THEJOURNAL OF BIOLOGICALCHEMISTRY Vol. 267, No. 35, Issue of December 15, pp. 2548&25493,1992 Q 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S. A, Genomic Organizationof the Mouse Granzyme A Gene TWO mRNAs ENCODE THE SAME MATURE GRANZYME A WITH DIFFERENT LEADER PEPTIDES* (Received for publication, June 23, 1992) R. Jane HershbergerS, Howard. K. Gershenfeld, Irving L. Weissman, and LishanSug From the Departments of Patholom and ~~euebD~n~~Biobgy and the Howard Hughes Medical Institute, S~anford~ed~~ Center, Stanjbrd, California 94305- Granzyme A is a serine protease that, together with and viral infection (5). Granzyme A, also named Hanukah the other granularcomponents of cytotoxic T lympho- Factor (HF, Ref. 6), BLT esterase (71, TSP-1 (81,SE-1 (9), cyte (CTL) cells, has been implicated in the cytolysis and CTLA-3 (lo), is a neutral serine protease that cleaves process. We report here two different messages and after Arg or Lys residues. It also cleaves the colorimetric the genomic organization of the mouse granzyme A substrate benzyloxycarbonyllysyl thioester (BLT).BLT gene. The granzyme A gene is composed of six exons cleavage activity has been shown to be released when CTLs spanning 7 kilobases. Alternative splicing of the second are incubated with their target cells (11) or with specific anti- exon results in the two transcripts. The two mRNA T cell receptor antibodies (12), or with some calcium iono- species encode the same mature granzyme A protein phore (9). but with different leader sequences. The first (HFl) encodes a typical leader signal sequence similar to Protease activity has longbeen implicated inthe cell- other granzymes, but the second (HF2) putative leader mediated killing process (13-15). A potent inhibitor of BLT sequence is different and less hydrophobic. Both mes- activity has been shown to inhibitthe killing ability of a CTL sages are present in cultured CTL cell lines and in (16). More recently, a CTL cell line whose granzyme A gene normal lymphoid tissues. They are both induced when expression has been inhibited by an antisense gene shows CTL cells are activated in vitro or in vivo. Both mes- reduced killing potential.’ Using purified granzyme A, it has sages can be translated in uitro, although the HF1 been shown that it can induce DNA degradation (18), a message appears to be much more efficient as a tem- process associated with cell-mediated cytolysis (19). plate. The putative5’ promoter region of the HF gene Many serine proteases are synthesized with an amino- sequenced (500 base pairs of upstream sequences) con- terminal peptide of 20-30 amino acids that is absentfrom the tains no well defined promoter sequences aside from mature form. This leader peptide consists of a signal (pre-) the TATA box. The results suggest that (a)granzyme peptide, which directs the nascent polypeptide into thelumen A may be produced with putative different leader se- of the rough endoplasmic reticulum, and an activation (pro-) quences from two differentmRNAs; (6)this may pro- peptide, which keeps the protease in an inactive state (20). vide amodel system for studyingalternate splicing and Granzyme A, like the other granzymes, doesnot have a leader the evolution of a complex enzymatic system in an peptide when it is isolated from the granules (21). It has been organelle; and (c) the genomic DNA reported will be proposed that the acidic pH of the granule environment (8) useful for studying transcription regulationsinvolved and/or binding to the granule proteoglycans (22) keeps it in controlling the specific expression pattern of this inactive. gene. We have clonedthe granzyme A gene and characterized its genomic organization. By analysis of its mRNA, we found that it expresses two different granzyme A mRNAs. They encode the same mature protein with two different leader Cytotoxic T lymphocytes (CTLs)’ and natural killer cells peptides. AIternative splicing of the second exon of the gran- may have cytoplasmic granules that are exocytosedupon zyme A gene produces the two types of mRNA. One leader binding to the target cells. These granules, which have been sequence is very similar to thatof the other granular proteins implicated in the killing process, contain perforin (cytolysin) such as perforin and the other granzymes. The other isdiffer- (1,2) and at least seven serine proteases called granzymes A ent and less hydrophobic. Both messages are expressed in through G (3). In vivo, the mRNAs for granzymes A and B CTL lines and in normal lymphoid tissues, although the one are expressed in the cellular infiltrates in graft rejection (4) with the typical granzyme leader sequence is muchmore abundant. Bothmessages are induced in vivo or in uitro during * This work was supported in part by National Institutes of Health activation of CTL function. Grant AI-19512 and by the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be hereby MATERIALS AND METHODS marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 Isolation of Full-length cDNAs-A subclone of the HF