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• W2107172000 abstract "Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (;II, ;I, ;III, and ;I), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1′ or S2′ binding pocket and results in the complete loss of the “47 loop,” reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates. Tryptases are serine proteases that are thought to be uniquely and proteolytically active as tetramers. Crystallographic studies reveal that the active tetramer is a flat ring structure composed of four monomers, with their active sites arranged around a narrow central pore. This model explains why many of the preferred substrates of tryptase are short peptides; however, it does not explain how tryptase cleaves large protein substrates such as fibronectin, although a number of studies have reported in vitro mechanisms for generating active monomers that could digest larger substrates. Here we suggest that alternate mRNA splicing of human tryptase genes generates active tryptase monomers (or dimers). We have identified a conserved pattern of alternate splicing in four tryptase alleles (;II, ;I, ;III, and ;I), representing three distinct tryptase gene loci. When compared with their full-length counterparts, the splice variants use an alternate acceptor site within exon 4. This results in the deletion of 27 nucleotides within the central coding sequence and 9 amino acids from the translated protein product. Although modeling suggests that the deletion can be easily accommodated by the enzymes structurally, it is predicted to alter the specificity by enlarging the S1′ or S2′ binding pocket and results in the complete loss of the “47 loop,” reported to be critical for the formation of tetramers. Although active monomers can be generated in vitro using a range of artificial conditions, we suggest that alternate splicing is the in vivo mechanism used to generate active tryptase that can cleave large protein substrates. Tryptases belong to a family of serine proteases and are named based on their similarity to the pancreatic enzyme trypsin. The most intensively studied tryptases are the ;/; tryptases (now known to be the products of two separate gene loci); they are reported to be selectively expressed in mast cells, where they are stored in granules in association with proteoglycans. Large quantities of these proteases are produced by mast cells, often representing around a quarter of the total cellular protein content (1Schwartz L. Lewis R. Austen K. J. Biol. Chem. 1981; 256: 11939-11943Abstract Full Text PDF PubMed Google Scholar).Recently published reports by Caughey and co-workers (2Vanderslice P. Ballinger S. Tam E. Goldstein S. Craik C. Caughey G. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3811-3815Crossref PubMed Scopus (201) Google Scholar, 3Caughey G.H. Raymond W.W. Blount J.L. Hau L.W.-T. Pallaoro M. Wolters P.J. Verghese G.M. J. Immunol. 2000; 164: 6566-6575Crossref PubMed Scopus (103) Google Scholar) (supported by data from the public and private genome data bases) indicate the presence of multiple gene loci on chromosome 16p13.3 that encode human tryptases. The first locus encodes a transmembrane tryptase called ; tryptase (3Caughey G.H. Raymond W.W. Blount J.L. Hau L.W.-T. Pallaoro M. Wolters P.J. Verghese G.M. J. Immunol. 2000; 164: 6566-6575Crossref PubMed Scopus (103) Google Scholar). The second is a locus whose allelic variants are ;II and ;III tryptase (4Pallaoro 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). The third has allelic variants that include ;I tryptase, ;II tryptase, and probably ;I tryptase (4Pallaoro 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). The fourth is a locus encoding ; tryptase (5Wang 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), and the fifth is a more distantly related member named ? tryptase (6Wong G.W. Yasuda S. Madhusudhan M.S. Li L. Yang Y. Krilis S.A. Sali A. Stevens R.L. J. Biol. Chem. 2001; 276: 49169-49182Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). With