Mouse Chromosome 17A3.3 Contains 13 Genes That Encode Functional Tryptic-like Serine Proteases with Distinct Tissue and Cell Expression Patterns
2004; Elsevier BV; Volume: 279; Issue: 4 Linguagem: Inglês
10.1074/jbc.m308209200
ISSN1083-351X
AutoresG. William Wong, Shinsuke Yasuda, Nasa Morokawa, Lixin Li, Richard L Stevens,
Tópico(s)Chemical Synthesis and Analysis
ResumoProbing of the mouse EST data base at GenBank™ with known tryptase cDNAs resulted in the identification of undiscovered serine protease transcripts whose genes reside at a 1.5-Mb complex on mouse chromosome 17A3.3. Mouse tryptase-5 (mT5), tryptase-6 (mT6), and mast cell protease-11 (mMCP-11) are new members of this serine protease superfamily whose amino acid sequences are 36–54% identical to each other and to their other 10 family members. The 13 functional mouse proteases can be subdivided into two subgroups based on conserved features in their propeptides. Of the three new serine proteases, mT6 is most widely expressed in tissues. mT5 is preferentially expressed in smooth muscle, whereas mMCP-11 is preferentially expressed in the spleen and bone marrow. In contrast to mT5 and mT6, mMCP-11 is also expressed in mast cells. Although mT6 and mMCP-11 are constitutively secreted when expressed in mammalian and insect cells, mT5 remains membrane-associated. The fact that recombinant mT5, mT6, and mMCP-11 possess non-identical expression patterns and substrate specificities suggests that each protease has a unique function in vivo. Of the 13 functional mouse tryptase genes identified at the complex, 12 have orthologs that reside in the syntenic region of human chromosome 16p13.3. The establishment of these ortholog pairs helps clarify the evolutionary relationship of the serine protease locus in the two species. This information provides a useful framework for the functional analysis of each protease using gene targeting and other molecular approaches. Probing of the mouse EST data base at GenBank™ with known tryptase cDNAs resulted in the identification of undiscovered serine protease transcripts whose genes reside at a 1.5-Mb complex on mouse chromosome 17A3.3. Mouse tryptase-5 (mT5), tryptase-6 (mT6), and mast cell protease-11 (mMCP-11) are new members of this serine protease superfamily whose amino acid sequences are 36–54% identical to each other and to their other 10 family members. The 13 functional mouse proteases can be subdivided into two subgroups based on conserved features in their propeptides. Of the three new serine proteases, mT6 is most widely expressed in tissues. mT5 is preferentially expressed in smooth muscle, whereas mMCP-11 is preferentially expressed in the spleen and bone marrow. In contrast to mT5 and mT6, mMCP-11 is also expressed in mast cells. Although mT6 and mMCP-11 are constitutively secreted when expressed in mammalian and insect cells, mT5 remains membrane-associated. The fact that recombinant mT5, mT6, and mMCP-11 possess non-identical expression patterns and substrate specificities suggests that each protease has a unique function in vivo. Of the 13 functional mouse tryptase genes identified at the complex, 12 have orthologs that reside in the syntenic region of human chromosome 16p13.3. The establishment of these ortholog pairs helps clarify the evolutionary relationship of the serine protease locus in the two species. This information provides a useful framework for the functional analysis of each protease using gene targeting and other molecular approaches. The serine protease gene cluster at chromosome 16p13.3 contains the genes that encode human tryptase α (1.Miller J.S. Westin E.H. Schwartz L.B. J. Clin. Investig. 