding mouse mast cell protease 8 (mMCP-8), and a comparative analysis of hematopoietic serine protease genes

Serine proteases are important granule constituents in several of the major hematopoietic cell lineages. We present here the nucleotide sequence of the gene encoding mouse mast cell protease 8 (mMCP-8). mMCP-8 was initially isolated as a cDNA from a mouse mast cell line, but has recently been found to be expressed primarily by mouse basophils. mMCP-8 and its rat homologues, rMCP-8, -9, and -10, form a new group of mast cell/basophil proteases, which are more closely related to the T-cell granzymes and neutrophil cathepsin G than to the mast cell tryptases and chymases. A dot matrix comparison of the mMCP-8 gene with other closely related hematopoietic serine protease genes shows detectable homology only in the exonic regions of the genes. No indication for conservation in the promoter region or introns was observed. This latter finding indicates that the upstream regulatory region has evolved at a relatively high rate. However, despite the low degree of direct sequence conservation, no major differences in the sizes of introns or exons were observed between mMCP-8 and genes for the closest related hematopoietic serine proteases, the mouse T-cell granzymes and cathepsin G, indicating that after evolutionary separation from the T-cell granzymes and cathepsin G, the majority of mutations primarily involved single base pair substitutions or short insertions or deletions.     

The serpin MNEI inhibits elastase-like and chymotrypsin-like serine proteases through efficient reactions at two active sites.

MNEI (monocyte/neutrophil elastase inhibitor) is a 42 kDa serpin superfamily protein characterized initially as a fast-acting inhibitor of neutrophil elastase. Here we show that MNEI has a broader specificity, efficiently inhibiting proteases with elastase- and chymotrypsin-like specificities. Reaction of MNEI with neutrophil proteinase-3, an elastase-like protease, and porcine pancreatic elastase demonstrated rapid inhibition rate constants >10(7) M(-1) s(-1), similar to that observed for neutrophil elastase. Reactions of MNEI with chymotrypsin-like proteases were also rapid: cathepsin G from neutrophils (>10(6) M(-1) s(-1)), mast cell chymase (>10(5) M(-1) s(-1)), chymotrypsin (>10(6) M(-1) s(-1)), and prostate-specific antigen (PSA), which had the slowest rate constant at approximately 10(4) M(-1) s(-1). Inhibition of trypsin-like (plasmin, granzyme A, and thrombin) and caspase-like (granzyme B) serine proteases was not observed or highly inefficient (trypsin), nor was inhibition of proteases from the cysteine (caspase-1 and caspase-3) and metalloprotease (macrophage elastase, MMP-12) families. The stoichiometry of inhibition for all inhibitory reactions was near 1, and inhibitory complexes were resistant to dissociation by SDS, further indicating the specificity of MNEI for elastase- and chymotrypsin-like proteases. Determination of the reactive site of MNEI by N-terminal sequencing and mass analysis of reaction products identified two reactive sites, each with a different specificity. Cys(344), which corresponds to Met(358), the P(1) site of alpha1-antitrypsin, was the inhibitory site for elastase-like proteases and PSA, while the preceding residue, Phe(343), was the inhibitory site for chymotrypsin-like proteases. This study demonstrates that MNEI has two functional reactive sites corresponding to the predicted P(1) and P(2) positions of the reactive center loop. The data suggest that MNEI plays a regulatory role at extravascular sites to limit inflammatory damage due to proteases of cellular origin.     

Chymase and tryptase in dog mastocytoma cells: asynchronous expression as revealed by enzyme cytochemical staining

Mast cell populations can be distinguished by differences in the content and substrate specificity of their two major cytoplasmic granule proteases, the chymases and the tryptases. To explore the origins of differences in the types of proteases present in mast cells, we used a double cytochemical staining technique to reveal both chymase and tryptase in cells from four lines of dog mast cell tumors containing both enzymes. We expected that if chymase and tryptase were expressed together during cell development the relative staining intensity of chymase compared to tryptase would be constant among different cells of each tumor. Instead, we found substantial variation in the relative intensity of chymase and tryptase staining among cells of a given mastocytoma line, each of which contained cells presumed to be monoclonal in origin but heterogeneous with respect to cell development. The overall staining intensity for chymase or tryptase correlated with the amount of protease activity in extracts of tumor homogenates. Staining specificity was established by use of selective inhibitors and competitive substrates and was tested on various types of dog cells obtained by bronchoalveolar lavage. The results suggest that active chymase and tryptase may be expressed differently during mast cell differentiation and support the possibility of a close developmental relationship between mast cells differing in protease phenotype. Moreover, the success of the staining procedures applied to mastocytoma cells suggests that they may be of general utility in phenotyping of mast cells according to the protease activities present in their granules.     

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.     