the recent report concerning the in vivo expression of ; tryptase (5Wang 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), mRNA and protein products for all five loci have now been detected.Native tryptase protein purified from tissue is biochemically heterogeneous (1Schwartz L. Lewis R. Austen K. J. Biol. Chem. 1981; 256: 11939-11943Abstract Full Text PDF PubMed Google Scholar, 7Harvima R.J. Harvima I.T. Dull D. Dunder U.K. Schwartz L.B. Arch. Dermatol. Res. 1999; 291: 73-80Crossref PubMed Scopus (14) Google Scholar, 8Peng Q. McEuen A.R. Benyon R.C. Walls A.F. Eur. J. Biochem. 2003; 270: 270-283Crossref PubMed Scopus (25) Google Scholar), but the basis of this has not been fully determined. It may be due to the presence of multiple tryptase gene products, the presence of different post-translational modifications such as glycosylation, or more likely a combination of causes.Tryptase has been implicated in the development of a number of clinical conditions, including asthma (9Clark J.M. Abraham W.M. Fishman C.E. Forteza R. Ahmed A. Cortes A. Warne R.L. Moore W.R. Tanaka R.D. Am. J. Respir. Crit. Care Med. 1995; 152: 2076-2083Crossref PubMed Scopus (199) Google Scholar, 10Sekizawa K. Caughey G. Lazarus S. Gold W. Nadel J. J. Clin. Investig. 1989; 83: 175-179Crossref PubMed Scopus (140) Google Scholar, 11Krishna M.T. Chauhan A. Little L. Sampson K. Hawksworth R. Mant T. Djukanovic R. Lee T. Holgate S. J. Allergy Clin. Immunol. 2001; 107: 1039-1045Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), inflammatory bowel disease (12Tremaine W.J. Brzezinski A. Katz J.A. Wolf D.C. Fleming T.J. Mordenti J. Strenkoski-Nix L.C. Kurth M.C. Aliment. Pharmacol. Ther. 2002; 16: 407-413Crossref PubMed Scopus (87) Google Scholar), and inflammatory arthritis (13He S. Gaca M.D.A. Walls A.F. Eur. J. Pharmacol. 2001; 412: 223-229Crossref PubMed Scopus (32) Google Scholar, 14Gotis-Graham I. McNeil H.P. Arthritis Rheum. 1997; 40: 479-489Crossref PubMed Scopus (112) Google Scholar). Furthermore, the detection of different forms of tryptase can be used as a diagnostic feature. For instance increased plasma levels of ; tryptase, apparently constitutively expressed by all mast cells, are indicative of the increased mast cell burden of mastocytosis (15Schwartz L.B. Sakai K. Bradford T.R. Ren S. Zweiman B. Worobec A.S. Metcalfe D.D. J. Clin. Investig. 1995; 96: 2702-2710Crossref PubMed Scopus (330) Google Scholar). Alternatively, the detection of ; tryptase indicates the presence of activated mast cells of allergic conditions and anaphylaxis (16Buckley M.G. Variend S. Walls A.F. Clin. Exp. Allergy. 2001; 31: 1696-1704Crossref PubMed Scopus (55) Google Scholar).Interest in the biology of tryptases has increased because of their proposed role in inflammatory diseases such as asthma. Although the underlying cause of many of these diseases is not fully understood, multiple lines of evidence support a link between tryptases and an inflammatory phenotype. For example, inflamed tissue is often characterized by an increase in mast cell numbers (and tryptase levels) when compared with uninflamed control tissue (17Holgate S.T. Hardy C. Robinson C. Agius R.M. Howarth P.H. J. Allergy Clin. Immunol. 1986; 77: 274-282Abstract Full Text PDF PubMed Scopus (90) Google Scholar), and both murine and human tryptases can act directly or indirectly to recruit inflammatory cells such as neutrophils and eosinophils (18Huang 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, 19He S. Peng Q. Walls A. J. Immunol. 1997; 159: 6216-6225PubMed Google Scholar).One of the most interesting findings that accompanied the publishing of the human genome was that the number of discrete genes was much lower than anticipated (20Gamba G. Am. J. Physiol. 2001; 281: F781-F794Crossref PubMed Google Scholar). Previously it was thought that the biochemical complexity of an organism was proportional to the size of its genome. Therefore, it was surprising that arguably the most complex organism, humans, possessed a genome not dramatically larger than that of an earthworm, and indeed significantly smaller than that of many plants (20Gamba G. Am. J. Physiol. 