Molecular cloning of the gene for human leukotriene C4 synthase. Organization, nucleotide sequence, and chromosomal localization to 5q35.

Leukotriene C4 (LTC4) synthase catalyzes the conjugation of LTA4 with reduced GSH to form LTC4, the parent of the receptor active cysteinyl leukotrienes implicated in the pathobiology of bronchial asthma. Previous cloning of the cDNA for human LTC4 synthase demonstrated significant homology of its amino acid sequence to that of 5-lipoxygenase activating protein (FLAP) but none to that of the GSH S-transferase super-family. Genomic cloning from a P1 library now reveals that the gene for LTC4 synthase contains five exons (ranging from 71 to 257 nucleotides in length) and four introns, which in total span 2.52 kilobase pairs in length. The intron/exon junctions of LTC4 synthase align identically with those of FLAP; however, the small size of the LTC4 synthase gene contrasts with the > 31-kilobase pair size reported for FLAP. Confirmation of the LTC4 synthase gene size to ensure that no deletions had occurred during the cloning was obtained by two overlapping polymerase chain reactions from genomic DNA, which provided products of the predicted sizes. Primer extension analysis with poly(A)+ RNA from culture-derived human eosinophilic granulocytes or the KG-1 myelogenous cell line revealed multiple transcriptional start sites with prominent signals at 66, 69, and 96 base pairs 5' of the ATG translation start site. The 5'-flanking region revealed a GC-rich promoter sequence consistent with an SP-1 site and consensus sequences for AP-1 and AP-2 enhancer elements, 24, 807, and 877 bp, respectively, 5' from the first transcription initiation site. Southern blot analysis of a genomic DNA (with full-length cDNA as well as 5' and 3' oligonucleotide probes) confirmed the size of the gene and indicated a single copy gene in normal human genomic DNA. Fluorescent in situ hybridization mapped LTC4 synthase to chromosomal location 5q35, which is in close proximity to the cluster of genes for cytokines and receptors involved in the regulation of cells central to allergic inflammation and implicated in bronchial asthma.


Leukotriene C 4 (LTC 4 ) synthase catalyzes the conjugation of LTA 4 with reduced GSH to form LTC
, the parent of the receptor active cysteinyl leukotrienes implicated in the pathobiology of bronchial asthma. Previous cloning of the cDNA for human LTC 4 synthase demonstrated significant homology of its amino acid sequence to that of 5-lipoxygenase activating protein (FLAP) but none to that of the GSH S-transferase superfamily. Genomic cloning from a P1 library now reveals that the gene for LTC 4 synthase contains five exons (ranging from 71 to 257 nucleotides in length) and four introns, which in total span 2.52 kilobase pairs in length. The intron/exon junctions of LTC 4 synthase align identically with those of FLAP; however, the small size of the LTC 4 synthase gene contrasts with the >31-kilobase pair size reported for FLAP. Confirmation of the LTC 4 synthase gene size to ensure that no deletions had occurred during the cloning was obtained by two overlapping polymerase chain reactions from genomic DNA, which provided products of the predicted sizes. Primer extension analysis with poly(A) ؉ RNA from culture-derived human eosinophilic granulocytes or the KG-1 myelogenous cell line revealed multiple transcriptional start sites with prominent signals at 66, 69, and 96 base pairs 5 of the ATG translation start site. The 5-flanking region revealed a GC-rich promotor sequence consistent with an SP-1 site and consensus sequences for AP-1 and AP-2 enhancer elements, 24, 807, and 877 bp, respectively, 5 from the first transcription initiation site. Southern blot analysis of a genomic DNA (with fulllength cDNA as well as 5 and 3 oligonucleotide probes) confirmed the size of the gene and indicated a single copy gene in normal human genomic DNA. Fluorescent in situ hybridization mapped LTC 4

synthase to chromosomal location 5q35, which is in close proximity to the cluster of genes for cytokines and receptors involved in the regulation of cells central to allergic inflammation and implicated in bronchial asthma.
