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• W1978176375 abstract "Tryptases α and β/II were expressed in insect cells to try to ascertain why human mast cells express these two nearly identical granule proteases. In contrast to that proposed by others, residue −3 in the propeptide did not appear to be essential for the three-dimensional folding, post-translational modification, and/or activation of this family of serine proteases. Both recombinant tryptases were functional and bound the active-site inhibitor diisopropyl fluorophosphate. However, they differed in their ability to cleave varied trypsin-susceptible chromogenic substrates. Structural modeling analyses revealed that tryptase α differs from tryptase β/II in that it possesses an Asp, rather than a Gly, in one of the loops that form its substrate-binding cleft. A site-directed mutagenesis approach was therefore carried out to determine the importance of this residue. Because the D215G derivative of tryptase α exhibited potent enzymatic activity against fibrinogen and other tryptase β/II-susceptible substrates, Asp215dominantly restricts the substrate specificity of tryptase α. These data indicate for the first time that tryptases α and β/II are functionally different human proteases. Moreover, the variation of just a single amino acid in the substrate-binding cleft of a tryptase can have profound consequences in the regulation of its enzymatic activity and/or substrate preference. Tryptases α and β/II were expressed in insect cells to try to ascertain why human mast cells express these two nearly identical granule proteases. In contrast to that proposed by others, residue −3 in the propeptide did not appear to be essential for the three-dimensional folding, post-translational modification, and/or activation of this family of serine proteases. Both recombinant tryptases were functional and bound the active-site inhibitor diisopropyl fluorophosphate. However, they differed in their ability to cleave varied trypsin-susceptible chromogenic substrates. Structural modeling analyses revealed that tryptase α differs from tryptase β/II in that it possesses an Asp, rather than a Gly, in one of the loops that form its substrate-binding cleft. A site-directed mutagenesis approach was therefore carried out to determine the importance of this residue. Because the D215G derivative of tryptase α exhibited potent enzymatic activity against fibrinogen and other tryptase β/II-susceptible substrates, Asp215dominantly restricts the substrate specificity of tryptase α. These data indicate for the first time that tryptases α and β/II are functionally different human proteases. Moreover, the variation of just a single amino acid in the substrate-binding cleft of a tryptase can have profound consequences in the regulation of its enzymatic activity and/or substrate preference. mast cell diisopropyl fluorophosphate enterokinase mouse MC protease p-nitroanilide polyacrylamide gel electrophoresis Mast cells (MCs)1 reside in connective tissue matrices and epithelial surfaces and are important effector cells in acquired and innate immunity. Human MCs express at least four closely related tryptases (designated human tryptases I, β/II, III, and α) 2Although the initial publication (2Miller J.S. Westin E.H. Schwartz L.B. J. Clin. Invest. 1989; 84: 1188-1195Crossref PubMed Scopus (177) Google Scholar) that described the nucleotide and amino acid sequences of human tryptase α contained a number of errors, the correct sequences (5Huang R. Åbrink M. Gobl A.E. Nilsson G. Aveskogh M. Larsson L.G. Nilsson K. Hellman L. Scand. J. Immunol. 1993; 38: 359-367Crossref PubMed Scopus (35) Google Scholar) have been deposited recently in GenBankTM (accession number M30038). Tryptases β (3Miller J.S. Moxley G. Schwartz L.B. J. Clin. Invest. 1990; 86: 864-870Crossref PubMed Scopus (153) Google Scholar) and II (4Vanderslice P. Ballinger S.M. Tam E.K. Goldstein S.M. Craik C.S. Caughey G.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3811-3815Crossref PubMed Scopus (202) Google Scholar) were independently cloned by different groups. Nevertheless, they are the same MC protease. (1Schwartz L.B. Lewis R.A. Austen K.F. J. Biol. Chem. 1981; 256: 11939-11943Abstract Full Text PDF PubMed Google Scholar, 2Miller J.S. Westin E.H. Schwartz L.B. J. Clin. Invest. 1989; 84: 1188-1195Crossref PubMed Scopus (177) Google Scholar, 3Miller J.S. Moxley G. Schwartz L.B. J. Clin. Invest. 1990; 86: 864-870Crossref PubMed Scopus (153) Google Scholar, 4Vanderslice P. Ballinger S.M. Tam E.K. Goldstein S.M. Craik C.S. Caughey G.