2001; 281: F781-F794Crossref PubMed Google Scholar). On closer inspection the relationship between the perceived complexity of an organism and the size of its genome holds true only for the so-called “lower organisms,” where there is roughly a 1:1 ratio between the number of individual genes and their resulting protein products. What becomes obvious is that in the “higher” organisms, complexity may be facilitated by increasing the number of distinct protein products that can be generated from a single gene. This may be accomplished by processes such as post-translational modifications or by generating multiple forms of mRNA from a single primary transcript, the process of alternate splicing. The human transcriptome is apparently distinguished from that of other organisms by the dramatically increased number of alternately spliced transcripts (20Gamba G. Am. J. Physiol. 2001; 281: F781-F794Crossref PubMed Google Scholar).Here we report the cloning and initial characterization of alternately spliced forms of human ;II, ;I, ;III, and ;I tryptase. The pattern of alternate splicing is identical in all four tryptases, and it results in the loss of 27 nucleotides from the mature transcript and 9 amino acids from the translated protein product, when compared with their full-length counterparts. The resulting proteases are predicted to have altered substrate specificities, at least partly due to the predicted inability of the splice variant tryptases to form tetramers. These splice variant tryptases were shown to be expressed in multiple human tissues. Alternate splicing is a mechanism that results in an increase in the number of functionally distinct human tryptases.EXPERIMENTAL PROCEDURESSources of RNA—Total RNA from adult lung, heart, stomach, spleen, skin, and colon, fetal heart and fetal lung, and poly(A+) RNA isolated from human lung were obtained from commercial sources (Invitrogen). In addition, total RNA was isolated from human mast cell-1 (HMC-1) 3The abbreviations used are: HMC-1, human mast cell-1; BAPNA, N;-benzoyl-dl-arginine p-nitroanilide; EK, enterokinase; SV, splice variant; tosyl-GPR-pNA, N-(p-tosyl)-Gly-Pro-Arg-p-nitroanilide; RT, reverse transcription; TBS, Tris-buffered saline; MES, 4-morpholineethanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. cells (5 × 106, a kind gift from Dr. J. H. Butterfield) using TRI Reagent™ (Sigma), according to the manufacturer's instructions, dissolved in distilled H2O, and stored at -80 °C until required.Preparation of cDNA—First strand cDNAs were generated using the cDNA Cycle® kit (Invitrogen). Poly(A+) RNA (300 ng) or total RNA (1.5 ;g) and 1 ;l of oligo(dT) primer were heated to 65 °C for 10 min to remove secondary structure. Reverse transcription was performed for 1 h at 42 °C in a solution containing 1 ;l of RNase inhibitor, 4 ;l of 5× RT buffer, 1 ;l of 100 mm dNTPs, 1 ;l of 80 mm sodium pyrophosphate, and 0.5 ;l of avian myeloblastosis virus reverse transcriptase. The reaction was terminated by incubating the mixture at 95 °C for 2 min and was then placed immediately on ice.PCR Amplification and Cloning of ;II and ;I Tryptase cDNAs—PCR amplification of first strand cDNA was performed within 2 h of the reverse transcription reaction. Oligonucleotide primers used to amplify cDNAs were designed based on the published sequences of the ;II and ;I tryptase genes: ;II, A2 forward, 5′-AGT GGC CAG GAT GCT GAG C-3′, and A2 reverse, 5′-GGA AGC AGT GGT GTT TTG GAC AG-3′; ;II, nested NA2 forward, 5′-GCC TAC GCG GCC CCT GCC CCA GTC-3′, and NA2 reverse, 5′-CCC AGG TGG ACA CCC CAG GCC TGA-3′; ;I, DF1, 5′-TGC AGC AAA CGG GCA TTG TTG-3′, and DR1 5′-AAA GCT GTG GCC CGT ATG GAG-3′.All PCR amplifications were performed with a GeneAmp PCR system 2400 (PerkinElmer Life Sciences). The PCRs were carried out with 2.5 units of AmpliTaq Gold™ (PerkinElmer Life