Leukotriene C 4 (LTC 4 ) 1 and its active metabolites, LTD 4 and LTE 4 , are the major components of the biologic activity previously known as the slow reacting substance of anaphylaxis. When inhaled, these arachidonic acid-derived lipid mediators exert profound smooth muscle constrictor effects on the airways of individuals with and without asthma (1,2). The cysteinyl leukotrienes are further implicated in the pathogenesis of asthma by the presence of their metabolites in the urine of patients with acute severe asthma (3). Moreover, cysteinyl leukotriene synthesis inhibitors or receptor antagonists significantly ameliorate the persistent pulmonary function abnormalities of individuals with asthma (4) and the exacerbations of bronchial asthma elicited by exercise (5), inhalation of specific allergens (6), and the idiosyncratic response to aspirin (7,8).
The formation of the cysteinyl leukotrienes is initiated by transmembrane stimuli that increase the levels of intracellular calcium, leading to the translocation of cytosolic phospholipase A 2 (9) and 5-lipoxygenase to the perinuclear membrane (10). Cytosolic phospholipase A 2 liberates arachidonic acid from phospholipids (11) for presentation to 5-lipoxygenase by 5-lipoxygenase activating protein (FLAP) (12,13), an integral perinuclear membrane protein (10). 5-Lipoxygenase catalyzes the sequential formation of 5-hydroperoxyeicosatetraenoic acid and the unstable epoxide, LTA 4 (14,15). LTC 4 synthase then catalyzes the conjugation of LTA 4 with reduced GSH to form intracellular LTC 4 (16,17). LTC 4 synthase is an 18-kDa integral membrane protein, which has been localized to the perinuclear region of alveolar macrophages (18) and recognized either by enzymatic function and/or by SDS-polyacrylamide gel electrophoresis immunoblot analysis in some hematopoietic cell populations such as eosinophils, basophils, mast cells, and platelets (18 -21). After carrier-mediated export of LTC 4 (22), the GSH adduct is cleaved sequentially by ␥-glutamyl transpeptidase to form LTD 4 (23) and by dipeptidases to yield LTE 4 (24), both of which are biologically active metabolites.
The cDNA and consensus amino acid sequence of LTC 4 synthase bear no homology to that of any member of the GSH S-transferase family, but instead, the deduced amino acid sequence shows significant homology to the amino acid sequence of FLAP (25,26). The predicted secondary structure of LTC 4 synthase contains three hydrophobic domains and two hydrophilic loops, which align identically with the predicted secondary structure of FLAP (25). A sequence of 22 amino acid residues at the carboxyl terminus of the first hydrophilic loop of FLAP is believed to bind the released arachidonic acid and is the site at which FLAP inhibitors act to prevent cellular 5-li-poxygenase function (27). LTC 4 synthase displays a high degree of amino acid identity to this hydrophilic region of FLAP, with 8 of 11 amino acids being identical in one sequence, and is functionally inhibited by the FLAP inhibitor, MK-886 (25). It is thus presumed that this FLAP-like domain of LTC 4 synthase binds LTA 4 for conjugation with GSH and that these two proteins belong to a functionally related gene family.
We have now isolated and sequenced the nucleotides of the gene for human LTC 4 synthase and demonstrate that the genomic organization of the LTC 4 synthase gene and FLAP gene are highly conserved. However, unlike FLAP, LTC 4 synthase is a small gene located on the long arm of human chromosome 5 in close proximity to a cluster of genes that are implicated in the asthmatic phenotype. Genomic Cloning of LTC 4 Synthase-A P1 genomic library was screened by Human Genome Systems (St. Louis, MO) with a polymerase chain reaction (PCR) product generated from oligonucleotides designed from the cDNA for human LTC 4 synthase. The sense oligonucleotide, 5Ј-CGTGGGCCTGAGACCAAG-3Ј, and the antisense oligonucleotide, 5Ј-CGGTCACTAGAACTTTAATGATAGAG-3Ј, corresponded to nucleotides 496 -518 and 622-597, respectively, of the cDNA. A positive genomic P1 clone for LTC 4 synthase was identified, and its plasmid DNA was digested with various restriction enzymes (BamHI, TaqI, SalI, HindIII, EcoRI, HaeIII, XbaI, XhoI, ApaI, SacI, EagI) (New England Biolabs, Beverly, MA). The reaction products were separated by electrophoresis in a 1% agarose gel, transferred to a nylon membrane (Millipore, Bedford, MA), and probed with a fluorescent labeled full-length cDNA for LTC 4 synthase using a chemiluminescent development method (Stratagene, La Jolla, CA). The blot was stripped and sequentially probed with ␥-32 P (3000 Ci/mmol, 10 mCi/ml, DuPont) end-labeled oligonucleotides from the 5Ј-(5Ј-AGCTCGCCTTCACACA-CAGCCCG-3Ј, corresponding to nucleotides 11-33) and 3Ј-(5Ј-CGGTCACTAGAACTTTAATGATAGAG-3Ј, corresponding to nucleotides 622-597) untranslated regions of the LTC 4 synthase cDNA. DNA was subcloned into the pT7T3 Vector (Pharmacia, Uppsala, Sweden) and was sequenced by the method of Sanger et al. (29), using dyelabeled dideoxy nucleotides as terminators (30) and an applied Biosystems 373A automated DNA sequencer in the Dana-Farber Cancer Institute Molecular Biology Core Facility.