A key role for mast cell chymase in the activation of pro-matrix metalloprotease-9 and pro-matrix metalloprotease-2

Chymases, serine proteases exclusively expressed by mast cells, have been implicated in various pathological conditions. However, the basis for these activities is not known, i.e. the in vivo substrate(s) for mast cell chymase has not been identified. In this study we show that mice lacking the chymase mouse mast cell protease 4 (mMCP-4) fail to process pro-matrix metalloprotease 9 (pro-MMP-9) to its active form in vivo, whereas both the pro and active form of MMP-9 was found in tissues of wild type mice. Moreover, the processing of pro-MMP-2 into active enzyme was markedly defective in mMCP-4 null animals. Histological analysis revealed an increase in collagen in the ear tissue of mMCP-4-deficient animals accompanied by increased ear thickness and a higher content of hydroxyproline. Furthermore, both lung and ear tissue from the knock-out animals showed a markedly increased staining for fibronectin. MMP-9 and MMP-2 are known to have a range of important activities, but the mechanisms for their activation in vivo have not been clarified previously. The present study thus indicates a key role for mast cell chymase in the regulation of pro-MMP-2 and -9 activities. Moreover, the results suggest an important role for mast cell chymase in regulating connective tissue homeostasis.     

Mouse mast cell protease-1 cleaves angiotensin I to form angiotensin II

The ability to convert angiotensin (Ang) I to Ang II was compared between human alpha-chymase and two mouse beta-chymases, mouse mast cell protease (mMCP)-1 and mMCP-4. Human chymase hydrolyzed Ang I to produce Ang II without further degradation. mMCP-1 similarly generated Ang II from Ang I in a time-dependent manner and the formation of the fragment other than Ang II was marginal. In contrast, mMCP-4 hydrolyzed Ang I at two sites, Tyr(4)-Ile(5) and Phe(8)-His(9), with Ang II formation being tentative. Consistently, mMCP-4 but not human chymase hydrolyzed Ang II and mMCP-1 showed little hydrolytic activity against Ang II. These data suggest that not only human chymase but also mMCP-1 might possess a physiological role in Ang II formation. Our findings also imply that the Ang-converting activity of chymase may not be related to the categorization of chymase into alpha- or beta-type based on their primary structure.     

Altered storage of proteases in mast cells from mice lacking heparin: a possible role for heparin in carboxypeptidase A processing

Heparin-deficient mice, generated by gene targeting of N-deacetylase/N-sulfotransferase-2 (NDST-2), display severe mast cell defects, including an absence of stored mast cell proteases. However, the mechanism behind these observations is not clear. Here we show that NDST-2+/+ bone marrow-derived mast cells cultured in the presence of IL-3 synthesise, in addition to highly sulphated chondroitin sulphate (CS), small amounts of equally highly sulphated heparin-like polysaccharide. The corresponding NDST-2-/- cells produced highly sulphated CS only. Carboxypeptidase A (CPA) activity was detected in NDST+/+ cells but was almost absent in the NDST-/- cells, whereas tryptase (mouse mast cell protease 6; mMCP-6) activity and antigen was detected in both cell types. Antigen for the chymase mMCP-5 was detected in NDST-2+/+ cells but not in the heparin-deficient cells. Northern blot analysis revealed mRNA expression of CPA, mMCP-5 and mMCP-6 in both wild-type and NDST-2-/- cells. A approximately 36 kDa CPA band, corresponding to proteolytically processed active CPA, as well as a approximately 50 kDa pro-CPA band was present in NDST-2+/+ cells. The NDST-2-/- mast cells contained similar levels of pro-CPA as the wild-type mast cells, but the approximately 36 kDa band was totally absent. This indicates that the processing of pro-CPA to its active form may require the presence of heparin and provides the first insight into a mechanism by which the absence of heparin may cause disturbed secretory granule organisation in mast cells.     

Cooperation between mast cell carboxypeptidase A and the chymase mouse mast cell protease 4 in the formation and degradation of angiotensin II

The octapeptide angiotensin II (Ang II) exerts a wide range of effects on the cardiovascular system but has also been implicated in the regulation of cell proliferation, fibrosis, and apoptosis. Ang II is formed by cleavage of Ang I by angiotensin-converting enzyme, but there is also evidence for non-angiotensin-converting enzyme-dependent conversion of Ang I to Ang II. Here we address the role of mast cell proteases in Ang II production by using two different mouse strains lacking mast cell heparin or mouse mast cell protease 4 (mMCP-4), the chymase that may be the functional homologue to human chymase. Ang I was added to ex vivo cultures of peritoneal cells, and the generation of Ang II and other metabolites was analyzed. Activation of mast cells resulted in marked increases in both the formation and subsequent degradation of Ang II, and both of these processes were strongly reduced in heparin-deficient peritoneal cells. In the mMCP-4(-/-) cell cultures no reduction in the rate of Ang II generation was seen, but the formation of Ang-(5-10) was completely abrogated. Addition of a carboxypeptidase A (CPA) inhibitor to wild type cells caused complete inhibition of the formation of Ang-(1-9) and Ang-(1-7) but did not inhibit Ang II formation. However, when the CPA inhibitor was added to the mMCP-4(-/-) cultures, essentially complete inhibition of Ang II formation was obtained. Taken together, the results of this study indicate that mast cell chymase and CPA have key roles in both the generation and degradation of Ang II.     