Sciences) and 1 ;l of the reverse transcriptase reaction mixture. The total reaction volume was 50 ;l with a final concentration of 10 mm Tris/HCl, pH 8.3, 50 mm KCl, 2 mm MgCl2, 0.2 mm dNTPs, and 0.1 ;m of the appropriate 5′ and 3′ primers. After an initial incubation for 5 min at 95 °C, samples were subjected to 35 cycles of PCR (45 s at 95 °C, 60 s at 58 °C, and 60s at 72 °C), followed by a final extension step of 72 °C for 10 min.PCR products (10 ;l) were visualized on a 1% agarose gel. Appropriately sized products were excised from the gel, purified with QIAquick gel extraction kit (Qiagen, Valencia, CA), and ligated into the plasmid vector pCR®2.1-TOPO (Invitrogen). The ligation mixture was then used to transform TOP10 Escherichia coli cells (Invitrogen). The transformation mixture was plated onto LB/agar plates containing ampicillin (50 ;g/ml) and coated with 5-bromo-4-chloro-3-indolyl-;-d-galactopyranoside (X-gal) to enable blue/white color selection.Screening of Plasmid Colonies—Plasmids containing the appropriate sized inserts were screened by PCR for the presence of ;II and ;I tryptase inserts using the appropriate primer sets listed above. Plasmid DNA was then purified using a commercial kit (Qiagen). Nucleotide sequencing was performed either in-house using an ABI Prism® BigDye terminator cycle sequencing ready reaction kit and an ABI377 PRISM DNA sequencer (PerkinElmer Life Sciences) or at a core facility (SUPAMAC, Sydney, Australia).dbEST Data Base Screening—EST databases accessed through the National Institutes of Health information website (www.ncbi.org) were screened using the sequences of the clones identified above.Real Time PCR Assays—A real time fluorescence-based RT-PCR assay was designed to detect all alternatively spliced (G234–G260) tryptase transcripts isolated in this study. To estimate the approximate ratio of alternatively spliced to full-length tryptase transcripts, an additional assay was designed to detect splice variant ;/;/; tryptase transcripts. Specificity was conferred by designing the forward primer spanning the junction of the deleted nucleotides (splice variant) or locating them in the deleted region (full length). The full-length primer was as follows: forward, 5′-CAC CCT CAG GGT GCA ACT G-3′, and reverse primer, 5′-ATG TAG AAC TGT GGG TGC ACG AT-3′; the splice variant was as follows: forward primer, 5′-TGG GAC CGG TGC AAC TG-3′, and reverse primer, 5′-TAG AAC TGT GGG TGC ACG AT-3′; Taqman probe was as follows: 5′-6-FAM-AGC AGC ACC TCT ACT ACC AGG ACC AGC TG-TAMRA-3′.Reverse transcription of total human lung, heart, stomach, spleen, and skin RNA (1 ;g) was performed using MultiScribe reverse transcriptase and random hexamers (PerkinElmer Life Sciences). The PCR was performed using the ABI Prism 7700 sequence detection system (PerkinElmer Life Sciences). The PCRs were carried out in a 25-;l mixture containing 2 ;l of reverse transcript sample, 12.5 ;l of 2× universal master mix buffer (PerkinElmer Life Sciences), 600 nm reverse primer, 600 nm forward primer, and 250 nm probe. After initial heating at 50 °C for 2 min, PCR consisted of 45 cycles, including a 15-s denaturation step at 95 °C and a 1-min annealing-extension at 59 °C. Each sample was tested in triplicate. Using the sequence detector system (version 1.7, PerkinElmer Life Sciences), fluorescence intensity was plotted against cycle number. Standard curves were constructed with serial dilutions of plasmid DNA containing a normally spliced (;II) or a splice variant (;IISV) tryptase insert, allowing the copy number to be calculated for each sample using appropriate software (Copy Calculator, version 1.1). Human tryptase transcripts resulting from the alternate splicing pattern described in this study will be annotated hereafter with superscript SV, e.g. ;IISV tryptase, ;ISV tryptase, etc.The specificity of the primers was tested by examining their ability to amplify cloned templates of full-length or splice variant tryptase cDNAs. Control amplifications were performed in the absence of template DNA.Three-dimensional Protein Modeling—Three-dimensional structures of the novel tryptase sequences were modeled from the reported 3.0 Á x-ray structure of human ;II tryptase (Protein Data Bank identification code 1AOL) (21Pereira P.J.B. Bergner A. Macedo-Ribeiro S. Huber R. Matschiner G. Fritz H. Sommerhoff C.P. Bode W. Nature. 