Southern Blot Analysis of Total Genomic DNA-One 30-g sample of DNA isolated from human peripheral blood leukocytes was digested with the restriction enzyme SacI, and another sample was digested with KpnI. Two 200-ng samples of P1 genomic DNA were treated in the same manner. The DNA preparations from both digests were resolved in a 0.75% agarose gel and transferred by capillary electrophoresis to a nylon membrane. The membrane was hybridized (31) with a 32 P-labeled full-length cDNA for LTC 4 synthase and developed with Kodak XAR film with two intensifying screens for 5 days.
Primer Extension Analysis-Poly(A) ϩ RNA was isolated directly from 1 ϫ 10 8 KG-1 cells with oligo(dT) cellulose resin (Invitrogen, San Diego, CA) as described by the manufacturer. Poly(A) ϩ RNA was also obtained from 5 ϫ 10 8 in vitro derived eosinophilic granulocytes cultured for 14 days with the use of TRI-reagent (Molecular Research Inc., Cincinnati, OH) to extract 300 g of total RNA and subsequent selection with oligo(dT) cellulose resin. A synthetic antisense oligonucleotide 5Ј-AG-TAGAGGTACCTCGTCCTTCATG-3Ј (nucleotides 78 -54 of the LTC 4 synthase cDNA) was labeled with ␥-32 P using T4 polynucleotide kinase (Promega, Madison, WI). A 10-pmol sample of the labeled primer was annealed to 8 g of poly(A) ϩ RNA from KG-1 cells or from in vitro derived eosinophilic granulocytes with a primer extension kit (Promega, Madison, WI) in 16-l reaction volumes and incubated at 70°C for 10 min and then at 41°C for 5 min. Reverse transcription with the addition of 1 unit of avian myeloblastosis virus reverse transcriptase was carried out at 41°C for 30 min. The reaction products were precipitated by the addition of 2 g of glycogen, 7.5 l of 4 M NH 4 OAc, and 100 l of 100% ethanol. The reaction products were resuspended in 4 l of sample buffer, boiled for 10 min, and resolved in a 6% acrylamide, 6.7 M urea gel at 60 watts for ϳ1 h. Additionally, for identification of the specific nucleotides at the transcription initiation sites, a genomic sequencing ladder was generated with the same primer as in the extension reaction to sequence the LTC 4 synthase genomic clone according to the dideoxy chain termination method of Sanger et al. (29). These reaction products and 32 P-labeled molecular weight standards were run in parallel lanes of the same gel. The gel was dried and exposed to Kodak XAR film with two intensifying screens for 5 days.