1989; 84: 1188-1195Crossref PubMed Scopus (177) Google Scholar), tryptase βI (2.Vanderslice 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), tryptase βII (2.Vanderslice 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, 3.Miller J.S. Moxley G. Schwartz L.B. J. Clin. Investig. 1990; 86: 864-870Crossref PubMed Scopus (153) Google Scholar), tryptase βIII (2.Vanderslice 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), transmembrane tryptase (TMT) 1The abbreviations used are: TMT, transmembrane tryptase; Bssp-4, brain-specific serine protease-4; Disp, distal intestinal serine protease; Esp-1, eosinophil serine protease-1; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glycosylphosphatidylinositol; IL, interleukin; MC, mast cell; Isp, implantation serine protease; mMCP, mouse MC protease; mT4, mouse tryptase-4; mT5, mouse tryptase-5; mT6, mouse tryptase-6; PPACK, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone; PRSS, protease serine member S; Tessp1, testis serine protease-1; PNGaseF, peptide:N-glycosidase; pNA, p-nitroanilide; mBMMCs, mouse bone marrow-derived MCs.1The abbreviations used are: TMT, transmembrane tryptase; Bssp-4, brain-specific serine protease-4; Disp, distal intestinal serine protease; Esp-1, eosinophil serine protease-1; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glycosylphosphatidylinositol; IL, interleukin; MC, mast cell; Isp, implantation serine protease; mMCP, mouse MC protease; mT4, mouse tryptase-4; mT5, mouse tryptase-5; mT6, mouse tryptase-6; PPACK, d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone; PRSS, protease serine member S; Tessp1, testis serine protease-1; PNGaseF, peptide:N-glycosidase; pNA, p-nitroanilide; mBMMCs, mouse bone marrow-derived MCs./tryptase γ (4.Wong G.W. Tang Y. Feyfant E. Šali A. Li L. Li Y. Huang C. Friend D.S. Krilis S.A. Stevens R.L. J. Biol. Chem. 1999; 274: 30784-30793Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 5.Caughey G.H. Raymond W.W. Blount J.L. Hau L.W. Pallaoro M. Wolters P.J. Verghese G.M. J. Immunol. 2000; 164: 6566-6575Crossref PubMed Scopus (103) Google Scholar), tryptase δ (6.Pallaoro M. Fejzo 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), tryptase ϵ/protease serine member S22 (PRSS22) (7.Wong G.W. Yasuda S. Madhusudhan M.S. Li L. Yang Y. Krilis S.A. Šali A. Stevens R.L. J. Biol. Chem. 2001; 276: 49169-49182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), pancreasin/marapsin/PRSS27 (8.Bhagwandin V.J. Hau L.W. Mallen-St. Clair J. Wolters P.J. Caughey G.H. J. Biol. Chem. 2003; 278: 3363-3371Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), eosinophil serine protease-1 (Esp-1)/testisin/PRSS21 (9.Inoue M. Kanbe N. Kurosawa M. Kido H. Biochem. Biophys. Res. Commun. 1998; 252: 307-312Crossref PubMed Scopus (36) Google Scholar, 10.Hooper J.D. Nicol D.L. Dickinson J.L. Eyre H.J. Scarman A.L. Normyle J.F. Stuttgen M.A. Douglas M.L. Loveland K.A. Sutherland G.R. Antalis T.M. Cancer Res. 1999; 59: 3199-3205PubMed Google Scholar), and EOS (11.Chen C. Darrow A.L. Qi J.S. D'Andrea M.R. Andrade-Gordon P. Biochem. J. 2003; 374: 97-107Crossref PubMed Scopus (31) Google Scholar). There are five additional nonpeptidase homolog genes (currently designated as hSPL-2, -3, -4, -6, and -7) within the locus that probably encode non-functional proteins due to the presence of premature translation-termination codons (7.Wong G.W. Yasuda S. Madhusudhan M.S. Li L. Yang Y. Krilis S.A. Šali A. Stevens R.L. J. Biol. Chem. 2001; 276: 49169-49182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The subtelomeric region of human chromosome 16 where these genes reside is mutating at a rate ∼300-fold faster than the rest of the genome in males (12.Badge R.M. Yardley J. Jeffreys A.J. Armour J.A. Hum. Mol. Genet. 