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3811-3815Crossref PubMed Scopus (202) Google Scholar), and this family of serine proteases has been implicated in asthma and other allergy-related disorders. Although the amino acid sequences of the varied tryptases are ≥93% identical, there are at least four genes on human chromosome 16 that encode related but distinct tryptases (6Pallaoro M. Feizo M.S. Shayesteh L. Blount J.L. Caughey G.H. J. Biol. Chem. 1999; 274: 3355-3362Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 7Wong G.W. Tang Y. Stevens R.L. Int. Arch. Allergy Immunol. 1999; 118: 419-421Crossref PubMed Scopus (13) Google Scholar). It has been shown recently that the two related tryptases designated mouse MC protease (mMCP)-6 (8Reynolds D.S. Stevens R.L. Lane W.S. Carr M.H. Austen K.F. Serafin W.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3230-3234Crossref PubMed Scopus (172) Google Scholar, 9Reynolds D.S. Gurley D.S. Austen K.F. Serafin W.E. J. Biol. Chem. 1991; 266: 3847-3853Abstract Full Text PDF PubMed Google Scholar) and mMCP-7 (10McNeil H.P. Reynolds D.S. Schiller V. Ghildyal N. Gurley D.S. Austen K.F. Stevens R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11174-11178Crossref PubMed Scopus (127) Google Scholar, 11Johnson D.A. Barton G.J. Protein Sci. 1992; 1: 370-377Crossref PubMed Scopus (66) Google Scholar) are metabolized differently in vivo (12Ghildyal N. Friend D.S. Stevens R.L. Austen K.F. Huang C. Penrose J.F. S̆ali A. Gurish M.F. J. Exp. Med. 1996; 184: 1061-1073Crossref PubMed Scopus (75) Google Scholar) and have dissimilar substrate specificities (13Huang C. Wong G.W. Ghildyal N. Gurish M.F. S̆ali A. Matsumoto R. Qiu W.-T. Stevens R.L. J. Biol. Chem. 1997; 272: 31885-31893Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 14Huang C. Friend D.S. Qiu W.-T. Wong G.W. Morales G. Hunt J. Stevens R.L. J. Immunol. 1998; 160: 1910-1919PubMed Google Scholar). Nevertheless, it is presently unclear why human MCs express so many homologous tryptases. Native (15Schwartz L.B. Bradford T.R. Littman B.H. Wintroub B.U. J. Immunol. 1985; 135: 2762-2767PubMed Google Scholar) and recombinant (16Ren S. Lawson A.E. Carr M. Baumgarten C.M. Schwartz L.B. J. Immunol. 1997; 159: 3540-3548PubMed Google Scholar) human tryptase β/II can degrade fibrinogen but whether or not human tryptase α is a functional enzyme is controversial. While normal human basophils contain a small amount of tryptase α protein (17Castells M.C. Irani A.M. Schwartz L.B. J. Immunol. 1987; 138: 2184-2189PubMed Google Scholar) and mRNA (18Xia H.Z. Kepley C.L. Sakai K. Chelliah J. Irani A.M. Schwartz L.B. J. Immunol. 1995; 154: 5472-5480PubMed Google Scholar), substantial numbers of tryptase α+ cells have been found in the blood of patients with asthma, chronic allergies, or adverse drug reactions (19Li L. Li Y. Reddel S.W. Cherrian M. Friend D.S. Stevens R.L. Krilis S.A. J. Immunol. 1998; 161: 5079-5086PubMed Google Scholar). The level of tryptase α is also elevated in the sera of patients with systemic mastocytosis (20Schwartz L.B. Sakai K. Bradford T.R. Ren S. Zweiman B. Worobec A.S. Metcalfe D.D. J. Clin. Invest. 1995; 96: 2702-2710Crossref PubMed Scopus (332) Google Scholar). Thus, whether or not tryptase α is a functional neutral protease in humans is of critical importance. Using an expression/site-directed mutagenesis approach, we now show that tryptases α and β/II are functional enzymes but that tryptase α exhibits a more restricted substrate specificity due to an alteration in one of the loops that forms its S1, S2, and S3 sites. Thus, like in the mouse, a primordial human tryptase gene duplicated a number of times to generate a family of functionally distinct granule proteases. Using a reverse transcriptase polymerase chain reaction approach, cDNAs were isolated from human lung RNA (CLONTECH) that encode tryptases α and β/II. In each instance, synthesis of the first DNA strand was accomplished using total RNA, random primers, and avian myeloblastosis virus-derived reverse transcriptase. The tryptase α cDNA was amplified from the multitude of lung cDNAs in the preparation using oligonucleotides (5′-ATGCTGAGCCTGCTGCTG-3′ and 5′-TGACTCACGGCTTTTTGGG-3′) that correspond to relatively conserved sequences in the hydrophobic signal peptide and the C terminus of the tryptase transcript. The tryptase β/II cDNA was amplified using oligonucleotides 5′-GTGGCCAGGATGCTGAATCTG-3′ and 5′-TGACTCACGGCTTTTTGGG-3′. Each cycle of the polymerase chain reaction consisted of a 1-min denaturing step at 90 °C, a 2-min annealing step at 60 °C, and a 3-min extension step at 72 °C. The polymerase chain reaction products were purified on a 1% low melting point gel and ligated into the TA-cloning vector (Novagen, Milwaukee, WI). After Novablue competent cells were transformed with the plasmids, clones were isolated, and the nucleotide sequences of their inserts were determined to confirm that the isolated cDNAs encode authentic tryptases α and β/II. Recombinant human tryptases α and β/II were expressed as pseudozymogens in baculovirus-infected High FiveTM insect cells (Invitrogen, San Diego, CA), as described previously for mMCP-6 (14Huang C. Friend D.S. Qiu W.