Chromosomal Localization-A genomic DNA blot from 25 humanrodent somatic hybrid DNAs that had been digested with TaqI (Biosys, New Haven, CT) was probed with a 32 P-labeled full-length cDNA for LTC 4 synthase and then exposed to Kodak XAR film for 2 weeks. A fluorescent in situ hybridization technique for the chromosomal localization of the LTC 4 synthase gene was performed by Human Genome Systems. Briefly, purified DNA of the P1 clone from which the entire genomic sequence was obtained was labeled with digoxigenin dUTP by nick translation. Labeled probe was hybridized to normal metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes, and specific hybridization signals were detected by incubating the hybridized slides with fluoresceinated antidigoxigenin antibodies followed by counterstaining with propidium iodide. 4 Synthase-A 5.5-kbp SacI-digested fragment liberated from the P1 plasmid hybridized with the full-length cDNA for LTC 4 synthase and with oligonucleotide primers from the 5Ј end and the 3Ј end of the cDNA, indicating that the full-length gene was contained within this fragment. This DNA fragment was subcloned and sequenced. The entire nucleotide sequence (Fig. 1) of the five exons and four introns, which spanned 2.52 kbp, was sequenced in both directions. 1.35 kbp of the 5Ј-flanking region and 0.59 kbp of the 3Ј-flanking region sequence were also obtained in both directions (Fig. 1). The intron/exon junctions were determined by nucleotide sequence comparison with the cDNA and obey the GT-AG rule (32) (Fig. 2). The exon sequence exhibits 100% identity to that of the reported cDNA (25). The size of the exons ranged from 71 to 257 bp, and that of the introns ranged from 84 to 1445 bp (Table I).

Characterization of Genomic Clone for Human LTC
To confirm the presence of the full-length gene, two overlapping PCRs were performed, using human genomic DNA as a template (Fig. 3). The expected PCR products of 2.1 and 0.86 kbp, respectively, were obtained and spanned the complete length of the LTC 4 synthase gene (Fig. 3).
A DNA blot was prepared from human genomic DNA and from P1 plasmid DNA, each of which was digested with KpnI and SacI, and probed with the full-length cDNA for LTC 4 synthase (Fig. 4). The patterns of hybridization in the P1 and the human genomic DNA were identical.
Analysis of the Transcription Initiation Sites-The transcription initiation sites were determined by primer extension of poly(A) ϩ RNA derived from KG-1 cells and from in vitro derived hybrid granulocytes, in comparison with parallel lanes containing the sequence of the LTC 4 synthase genomic clone and molecular weight markers, respectively (Fig. 5). Both cellular sources revealed three predominant transcription initiation start sites 66, 69, and 96 nucleotides upstream from the ATG translation start site.
Chromosomal Localization of the Gene for Human LTC 4 Synthase-The cDNA for LTC 4 synthase hybridized to the genomic DNA from 8 of the 26 human-rodent somatic cell hybrids. The only chromosome common to these hybrids was chromosome 5.
Fluorescent in situ hybridization with the P1 plasmid clone containing the gene for LTC 4 synthase was performed to confirm the chromosomal assignment and to localize the region on the chromosome. The fluorescent in situ hybridization specifically labeled the long arm of a group B chromosome (chromosome 4 or 5) (Fig. 6A). In a subsequent experiment, a probe associated with the cri-du-chat locus, previously mapped to 5q21 by Genome Systems, and the P1 clone for LTC 4 synthase were simultaneously hybridized to chromosome 5 (Fig. 6B). In that experiment, 73 of a total of 80 cells in metaphase that were analyzed exhibited specific labeling for the human LTC 4 synthase gene. Measurements of 10 specifically hybridized chromosomes demonstrated that the P1 clone localized at a position 98% of the distance from the centromere to the telomere of chromosome arm 5q, an area that corresponds to band 5q35. DISCUSSION The cloning and sequencing of the gene for human LTC 4 synthase (Fig. 1) have revealed that its intron size and chromosomal location are prominently different from the gene for FLAP, which encodes the only known homologous protein. The exon sequence of the gene has 100% identity with that of the previously reported cDNA, which encodes the 18-kDa LTC 4

FIG. 2. Comparison of the intron/exon junctions of the encoded regions of the human genes for LTC 4 synthase and FLAP.