2000; 9: 1239-1244Crossref PubMed Scopus (59) Google Scholar). One explanation for this finding is that there is strong evolutionary pressure to expand some of the genes and delete others because of the respective beneficial and adverse roles. The corresponding serine protease locus in the mouse genome resides at chromosome 17A3.3. When this study was initiated, 10 genes had been identified at the site that encode mouse mast cell protease (mMCP) 6 (13.Reynolds D.S. Gurley D.S. Austen K.F. Serafin W.E. J. Biol. Chem. 1991; 266: 3847-3853Abstract Full Text PDF PubMed Google Scholar), mMCP-7 (14.McNeil 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), mTMT (4.Wong G.W. Tang Y. Feyfant E. Šali A. Li L. Li Y. Huang C. Friend D.S. Krilis S.A. Stevens R.L. J. Biol. Chem. 1999; 274: 30784-30793Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), tryptase-4 (mT4)/mEsp-1/mTesp5/mTestisin/mPrss21 (15.Wong G.W. Li L. Madhusudhan M.S. Krilis S.A. Gurish M.F. Rothenberg M.E. Šali A. Stevens R.L. J. Biol. Chem. 2001; 276: 20648-20658Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 16.Scarman A.L. Hooper J.D. Boucaut K.J. Sit M.L. Webb G.C. Normyle J.F. Antalis T.M. Eur. J. Biochem. 2001; 268: 1250-1258Crossref PubMed Scopus (34) Google Scholar, 17.Honda A. Yamagata K. Sugiura S. Watanabe K. Baba T. J. Biol. Chem. 2002; 277: 16976-16984Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar), testis serine protease-1 (mTessp1), 2Although mBssp-4 and mTessp1 cDNAs have not been described in any scientific publication at the time of submission of this paper, their nucleotide sequences have the GenBank™ accession numbers BAB20262 and BAB68561, respectively.2Although mBssp-4 and mTessp1 cDNAs have not been described in any scientific publication at the time of submission of this paper, their nucleotide sequences have the GenBank™ accession numbers BAB20262 and BAB68561, respectively. distal intestinal serine protease (mDisp) (18.Shaw-Smith C.J. Coffey A.J. Leversha M. Freeman T.C. Bentley D.R. Walters J.R. Biochim. Biophys. Acta. 2000; 1490: 131-136Crossref PubMed Scopus (10) Google Scholar), brain-specific serine protease-4 (mBssp-4) 2Although mBssp-4 and mTessp1 cDNAs have not been described in any scientific publication at the time of submission of this paper, their nucleotide sequences have the GenBank™ accession numbers BAB20262 and BAB68561, respectively./4733401N09Rik, pancreasin (8.Bhagwandin V.J. Hau L.W. Mallen-St. Clair J. Wolters P.J. Caughey G.H. J. Biol. Chem. 2003; 278: 3363-3371Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), implantation serine protease (mIsp) 1 (19.O'Sullivan C.M. Rancourt S.L. Liu S.Y. Rancourt D.E. Reproduction. 2001; 122: 61-71Crossref PubMed Scopus (57) Google Scholar), and mIsp-2 (20.O'Sullivan C.M. Liu S.Y. Rancourt S.L. Rancourt D.E. Reproduction. 