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Biochem. J. (2004) 381, 113–123 (Printed in Great Britain)
113
Murine cystathionine γ -lyase: complete cDNA and genomic sequences,
promoter activity, tissue distribution and developmental expression
Isao ISHII*
1
, Noriyuki AKAHOSHI*, Xiao-Nian YU*†, Yuriko KOBAYASHI*, Kazuhiko NAMEKATA*, Gen KOMAKI†
and Hideo KIMURA*
*Department of Molecular Genetics, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Ogawahigashi 4-1-1, Kodaira, Tokyo 187-8502, Japan,
and †Division of Psychosomatic Research, National Institute of Mental Health, NCNP (National Center of Neurology and Psychiatry), Ichikawa, Chiba 272-0827, Japan
Cystathionine γ -lyase (CSE) is the last key enzyme in the trans-
sulphuration pathway for biosynthesis of cysteine from methi-
onine. Cysteine could be provided through diet; however, CSE has
been shown to be important for the adequate supply of cysteine
to synthesize glutathione, a major intracellular antioxidant.
With a view to determining physiological roles of CSE in mice, we
report the sequence of a complete mouse CSE cDNA along with
its associated genomic structure, generation of specific polyclonal
antibodies, and the tissue distribution and developmental expres-
sion patterns of CSE in mice. A 1.8 kb full-length cDNA contain-
ing an open reading frame of 1197 bp, which encodes a
43.6 kDa protein, was isolated from adult mouse kidney. A 35 kb
mouse genomic fragment was obtained by λ genomic library
screening. It contained promoter regions, 12 exons, ranging in
size from 53 to 579 bp, spanning over 30 kb, and exon/intron
boundaries that were conserved with rat and human CSE.The
GC-rich core promoter contained canonical TATA and CAAT
motifs, and several transcription factor-binding consensus seq-
uences. The CSE transcript, protein and enzymic activity were
detected in liver, kidney, and, at much lower levels, in small
intestine and stomach of both rats and mice. In developing mouse
liver and kidney, the expression levels of CSE protein and activity
gradually increased with age until reaching their peak value at
3 weeks of age, following which the expression levels in liver
remained constant, whereas those in kidney decreased signi-
ficantly. Immunohistochemical analyses revealed predominant
CSE expression in hepatocytes and kidney cortical tubuli. These
results suggest important physiological roles for CSE in mice.
Key words: cystathionase, cystathionine γ -lyase, cystathion-
inaemia, cysteine, methionine, trans-sulphuration.
INTRODUCTION
In mammals, cysteine is provided through the diet or the trans-
sulphuration pathway in which
L-cysteine is synthesized by sul-
phur transfer from
L-methionine to L-serine. Several catalytic
enzymes are involved in the transsulphuration, and cystathionine
γ -lyase (CSE, γ -cystathionase; EC 4.4.1.1), a PLP (pyridoxal
5
-phosphate)-dependent enzyme, catalyses its final essential step,
the conversion of
L-cystathionine into L-cysteine, α-ketobutyrate
and ammonia. Cysteine is further irreversibly metabolized in liver
to yield glutathione, taurine or inorganic sulphate by other en-
zymes, although CSE itself is capable of metabolizing cyst(e)ine
and produces hydrogen sulphide [1–3], a gaseous neuromodulator
[4] or smooth-muscle relaxant [5–7].
Deficiency of the CSE activity in humans is presumed to cause
cystathioninaemia (cystathionineuria; MIM 219500), an auto-
somal recessive inborn error probably with no consistent clinical
consequences [8]. Multiple mutations in the human CSE gene
were recently found in patients with cystathioninaemia [9]. Since
the CSE activity in rat liver is five times as high as that in human
liver [10,11], it is possible that CSE may play more important roles
in rodents. In rats, the CSE expression is restricted to specific
tissues; CSE is highly expressed in liver and kidney with very
low expression in brain [12–14]. Using
DL-propargylglycine, a
Abbreviations used: CBS, cystathionine β-synthase; CSE, cystathionine γ-lyase; E15.5, embryonic day 15.5; HEK-293 cells, human embryonic kidney
293 cells; MZF1, myeloid zinc finger protein 1; ORF, open reading frame; PFA, paraformaldehyde; PLP, pyridoxal 5
-phosphate; poly(A)
+
, polyadenylated;
RACE, rapid amplification of cDNA ends; Sp1, specificity protein 1; USF-1, upstream stimulatory factor-1; UTR, untranslated region.
1
To whom correspondence should be addressed (e-mail isao@ncnp.go.jp). A request for antibodies should be addressed to H. Kimura
(e-mail kimura@ncnp.go.jp).