The exons are in uppercase letters and within the boxed regions, with deduced protein sequences provided in three-letter code above or below the respective nucleotide sequence.  (Fig. 3), which spanned the 2.52-kbp gene and 0.44 kbp of the 5Ј-flanking region. Second, genomic DNA blot restriction enzyme digests of both the P1 plasmid clone and normal human genomic DNA probed with the full-length cDNA for LTC 4 synthase (Fig. 4) demonstrated the same pattern of hybridization. The genomic structure revealed five exons and four introns, which span 2.52 kbp ( Fig. 1 and Table I). The exon sequences responsible for encoding the LTC 4 synthase protein are small, ranging from 71 to 257 nucleotides, and are interspersed with small introns (Table I). Analysis of the human LTC 4 synthase 5Ј-flanking region reveals three putative transcription initiation sites, located 66, 69, and 96 nucleotides upstream of the ATG translation start site (Fig. 5). The presence of multiple transcription initiation sites is consistent with the observation that other genes lacking TATA sequences also have multiple transcription start sites (33,34). LTC 4 synthase contains the typical features of genes that have been identified with multiple transcription initiation sites: a high G/C content and at least one consensus sequence (GGGCGG) that binds SP-1, a ubiquitous transcription factor identified in many housekeeping genes (35). The SP-1 site resides 120 nucleotides upstream from the ATG translation start site (corresponding to a position 24 nucleotides upstream from the first transcription initiation site). Other proteins in the pathways of lipid mediator biosynthesis, such as 5-, 12-, and 15-lipoxygenases also exhibit this pattern (36,37). Additionally, consensus sequences for an AP-1 site (TGAGTCAG) (38), and an AP-2 site (TC-CCCCTCCC) (39) were identified 807 and 877 nucleotides 5Ј of the first transcription initiation site. Both of these elements are responsive to the activation of protein kinase C by phorbol 12-myristate 13-acetate and are consistent with the observation that LTC 4 synthase activity is induced in HL-60 cells (40) and human erythroleukemia cells after treatment with phorbol 12-myristate 13-acetate (41). In contrast, the FLAP gene has both a transcription initiation site residing in an A residue 74 nucleotides upstream from the ATG start codon and a modified TATA box (42). When the entire genomic sequence, exon sequence, and amino acid sequence were each analyzed for similarity to sequences in the EMBL data base with blast programs (43), the only significant protein homologies were with the family of FLAP molecules from different species. The human gene for FLAP has been cloned and found to be Ͼ31 kbp in size. The FLAP gene also contains 5 small exons but four large introns (Table I) for which sequence data are limited to the intron/exon junctions (42). The exons of the gene for LTC 4 synthase are identical in size to those of FLAP with the exception of the first and fifth, which are affected minimally by the number of nucleotides in the 5Ј-and 3Ј-untranslated regions (Table I). In addition, the exons of LTC 4 synthase and FLAP align identically with regard to the amino acids that they encode (Fig. 2). This fact also allows the deduced amino acids with the respective predicted secondary structures of the two molecules to be aligned as previously shown (25). The identical intron/exon organization of LTC 4 synthase and FLAP suggests the evolution of these two molecules from the process of gene duplication, as has been proposed for the ancient gene family of glyceraldehyde-3-phosphate dehydrogenases, which share five identical intron positions (44). Identical intron/exon overlap has also been shown for the 5-, 12-, and 15-lipoxygenase genes; the more closely related 12-and 15-lipoxygenases are on chromosome 17, and the less related 5-lipoxygenase is on chromosome 10 (37). Thus, although LTC 4 synthase and FLAP are both 18-kDa integral membrane proteins involved in the synthesis of leukotrienes and appear by homology at both the protein and cDNA levels and by genomic organization to be related members within a novel family, their evolutionary divergence is significant in terms of intron size, 5Ј-flanking regions, and chromosomal location.
The human gene for LTC 4 synthase has been localized with fluorescent in situ hybridization to the q35 region of chromosome 5 (Fig. 6). This finding contrasts with a report of the FIG. 3. Map of the gene for LTC 4 synthase obtained by overlapping PCR products. PCR product 1 extends from oligonucleotide Ϫ530 to ϩ1609, with nucleotide 1 corresponding to the ATG start site, and encompasses some of the 5Ј-flanking region, the first exon, and most of the second exon. PCR product 2, corresponding to nucleotides 1573-2429, overlaps PCR product 1 in the second exon and extends through exon 5.