2001; 122: 235-244Crossref PubMed Scopus (37) Google Scholar). Of these proteases, only mMCP-6, mMCP-7, and mT4 have been expressed and the recombinant proteases functionally characterized. Nevertheless, each member of this family of serine proteases appears to contain a unique substrate-binding cleft that also is more restricted than that of pancreatic trypsin. Screening of phage display peptide libraries and varied chromogenic substrates confirmed that recombinant mMCP-6 and mMCP-7 possess different substrate specificities (21.Huang C. Wong G.W. Ghildyal N. Gurish M.F. Šali 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, 22.Huang 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). hTryptases α, βI, TMT/γ, δ, and ϵ also have been shown to be functionally different (7.Wong G.W. Yasuda S. Madhusudhan M.S. Li L. Yang Y. Krilis S.A. Šali A. Stevens R.L. J. Biol. Chem. 2001; 276: 49169-49182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 23.Huang C. Li L. Krilis S.A. Chanasyk K. Tang Y. Li Z. Hunt J.E. Stevens R.L. J. Biol. Chem. 1999; 274: 19670-19676Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 24.Huang C. De Sanctis G.T. O'Brien P.J. Mizgerd J.P. Friend D.S. Drazen J.M. Brass L.F. Stevens R.L. J. Biol. Chem. 2001; 276: 26276-26284Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 25.Harris J.L. Niles A. Burdick K. Maffitt M. Backes B.J. Ellman J.A. Kuntz I. Haak-Frendscho M. Craik C.S. J. Biol. Chem. 2001; 276: 34941-34947Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 26.Wong G.W. Foster P.S. Yasuda S. Qi J.C. Mahalingam S. Mellor E.A. Katsoulotos G. Li L. Boyce J.A. Krilis S.A. Stevens R.L. J. Biol. Chem. 2002; 277: 41906-41915Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 27.Wang H.W. McNeil H.P. Husain A. Liu K. Tedla N. Thomas P.S. Raftery M. King G.C. Cai Z.Y. Hunt J.E. J. Immunol. 2002; 169: 5145-5152Crossref PubMed Scopus (37) Google Scholar). In regard to their in vivo function, administration of recombinant mMCP-6 or hTryptase βI into the lungs significantly enhances the ability of MC-deficient W/Wv mice to combat a Klebsiella pneumoniae infection (24.Huang C. De Sanctis G.T. O'Brien P.J. Mizgerd J.P. Friend D.S. Drazen J.M. Brass L.F. Stevens R.L. J. Biol. Chem. 2001; 276: 26276-26284Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). This protective effect is mediated, in part, by the ability of both hTryptase βI and mMCP-6 to selectively recruit large numbers of neutrophils (22.Huang 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, 24.Huang C. De Sanctis G.T. O'Brien P.J. Mizgerd J.P. Friend D.S. Drazen J.M. Brass L.F. Stevens R.L. J. Biol. Chem. 2001; 276: 26276-26284Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 28.Hallgren J. Karlson U. Poorafshar M. Hellman L. Pejler G. Biochemistry. 2000; 39: 13068-13077Crossref PubMed Scopus (67) Google Scholar). In support of these in vivo findings, Oh et al. (29.Oh S.W. Pae C.I. Lee D.K. Jones F. Chiang G.K. Kim H.O. Moon S.H. Cao B. Ogbu C. Jeong K.W. Kozu G. Nakanishi H. Kahn M. Chi E.Y. Henderson Jr, W.R. J. Immunol. 2002; 168: 1992-2000Crossref PubMed Scopus (122) Google Scholar) noted that neutrophil recruitment can be inhibited substantially in ovalbumin-treated mice by the tryptase tetramer inhibitor MOL-6131. Although the amino acid sequences of mMCP-6 (13.Reynolds 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 (14.McNeil 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) are 71% identical, these two tryptases exhibit different biologic activities in vivo. When placed into the peritoneal cavities of mice, mMCP-7 preferentially induces the extravasation of eosinophils (24.Huang C. De Sanctis G.T. O'Brien P.J. Mizgerd J.P. Friend D.S. Drazen J.M. Brass L.F. Stevens R.L. J. Biol. Chem. 2001; 276: 26276-26284Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The α chain of fibrinogen is a physiologic substrate of mMCP-7 (21.Huang C. Wong G.W. Ghildyal N. Gurish M.F. Šali 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). Thus, the latter mouse tryptase also appears to help prevent the formation of