-T. Wong G.W. Morales G. Hunt J. Stevens R.L. J. Immunol. 1998; 160: 1910-1919PubMed Google Scholar) and mMCP-7 (13Huang C. Wong G.W. Ghildyal N. Gurish M.F. S̆ali A. Matsumoto R. Qiu W.-T. Stevens R.L. J. Biol. Chem. 1997; 272: 31885-31893Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) pseudozymogens. We have shown previously that the C terminus does not influence substantially the enzymatic activity of mMCP-6 and mMCP-7 (13Huang C. Wong G.W. Ghildyal N. Gurish M.F. S̆ali A. Matsumoto R. Qiu W.-T. Stevens R.L. J. Biol. Chem. 1997; 272: 31885-31893Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 14Huang C. Friend D.S. Qiu W.-T. Wong G.W. Morales G. Hunt J. Stevens R.L. J. Immunol. 1998; 160: 1910-1919PubMed Google Scholar). Thus, the 8-residue FLAG peptide was attached to the C terminus in order to facilitate the purification of each human tryptase pseudozymogen from the insect cell-conditioned medium. To enable the recombinant pseudozymogen to be activated after its purification at low pH, the enterokinase (EK)-susceptible peptide Asp-Asp-Asp-Asp-Lys was also placed in between the natural propeptide and the mature portion of the human tryptase. Purified plasmid DNA (∼5 μg) was mixed with ∼0.5 μg of linearized BaculoGoldTM DNA (PharMingen, San Diego, CA) and calcium phosphate. The resulting DNA solution was added to 3 × 106 adherent Spodoptera frugiperda 9 insect cells (Invitrogen) that were in their log phase of growth, and the infected cells were cultured for 7 days at 27 °C in medium (Invitrogen) supplemented with 10% heat-inactivated FCS (Sigma). Recombinant virus particles (≥3 × 107) from these insect cells were added to a new culture dish containing 6 × 106 Trichoplusia ni High FiveTMinsect cells (Invitrogen) in their log phase of growth, and then the infected cells were cultured in serum-free, Xpress medium (BioWhittaker, Walkersville, MD). Generally 4 days later, the conditioned medium was centrifuged at 1500 × g for 15 min at room temperature before purification of the secreted recombinant protein was attempted. Asp215 resides in one of the loops predicted to form the substrate-binding cleft of tryptase α based on the crystallographic structures of pancreatic trypsin (21Walter J. Steigemann W. Singh T.P. Bartunik H. Bode W. Huber R. Acta Crystallogr. Sect. B Struct. Sci. 1982; 38: 1462-1472Crossref Google Scholar) and tryptase β/II (22Pereira P.J. Bergner A. Macedo-Ribeiro S. Huber R. Matschiner G. Fritz H. Sommerhoff C.P. Bode W. Nature. 1998; 392: 306-311Crossref PubMed Scopus (283) Google Scholar). To evaluate the functional role of this amino acid, the bioengineered cDNA that encodes the pseudozymogen form of the tryptase was subcloned into the pALTER®-1 vector (Promega, Madison, WI). Site-directed mutagenesis was performed using the Altered Sites® II in vitro mutagenesis system (Promega), according to the manufacturer's instructions. The mutagenic oligonucleotide 5′-GTCAGCTGGGGCGAGGGCTGT-3′ (corresponding to nucleotides 724–744 in the isolated tryptase α cDNA) was used to convert Asp215 to Gly215. A similar site-directed mutagenesis approach was used to evaluate whether or not Arg−3 was essential for the proper folding and/or activation of recombinant tryptase β/II in our expression system. In these latter experiments, the mutagenic oligonucleotide 5′-CAGGCCCTGCAGCAAGTGGGC-3′ was used to convert Arg−3 to Gln−3 in the tryptase β/II pseudozymogen. MC tryptases bind to heparin in a conformation-dependent manner due to the alignment of a number of positively charged residues on the surface when the serine protease is properly folded (23Matsumoto R. S̆ali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Analogous to that carried out on recombinant pro-mMCP-7 (23Matsumoto R. S̆ali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), heparin-Sepharose chromatography was therefore used to evaluate whether or not recombinant tryptase β/II, tryptase α, and the D215G mutant of tryptase α were properly folded. Samples were applied to 3-ml columns of heparin-Sepharose (Amersham Pharmacia Biotech) that had been equilibrated in 100 mm NaCl and 50 mm sodium acetate, pH 5.0. After each column was washed with 10 ml of equilibration buffer, the column was subjected to a 40-ml gradient in which the NaCl concentration was increased linearly from 0.1 to 4.0 m. The 1-ml eluate fractions were evaluated for their protein content; samples were also subjected to immunoblot analysis using anti-FLAG Ig. An immunoaffinity column (24Brizzard B.L. Chubet R.G. Vizard D.L. BioTechniques. 