FIG. 4. Analysis of genomic DNA isolated from the P1 plasmid and from peripheral blood leukocytes of a normal donor. Two hundred nanograms of P1 plasmid DNA from a clone known to contain the gene for LTC 4 synthase and 30 g of genomic DNA from peripheral blood leukocytes were individually digested with the SacI and KpnI restriction enzymes, resolved in a 1% agarose gel, transferred to a nitrocellulose membrane, and probed with a 32 P-labeled full-length cDNA for LTC 4 synthase. localization of the human FLAP gene on chromosome 13 (45) and the 5-lipoxygenase gene on chromosome 10 (37). The long arm of the fifth chromosome has also been identified as the site at which many of the genes encoding growth factors, cytokines, and receptors relating to the asthmatic phenotype are localized. These include IL-3, IL-4, IL-5, and granulocyte-macrophage colony-stimulating factor, as well as IL-9, IL-13, and fibroblast growth factor-acidic, all localized within a gene cluster at 5q23-5q31 (46,47). Receptors that have been localized more distally in the 5q31-q32 region include the ␤2 adrenergic receptor and the lymphocyte-specific corticosteroid receptor, whereas colony-stimulating factor receptor-1, monocyte colonystimulating factor receptor, and platelet-derived growth factor receptor are in the 5q33-q35 region (47,48). The most distal genes located in the 5q34-q35 region include dopamine receptor 1 and ␥-butyric acid A receptor (47).
The inflammatory changes of bronchial asthma demonstrated by biopsies are characterized by degranulation of mast cells as well as by infiltration of eosinophils and TH2 cells, all of which express markers of activation (49,50). These findings implicate the products of genes residing on the long arm of chromosome 5. Interleukin-3, IL-5, and granulocyte-macrophage colony-stimulating factor not only regulate eosinophilopoiesis (28,51,52), but act on mature eosinophils to attenuate steroid-induced apoptosis (53). Furthermore, these cytokines convert eosinophils to a phenotype similar to that associated with disease in which the cells are primed for ligand-initiated generation of LTC 4 and target cell cytotoxicity (54 -56). IL-4 mediates immunoglobulin isotype switching in general and IgE biosynthesis by B cells in particular (57). IgE sensitizes mast cells and basophils through their high affinity receptors for allergen-specific activation, providing an additional mechanism for LTC 4 generation. Importantly, IL-4 perpetuates the inflammatory reaction by favoring T cell maturation and differentiation to the TH2 phenotype, which provides IL-4 and the eosinophilopoietic cytokine triad (58).
In addition to the genomic localization of cytokines that amplify and perpetuate the asthmatic response to the long arm of the fifth chromosome, substantial clinical evidence supports the linkage of specific allelic oligonucleotide markers from this region of chromosome 5 to the atopic/asthmatic state. Atopy describes a heritable condition in which specific IgE is synthesized after exposure to specific allergens, and bronchial asthma is associated with bronchial hyperresponsiveness defined by compromised pulmonary function in response to environmental stimuli or concentrations of defined agonists that are inactive in the unaffected population. Bronchial hyperresponsiveness assessed by methacholine inhalation correlates with circulating levels of total IgE in individuals with asthma (59), and both of these coinherited features demonstrate significant linkage to the IL-4 gene, localizing specifically to position 5q31.1 (60). Fibroblast growth factor-acidic and colony-stimulating factor 1 receptor have also been shown by linkage analysis to be disproportionately associated with bronchial hyperresponsiveness in sibling pairs (61). Specific polymorphisms have been identified in the enhancer sequences (a C to T exchange at position Ϫ590 from the open reading frame) of IL-4 and correlate with increased IL-4 level activity manifested by higher total serum IgE in atopic asthmatic kindreds (62). The immediate improvement of pulmonary function in individuals with asthma who receive an initial dose of agents that are devoid of intrinsic bronchodilatory activity but selectively attenuate the formation or action of the cysteinyl leukotrienes indicates that  chronic overproduction of the cysteinyl leukotrienes occurs in the natural disease (4). The finding that the gene for LTC 4 synthase resides in the terminal region of the long arm of chromosome 5 adds another important candidate gene for asthma to the previously recognized cytokine genes clustered at the locus, i.e. one related to the generation of lipid mediators.