1998; 392: 306-311Crossref PubMed Scopus (281) Google Scholar). The nine-residue segment DVKDLATLR missing in the splice variants was deleted from a single monomer of the structure before energy minimization with AMBER 4.1 (22Pearlman D. Case D. Caldwell J. Ross W. Cheatham T. Ferguson D. Seibel G. Singh U. Weiner P. Kollman P. AMBER. Version 4.1, University of California, San Francisco, CA1995Google Scholar) to produce a new model. Modified monomers were superimposed on the wild-type tetramer structure for visualization.Generation of Polyclonal Anti-peptide Antibodies to ;IISV Tryptase—To determine whether splice variant tryptases are translated into protein products in vivo, we developed an antibody that recognized ;IISV and ;ISV tryptase, but which may also cross-react with splice variant ;II and ; tryptases. Anti-;IISV antibodies were raised in New Zealand White rabbits (Institute of Medical and Veterinary Science, Gilles Plains, SA, Australia) by immunizing with an ;IISV-specific peptide, based on the “novel” juxtaposition of residues Pro48 and Val58. Therefore, an immunizing 10-mer peptide, His44-Cys45-Leu46-Gly47-Pro48-Val58-Gln59-Leu60-Arg61-Glu62, was designed consisting of the five amino acid residues on either side of the deletion, conjugated to diphtheria toxin (Mimotopes, Melbourne, Australia). Anti-;IISV antibodies were affinity-purified from antisera using the immunizing peptide conjugated to thiopropyl-Sepharose.Western Blot Analysis—The specificity of the antibody was confirmed by Western blot. 1 ;g of purified recombinant r;ISV tryptase and r;I tryptase (Escherichia coli) and recombinant ;II tryptase (Promega, Madison, WI) were separated on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking overnight at 4 °C with 5% skim milk powder, phosphate-buffered saline, 0.1% Tween 20, membranes were incubated with affinity-purified ;IISV tryptase anti-peptide antibody (1 ;g/ml in phosphate-buffered saline, 0.1% Tween 20, 1 h, room temperature). Bound primary antibody was detected using a goat anti-rabbit horseradish peroxidase-conjugated second antibody (Dako, Glostrup, Denmark) diluted 1:1000 for 30 min at room temperature, followed by exposure to a horseradish peroxidase chemiluminescence substrate for 30 s (ECL Western blotting substrate; Pierce). The resulting bands were visualized by exposure to Biomax ML photographic film (Eastman Kodak Co.). The blot was stripped (62.5 mm Tris/HCl, 2% SDS, 10 mm ;-mercaptoethanol, pH 6.8) and re-probed with the mouse monoclonal anti-tryptase antibody AA1 diluted 1:1000 (Dako) followed by goat anti-mouse horseradish peroxidase conjugate.Immunohistochemistry—Immunohistochemistry was performed on 4-;m serial sections cut from formalin-fixed and paraffin-embedded samples of human aorta adventitia, lung, colon, spleen, lymph node, and breast cancer. Sections were deparaffinized, dehydrated, and rinsed in tap water. Antigen retrieval was performed by microwaving the sections in citrate buffer (10 mm trisodium citrate, 0.05% Tween 20, pH 6.0) for 10 min on high and then allowed to cool for 10 min. Sections were then rinsed with TBS and blocked with 20% normal goat serum/TBS at room temperature for 20 min. Sections were incubated with primary antibody diluted in TBS, 2% bovine serum albumin (;IISV tryptase = 4 ;g/ml overnight at 4 °C, normal rabbit IgG = 4 ;g/ml overnight at 4 °C, and AA1 anti-tryptase antibody = 1:1000-dilution for 1 h at room temperature). Sections were then washed four times for 5 min in TBS and then incubated at room temperature for 30 min with the appropriate biotinylated secondary antibody diluted 1:200 in TBS, 2% bovine serum albumin/goat