The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank
®
and GSDB Nucleotide Sequence Databases under the accession
numbers AY083352 (for C57BL/6J mouse CSE cDNA) and AY262829 (for 129/SvJ mouse
CSE
).
specific irreversible CSE inhibitor [15], CSE has been shown to
be essential in rat liver, kidney and cultured hepatocytes for an
adequate supply of cysteine to synthesize glutathione [11,16,17],
a major intracellular antioxidant that protects cells from oxidative
stress. Cysteine is also utilized for biosynthesis of taurine, the
most abundant intracellular free amino acid, which has numerous
biological functions and also could act as an antioxidant. Regard-
less of its role as the precursor of such bioactive molecules,
cysteine itself could up-regulate the expression of cysteine dioxy-
genase that mediates taurine production and down-regulate the
expression of γ -glutamylcysteine synthetase, which mediates
glutathione production in cultured rat hepatocytes and intact rats
[18,19]. Moreover, high cysteine concentrations could be cyto-
toxic and neurotoxic in rats [20,21] and high plasma cysteine
concentrations in humans were associated with pre-eclampsia,
premature delivery, low birth weight and cardiovascular diseases
[22–24]. These lines of evidence suggest important roles for
CSE as the regulator of cysteine homoeostasis and the gluta-
thione–taurine rheostat.
Unfortunately, very little is known about mouse CSE at present.
This study was performed to characterize CSE gene and protein in
mice. We first cloned, sequenced and characterized the full-length
mouse CSE cDNA and the complete mouse CSE gene. Anti-CSE
polyclonal antibodies were generated and tissue d istribution of the
c
2004 Biochemical Society
114 I. Ishii and others
CSE transcript, protein and enzymic activity was examined in rats
and mice. Developmental expression was investigated and specific
CSE localization was revealed by immunohistochemistry, in both
mouse liver and kidney. These results should contribute to uncover
novel physiological functions of CSE and the transsulphuration
pathway in mice.
EXPERIMENTAL
Materials
Molecular biology and cell-culture reagents were purchased from
Invitrogen. All other reagents were obtained from Sigma, unless
otherwise mentioned. Mice (C57BL/6J Jcl) and rats [Jcl:SD
(Sprague–Dawley)] were purchased from Clea Japan (Tokyo,
Japan). The use of animals was in compliance with the guidelines
established by the Animal Care Committee of our Institute.
Mouse CSE cDNA cloning
Kidney was quickly removed from an 8-week-old male mouse
and homogenized in TRIzol
®
with the Polytron homogenizer
(Kinematica AG, Lucerne, Switzerland). Total RNA was isolated
according to the manufacturer’s instructions, and the first-strand
cDNA was synthesized from 10 µg of total RNA using the avian
myeloblastosis virus Reverse Transcriptase First-strand cDNA
Synthesis kit (Invitrogen) and the NotI-adaptor primer (5
-AA-
CTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTT-
TTTT-3
). This cDNA was used as a template for the first PCR with
the adaptor primer (5
-TGGAAGAATTCGCGGCCGCAG-3
)
and the CSE-e0 primer (5
-GCAAGACGTCGCACTCCTGCC-
3
), which contains the 5
-UTR (untranslated region) sequences
conserved within several mouse expressed sequence tags
(GenBank
®
accession numbers AI891806, AI099398, AI226268
and AI316238); the PCR condition was 25 cycles of 94
◦
Cfor
15 s, 55
◦
C for 30 s and 68
◦
C for 4 min. The PCR product was
further used as a template for the second PCR to obtain an entire
ORF (open reading frame) with the primers CSE-e1-XhoI(5
-
ATGCCTCGAGATGCAGAAGGACGCCTCTTTGAG-3
)and
CSE-NotI-Cter (5
-ATGCATGCGCGGCCGCTTAAGGGTGCG-
CTGCCTTCAA-3
). The second PCR product was digested
with XhoIandNotI, and subcloned into XhoI–NotI sites of
the pME18S mammalian expression vector [25], producing the
pME18S-mCSE vector. The full-length rat CSE ORF (a gift
from Dr Nishi, Kagawa Medical School, Kagawa, Japan) [12]
was similarly subcloned into the pME18S, producing the
pME18S-rCSE vector. The 5
-and3
-UTRs were isolated by 5
-
and 3
-RACE (rapid amplification o f cDNA ends) respectively
using the Gene Racer Superscript II Reverse Transcriptase kit
(Invitrogen). The following two primer sets were used: Gene
Racer 5
-primer (Invitrogen) and CSE-e2-2 (5
-GTGCTTTGCC-
CCATCCAACGCAG-3
)for5
-RACE or CSE-e9-1 (5
-TCTC-
ACCCTCAGCATGAGCT-3
) and Gene Racer 3
-primer (Invitro-
gen) for 3
-RACE. More than ten independent cDNA clones from
each PCR were confirmed by sequencing and the overlapping
sequences were identical within all those clones.
Isolation of mouse
CSE
gene
Two p robes, probes A and B, were used to screen total 5×10
5
independent plaques from the 129/SvJ mouse λ genomic library
(Stratagene) with a conventional plaque hybridization method.
Both probes were prepared by PCR using R1 embryonic stem
cell genomic DNA [26,27] as a template. The 1.7 kb probe A,
which spans exons 3–4, was amplified by PCR with the primers
CSE-e3-1 (5
-GGCCTTTGCATCGGGTCTTGCTGC-3
)and
CSE-e4-2 (5
-GTAATCGCTGCCTCTAGCAATTTG-3
). For the
preparation of probe B, the 1.3 kb fragment that resides in
exons 11–12, was amplified by PCR with the primers CSE-e11-3
(5
-TGTCACTTGCTTGTCAACACTG-3
) and CSE-rev-2 (5
-
CAGAACAACCTGTTAGTTAGAAGA-3
) and subcloned into
the pCR-TOPO vector (Invitrogen). The 406 bp EcoRI–XhoI
fragment neighbouring exon 12 was excised as the probe B.
