Human Mast Cell Tryptase: Multiple Cdnas and Genes Reveal a Multigene Serine Protease Family PETER VANDERSLICE*, SUSAN M
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Proc. Natl. Acad. Sci. USA Vol. 87, pp. 3811-3815, May 1990 Biochemistry Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family PETER VANDERSLICE*, SUSAN M. BALLINGER*, ELIZABETH K. TAM*t, SANFORD M. GOLDSTEIN*, CHARLES S. CRAIK§, AND GEORGE H. CAUGHEY*111 *Cardiovascular Research Institute and Departments of tDermatology, §Pharmaceutical Chemistry, and 1Medicine, University of California, San Francisco, CA 94143 Communicated by John A. Clements, February 13, 1990 ABSTRACT Three different cDNAs and a gene encoding contains an active site (3). Tryptases from different tissue human skin mast cell tryptase have been cloned and sequenced sources are antigenically cross-reactive (17, 20, 21). How- in their entirety. The deduced amino acid sequences reveal a ever, differences in kcat and Km toward synthetic peptide 30-amino acid prepropeptide followed by a 245-amino acid substrates and in susceptibility towards protease inhibitors catalytic domain. The C-terminal undecapeptide of the human have been observed among preparations of tryptase from preprosequence is identical in dog tryptase and appears to be lung, skin, and pituitary gland, raising the possibility of part of a prosequence unique among serine proteases. The functional differences between mast cell tryptases in different differences among the three human tryptase catalytic domains locations (4, 17). include the loss of a consensus N-glycosylation site in one To correlate the structure of tryptases with their unique cDNA, which may explain some ofthe heterogeneity in size and properties and to explore the potential genetic basis of susceptibility to deglycosylation seen in tryptase preparations. heterogeneity and tissue differences in human tryptases, we All three tryptase cDNAs are distinct from a recently reported cloned and sequenced three different human tryptase cDNAs cDNA obtained from a human lung mast cell library. A skin and a gene corresponding to one ofthe cDNAs.** A genomic tryptase cDNA was used to isolate a human tryptase gene, the DNA blot probed with a fragment ofthe cloned gene revealed exons of which match one of the skin-derived cDNAs. The the presence of multiple tryptase genes. organization of the -1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions ofthe gene MATERIALS AND METHODS share features with those of other serine proteases, including Construction and Screening of a Skin cDNA Library. A mast cell chymase, but are unusual in being separated from the sample of 4 x 106 mast cells (1.1% of total cells) from scalp protein-coding sequence by an intron. High-stringency hybrid- skin of a single donor was obtained (22), and poly(A)+ RNA ization of a human genomic DNA blot with a fragment of the was isolated by LiCl precipitation (23) and oligo(dT)- tryptase gene confirms the presence ofmultiple tryptase genes. cellulose chromatography (24). A cDNA library containing 8 These rmdings provide genetic evidence that human mast cell x 105 unique recombinants was constructed in phage A ZAP tryptases are the products of a multigene family. II vector (Stratagene) (25) and was amplified once in Esch- erichia coli XL1-Blue cells (26) prior to screening. Approx- Tryptase, a major constituent ofmast cell secretory granules, imately 106 plaque-forming units from the library were has emerged recently as the most specific marker in vivo of screened at 420C with a dog tryptase cDNA (15) labeled to 2 mast cell activation in humans (1, 2). Tryptase is a trypsin- x 108 cpm/,Fg by nick-translation (27) as described, except like serine protease that hydrolyzes peptide bonds on the for use of 30% formamide in the hybridization solution (27). C-terminal side of basic amino acids. Unlike trypsin, how- Three positive recombinants were identified by autoradiog- ever, tryptase is highly selective in hydrolyzing its peptide raphy, plaque-purified, and reprobed. Phagemids containing and protein targets and resists inactivation by circulating inserts that hybridized to the cDNA probe were excised from inhibitors (3-6). In vitro studies using purified dog and human the ZAP II A phage vector by using R408 helper phage (28), tryptase preparations suggest potential roles in the lung, skin, transformed into E. coli XL1-Blue cells, and purified by and other tissues as a local anticoagulant (7, 8), activator of alkaline lysis (29). The sequence of the cDNA inserts was collagenase (9), promoter of smooth muscle contraction (10), determined by dideoxy chain termination (30) modified for and modulator of neuropeptide activity (11, 12). double-stranded DNA (31) by using Sequenase (United The distinctive properties of tryptase have yet to be States Biochemical) (32). The M13 forward, reverse, and KS adequately understood in molecular terms. Although primers (Stratagene) were used initially. Subsequent se- tryptase is stored and released from the mast cell granule in quencing reactions used oligonucleotide primers designed an active form (13, 14), the tryptase preprosequence deduced from previously determined sequence. from dog and human cDNAs suggests that tryptase may be Genomic Library Screening. Human tryptase clone II synthesized as a proenzyme (15, 16). Preparations of human cDNA (see Results) was used to screen -6 x 105 plaque- and dog tryptase with a single N-terminal sequence are inhomogeneous, yielding two or more closely spaced bands on sodium Abbreviation: RMCP II, rat mast cell proteinase II. dodecyl sulfate/polyacrylamide gel electrophore- tPresent address: Division ofPulmonary and Critical Care Medicine, sis (SDS/PAGE) (3, 5, 6, 17). Some, but not all, of this 111J, La Jolla Veterans Administration Medical Center, La Jolla, heterogeneity is due to variable N-glycosylation (15, 17). The CA 92161. active form of dog and human tryptase is a tetramer (6, 18, ITo whom reprint requests should be addressed at: Cardiovascular 19), each subunit ofwhich shares antigenic determinants and Research Institute, University of California, San Francisco, CA 94143-0130. **The sequences reported in this paper have been deposited in the The publication costs of this article were defrayed in part by page charge GenBank data base [accession nos. M33491 (for tryptase I cDNA), payment. This article must therefore be hereby marked "advertisement" M33492 (for tryptase II cDNA), M33493 (for tryptase III cDNA), in accordance with 18 U.S.C. §1734 solely to indicate this fact. and M33494 (for tryptase I gene)]. 3811 Downloaded by guest on September 30, 2021 3812 Biochemistry: Vanderslice et al. Proc. Nad. Acad. Sci. USA 87 (1990) a -30 -20 forming units from human placental genomic library in Met Leu Asn Leu Leu Leu Leu Ala Leu Pro Val Leu Ala Ser Arg EMBL-3 (Clontech). The cDNA was labeled with biotin- ATG CTG AAT CTG CTG CTG CTG GCG CTG CCC GTC CTG GCG AGC CGC 45 II I III 7-dATP by nick-translation and was hybridized at 50°C to the -10 immobilized phage DNA (33). Positive clones were visualized Ala Tyr Ala Ala Pro Ala Pro Gly Gln Ala Leu Gln Arg Val Gly by using the BluGENE nonradioactive nucleic acid detection GCC TAC GCG GCC CCT GCC CCA GGC CAG GCC CTG CAG CGA GTG GGC 90 1 10 system (Bethesda Research Laboratories) (33). Nine of the Ile Val Glv Gly Gln Glu Ala Pro Aro Ser Lvs Tzm Pro Tn, Gin ATC GTC GGG GGT CAG GAG GCC CCC AGG AGC AAG TGG CCC TGG CAG 135 most strongly hybridizing clones were plaque-purified and II T III T rescreened. Phage DNA was purified by the plate lysate 20 30 method (27) and digested with BamHI to yield genomic Val Ser Leu Ara Val His plPro Tyr Trp Met His Phe Cys Gly GTG AGC CTG AGA GTC CAC GGC CCA TAC TGG ATG CAC TTC TGC GGG 180 fragments, which were separated by agarose gel electropho- III G A G Arq Asp Arq resis, transferred to nitrocellulose (34), and hybridized to 40 tryptase cDNA. Hybridizing fragments from two ofthe clones Gly Ser Leu Ile His Pro Gln Trp Val Leu Thr Ala AlarHis Cys were ligated into the BamHI site of pBluescript KS+ ph- GGC TCC CTC ATC CAC CCC CAG TGG GTG CTG ACC GCA GCG CAC TGC 225 50 60 agemid (Stratagene), and the nucleotide sequence was deter- Val Gly Pro Asp Val Lys Asp Leu Ala Ala Leu Arq Val Gln Leu mined as described for the cDNAs. The nucleotide sequence GTG GGA CCG GAC GTC AAG GAT CTG GCC GCC CTC AGG GTG CAA CTG 270 70 of both strands of the gene shown in Fig. 2 was determined Arg Glu Gln His Leu Tyr Tyr Gln Asp Gln Leu Leu Pro Val Ser with the exception of the sequence of the 3' 179 nucleotides, CGG GAG CAG CAC CTC TAC TAC CAG GAC CAG CTG CTG CCG GTC AGC 315 which was determined in one direction. The sequence of 521 80 90 Arq Ile Ile Val His Pro Gln Phe Tyr Thr Ala Gln Ile Gly Ala bases of the second clone was determined in one direction. AGG ATC ATC GTG CAC CCA CAG TTC TAC ACC GCC CAG ATC GGA GCG 360 Genomic DNA Blotting. Human lymphocytes from a single 100 * individual were purified from whole blood by centrifugation (ij Ile Ala Leu Leu Glu Lou Glu Glu Pro Val Asn Val Ser Ser I ATC GCC CTG CTG GAG CTG GAG GAG CCG GTG AAC GTC TCC AGC 405 on Ficoll-Paque (Pharmacia) (35), and genomic DNA was II G Lys isolated by proteinase K treatment and phenol extraction 110 120 (36). Samples of 10 ,ug of DNA were digested to completion His Val His Thr Val Thr Leu Pro Pro Ala Ser Glu Thr Phe Pro with Ava II, Bal I, and BamHI (27).