1994; 16: 730-735PubMed Google Scholar) (International Biotechnol. Inc., New Haven, CT) was used to purify recombinant tryptase α, the D215G mutant of tryptase α, tryptase β/II, and the R-3Q mutant of tryptase β/II from the insect cell-conditioned medium. Each column (2 ml) was equilibrated in 50 mmTris-HCl and 150 mm NaCl, pH 7.4. After application of ∼200 ml of the insect cell-conditioned medium, the column was washed with the pH 7.4 buffer. Bound recombinant protein was eluted with 0.1m glycine, pH 4.0. The final eluate was collected into tubes that contained 0.1 m Tris-HCl, pH 7.0, to minimize acid-mediated denaturation of the recombinant pseudozymogen. The protein concentration of the eluate was estimated using a Bio-Rad colorimetric assay or by measuring its optical density at 280 nm. Purified pseudozymogen (∼100 μg in 100 μl) was placed in 100 μl of a pH 5.5 buffer containing 10 mm Tris-HCl, 5 mm calcium chloride, and 0.1% Triton X-100, with or without 50 μg of heparin glycosaminoglycan. The heparin (Sigma) used in the study had been subjected to a guanidine HCl/CsCl density-gradient centrifugation step (14Huang C. Friend D.S. Qiu W.-T. Wong G.W. Morales G. Hunt J. Stevens R.L. J. Immunol. 1998; 160: 1910-1919PubMed Google Scholar) to denature and remove any trace protein contaminants that might be present in the commercial preparation of this glycosaminoglycan. In most cases, a 1-μl solution containing 0.4 units of recombinant EK (New England Biolabs) was added, and the mixture was incubated at room temperature for 17 h to allow EK to activate each tryptase pseudozymogen. Samples from insect cell-conditioned medium infected with varied recombinant baculovirus or proteins purified using the immunoaffinity column were diluted in SDS-polyacrylamide gel electrophoresis (PAGE) buffer (1% SDS, 1% β-mercaptoethanol, 0.1% bromphenol blue, and 500 mm Tris-HCl, pH 6.8) and boiled for 5 min before being loaded onto a 12% polyacrylamide gel. After electrophoresis, the gel was stained with Coomassie Blue or was placed in a Bio-Rad immunoblotting apparatus. In the latter instance, the resolved proteins were transferred for 2–4 h at 200 mA to an Immobilon-P membrane in a solution consisting of 20% methanol, 16 mm Tris-HCl, and 120 mm glycine, pH 8.3. The resulting protein blot was washed three times with Tris-buffered saline and then incubated for 1 h at room temperature in Tris-buffered saline containing 5% nonfat milk and 0.02% Tween 20 (TBST buffer). After three washes with TBST buffer, the protein blot was incubated with a 1:500 dilution of affinity-purified anti-human tryptase immunoglobulin (Ig) (∼1.5 μg/ml final concentration) (25Schwartz L.B. J. Immunol. 1985; 134: 526-531PubMed Google Scholar) (Chemicon, Temecula, CA) or with mouse anti-FLAG M2 Ig (∼5 μg/ml final concentration) (Sigma) in TBST buffer for 1 h at room temperature. After three washes with TBST buffer, the protein blot was incubated for 1 h in a 1:1000 dilution of an alkaline phosphatase conjugate of goat anti-rabbit Ig or goat anti-mouse Ig (∼1 ng/ml final concentration) in TBST buffer. Immunoreactive proteins were visualized with nitro blue tetrazolium (0.2 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.1 mg/ml) as substrates. For N-terminal amino acid analysis, relevant SDS-PAGE protein blots were briefly stained with Ponceau S red (Sigma). The visualized protein bands were isolated and then were subjected to automated Edman degradation by the Harvard Microchemistry Facility (Harvard Biological Laboratories, Cambridge, MA). After EK activation, recombinant tryptase α, the D215G mutant of tryptase α, and tryptase β/II (∼16 μg) were suspended in 33 μl of 0.15 m NaCl, 20 mmTris, pH 7.4, buffer containing ∼8 μCi of [3H]diisopropyl fluorophosphate (DFP) (4 Ci/nmol; Ci = 37 Gbq; Amersham Pharmacia Biotech). The samples were incubated for 60 min at 37 °C. SDS-PAGE loading buffer was added (33 μl), and each sample was boiled for 5 min before subjecting the [3H]DFP-labeled proteins to SDS-PAGE analysis. The resulting gel was treated with EN3HANCE (NEN Life Science Products), dried, and exposed to x-ray film. A duplicate gel was stained with Coomassie Blue to confirm that similar amounts of protein were placed in each lane. Tryptase α, the D215G mutant of tryptase α, tryptase β/II, and the R-3Q mutant of tryptase β/II were incubated at room temperature or 37 °C for up to 24 h to determine the stability of each recombinant protease. At various times, samples of the digest were evaluated for the ability of the tryptase to cleave the trypsin-susceptible p-nitroanilide (pNA) chromogenic substrates Ile-Phe-Lys-pNA,N-acetyl-Ile-Glu-Ala-Arg-pNA,N-benzoyl-Arg-pNA,N-benzoyl-Ile-Glu-Gly-Arg-pNA,N-benzoyl-Phe-Val-Arg-pNA,N-benzoyl-Pro-Phe-Arg-pNA, tosyl-Gly-Pro-Lys-pNA, tosyl-Gly-Pro-Arg-pNA (Sigma), and H-d-HHT-Ala-Arg-pNA (American Diagonstica Inc., Greenwich, CT), as described previously for recombinant mouse tryptases (13Huang C. Wong G.W. Ghildyal N. Gurish M.F. S̆ali A. Matsumoto R. Qiu W.