Two overlapping clones (λCSE-1 and λCSE-2) and a single clone
(λCSE-3) were isolated after the tertiary screening with probes A
and B respectively. The λ phage DNA was prepared with QIAGEN
λ system (Qiagen) and sequenced.
CSE promoter analyses
A 3 .5 kb genomic DNA fragment upstream of the transcriptional
start site (− 3498 to + 18) was isolated by PCR using λCSE-1
as a template with the two primers CSE-pro-1 (5
-ATGGGTA-
CCACTTAGCATAATACTTAGAC-3
) and CSE-pro-rev (5
-AT-
GCCTCGAGGTGTTGCTTTGGCTAA-3
). The PCR product
was digested with KpnIandXhoI, subcloned into the promoterless
pGL3-Enhancer vector (Promega) that contains a firefly luciferase
gene driven by the promoter activity of inserted sequences,
and sequenced for confirmation, producing the pGL3-CSE-pro-1
vector. This vector was used as the PCR template to generate
pGL3-CSE-pro-2–22 vectors that contain different lengths within
the − 3498 bp sequences. Reporter gene assay was conducted
in transiently transfected Cos-7 and HEK-293 cells (human em-
bryonic kidney 293 cells) that were maintained in phenol red-free
Dulbecco’s modified Eagle’s medium supplemented with 10 %
(v/v) heat-inactivated foetal bovine serum (Hyclone Laboratories,
Logan, UT, U.S.A.) and antibiotics. At 24 h before the trans-
fection, 2×10
4
(for Cos-7) or 4×10
4
(for HEK-293) cells were
seeded into each well of the ViewPlate-96 White (Packard,
Meriden, CT, U.S.A.). On the day of transfection, 0.175 µg
(12.6 nmol) of the pRL-TK vector (Promega) that contains a
Renilla luciferase gene driven by the herpes simplex virus thy-
midine kinase promoter and 0.852 nmol (equivalent to 0.025 µg
of the pGL3-CSE-pro-1 vector) of the pGL3-Enhancer (or pGL3-
CSE-pro-1–22 vectors) were combined, and then the DNA mix-
ture was incubated with
LIPOFECTAMINE
TM
2000 (Invitrogen) at
the ratios of 2 µlof
LIPOFECTAMINE
TM
2000/1 µgofDNA.
Transfection was performed according to the manufacturer’s
instructions and the transfected cells were assayed for both firefly
and Renilla luciferase activities after 48 h of incubation. The lu-
minescence was measured using the FireLite Dual Luminescence
Reporter Gene Assay System (PerkinElmer) with the Fusion Uni-
versal Multiplate Analyzer (PerkinElmer). Both the (firefly and
Renilla) luciferase activities were measured and the promoter
activity was expressed as multiples of induction relative to the ac-
tivity (the ratio between firefly and Renilla luciferase activities)
when promoterless pGL3-Enhancer was transfected.
The TFSearch program (established by Y. Akiyama, Real World
Computing Partnership, Japan) was used to search the Transfac
4.0 database [28] for transcriptional factor-binding sites within
the sequences. T he transactivating factors with threshold scores
over 88.0 were considered to be important for the transcriptional
regulation.
Northern-blot analyses
Total RNA was isolated from various tissues of an 8-week-old rat
or mouse with TRIzol
®
and 10 µg of each was separated on
6 % (w/v) formaldehyde/1 % agarose gels. After transferring
on to the Hybond-XL nylon membrane (Amersham Biosciences),
c
2004 Biochemical Society
Characterization of mouse γ -cystathionase 115
hybridization was performed as described previously [29]. The
entire ORFs of rat [12] and mouse CSE cDNAs were used
as the specific probes against rat and mouse CSE respectively.
The probes were radiolabelled with [α-
32
P]dCTP (Amersham
Biosciences) and the hybridized blots were scanned with the Bio-
Imaging Analyzer Bas2500 (Fuji Photo Film, Tokyo, Japan). As
loading controls, 18 S rRNA was stained with ethidium bromide.