fibrin/platelet clots at inflammatory sites. Unlike mMCP-6 and mMCP-7, mTMT has a membrane-spanning domain at its C terminus that anchors this tryptase in the plasma membrane when MCs degranulate (4.Wong G.W. Tang Y. Feyfant E. Šali A. Li L. Li Y. Huang C. Friend D.S. Krilis S.A. Stevens R.L. J. Biol. Chem. 1999; 274: 30784-30793Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 26.Wong G.W. Foster P.S. Yasuda S. Qi J.C. Mahalingam S. Mellor E.A. Katsoulotos G. Li L. Boyce J.A. Krilis S.A. Stevens R.L. J. Biol. Chem. 2002; 277: 41906-41915Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). MCs physically contact T cells (30.Mekori Y.A. Metcalfe D.D. J. Allergy Clin. Immunol. 1999; 104: 517-523Abstract Full Text Full Text PDF PubMed Google Scholar), and exposure of Jurkat T cells to recombinant hTMT results in altered gene expression (26.Wong G.W. Foster P.S. Yasuda S. Qi J.C. Mahalingam S. Mellor E.A. Katsoulotos G. Li L. Boyce J.A. Krilis S.A. Stevens R.L. J. Biol. Chem. 2002; 277: 41906-41915Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). TMT therefore appears to be a novel granule mediator that mouse and human MCs use to preferentially regulate adjacent cell types that they physically contact. TMT can adversely affect lung function due to its ability to activate IL-13/IL-4Rα/STAT6-dependent signaling pathways. It was discovered recently that pro-urokinase-type plasminogen activator is a preferred substrate of recombinant hTryptase ϵ (31.Yasuda S. Wong G.W. Krilis S.A. Stevens R.L. FASEB J. 2003; 17: 12Google Scholar). The potential roles of the tryptase family of serine proteases in varied inflammatory disorders has prompted the pharmaceutical industry to begin generating low molecular weight protease inhibitors before the number of family members had been deduced. The ability to create inhibitors that selectively inactivate a harmful protease is dependent on our knowledge of how many related protease genes exist in humans, where and when they are expressed, and what are their beneficial and adverse roles in vivo. This knowledge becomes attainable, in part, with the generation of the complete sequence of the human genome and its transcripts. To deduce the function of these proteases in mouse models of human disease, it is imperative that their corresponding orthologs be identified. It is presently unclear how many functional tryptic genes of this family are present in the mouse genome. Even considering the known mouse protease genes on chromosome 17A3.3, it was difficult to assign unambiguously each one to its corresponding human ortholog. For this reason, it was not obvious which mouse protease studies would be relevant to humans. In the present study, we attempt to resolve these issues by comparative genome analysis of the mouse and human loci where these tryptic genes reside. In this process, we describe three new functionally distinct mouse tryptic proteases that are not coordinately expressed in vivo. We also establish that 12 mouse serine protease genes at chromosome 17A3.3 have human orthologs. This information provides a useful framework for the systematic functional analysis of each mouse/human protease pair using transgenic approaches. Analysis of the Serine Protease Gene Cluster on Mouse Chromosome 17A3.3 and Cloning of mT5, mT6, and mMCP-11 cDNAs—The nucleotide sequences of the mMCP-6, mMCP-7, mT4, and mTMT transcripts were used as templates to search for novel, but related, mouse ESTs or genomic sequences in the varied data bases of GenBank™. As noted under "Results," this approach resulted in the identification of three new members of the tryptase superfamily (designated as mT5, mT6, and mMCP-11). Based on the nucleotide sequences of the identified EST clones, the entire coding regions of the mT5, mT6, and mMCP-11 transcripts were obtained using cDNA libraries from BALB/c mouse skeletal muscle, spleen (Clontech, Palo Alto, CA), and IL-3-developed BALB/c mouse bone marrow-derived MCs (mBMMCs) (32.Razin E. Ihle J.N. Seldin D. Mencia-Huerta J.M. Katz H.R. LeBlanc P.A. Hein A. Caulfield J.P. Austen K.F. Stevens R.L. J. Immunol. 