-T. Stevens R.L. J. Biol. Chem. 1997; 272: 31885-31893Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 14Huang C. Friend D.S. Qiu W.-T. Wong G.W. Morales G. Hunt J. Stevens R.L. J. Immunol. 1998; 160: 1910-1919PubMed Google Scholar, 26Svendsen L. Blombäck B. Blombäck M. Olsson P.I. Thromb. Res. 1972; 1: 267-278Abstract Full Text PDF Scopus (209) Google Scholar). One unit is defined as the amount of enzymatic activity that induces an 0.001 change in optical density at 405 nm/5 min when tosyl-Gly-Pro-Arg-pNA is used as a substrate. The ability of recombinant tryptase α, the D215G mutant of tryptase α, tryptase β/II, and the R-3Q mutant of tryptase β/II to degrade the more physiologic substrate fibrinogen was also evaluated. Human fibrinogen (Sigma) (∼40 μg), 50 μl of digestion buffer (25 mm sodium phosphate, 1 mm EDTA, pH 7.4), and 10 μl of mature tryptase β/II (∼2 μg), the tryptase β/II mutant (∼2 μg), tryptase α (∼2 μg), or the tryptase α mutant (∼2 μg corresponding to ∼20 units of tryptase β/II) were mixed and incubated for 1–2 h at 37 °C. The resulting digests were subjected to SDS-PAGE analysis, as described previously for the evaluation of the mMCP-7-mediated digestion of mouse fibrinogen (13Huang C. Wong G.W. Ghildyal N. Gurish M.F. S̆ali A. Matsumoto R. Qiu W.-T. Stevens R.L. J. Biol. Chem. 1997; 272: 31885-31893Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Recombinant tryptase α, tryptase β/II, and the R-3Q mutant of tryptase β/II were generated in insect cells to determine whether residue −3 was critical for the three-dimensional folding of their zymogens, as well as to compare the substrate specificities of these tryptases in their mature forms. Insect cells could be induced to express large amounts (up to 10 μg/ml conditioned medium) of pseudozymogen forms of tryptase α (Fig.1 A), tryptase β/II (Fig.1 B), and the tryptase β/II mutant (data not shown) that, in each instance, contained an EK-susceptible site in its propeptide and the FLAG peptide in its C terminus. The recombinant pseudozymogens were secreted into the conditioned media and were recognized by an anti-human tryptase antibody. Because the recombinant tryptases also bound to an anti-FLAG immunoaffinity column (Figs. 1 A and1 B) and a heparin-Sepharose column in a conformation-dependent manner (data not shown), all three expressed pseudozymogens appeared to be properly folded. After EK treatment, the activated recombinant tryptase monomers exhibited the appropriate molecular mass in an SDS-PAGE gel (Fig.1 C). The mature forms of tryptases α and β/II also possessed the expected N-terminal amino acid sequence of Ile-Val-Gly-Gly-Gln-Glu-Ala-Pro-Arg. The initially expressed mutated tryptases β/II zymogen secreted into the insect cell culture media also possessed the expected N-terminal amino acid sequence of Ala-Pro-Ala-Pro-Gly-Gln-Ala-Leu-Gln-Gln-Val-Gly which corresponds to residues −12 to −1. Mature tryptase α (Fig.2) and β/II (data not shown), but not the inactive pseudozymogens, readily bound the active site inhibitor DFP. Recombinant tryptase β/II and its R-3Q mutant were both able to degrade tosyl-Gly-Pro-Lys-pNA and tosyl-Gly-Pro-Arg-pNA, but not Ile-Phe-Lys-pNA,N-acetyl-Ile-Glu-Ala-Arg-pNA,N-benzoyl-Arg-pNA,N-benzoyl-Ile-Glu-Gly-Arg-pNA,N-benzoyl-Phe-Val-Arg-pNA, orN-benzoyl-Pro-Phe-Arg-pNA. Thus, residue −3 is not essential for generating active enzyme in this in vitrosystem. Recombinant mature tryptase α was able to cleave H-d-HHT-Ala-Arg-pNA and was able to cleave tosyl-Gly-Pro-Arg-pNA, but only if the digestion reaction was carried at room temperature for at least 4 h. The observation that tryptase α cleaves H-d-HHT-Ala-Arg-pNA much less efficiently than trypsin and cleaves tosyl-Gly-Pro-Arg-pNA much less efficiently than tryptase β/II suggests that tryptase α possess a substrate specificity that is even more restricted than that of tryptase β/II. Seven loops form the substrate-binding cleft of each tryptase (Fig.3 A). When the substrate-binding cleft of tryptase α was compared