anti-rabbit for ;IISV tryptase and normal rabbit IgG, and goat anti-mouse for AA1 tryptase antibody. Sections were washed four times for 5 min in TBS, incubated with avidin-conjugated alkaline phosphatase (Vector Laboratories, Burlingame, CA) for 30 min at room temperature, and then washed four times for 5 min in TBS. The sections were incubated in the dark for ∼15 min with alkaline phosphatase substrate (Vector Red, Vector Laboratories), which gives a red reaction product in the presence of alkaline phosphatase. All incubations were performed in a humidified chamber. Sections were rinsed in tap water, counterstained with hematoxylin for 30 s, rinsed in tap water and coverslipped with CrystalMount (Biomeda, Foster City, CA). Stained sections were examined using an Olympus BX-60 microscope and images captured using a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI).Generation of Recombinant ;I Tryptase and ;ISV Tryptase—When compared with their full-length counterparts, the splice variant tryptases identified in this study have a 9-amino acid deletion, Asp49–Arg57. To determine whether a splice variant tryptase may be proteolytically active, recombinant ;I tryptase and its splice variant ;ISV were expressed in bacterial cells to test splice variant antibody specificity and in yeast to test for the ability to cleave a trypsin-sensitive substrate.The recombinant fusion protein included an N-terminal His6 tag (to enable purification), an enterokinase (EK) recognition site (to allow activation of the pro-enzyme), and the mature ;Ior ;ISV tryptase sequence. The construct was made in bacteria (E. coli) using the pET30 EK/ligation-independent cloning vector (Merck), and the following primers: forward, 5′-GAC GAC GAC AAG AAT ATC GTC GGG GGT CAG-3′, and reverse; 5′-GAG GAG AAG CCC GGT TCA CGG CTT TTT GGG-3′, using ligation independent cloning, and the construct was used to transform BL21 DE3 cells. Following the addition of isopropyl ;-thiogalactopyranoside (0.5 mm final concentration), the bacterial cells were incubated for 6 h at 37 °C while being agitated vigorously. The cells were pelleted by centrifugation and resuspended in lysis buffer (Cellytic B, Sigma). The lysate was centrifuged to remove cellular debris, and the His-6 tagged recombinant protein was purified from the supernatant using a nickel-nitrilotriacetic acid column (Qiagen).The construct was made in yeast (Pichia pastoris) by insertion of the mature ;Ior ;ISV tryptase sequence into the pPIC9; vector (Invitrogen), similar to a method described previously (23Niles A.L. Maffitt M. Haak-Frendscho M. Wheeless C.J. Johnson D.A. Biotechnol. Appl. Biochem. 1998; 28: 125-131PubMed Google Scholar), using the following primers: EKBI forward, 5′-GAT GAC GAC GAC AAG ATC GTT GGG GGT-3′, and BII reverse, 5′-ACG ACG GCG GCC GCT TCA CGG CTT TTT GGG GAC ATA GTG-3′; HISEKF forward, 5′-CAT CAT CAT CAT CAT CAC GAT GAC GAC GAC AAG-3′; KEXHIS forward, 5′-GGG CCC CTC GAG AAA AGA CAT CAT CAT CAT CAT CAT-3′. These primers were used to amplify ;II tryptase sequence and to add a His6 and EK cleavage site upstream of the mature tryptase sequence. The KEXHIS forward primer encodes the KEXI cleavage site, as well as an XhoI restriction site. The reverse primer encodes a NotI restriction site and the mature ; tryptase sequence. The vectors were linearized with SalI, and GS115 cells were transformed using electroporation (XCell II, Bio-Rad). These cells were grown on minimal dextrose/agar plates, followed by minimal methanol/agar plates to screen for colonies expressing ;ISV tryptase, as described previously (24Wung J.L. Gascoigne N.R. BioTechniques. 