cDNA (6) solely to indicate this fact. lacking the poly(A) tail was used to screen 106 recombin~tbacteri- The nucleotide sequence& reported in this paper has been submitted ophage each from the AR1 and 1E4 CTL cDNA libraries (6).Twenty to the GenBankTM/EMBL Data Bank with accession numberb) positive clones were purified and theirlengths determined by restric- LoO631. tion mapping. The two longest clones were subcloned into M13mp18 § Postdoctoral fellow of the Irvington Institute for Medical Re- or M13mp19 (23) for sequencing. The nucleotide sequence was deter- search. To whom correspondence should be addressed Dept. of Pa- thology, Howard Hughes Medical Institute, StanfordMedical Center, Stanford, CA 94305. Tel.: 415-723-7389; Fax: 415-723-1399. ‘A. Tabnto, M. Nguyen, s. Law, J. K. Wu, M. Poe, J. T. Blake, The abbreviations used are: CTL(s), cytotoxic lymphocyte(s); HF, M. Patel, T. J. Wu, C. Manyak, M. Silberklong, G.Mark, M. Springer, Hanukah Factor; BLT, benzyloxycarbonyl lysyl thioester; kb, kilo- N. H. Sigal, I. Weissman, C. Bleackley, E. Podack, M. Tykocinski, base(s); PCR,polymerase chain reaction; bp, base pair(s). and G. C. Koo, submitted for publication. 25488 GenomicOrganization ofMouse theGranzyme A Gene 25489 mined on both strands by the dideoxynucleotide chain termination to Genetran (Plasco), and probed with an isolated fragment corre- method (24), and the Genepro program (Riverside Scientific) was sponding to exon 4. used to analyze the sequence data andcalculate sequence similarities. In Vitro Transcription and Translation-The HF1 andHF2 cDNAs Isolation of Genomic Clones-A murine genomic library in lEMBL3 were cloned into pBluescript (Stratagene). The transcription and (a kind gift of T. St. John, ICOS, Seattle, WA) wasscreened with the translation reactions were performed essentially as described (33), HF subclone described above. The 6 X lo5 recombinant phages except that 14C-labeledleucine and a rabbit reticulocyte system (Be- screened contained two classes of hybridizing inserts; the longest thesda Research Laboratories) were used in the translation reaction. representative of each class was characterized further. Restriction Equal amounts of RNA transcripts were used in the translation mapping was done according to standard methods, and the digested reactions. The translation products were examined by electrophoresis DNA was transferred to Genetran (Plasco). The locations of the exon in a 12% polyacrylamide gel (30 acrylamide, 0.8 bisacrylamide) con- boundaries in the HF2 cDNA (described below) were approximated taining sodium dodecyl sulfate (34). A 14C-methylatedprotein mixture by comparison with the rat mast cell protease I1 gene (25), and (Bethesda Research Laboratories) was used as the molecular weight restriction fragments corresponding to the putative exons were sub- standard. After electrophoresis, the gelwas fixed, treated with cloned. These fragments, used as probes on the genomic clone blots, En3Hance (Du Pont-New England Nuclear), and autoradiographed. gave a rough map of the genomic organization. One of the genomic Transient Expression in COS-7 Cells-The HF1 and HF2 cDNAs clones had a 13-kb insert that included the entire gene and 6 kb of were cloned into pSRa.SD2 (kindly provided by Dan Denney, Stan- upstream sequences. The other clone had 11 kb upstream and 4 kb ford, CA). The transfection procedure was essentially as described in of the gene, terminating between the third andfourth exons. Restric- the lipofectin (Bethesda Research Laboratories) information sheet. tion fragments covering all of the exons and 1 kb upstream of the Briefly, 5 pgof the appropriate DNAs were mixed with 1.5 mlof first exon were subcloned into M13 for sequencing. The sequences Optimum (Bethesda Research Laboratories) medium and thenmixed were determined on both strands asdescribed above. with 1.5ml of the same medium containing 40pg of lipofectin RNA Sources-RNA from the following cell lines was isolated in (Bethesda Research Laboratories). The mixture was overlaid onto a the presence of guanidium isothiocyanate and purified on CsCl gra- 6-cm plate with COS-7 cells at 70-80% confluence. After incubation dients (26): AR1, an H-2d-specificCTL line derived from the C57L at 37 "C for 6 h, 3 ml of Dulbecco's modifiedEagle's medium contain- mouse strain (6); 1E4, another C57L CTL line that kills Abelson ing 20% FCS was added to each plate, and theplates were incubated virus-infected H-2btargets (6); RAW112, an H-2dexpressing, Abelson at 37 "C for 20 more h. The medium was then replaced with 5 ml of virus-induced B cell lymphoma used as theAR1 target (6); and PC60, Dulbecco's modified Eagle's mediumcontaining 10% fetal calf serum. a cytolytic T cell hybridoma with inducible cytolytic activity by At about 48 h post-transfection,the plate was washed with phosphate- culturing for 3 days in Dulbecco's modifiedEagle's medium with 25% buffered saline twice, and the cells were scraped into 1 ml of phos- supernatant from concanavalin A-stimulated rat spleen cells (27).