with that of tryptases I, β/II, and III, 9 amino acid differences were seen in loops A, B, C, 1, and 2 (Fig. 3 A). The discovery that a novel charged residue in loop 2 dominantly controls the substrate specificity of granzyme B, coupled with discovery that most MC tryptases cloned from different species have a noncharged Gly at residue 215 (Fig. 3 B), raised the possibility that Asp215 restricts the substrate specificity of tryptase α. A site-directed mutagenesis approach was therefore carried out to convert Asp215 in tryptase α to Gly. The mutant was expressed in insect cells, and after EK treatment, it was able to cleave tosyl-Gly-Pro-Lys-pNA and tosyl-Gly-Pro-Arg-pNA (Fig.4), but not Ile-Phe-Lys-pNA,N-acetyl-Ile-Glu-Ala-Arg-pNA,N-benzoyl-Arg-pNA,N-benzoyl-Ile-Glu-Gly-Arg-pNA,N-benzoyl-Phe-Val-Arg-pNA, orN-benzoyl-Pro-Phe-Arg-pNA. Using tosyl-Gly-Pro-Arg-pNA as the substrate in reactions carried out at room temperature, the estimated K m,k cat, andk cat/K mvalues 3In these comparative studies, similar amounts (as measured by the Bio-Rad protein bioassay) of tryptase β/II pseudozymogen and the D215G mutant of tryptase α pseudozymogen were exposed to EK. The processed recombinant tryptases were then examined for their relative ability to cleave tosyl-Gly-Pro-Arg-pNA. Even when comparing tryptases whose amino acid sequences are 93% identical, such as these two proteases, obtained K m, kcat, and kcat/K m values can only be estimated, because a number of assumptions have to be made. Studies carried out on recombinant mouse tryptases have revealed that this family of serine proteases must spontaneously form a tetramer to become enzymatically active. It is therefore assumed that the percentage of pseudozymogen in each preparation which is converted by EK into active enzyme is the same. It is also assumed that the rate of conversion of inactive monomer to active tetramer and then back to inactive monomer is the same for both tryptases. Finally, it is assumed that each tryptase tetramer has the same number of active sites. of the D215G mutant of tryptase α were 120 μm, 1.9 s−1, and 0.016 μm−1 s−1, respectively, whereas the corresponding values of tryptase β/II were 49 μm, 21 s−1, and 0.43 μm−1 s, respectively. Because tryptase β/II cleaved tosyl-Gly-Pro-Arg-pNA ∼25-fold better than the D215G mutant of tryptase α, it is likely that pocket residues 21, 22, 23, 46, 84, 85, 88, and/or 91 (Fig. 3) contribute to the different substrate specificities of the two human tryptases.Figure 4Heparin-dependent activation and stabilization of the D215G mutant of recombinant human tryptase α. The purified D215G mutant of tryptase α was incubated at room temperature (♦, ▪) or 37 °C (○, ▴) for up to 24 h with EK in the presence (▴, ♦) or absence (○, ▪) of heparin. At the indicated times, samples were removed from the activation mixture and assayed for their ability to cleave tosyl-Gly-Pro-Arg-pNA during a 5-min incubation.View Large Image Figure ViewerDownload (PPT) Kinetic experiments revealed that the enzymatic activity of the D215G mutant was maintained for at least 24 h if heparin was present (Fig. 4). In addition, there was no evidence of autolysis of the recombinant protein by SDS-PAGE analysis (Fig. 1 C). Wild type tryptase β/II and its R-3Q mutant were able to cleave the α and β chains of fibrinogen but not the γ chain of this plasma protein (Fig. 5 A). Although fibrinogen was not susceptible to wild type tryptase α, its D215G mutant was able to degrade the α chain of fibrinogen (Fig.5 B). Using a bioengineering approach, pseudozymogen forms of human tryptases α and β/II were expressed in insect cells that could be activated and studied immediately (Fig. 1). Although Mirza et al. (31Mirza H. Schmidt V.A. Derian C.K. Jesty J. Bahou W.F. Blood. 1997; 90: 3914-3922Crossref PubMed Google Scholar) were able to obtain enzymatically active tryptase α in transiently transfected COS cells, Sakaiet al. (32Sakai K. Ren S. Schwartz L.B. J. Clin. Invest. 1996; 97: 988-995Crossref PubMed Scopus (139) Google Scholar) concluded that this human MC protease probably is not functional in vivo. The latter investigators noted that the propeptide of tryptase α differs from that of mouse, rat, and other human tryptase zymogens at residue −3. Based on their inability to obtain active enzyme in an insect cell expression system, Sakai et al. (32Sakai K. Ren S. Schwartz L.B. J. Clin. Invest. 