title: Mechanism for activation of mouse mast cell tryptase: dependence on heparin and acidic pH for formation of active tetramers of mouse mast cell protease 6.
abstract: Tryptase, a serine protease with trypsin-like substrate cleavage properties, is one of the key effector molecules during allergic inflammation. It is stored in large quantities in the mast cell secretory granules in complex with heparin proteoglycan, and these complexes are released during mast cell degranulation. In the present paper, we have studied the mechanism for tryptase activation. Recombinant mouse tryptase, mouse mast cell protease 6 (mMCP-6), was produced in a mammalian expression system. The mMCP-6 fusion protein contained an N-terminal 6 x His tag followed by an enterokinase (EK) site replacing the native activation peptide (6xHis-EK-mMCP-6). In the absence of heparin, barely detectable enzyme activity was obtained after enterokinase cleavage of 6xHis-EK-mMCP-6 over a pH range of 5.5-7.5. However, when heparin was present, 6xHis-EK-mMCP-6 yielded active enzyme when enterokinase cleavage was performed at pH 5.5-6.0 but not at neutral pH. Affinity chromatography analysis showed that mMCP-6 bound strongly to heparin-Sepharose at pH 6.0 but not at neutral pH. After enterokinase cleavage of the sample at pH 6.0, mMCP-6 occurred in inactive monomeric form as shown by FPLC analysis on a Superdex 200 column. When heparin was added at pH 6.0, enzymatically active higher molecular weight complexes were formed, e.g., a dominant approximately 200 kDa complex that may correspond to tryptase tetramers. No formation of active tetramers was observed at neutral pH. When injected intraperitoneally, mMCP-6 together with heparin caused neutrophil influx, but no signs of inflammation were seen in the absence of heparin. The present paper thus indicates a crucial role for heparin in the formation of active mast cell tryptase.
authors.affiliation: Swedish University of Agricultural Sciences, Department of Veterinary Medical Chemistry, The Biomedical Center, Box 575, 751 23 Uppsala, Sweden.
authors.forename: J
authors.initials: J
authors.lastname: Hallgren
authors.forename: U
authors.initials: U
authors.lastname: Karlson
authors.forename: M
authors.initials: M
authors.lastname: Poorafshar
authors.forename: L
authors.initials: L
authors.lastname: Hellman
authors.forename: G
authors.initials: G
authors.lastname: Pejler
mesh_terms: D000143:Acids; D000818:Animals; D015550:Cell Degranulation; D053818:Chymases; D004765:Enteropeptidase / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D006493:Heparin / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D018836:Inflammation Mediators / Q000008:administration & dosage; D007274:Injections, Intraperitoneal; D008407:Mast Cells / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000096:biosynthesis / Q000378:metabolism; D012697:Serine Endopeptidases / Q000008:administration & dosage / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D053802:Tryptases
chemical_list: D000143:Acids; D018836:Inflammation Mediators; D011993:Recombinant Fusion Proteins; C509190:Tpsb2 protein, mouse; D006493:Heparin; D012697:Serine Endopeptidases; C083294:chymase 2; D053818:Chymases; C509222:Tpsab1 protein, mouse; D053802:Tryptases; D004765:Enteropeptidase