1984; 132: 1479-1486PubMed Google Scholar), respectively. The oligonucleotides used in these PCRs were 5′-GCAGGTGTACTATGGAGCTGGCTCTG-3′ and 5′-TTAGGGTCCCAGGAGGAAGAAGGCGCTACTG-3′ for the mT5 transcript, 5′-CAATGAGGGGTGCTTCCCACCTCCAG-3′ and 5′-GAGGCTCAGGCGAGCCTGGATCCAG-3′ for the mT6 transcript, and 5′-GATATGTGCTTGGGGATGCTCTGG-3′ and 5′-AGAAAGTGAGGCTGGACCAGAAGG-3′ for the mMC-P-11 transcript. The resulting products were purified on 1% agarose gels, subcloned, and sequenced to confirm their identities. The nucleotide sequences of the cDNAs that encode mT4, mT5, mT6, mMCP-6, mMCP-7, mMCP-11, mTMT, mTessp1, mDisp, mBssp-4, mPancreasin, mIsp-1, and mIsp-2 were aligned against the sequence of chromosome 17A3.3 (from the Mouse Genome Project) with the "blastn" algorithm available at the web site for the National Center for Biotechnology Information. The draft sequence of mouse chromosome 17A3.3 also was searched with the translated tblastn algorithm for Spl genes that encode conserved peptide sequences in serine proteases. Transcript Analysis—A semi-quantitative PCR approach was used to screen multiple tissue cDNA libraries (Clontech) for the presence of the varied mouse tryptase transcripts. The oligonucleotides used in these transcript studies and the sizes of the resulting PCR products are summarized in Table I. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH)-specific oligonucleotides (Clontech) were used as positive controls in these transcript profiling studies. MCs express mMCP-6 (13.Reynolds D.S. Gurley D.S. Austen K.F. Serafin W.E. J. Biol. Chem. 1991; 266: 3847-3853Abstract Full Text PDF PubMed Google Scholar), mMCP-7 (14.McNeil 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), and mTMT (4.Wong G.W. Tang Y. Feyfant E. Šali A. Li L. Li Y. Huang C. Friend D.S. Krilis S.A. Stevens R.L. J. Biol. Chem. 1999; 274: 30784-30793Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Thus, total RNA was isolated from BALB/c, C57BL/6, 129Sv, and WBB6F1-W/Wv mBMMCs cultured in the presence of IL-3 for 1–5 weeks (32.Razin E. Ihle J.N. Seldin D. Mencia-Huerta J.M. Katz H.R. LeBlanc P.A. Hein A. Caulfield J.P. Austen K.F. Stevens R.L. J. Immunol. 1984; 132: 1479-1486PubMed Google Scholar). At week 3, >99% of the cells in these cultures are MCs. A semi-quantitative reverse transcriptase-PCR approach (30–32 cycles) was then used to assess the relative levels of the mT5, mT6, and mMCP-11 transcripts in the different populations of MCs. As noted under "Results," IL-3-developed mBMMCs transiently express mMCP-11 mRNA. Thus, to investigate the influence of MC-regulatory cytokines on the expression of mMCP-11, additional experiments were carried out using BALB/c-derived mBMMCs that had been developed by culturing progenitors in the presence of IL-3 with varied combinations of IL-4, IL-9, IL-10, IL-12, or kit ligand as described in other MC studies (33.Gurish M.F. Ghildyal N. McNeil H.P. Austen K.F. Gillis S. Stevens R.L. J. Exp. Med. 1992; 175: 1003-1012Crossref PubMed Scopus (167) Google Scholar, 34.Ghildyal N. Friend D.S. Nicodemus C.F. Austen K.F. Stevens R.L. J. Immunol. 