1996; 97: 988-995Crossref PubMed Scopus (139) Google Scholar) concluded that all tryptases probably undergo an unusual multistep activation process in MCs that is exquisitely heparin-dependent. To explain their in vitro findings, these investigators proposed that a conformation-dependent change occurs when a tryptase zymogen initially binds to heparin, which in turn causes a partial autocatalytic event resulting in the removal of the first 8 residues of the propeptide. It was then proposed that dipeptidyl peptidase I removes the remaining 2 residues of the zymogen in the granule to create the mature enzyme. Unexplained in the Sakai et al.(32Sakai K. Ren S. Schwartz L.B. J. Clin. Invest. 1996; 97: 988-995Crossref PubMed Scopus (139) Google Scholar) mechanism of tryptase activation is how a heparin-bound tryptase zymogen can partially activate itself yet still fail to cleave varied low molecular weight substrates. We have noted that certain recombinant mouse MC chymases denature spontaneously during their expression in insect cells. 4C. Huang and R. L. Stevens, unpublished results. Because the propeptide often is needed for the proper folding of a serine protease (33Urata H. Karnik S.S. Graham R.M. Husain A. J. Biol. Chem. 1993; 268: 24318-24322Abstract Full Text PDF PubMed Google Scholar), we examined whether or not Arg−3 is essential for the proper folding of tryptases in insect cells. Using a site-directed mutagenesis approach, Arg−3 in tryptase β/II was converted to Gln−3. Analogous to wild type tryptase β/II, the R-3Q derivative of the pseudozymogen could be readily activated in our in vitro system. While this finding alone does not prove that tryptase α is post-translationally processed into a functional enzyme in human MCs, a tryptase has been cloned from gerbil jejunal MCs (30Murakumo Y. Ide H. Itoh H. Tomita M. Kobayashi T. Maruyama H. Horii Y. Nawa Y. Biochem. J. 1995; 309: 921-926Crossref PubMed Scopus (21) Google Scholar), which is enzymatically active (34Nawa Y.Y. Horii Y. Okada M. Arizono N. Int. Arch. Allergy Immunol. 1994; 104: 249-254Crossref PubMed Scopus (31) Google Scholar) even though its zymogen possesses a Glu at residue −3. In addition, Wang et al. (35Wang Z.-M. Walter M. Selwood T. Rubin H. Schechter N.M. Biol. Chem. 1998; 379: 167-174Crossref PubMed Scopus (40) Google Scholar) recently generated active tryptase β/II in insect cells using a fusion protein approach in which the natural propeptide of human tryptase β/II was replaced by that which encodes ubiquitin followed by a short EK-susceptible peptide. More definitive data relevant to this issue have been obtained recently during analysis of the transgenic mouse we created which is unable to express heparin due to targeted disruption of theN-deacetylase/N-sulfotransferase-2 gene. Although the MCs developed from these heparin-null mice are unable to store the chymase mMCP-5 and the exopeptidase mMC-CPA in their granules, they do contain substantial amounts of enzymatically active tryptase. 5D. E. Humphries and R. L. Stevens, unpublished results. The cumulative findings from all of these studies now indicate that heparin is not essential for tryptase expression in MCs and that Arg−3 is not essential for the proteolytic activation of tryptase zymogens. Because we concluded that Mirza et al. (31Mirza H. Schmidt V.A. Derian C.K. Jesty J. Bahou W.F. Blood. 1997; 90: 3914-3922Crossref PubMed Google Scholar) are probably correct in their conclusion that human tryptase α is a functional enzyme in vivo, we expressed this tryptase and tryptase β/II in our insect cell expression system to try to address why human MCs produce so many nearly identical neutral proteases. Insect cell-derived recombinant tryptases α and β/II have all of the features of biologically active serine proteases (Fig.3 A). DFP binds to each mature tryptase but, importantly, not to their pseudozymogens (Fig. 2). These observations indicate that DFP is not binding to either tryptase in a nonspecific manner. In vitro and in vivo studies carried out in mice with recombinant mouse tryptases (13Huang C. Wong G.W. Ghildyal N. Gurish M.F. S̆ali A. Matsumoto R. Qiu W.-T. Stevens R.L. J. Biol. Chem. 1997; 272: 31885-31893Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 14Huang C. Friend D.S. Qiu W.-T. Wong G.W. Morales G. Hunt J. Stevens R.L. J. Immunol. 1998; 160: 1910-1919PubMed Google Scholar) have confirmed earlier structural (11Johnson D.A. Barton G.J. Protein Sci. 1992; 1: 370-377Crossref PubMed Scopus (66) Google Scholar, 22Pereira P.J. Bergner A. Macedo-Ribeiro S. Huber R. Matschiner G. Fritz H. Sommerhoff C.P. Bode W. Nature. 