The gene encoding the fibronectin‐binding protein (FNBP) from Staphylococcus aureus strain 8325‐4 was isolated from a gene bank in pBR322. The original clone, containing a 6.5‐kb insert, gave a functional product present in the periplasm of Escherichia coli. Analysis of polypeptides isolated after affinity chromatography on fibronectin‐Sepharose followed by ion‐exchange chromatography revealed two gene products, 87 and 165 kd in mol. wt. The amino acid compositions of these two polypeptides and a native FNBP from S. aureus strain Newman were very similar. Antibodies raised against the native FNBP from strain Newman precipitated the 125I‐labelled 165‐kd polypeptide, and unlabeled 165‐ and 87‐kd polypeptides as well as native FNBP inhibited the immunoprecipitation reactions. The region of the fnbp‐gene encoding the fibronectin‐binding activity has been identified and subcloned in an expression vector based on the staphylococcal protein A gene. The resulting product in E. coli is an extracellular fusion protein consisting of two IgG‐binding domains of protein A followed by a fibronectin‐binding region. The fusion protein binds to fibronectin and completely inhibits the binding of fibronectin to intact cells of S. aureus.

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...

No abstract

No abstract

No abstract