Dipeptidyl peptidase I is essential for activation of mast cell chymases, but not tryptases, in mice

Dipeptidyl peptidase I (DPPI) is the sole activator in vivo of several granule-associated serine proteases of cytotoxic lymphocytes. In vitro, DPPI also activates mast cell chymases and tryptases. To determine whether DPPI is essential for their activation in vivo, we used enzyme histochemical and immunohistochemical approaches and solution-based activity assays to study these enzymes in tissues and bone marrow-derived mast cells (BMMCs) from DPPI +/+ and DPPI -/- mice. We find that DPPI -/- mast cells contain normal amounts of immunoreactive chymases but no chymase activity, indicating that DPPI is essential for chymase activation and suggesting that DPPI -/- mice are functional chymase knockouts. The absence of DPPI and chymase activity does not affect the growth, granularity, or staining characteristics of BMMCs and, despite prior predictions, does not alter IgE-mediated exocytosis of histamine. In contrast, the level of active tryptase (mMCP-6) in DPPI -/- BMMCs is 25% that of DPPI +/- BMMCs. These findings indicate that DPPI is not essential for mMCP-6 activation but does influence the total amount of active mMCP-6 in mast cells and therefore may be an important, but not exclusive mechanism for tryptase activation.     

New, sensitive fluorogenic substrates for human cathepsin G based on the sequence of serpin-reactive site loops.

Cathepsin G has both trypsin- and chymotrypsin-like activity, but studies on its enzymatic properties have been limited by a lack of sensitive synthetic substrates. Cathepsin G activity is physiologically controlled by the fast acting serpin inhibitors alpha1-antichymotrypsin and alpha1-proteinase inhibitor, in which the reactive site loops are cleaved during interaction with their target enzymes. We therefore synthesized a series of intramolecularly quenched fluorogenic peptides based on the sequence of various serpin loops. Those peptides were assayed as substrates for cathepsin G and other chymotrypsin-like enzymes including chymotrypsin and chymase. Peptide substrates derived from the alpha1-antichymotrypsin loop were the most sensitive for cathepsin G with kcat/Km values of 5-20 mM-1 s-1. Substitutions were introduced at positions P1 and P2 in alpha1-antichymotrypsin-derived substrates to tentatively improve their sensitivity. Replacement of Leu-Leu in ortho-aminobenzoyl (Abz)-Thr-Leu-Leu-Ser-Ala-Leu-Gln-N-(2, 4-dinitrophenyl)ethylenediamine (EDDnp) by Pro-Phe in Abz-Thr-Pro-Phe-Ser-Ala-Leu-Gln-EDDnp produced the most sensitive substrate of cathepsin G ever reported. It was cleaved with a specificity constant kcat/Km of 150 mM-1 s-1. Analysis by molecular modeling of a peptide substrate bound into the cathepsin G active site revealed that, in addition to the protease S1 subsite, subsites S1' and S2' significantly contribute to the definition of the substrate specificity of cathepsin G.     

The inflammatory response after an epidermal burn depends on the activities of mouse mast cell proteases 4 and 5

A second-degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to nonmuscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-Kit(W)/Kit(Wv) mice, which lack MCs in a context of other defects due to a mutation of the Kit receptor. To address further an MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury after a second-degree scald burn, whereas mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficient mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first hour postburn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing nonredundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling.     

rMCP-2, the Major Rat Mucosal Mast Cell Protease,an Analysis of Its Extended Cleavage Specificity and Its Potential Role in Regulating Intestinal Permeability by the Cleavage of Cell Adhesion and Junction Proteins

Mast cells of the rat intestinal mucosa express three chymotryptic enzymes named rMCP-2, -3 and 4. rMCP-2, the most abundant of these enzymes, has been shown to increase the permeability of the intestinal epithelium, most likely by cleavage of cell adhesion and junction proteins and thereby play a role in intestinal parasite clearance. However, no target for this effect has yet been identified. To address this question we here present its extended cleavage specificity. Phage display analysis showed that it is a chymase with a specificity similar to the corresponding enzyme in mice, mMCP-1, with a preference for Phe or Tyr in the P1 position, and a general preference for aliphatic amino acids both upstream and downstream of the cleavage site. The consensus sequence obtained from the phage display analysis was used to screen the rat proteome for potential targets. A few of the most interesting candidate substrates were cell adhesion and cell junction molecules. To see if these proteins were also susceptible to cleavage in their native conformation we cleaved 5 different recombinant cell adhesion and cell junction proteins. Three potential targets were identified: the loop 1 of occludin, protocadherin alpha 4 and cadherin 17, which indicated that these proteins were at least partly responsible for the previously observed prominent role of rMCP-2 in mucosal permeability and in parasite clearance.