Anti-CSE polyclonal antibody production
Two different anti-rat CSE rabbit polyclonal antibodies were
constructed against the N- (amino acid numbers 1–193) or C-ter-
minal (amino acid numbers 194–398) portions of the recombinant
rat CSE protein consisting of 398 amino acids. The N-terminal
coding sequence was obtained by PCR using the entire rat
CSE cDNA as a template with the primers rCSE-BamHI-Nter1
(5
-ATGCGGATCCGCAGGAAGGACGCCTCCTCCAGC-3
)
and rCSE-XhoI-Cter1 (5
-A TGCCTCGAGAT ATGCAGACA TG-
AAAGTGTT-3
), digested with BamHI and XhoI, and subcloned
into the pET-21b(+) vector (Novagen), producing the pET-21b-
rCSE-Nter vector. The C-terminal coding sequence was also
isolated using the same template by PCR with the primers rCSE-
BamHI-Nter2 (5
-ATGCGGATCCGTTCCAGAGACCTTTGG-
CTCTG-3
)andrCSE-XhoI-Cter2 (5
-ATGCCTCGAGAGGGT-
GAGATGCCTTTAAAGC-3
), digested with BamHI and XhoI,
and subcloned into the same vector, producing the pET-21b-
rCSE-Cter vector. Each expression vector was transformed
into the BL21(DE3) Escherichia coli strain (Novagen) and the
recombinant protein was overexpressed in isopropyl β-
D-galacto-
pyranoside-treated bacterial culture. Both recombinant proteins
contain the His Tag (Novagen) at their N-termini, and were af-
finity-purified with the TALON Metal Affinity Resins (ClonTech)
under guanidine-denaturing conditions according to the manu-
facturer’s instructions. Rabbits were immunized with the purified
proteins three times and the anti-serum was prepared by con-
ventional procedures. Anti-CSE N-terminal serum was used for
Western blotting. Anti-CSE C-terminal serum was further purified
with HiTrap Protein G HP columns (Amersham Biosciences),
and the IgG fraction was used for immunohistochemistry.
Western-blot analyses
Tissues were quickly removed from an 8-week-old SD rat or
C57BL/6J mice of different ages, homogenized with a Teflon
tissue grinder in ice-cold buffer (100 mM sodium phosphate,
pH 7.8/1 mM PMSF), and sonicated with the Sonifier 450
(Branson Ultrasonics, Danbury, CT, U.S.A.). The homogenates
were centrifuged at 10 900 g for 5 min at 4
◦
C and the super-
natants were further centrifuged at 17 400 g for 20 min at 4
◦
C.
The resulting supernatants were quickly frozen in liquid nitrogen
and stored at − 80
◦
C until use. Tissue samples (5 µg) or trans-
fected Cos-7 samples (2 µg) were solubilized in the SDS-sample
buffer, boiled for 5 min, separated on a 10 % SDS/polyacrylamide
gel, and transferred on to the Immobilon PVDF transfer membrane
(0.45 µm, Millipore). The CSE protein was detected with
anti-CSE N-terminal serum (1:3000 dilution), horseradish-per-
oxidase-conjugated anti-rabbit IgG antibody and the ECL
®
Western-blotting system (Amersham Biosciences).
CSE activity measurement
CSE activity was determined by a sensitive method recently re-
ported by Ogasawara et al. [30] with minor modifications;
DL-pro-
pargylglycine (final 1 mM), instead of 4,4
-dithiodipyridine (final
3 mM), was used to inactivate CSE. This method utilizes
colorimetry for the determination of pyruvate produced from
β-chloro-
L-alanine by a CSE-catalysed β-elimination reaction,
coupling a colour enzymic reaction with pyruvate oxidase and per-
oxidase [30]. Briefly, CSE catalyses the pyruvate formation from
β-chloro-
L-alanine. This reaction is terminated by the addition
of
DL-propargylglycine. The produced pyruvate is oxidized by
pyruvate oxidase in the presence of thiamine pyrophosphate
and bivalent magnesium to liberate H
2
O
2
. A leuco dye, N-
(carboxymethylamino)-4,4
-bis(dimethylamino)-diphenylamine,
is oxidized by H
2
O
2
with peroxidase to produce Bindschedler’s
Green. The reaction was performed in 96-well dishes, and the
absorbance of green dye (727 nm) was measured by a Bio-Rad
Benchmark Plus microplate reader. The sample blank was simi-
larly prepared, except that the sample was added to the substrate
mixture after the addition of propargylglycine. The CSE-specific
activity was expressed as the balance (between sample and
sample blank) of absorbance at 727 nm/µg of protein.
Immunohistochemistry
Embryonic day 15.5 (E15.5) embryos and 8-week-old mice were
analysed for CSE expression by immunohistochemistry using
anti-CSE C-terminal antibody. The pregnant mice were eutha-
nized by ether and embryos were quickly removed and fixed
with 4 % (w/v) PFA (paraformaldehyde) in 0.1 M PBS (pH 7.3).
The adult (non-pregnant) female mice were anaesthetized with
pentobarbital (50 µg/g of body weight), and perfused through the
heart with PBS followed by 4 % PFA in PBS. Each tissue (liver,
kidney and spleen) was dissected out and postfixed overnight in
4 % PFA in PBS. Embryos or adult tissues were then embedded
in 30 % sucrose in PBS. After sinking, they were embedded in
Optimal Cutting Temperature compound (TISSUE-TEK; Sakura
Finetechnical, Tokyo, Japan), frozen and sectioned in a cryostat
at 10 µm. After washing with PBS, the sections were blocked
with Blocking Reagent (Roche Diagnostics) in PBS, and then
incubated with anti-CSE C-terminal antibody (IgG fraction;
diluted 1:100) or pre-immune serum in the blocking solution.