1996; 21 (810, 812): 808Crossref PubMed Scopus (21) Google Scholar). Positive colonies were grown in BMMY media, containing 1% methanol.Recombinant ;I and ;ISV tryptase purified as above were activated for 16 h at room temperature in cleavage buffer (10 mm MES, 150 mm NaCl, 200 mm CaCl2, pH 6.1) using 1 unit of EK. After enzyme activation, EK was removed using EKapture-agarose beads (EMD Biosciences-Novagen) as per the manufacturer's instructions.The enzymatic activity of the recombinant ;ISV tryptase was evaluated by testing its ability to cleave peptide substrates, and N;-benzoyl-dl-arginine p-nitroanilide (BAPNA) (Sigma) or N-(p-tosyl)-Gly-Pro-Arg p-nitroanilide (tosyl-GPR-pNA) (Sigma), or the physiological substrate human fibronectin. The cleavage pattern was compared with that of ;I tryptase.For the tosyl-GPR-pNA assay, ;I tryptase was purified using a nickel-nitrilotriacetic acid column (Qiagen), whereas ;ISV tryptase was purified using Affi-Gel Hz (Bio-Rad) conjugated to the AA1 tryptase antibody (Dako), as per the manufacturers' protocol. EK activated and unactivated (as described above) ;I and ;ISV tryptase (25 ;l), and an EK control was incubated with tosyl-GPR-pNA (0.5 ;l, 20 mg/ml) in 200 ;l of Dulbecco's phosphate-buffered saline, pH 7.4, for 2 h at room temperature. The plate was read at 405 nm. The EK control consisted of 1 unit of EK, which was then removed using Ekapture-agarose beads; this was done to control for any residual EK in the tryptase preparations activated with EK.For the BAPNA assay, yeast supernatants were concentrated five times using a Microcon centrifugal filter (Millipore, Billerica, MA), and 40 ;l of concentrated supernatant was added to 110 ;l of reaction buffer (2 mm BAPNA, 100 mm Tris, HCl, 1 m glycine, pH 8.0) in a 96-well plate and incubated for 1 h at 37 °C. The plate was read at 405 nm.Human fibronectin (1 ;g; Invitrogen) was added to the activated tryptases and incubated for 18 h at room temperature. To ensure tetramer formation, heparin (1 mg, Sigma) was added to the activated enzymes. Digests were then separated on a non-reduced 7.5% Tris-Tricine SDS-polyacrylamide gel, fixed, and visualized by silver staining.RESULTSIdentification and Cloning of Alternatively Spliced Human Tryptase cDNAs—RT-PCR was performed using ;II tryptase-specific primers on total RNA derived from human lung and using ; tryptase-specific primers on total RNA derived from the HMC-1 cell line. When the resulting PCR products were electrophoresed and visualized on an agarose gel, the amplimers from each reaction appeared to run as a “doublet.” One amplimer in each lane was of the expected size for a human tryptase, and the second, or doublet, was slightly smaller (data not shown). All PCR products were isolated from the gel, cloned into the pCR2.1 vector, and sequenced.Sequencing revealed that the larger cDNA generated using ;II tryptase primers was 884 nucleotides in length and included an ATG translation start codon and TGA stop codon at position 826. The sequence of this fragment (Fig. 1A, submitted as GenBank™ accession number AF206665) matched the predicted exonic sequence of the ;II tryptase gene (4Pallaoro 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). Sequencing of the smaller cDNA generated using these same primers revealed an 805-bp cDNA fragment that matched the ;II-tryptase cDNA exactly, except for a 27-nucleotide deletion from nucleotides 234 to 260 (Fig. 1A, submitted as GenBank™ accession number AF206666).Analysis of cloned PCR products generated using the ; tryptase-specific primers revealed similar results. The sequence of the larger PCR product matched that of ;I tryptase, as described by us previously (5Wang 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). The sequence of the smaller cDNA also matched that of ;I tryptase, except for the same 27-nucleotide deletion present in the truncated ;II cDNA described above (Fig. 1B, submitted as GenBank™ accession number AF421357).Using the truncated tryptase sequences as a query, a BLAST search of dbEST da" @default.
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• W2107172000 date "2008-12-01" @default.
• W2107172000 modified "2023-10-07" @default.
• W2107172000 title "Alternate mRNA Splicing in Multiple Human Tryptase Genes Is Predicted to Regulate Tetramer Formation" @default.
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