1998; 392: 306-311Crossref PubMed Scopus (283) Google Scholar) and biochemical studies (36Cromlish J.A. Seidah N.G. Marcinkiewicz M. Hamelin J. Johnson D.A. Chrétien M. J. Biol. Chem. 1987; 262: 1363-1373Abstract Full Text PDF PubMed Google Scholar), which had suggested that mouse MC tryptases possess substrate specificities that are considerably more restricted than that of trypsin. The physiologic function of tryptase α remains to be determined. However, the in vitro studies carried out in the present investigation now suggest that tryptase α also has a restricted substrate specificity and one that is different from that of tryptase β/II. Fibrinogen is a major protein constituent of blood. Although tissue edema can be quite pronounced during a MC-mediated inflammatory reaction, one generally does not see the deposition of large amounts of fibrin/platelet clots in affected tissues. Fibrinogen is a physiologic substrate of mMCP-7 (13Huang C. Wong G.W. Ghildyal N. Gurish M.F. S̆ali A. Matsumoto R. Qiu W.-T. Stevens R.L. J. Biol. Chem. 1997; 272: 31885-31893Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), and both native (15Schwartz L.B. Bradford T.R. Littman B.H. Wintroub B.U. J. Immunol. 1985; 135: 2762-2767PubMed Google Scholar) and recombinant tryptase β/II (16Ren S. Lawson A.E. Carr M. Baumgarten C.M. Schwartz L.B. J. Immunol. 1997; 159: 3540-3548PubMed Google Scholar) can cleave fibrinogen in vitro. Thus, tryptase β/II probably plays an important role in preventing the accumulation of clots that would physically hinder lymphocyte and granulocyte extravasation into the inflamed tissue. Although the amino acid sequences of tryptase α and β/II are 93% identical, recombinant tryptase α was unable to degrade human fibrinogen in vitro(Fig. 5). Seven loops form the substrate-binding cleft of each serine protease (37Perona J.J. Craik C.S. Protein Sci. 1995; 4: 337-360Crossref PubMed Scopus (759) Google Scholar). Although the loops that comprise the substrate-binding cleft of a human tryptase represent only ∼20% of the mature enzyme, 9 of the 18 amino acid differences between tryptase α and tryptase β/II reside precisely in this small portion of the protease (Fig.3 A). Because the substrate-binding cleft was selectively mutated during the evolution of these two human proteases, we reasoned that it probably occurred to generate functionally distinct tryptases. Based on the crystallographic structure of tryptase β/II (22Pereira P.J. Bergner A. Macedo-Ribeiro S. Huber R. Matschiner G. Fritz H. Sommerhoff C.P. Bode W. Nature. 1998; 392: 306-311Crossref PubMed Scopus (283) Google Scholar), loop 2 (corresponding to amino acid residues 211–218) contributes substantially to the enzyme's S1, S2, and S3 sites. A comparative analysis of their amino acid sequences revealed that tryptase α has an Asp at residue 215 rather than Gly, which is found in mouse, rat, dog, gerbil, and other human tryptases (Fig. 3 B). This observation raised the possibility that human tryptases α and β/II might be functionally different, in part, due to this amino acid substitution. To explore this possibility, a site-directed mutagenesis approach was carried out to change Asp215 in tryptase α to Gly (Fig. 1). Like wild type tryptase β/II, the resulting D215G mutant of tryptase α was able to effectively cleave the chromogenic substrate tosyl-Gly-Pro-Lys-pNA (Fig. 4). The mutant also was able to cleave the α chain of human fibrinogen (Fig. 5). It is well known that loop 2 impacts the substrate specificities of other serine proteases. For example (38Caputo A. James M.N. Powers J.C. Hudig D. Bleackley R.C. Nat. Struct. Biol. 1994; 1: 364-367Crossref PubMed Scopus (63) Google Scholar), the presence of an Arg in loop 2 causes granzyme B to prefer substrates which have an Asp at the P1 site rather than a hydrophobic residue preferred by many other members of the chromosome 14 family of serine proteases. Although the bulky negatively charged amino acid at residue 215 explains the restricted substrate specificity of tryptase α relative to that of tryptase β/II, it is likely that, in vivo, tryptase α still prefers substrates that have a positively charged amino acid at their P1 sites. Like mMCP-6 and mMCP-7 in the mouse, human tryptases α and β/II are functional proteases that differ in their substrate specificities. It now appears that these two neutral proteases evolved so that human MCs can degrade different extracellular proteins during an inflammatory reaction. We gratefully acknowledge the technical assistance of Mary Ferrazi (Brigham and Women's Hospital, Boston, MA) and the helpful suggestions of Dr. A. S̆ali (Rockefeller University, New York)." @default.
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• W1978176375 title "Human Tryptases α and β/II Are Functionally Distinct Due, in Part, to a Single Amino Acid Difference in One of the Surface Loops That Forms the Substrate-binding Cleft" @default.
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