After incubation with the primary antibody, the sections were
washed with PBS and reacted with the VECTASTAIN ABC
peroxidase kit for rabbit antibodies (Vector Laboratories,
Burlingame, CA, U.S.A.) followed by the chromogen diamino-
benzidine.
RESULTS
Cloning and sequencing of the full-length mouse CSE cDNA
It was previously shown that the CSE gene is highly expressed in
adult mouse kidney and liver [31]. Therefore we have cloned the
full-length CSE cDNA from adult mouse kidney using PCR and
RACE. The 1815 bp of the entire mouse CSE transcript consists
of 33 bp of 5
-UTR, 1197 bp of ORF and 585 bp of 3
-UTR, of
which 15 nt are poly(A)
+
(polyadenylated) (Figure 1). This was
the first report for mouse CSE cDNA cloning and the sequence
was deposited with GenBank
®
, accession number AY083352. The
nucleotide sequence of the mouse CSE ORF is 92.5% identical
with the rat sequence and was 81.6% identical with the human
sequence (Figure 2). CSE is one of the fundamental enzymes
for methionine catabolism and is evolutionarily conserved from
yeast to mammals (Figure 2). Possible gene duplication was
reported in Caenorhabditis elegans but not in yeast or mammals
(Figure 2). The mouse ORF encodes a 43.6 kDa protein, which
shares high amino acid identity with those of the rat and human
CSE (93.5 and 85.6% respectively) [7,32]. A putative PLP-bind-
ing site with an active lysine (Lys
211
) [33] is located in the middle
of the ORF (Figure 1), which is fully conserved with rat CSE.
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2004 Biochemical Society
116 I. Ishii and others
Figure 1 Full-length cDNA and predicted amino acid sequences of the mouse kidney CSE
The transcriptional start site shown by a broken arrow is set at 1. The 1815 bp sequence contains a 1197 bp ORF that extends over bases 34–1230 (*, a stop codon). All the 11 exon/
intron boundaries are indicated by arrows. A putative PLP-binding site and poly(A)
+
consensus signal sequences (ATTAAA or AATAAA) are boxed and underlined respectively.
Four polyadenylation consensus signal sequences (ATTAAA or
AATAAA) are found in the 3
-UTR.
Cloning and sequencing of the mouse
CSE
gene
BLAST searches of the NCBI (National Center for Biotechnology
Information) GenBank
®
for human CSE gene with the human
full-length cDNA (GenBank
®
accession number NM 153742) re-
vealed a 12-exon structure (Figure 3A). The searches for the rat
CSE gene with rat cDNA that lacks a part of 3
-UTR (GenBank
®
accession number AB052882), also suggested a structure with
12 exons (Figure 3A). A similar 12-exon structure with con-
served exon/intron boundaries was speculated for the mouse CSE
organization. Thus two DNA fragments were amplified by PCR
using the R1 cell [established from a (129/Sv × 129/J)F
1
origin]
genomic DNA as a template and the possible exon-spanning
primer sets, and two screening probes, A and B, were prepared.
Southern-blot analyses using these two probes and R1 genomic
DNA digested with several restriction enzymes suggested the
existence of a multiexon, single copy gene for the mouse CSE
(results not shown). By screening 5 × 10
5
independent plaques
from a 129/SvJ mouse λ genomic library, we obtained three
overlapping clones (λCSE-1–3) that cover the complete CSE gene
(Figure 3A).
Sequencing of the three clones localized 12 exons ranging in
size from 53 to 579 bp that span over 30 kb in which CSE ORF
is encoded within all 12 exons (Figures 1 and 3). A 35 246 bp
129/SvJ mouse CSE genomic sequence, including a 4 kb sequence
upstream of the transcriptional start site, 12 exons and 11 introns,
was deposited with GenBank
®
, accession number AY262829. It
contains exon/intron boundaries conserved with rat and human
CSE genes that follow the GT/AG rule for intron donor/acceptor
sites (Figure 3B). Genomic organization of mouse CSE gene
(such as distances between exons) resembles that of the rat CSE
rather than that of the human CSE (Figure 3A), reflecting the
evolutionary distances. The 35246 bp sequence of the 129/SvJ
origin is 99.8 % identical with that of the C57BL/6J mouse gene
that was recently filed in the GenBank
®
database. The full-length
CSE cDNA sequence isolated from C57BL/6J kidney (Figure 1)
is fully compatible with this 129/SvJ mouse genomic sequence.
Core regions of the mouse CSE promoter
To identify important regulatory regions for CSE gene expression,
deletion mutants of the 5
-flanking regions, fused to firefly
luciferase reporter gene, were generated (Figure 4). The 3.5 kb
genomic DNA fragment upstream from the transcriptional start
site (− 3498 to + 18), containing canonical TATA and CAAT
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2004 Biochemical Society
Characterization of mouse γ -cystathionase 117
Figure 2 Sequence identity of CSE
Table of percentage ORF nucleotide identity (in the upper right half) or percentage amino acid
identity (in the lower left half) of CSE sequences from mouse, rat, human,
C. elegans
(2H346 and
5Q581) and
Saccharomyces cerevisiae
(GenBank
®
accession numbers AY083352, AB052882,
NM
001902, NM 063048, NM 074652 and D14135 respectively). The number of amino acids
(aa) in ORFs and estimated molecular masses (kDa) are shown.
Figure 3 Structure of the mouse
CSE
gene
(A) Schematic representation of mammalian (mouse, rat and human)
CSE
genes. Organization
of mouse
CSE
gene was revealed by sequencing three overlapping λ phage clones (λCSE-1–3)
that were isolated by screening the mouse genomic library with probes A and B. Sequences of
rat and human
CSE
genes were obtained from the NCBI database. All three mammalian
CSE
genes consist of 12 exons with conserved exon/intron boundaries. A splice variant that skips
exon 5 was reported in human liver. (B) Exon/intron boundaries in the mouse
CSE
gene. Exons
(53–579 bp) are boxed with nucleotide numbers above when the transcriptional start site is set
at 1. Invariant AG/GT sequences (splicing consensuses) are shown in bold.
boxes (Figure 4A), was isolated by PCR from λCSE-1 and used
as a template for further mutant constructions. A much larger
amount (14.8-fold in mol) of the plasmid containing the thymidine
kinase promoter-driven Renilla luciferase gene was co-transfected
with the deletion constructs for the normalization of transfection
efficiency into two kidney cell lines, Cos-7 and HEK-293 cells.
In transfected Cos-7 cells, increasing the length of the 5
-flanking
sequence up to − 3498 bp enhanced the basal promoter activity
with a maximum of 67-fold enhancement (Figure 4B, left panel).
The − 357 bp GC-rich (G, 33.7 %; C, 30.1%) construct (referred
to as pro-9) displayed 52.4 % of the activity obtained with the
− 3498 bp construct (pro-1). In contrast, the − 137 bp sequence
conferred the highest promoter activity (33-fold enhancement)
in HEK-293 cells (Figure 4B, right panel). The core regulatory
regions also existed in the proximal regions (− 357 to +18)
in transfected HEK-293 cells (Figure 4B, right panel). The dif-
ferences between the two cell lines regarding the availability
of transcriptional factors may reflect the altered patterns in the
deletion analyses.
The database search for the transcriptional factor-binding
consensus identified STATx (signal transducers and activators of
transcription x), MZF1 (myeloid zinc finger protein 1), AML-1a
(acute myeloid leukaemia-1a), USF-1 (upstream stimulatory fac-
tor-1), N-Myc, Sp1 (specificity protein 1), HSF2 (heat shock
factor 2) and GATA-1 (GATA-binding factor 1) as important trans-
activating factors in the −357 bp sequence (Figure 4A). Deletion
of MZF-1 (in both cells) or Sp1 consensus (in HEK-293 cells)
significantly decreased the promoter activity (Figure 4B), sug-
gesting the involvement of these factors in the basal transcriptional
activity. In contrast, removal of AML-1a, USF-1, or N-Myc
consensus sequences increased the transcriptional activity in
HEK-293 cells, suggesting a role for these factors as repressive
elements. In both cells, deletion of the CAAX motif or GATA-1
consensus significantly reduced the basal promoter activity.
Various cell activators, including PMA (100 nM), dibutyryl cAMP
(1 mM), dexamethasone (100 nM) or glucagon (100 ng/ml) [34],
were tested to see whether they could regulate the CSE transcrip-
tion in pGL3-CSE-pro-1 vector-transfected Cos-7 cells, but none
of the tested reagents affected the transcriptional activity (results
not shown).
Distribution of the CSE transcript, protein and enzymic
activity in rat and mouse tissues
To determine CSE gene expression in rat and mouse tissues, total
RNA isolated from 12 different adult tissues was analysed by
Northern blot, using rat and mouse CSE cDNA fragments as
probes (Figure 5A). The 2 kb single transcripts were observed
in rat liver and kidney, and at a much lower level, in small
intestine. In mouse, the 2 kb transcripts were found mainly in liver
and kidney, and in lower abundance in adipose tissue, stomach and
small intestine. A faint expression of the 2 kb single transcripts
was observed in mouse brain, heart and lung. This transcript size
is in good agreement with that predicted from the full-length
cDNA (Figure 1) by adding approx. 200 bp poly(A)
+
, suggesting
the existence of the CSE single transcript with no splice variant
in those mouse tissues. Next, expression of the CSE protein was
investigated using anti-CSE N-terminal antibody developed in
this study. This antibody detected the approx. 44 kDa protein
in Cos-7 cells transfected with rat and mouse CSE expression
plasmids (pME18S-rCSE and pME18S-mCSE respectively), but
not in control vector (pME18S)-transfected cells (Figure 5B).
Thus it recognized both recombinant rat and mouse CSE. In both
rodents, major 44 kDa proteins were detected mainly in liver and
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2004 Biochemical Society
118 I. Ishii and others
Figure 4 Promoter activity of the mouse
CSE
gene
(A) Nucleotide sequence of the 5
-flanking region of the mouse
CSE
gene. Numbering is relative to the transcriptional start site. Transcriptional start sites, translational start sites in the
CSE
gene
(1) and the reporter plasmid (2), and the 5
-ends of the deletion constructs, are all shown by broken arrows. Putative transcriptional-factor-binding sites are indicated by arrows below the se-
quences. The consensus TATAA and CAAT motifs are boxed. (B) Defining core regions of the mouse CSE promoter by deletion analyses. Serial deletion constructs were inserted upstream of the
firefly luciferase gene in the pGL3-Enhancer reporter plasmid. Each deletion construct was transiently transfected into Cos-7 cells (left panel) or HEK-293 cells (right panel), together with larger
amounts of pRL-TK plasmid containing the
Renilla
luciferase gene. Both (firefly and
Renilla
) luciferase activities were measured 2 days later, and the promoter activity was expressed as multiples
of induction relative to the activity (the ratio between firefly and
Renilla
luciferase activities) when promoterless pGL3-Enhancer plasmid was transfected. Results shown are means
+
−
S.E.M. for
16 samples from four experiments.
kidney, with much reduced levels in small intestine and stomach
(Figure 5B). Finally, tissue distribution of the CSE activity was
examined with a sensitive method utilizing colorimetric reaction.
No significant activity was detected in control vector-transfected
Cos-7 cells but transient expression of mouse CSE cDNA
conferred the CSE activity (Figure 5C, right panel). In both rats
and mice, the highest levels of CSE activity were detected in liver
followed by kidney although very low but significant levels of
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2004 Biochemical Society
Characterization of mouse γ -cystathionase 119
Figure 5 Tissue distribution of the CSE transcript, protein and enzymic activity in rat and mouse
(A) Distribution of the CSE transcript. Total RNA isolated from each tissue of an adult male rat or mouse (both 8 weeks old) was analysed by Northern blot (10 µg/lane), and approx. 2 kb single
bands were detected. As a loading control, 18 S rRNA was stained with ethidium bromide. (B) Distribution of the CSE protein. Tissue extracts (5 µg/lane) or Cos-7 cell extracts (2 µg/lane) were
fractioned on SDS/PAGE (10 % gel). The 44 kDa proteins specific to CSE were detected by Western-blot analyses. (C) Distribution of CSE enzyme activity in tissue extracts. Boiled liver samples
were loaded as negative controls or reaction backgrounds. Results shown are means
+
−
S.E.M. for triplicate samples from a representative experiment. Cell extracts were prepared from Cos-7 cells
transfected with CSE expression vectors and used as positive controls in both Western-blot analyses (B) and enzyme assay (C). OD
727
=
A
727
.
activity were detected in small intestine and stomach (Figure 5C,
left panel). The activities in rat liver and kidney are approx.
2.5-fold higher than those in mouse liver and kidney respectively.
Heat-denatured (boiled at 100
◦
C for 10 min) liver samples did not
show any CSE activity (Figure 5C, left panel). The 2 kb transcript
was not detected in rat stomach (Figure 5A); however, more
sensitive reverse transcriptase–PCR revealed low expression of
the CSE transcripts in this tissue (results not shown), which might
explain the presence of CSE protein and activity in this tissue
(Figures 5B and 5C).
Developmental expression of CSE in mouse
Expression of CSE protein and activity were examined in mouse
liver and kidney at various developmental stages: E12.5, E15.5,
E18.5, postnatal day 3 (P3), P7, P14, P21, P28, P42, P56 and P84
(Figure 6). Embryonic liver and kidney could be identified and
removed as early as E12.5. Very low levels of the CSE activity
were detected at E12.5 in both liver and kidney; however, the
44 kDa proteins specific to CSE were detected by Western-blot
analyses only at very low levels (Figure 6). The expression of
CSE protein became apparent at E15.5, although there was no
simultaneous increase in CSE activity. After E18.5, the expression
of CSE protein and activity significantly increased in both tissues,
and thereafter, the CSE activity showed a good correlation with the
protein expression level. The CSE expression in liver gradually
increased with age until P21, the weaning age, and thereafter
remained constant at least by P84. In contrast, the CSE expression
in kidney gradually increased with age until reaching its peak at
P21 but then decreased to approx. 50 % of the peak level at P21.
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2004 Biochemical Society
120 I. Ishii and others
Figure 6 Developmental expression of the CSE protein and enzyme activity
in mouse liver (䊐) and kidney (䊏)
Cell extracts prepared from both tissues at various developmental stages (E12.5–P84) were
analysed for CSE protein expression by Western blotting (inset) and CSE enzyme activity
(histogram). Bars shown represent means
+
−
S.E.M. for samples from three to four mice. For
E12.5 liver and kidney, samples from three to four embryos were pooled because of their small
yields. OD
727
=
A
727
.
CSE immunostaining of mouse liver and kidney
Cryosections from E15.5 embryos and adult tissues (liver, kidney
and spleen) were stained with anti-CSE C-terminal antibody
using pre-immune serum as negative control (Figures 7A, 7C,
7F and 7K). Immunostaining of sagittal sections from an E15.5
embryo detected weak but significant levels of CSE expression
in liver (Figure 7B), which contrasts with the negative control
(Figure 7A). The CSE expression was much higher in adult liver
and kidney (Figures 7D, 7E and 7G–7J). The liver mainly consists
of hepatocytes that were highly stained, whereas interlobular
connective tissues surrounding interlobular hepatic artery were
not stained (Figure 7E). In adult kidney, the CSE protein was
expressed in cortex rather than in medulla (Figures 7G and 7H),
especially in cortical renal tubules of the inner cortex (Figures 7I
and 7J). CSE was absent in renal corpuscles that consist of
glomeruli and Bowman’s capsules (Figure 7I) or in adult spleen
(Figure 7L).
DISCUSSION
Mammalian cells are capable of synthesizing cysteine from
methionine by the trans-sulphuration pathway, giving the basis
for the nutritional concept that cysteine is one of the non-essential
amino acids among sulphur-containing amino acids. However,
several investigators have suggested that infants, especially pre-
mature infants, may require the dietary supplement of cysteine
because of lower CSE activity in infants when compared with
that in adults [10,11,35,36]; cysteine deficiency might contribute
to the low survival rates for extremely premature infants of very
low birth weight. This had led to concerns about the use of
cow’s milk protein in infant formulas, which has a lower ratio
of cysteine to methionine when compared with human milk [11].
In addition, CSE activity could be impaired in certain clinical
conditions such as hepatic cirrhosis [37,38] and sepsis [39] or
under surgical stress [40]. Therefore cysteine could be considered
as one of the conditionally essential amino acids [41]. In the
course of generating genetically engineered CSE-deficient mice
to reveal in vivo roles of CSE, this is the first study in mouse of
CSE cDNA and gene cloning, identification of the core promoter
regions, distribution of the transcript, protein and enzymic activity
within the 1 2 major organs, developmental expression in liver and
kidney and the immunohistochemistry on entire embryos, liver
and kidney sections.
The 1.8 kb full-length mouse kidney CSE cDNA contains a
1197 bp ORF encoding a 43.6 kDa protein and includes PLP-
binding consensus sequences (Figure 1). The CSE ORF is encoded
by all the 12 exons of the single CSE gene expanding over 30 kb
(Figures 1 and 3A). It shares high homology (81–92 % identity)
with rat and human CSE ORF nucleotide sequences (Figure 2) and
the exon/intron boundaries are all conserved within those three
mammalian species (Figure 3B), suggesting an identical evolu-
tionary origin. The mammalian CSE genes and proteins share
substantial identity (47–61 %) with those of C. elegans and
yeast (Figure 2), indicating the evolutionary conservation of this
enzyme.
The core promoter existed in the 5
-flanking region proximal
to the transcriptional start site (−357 and − 137 bp in Cos-7 and
HEK-293 cells respectively) that contains several putative trans-
criptional factor-binding sites (Figure 4), although the regulation
of basal activity was cell type specific. MZF-1 and Sp1 seem
to play major roles in the basal transcriptional activity. Regu-
latory mechanisms for CSE transcription remain to be determined.
Interestingly, promoter analyses of the human CBS (cystathionine
β-synthase) gene revealed that the binding of MZF-1, Sp1 and
USF-1 to their consensus sequences within the CBS core promoter
was important for the transcriptional activity [42–44]. CBS is
another PLP-dependent key enzyme of the transsulphuration
pathway, which is located just upstream of CSE and catalyses the
condensation of homocysteine and serine to form cystathionine.
Since CBS is also highly and mainly expressed in mouse liver
and kidney (results not shown), the transcription of these two
transsulphuration genes might be co-regulated by the similar sets
of transcriptional factors in vivo if the CBS promoter sequences
in human and mouse are similar.
In adult rat and mouse tissues, the CSE transcript was detected
mainly in liver followed by kidney as the 2 kb single band (Fig-
ure 5 A). Occurrence of the splice variant with altered functions
(the short form that skips exon 5; Figure 3A) has been reported
in human liver [32,45], but such shorter transcripts were not
observed in mouse tissues, as described previously [46]. Recent
crystallographic analyses of the yeast CSE [47] suggest that the
putative PLP-binding sites (boxed in Figure 1) are essential for
the formation of active CSE tetramer, and the deletion of exon 5
coding sequences (next to the PLP-binding consensus) in human
CSE may produce the loss-of-function variant [45]. We a lso did
not detect the 4.3 kb band that Nishi et al. [12] observed in rat
liver samples.
Two sets of anti-rat CSE polyclonal antibodies were generated
and utilized for the Western-blot analyses (Figures 5B and 6) and
immunohistochemistry (Figure 7). The anti-rat CSE N-terminal
antibody specifically recognized the recombinant 44 kDa rat and
mouse CSE in CSE expression vector-transfected Cos-7 cells but
not in control cells, and detected the major 44 kDa proteins in
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2004 Biochemical Society
Characterization of mouse γ -cystathionase 121
Figure 7 Expression o f the CSE protein in E15.5 embryos and adult mouse tissues: liver, kidney and spleen
Midsagittal sections of E15.5 embryos (A, B), 8-week-old male liver (C–E), kidney (F–J) and spleen (K, L). