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

Abstract

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...

Extended Substrate Specificity of Rat Mast Cell Protease 5, a Rodent α-Chymase with Elastase-like Primary Specificity

“…However, in contrast to substrate regions recognized by HC and, specifically, rMCP-1 (25), P3 aromatic aa are absent in the rMCP-5-susceptible substrate regions. The same phage library has previously been used successfully to determine the substrate specificity of a rat ␤-chymase (rMCP-4) with P1 specificity for aromatic aa (23). All of the sequenced rMCP-4-susceptible clones contained one or several aromatic amino acid residues, demonstrating that the lack of aromatic P3 residues in the rMCP-5-susceptible substrate regions is not a consequence of a limited library.…”

“…Determination of Cleavage Specificity by Phage-displayed Nonapeptides-The library of phage-displayed peptides was constructed as previously described (23). An aliquot of the amplified phages (ϳ10 9 plaqueforming units) were allowed to bind to 100 l of Ni-NTA-agarose beads…”

Identification of an ADAMTS-4 Cleavage Motif Using Phage Display Leads to the Development of Fluorogenic Peptide Substrates and Reveals Matrilin-3 as a Novel Substrate

“…Peptide phage display has been demonstrated to be a valid means of determining the consensus motifs for a number of proteases including MMP-11 (15), human kallikrein 2 (35), rat mast cell protease 4 (16), and outer membrane protein T (17). Motifs derived from such an analysis have been shown to be physiologically relevant and used to determine preferred as well as possible alternate protease substrates.…”

Implantation serine proteinase 2 is a monomeric enzyme with mixed serine proteolytic activity and can silence signalling via proteinase activated receptors

“…In addition to confirming the randomness of the library, we also validated our biopanning approach using thrombin as a test enzyme with previously known substrate specificity. Biopanning was performed using 2.0 units/mL of ISP2 enzyme per cycle in 25 mmol/L Tris.Cl, pH 8.0 (total volume of reaction mix: 0.5 mL) at 37°C overnight, subsequent to the binding of the amplified phage preparation (1 × 10 10 pfu) with 100 ul Ni-NTA agarose beads, as described in Karlson et al (2002). A control elution was performed using 500 mmol/L imidazole solution.…”

“…A library of random hexapeptides (6-mers) was generated as described by Karlson et al (2002) with minor modifications. The sequence of synthetic degenerate oligonucleotides inserted in the coding region of T7 phage capsid protein (employing T7 Select 1-1 vector arms, T7 Select system, Novagen, Canada), encoding a random hexamer followed by (His)6 tag is as follows: 59-AAT TCT CTC ACT CCA GGC GGC-(NNK)6-GGT GGT CAT CAC CAT CAC CAT CAC TAA-39 (N represents any nucleotide and K represents T or G).…”