(Received for publication, February 5, 1996; and in revised form, February 29, 1996)
From the
Leukotriene C (LTC
) synthase catalyzes
the conjugation of LTA
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
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
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
synthase align identically with those of
FLAP; however, the small size of the LTC
synthase gene
contrasts with the >31-kilobase pair size reported for FLAP.
Confirmation of the LTC
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 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 LTC
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 (LTC
) (
)and its
active metabolites, LTD
and LTE
, 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(9) and 5-lipoxygenase to the perinuclear
membrane(10) . Cytosolic phospholipase A
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
(14, 15) . LTC
synthase
then catalyzes the conjugation of LTA
with reduced GSH to
form intracellular LTC
(16, 17) . LTC
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, 19, 20, 21) . After
carrier-mediated export of LTC
(22) , the GSH adduct
is cleaved sequentially by
-glutamyl transpeptidase to form
LTD
(23) and by dipeptidases to yield
LTE
(24) , both of which are biologically active
metabolites.
The cDNA and consensus amino acid sequence of LTC 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
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-lipoxygenase
function(27) . LTC
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
synthase binds
LTA
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 synthase and demonstrate that the genomic organization of the
LTC
synthase gene and FLAP gene are highly conserved.
However, unlike FLAP, LTC
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.
Figure 1:
Complete, numbered nucleotide
sequence of the human gene for LTC synthase. The introns
and 1.35 and 0.59 kbp of the 5`- and 3`-flanking regions, respectively,
are shown in lowercase letters. The exons are depicted in uppercase letters and in large boxes. The putative
SP-1 promotor site and AP-1 and AP-2 enhancer elements are shown in small boxes, the three predominant transcription initiation
sites are circled, and the ATG translation initiation site is underlined.
Figure 2:
Comparison of the intron/exon junctions of
the encoded regions of the human genes for LTC 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.
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 synthase gene (Fig. 3).
Figure 3:
Map of the gene for LTC synthase obtained by overlapping PCR products. PCR product 1
extends from oligonucleotide -530 to +1609, with nucleotide
1 corresponding to the ATG start site, and encompasses some of the
5`-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.
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 synthase (Fig. 4). The
patterns of hybridization in the P1 and the human genomic DNA were
identical.
Figure 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 synthase and
30 µg 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
P-labeled
full-length cDNA for LTC
synthase.
Figure 5:
Transcription initiation site analysis for
the human LTC synthase gene. Samples of poly(A)
RNA isolated from KG-1 monocytic cells and from in vitro derived granulocytes were extended by reverse transcription with a
primer corresponding to nucleotides 78-54 of the LTC
synthase cDNA. A genomic sequencing ladder was created in
parallel and is shown on the left with the appropriate
radiolabeled nucleotides. Molecular weight markers are shown on the right.
Fluorescent in situ hybridization with the P1 plasmid clone
containing the gene for LTC 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
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
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.
Figure 6:
Fluorescent in situ chromosomal
localization of the human LTC synthase gene. Panel A shows the computer-enhanced image of hybridization of a P1 plasmid
clone containing LTC
synthase to a group B chromosome
(chromosome 4 or 5). Panel B shows a computer-enhanced image
of hybridizations of the probe for the cri-du-chat locus and the P1
plasmid clone containing the LTC
synthase gene,
respectively, to the q arm of chromosome 5.
The cloning and sequencing of the gene for human LTC 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
synthase protein. The entire
gene was contained in a 5.5-kbp SacI fragment that hybridized
with the full-length cDNA for LTC
synthase and
oligonucleotides corresponding to the 5` and 3` ends of the cDNA. That
no deletion or rearrangements had occurred during the cloning was
demonstrated in two ways. First, two overlapping PCRs on human genomic
DNA yielded the appropriately sized reaction products of 2.1 and 0.86
kbp (Fig. 3), which spanned the 2.52-kbp gene and 0.44 kbp of
the 5`-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
synthase (Fig. 4) demonstrated the same pattern of hybridization. The
genomic structure revealed five exons and four introns, which span 2.52
kbp ( Fig. 1and Table 1). The exon sequences responsible
for encoding the LTC
synthase protein are small, ranging
from 71 to 257 nucleotides, and are interspersed with small introns (Table 1). Analysis of the human LTC
synthase
5`-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
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 (TCCCCCTCCC) (39) were identified 807 and 877 nucleotides 5` 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
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 >31 kbp in size. The FLAP gene also contains 5 small exons but
four large introns (Table 1) for which sequence data are limited
to the intron/exon junctions(42) . The exons of the gene for
LTC 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 5`- and 3`-untranslated regions (Table 1). In addition, the exons of LTC
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
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
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,
5`-flanking regions, and chromosomal location.
The human gene for
LTC 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
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 colony-stimulating 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 and target cell
cytotoxicity(54, 55, 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
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 -590 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 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U50136[GenBank].