1993; 151: 3206-3214PubMed Google Scholar, 35.Eklund K.K. Ghildyal N. Austen K.F. Stevens R.L. J. Immunol. 1993; 151: 4266-4273PubMed Google Scholar, 36.Ochi H. Hirani W.M. Yuan Q. Friend D.S. Austen K.F. Boyce J.A. J. Exp. Med. 1999; 190: 267-280Crossref PubMed Scopus (301) Google Scholar). Whether or not two mouse MC lines (V3 and C1.MC/C57.1), two macrophage cell lines (RAW and WEHI-3), a T cell hybridoma (BY155), and a fibroblast cell line (mTc-1) contain mMCP-11 mRNA also was evaluated. The V3 and C1.MC/C57.1 cell lines have been maintained continuously for >1 decade. Thus, there is no contaminating cell type in these MC lines.Table IPrimer sets used to evaluate the expression of varied mouse tryptases in tissuesGene/transcriptDirectionPCR primersProduct sizebpmT4Forward5′-CAACAGCATGTGTAACCATATG-3′503Reverse5′-GCCTGAGCAGCCCATTGCGGATC-3′mT5Forward5′-CAAGCTATTCAGCGGACGAGCACAG-3′660Reverse5′-GCTGTGCTGGCCTGGAAGCCAGTTGAG-3′mT6Forward5′-TGGGAGCACTGAGTCTGGACGTCAG-3′617Reverse5′-GTGGGATGGACCAGGAAGCTCCAG-3′mMCP-6Forward5′-CTCTTCCGGGTGCAGCTTCGTGAGCAG-3′533Reverse5′-TATGTCACCCGGGTGTAGATGCCAGG-3′mMCP-7Forward5′-ATGACCACCTGATGACTGTGAGCCAG-3′563Reverse5′-AGGAACGGAGGTCATCCTGGATGTG-3′mMCP-11Forward5′-GCTGATGAAAGTGGTCAAGATCATCCG-3′610Reverse5′-AGGAGTGAATGGATCAATATGAGTGGCTG-3′mTMTForward5′-GATCATCATGTACACTGGCTCTCCAG-3′645Reverse5′-CTACACCTCATTCAGAGTTCCGAGG-3′mBssp-4Forward5′-GTGCTGCCTCACCCCAGGTATTCTTGG-3′574Reverse5′-CAGGAGGCTGCTCATCTTCAGATCCTAG-3′mTessp1Forward5′-ACCTACAACAAGGACATCCAGCCTG-3′573Reverse5′-AGCTATCCCTACAGTATATGGACTG-3′mDispForward5′-TACCTCTGGGCAGATGCGTCTAGCG-3′607Reverse5′-GAGGATCTAGAACTCTAGAGCTCACAG-3′mPancreasinForward5′-GAAGCTGCAGCAGCCAGGACCACACG-3′677Reverse5′-CTCCCAGGGCCAGCACCATTGCATGG-3′mIsp-1Forward5′-AGGACGCCGACCCAGCCGTATACCG-3′527Reverse5′-TGATGGCAGATTGTTGCTGCAATCG-3′mIsp-2Forward5′-GCAAGGAGCTGCTGAGTGTGAGCCG-3′508Reverse5′-CAGGGCAGGAAGGACTGTACACGTGC-3′ Open table in a new tab Expression of Recombinant Mouse Proteases in Mammalian and Insect Cells—Bioengineered forms of recombinant mT6, mMCP-11, and mBssp-4 were generated in mammalian cells that contained the His6 and V5 (Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr) peptides at their C termini, as described previously for recombinant hTryptase ϵ (7.Wong G.W. Yasuda S. Madhusudhan M.S. Li L. Yang Y. Krilis S.A. Šali A. Stevens R.L. J. Biol. Chem. 2001; 276: 49169-49182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In each instance, the coding region of the tryptase cDNA was placed in the mammalian expression vector pcDNA3.1/V5-His TOPO (Invitrogen). Vector lacking an insert served as a negative control in the transfection experiments. mT4, mT5, mPancreasin, mDisp, and mTessp1 possess predicted glycosylphosphatidylinositol (GPI) signal sequences at their C termini. Thus, in these instances, the expressed protease contained the 8-mer FLAG (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys) peptide inbetween the propeptide and the mature domain of the protease. African green monkey SV40-transformed kidney COS-7 cells (line CRL-1651; American Type Culture Collection (ATCC), Manassas, VA) and human embryonic kidney HEK 293T cells (line CRL-1573; ATCC) were cultured in DMEM containing 10% fetal calf serum. Transient transfections were performed in both cell types with SuperFect (Qiagen, Valencia, CA), according to the manufacturer's instructions. Cells were plated at a density of ∼2 × 105 cells/well in 6-well plates 24 h before transfection. Twenty four h after transfection, the treated cells were washed and then cultured in serum-free Opti-MEM I medium for another 24 h before the conditioned medium and cell pellets were collected. Recombinant mT5, mT6, and mMCP-11 also were generated in High Five insect cells using a modification of the expression system we developed previously to generate recombinant mMCP-6, mMCP-7 (21.Huang C. Wong G.W. Ghildyal N. Gurish M.F. Šali 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), mT4, and hTryptases α, βI, βII, TMT/γ, and ϵ. Insect cells lack the post-translational machinery needed to remove the propeptide of a mammalian serine protease zymogen. Thus, the FLAG peptide was placed inbetween the natural propeptide and first residue of the catalytic domain to allow activation of the recombinant bioengineered zymogen by enterokinase. The FLAG peptide also facilitates the purification of the recombinant proteases from the conditioned medium using an immunoaffinity column. In this expression system, the C-terminal hydrophobic domain in mT5 was removed. Protease-expressing insect cells were cultured at room temperature in serum-free, X-press medium for 6–7 days. Generally, 750 ml of conditioned medium were loaded onto a freshly prepared 1-ml column containing anti-FLAG M2 antibody (Sigma). After each column was washed with 250 ml of Tris-buffered saline (pH 7.0), 0.1 m glycine (pH 3.5) was used to elute the bound protease. In each instance, 10 1-ml fractions were collected into tubes containing 20 μl of 1 m Tris-HCl (pH 8.0). Samples of the resulting fractions were analyzed by SDS-PAGE for the presence of Coomassie Blue-stained proteins and for immunoreactive tryptases by using anti-FLAG M2 antibody (Sigma). Protease-enriched fractions were pooled and their protein contents estimated using the micro BCA protein assay reagent kit (Pierce). Evaluation of the Glycosylation Status and Substrate Specificities of Recombinant Mouse Proteases—Many mouse and human tryptases contain N-linked glycans (37.Cromlish J.A. Seidah N.G. Marcinkiewicz M. Hamelin J. Johnson D.A. Chretien M. J. Biol. Chem. 1987; 262: 1363-1373Abstract Full Text PDF PubMed Google Scholar, 38.Benyon R.C. Imai T. Abe T. Befus D. Int. Arch. Allergy Appl. Immunol. 1991; 94: 218-219Crossref PubMed Scopus (8) Google Scholar, 39.Ghildyal N. Friend D.S. Freelund R. Austen K.F. McNeil H.P. Schiller V. Stevens R.L. J. Immunol. 1994; 153: 2624-2630PubMed Google Scholar), and expression/site-directed mutagenesis analysis of mMCP-7 revealed that these glycans often are important in thermal stability (40.Huang C. Morales G. Vagi A. Chanasyk K. Ferrazzi M. Burklow C. Qiu W.T. Feyfant E. Šali A. Stevens R.L. J. Biol. Chem. 2000; 275: 351-358Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Analysis of their predicted primary amino acid sequences revealed potential N-linked glycosylation sites in mT5, mMCP-11, mBssp-4, mTessp1, and mPancreasin. Thus, to determine whether any of these proteases contain N-linked glycans, a sample of the conditioned medium or cell lysate from each transfectant was incubated with PNGaseF (New England Biolabs, Beverly, MA) according to the manufacturer's suggested conditions. The resulting digests were then subjected to SDS-PAGE/immunoblot analysis. The biotinylated d-phenylalanyl-l-prolyl-l-arginine chloromethyl ketone (PPACK) active-site assay developed by Williams et al. (41.Williams E.B. Krishnaswamy S. Mann K.G. J. Biol. Chem. 1989; 264: 7536-7545Abstract Full Text PDF PubMed Google Scholar) was used to evaluate whether or not the varied uncharacterized mouse proteases in this study
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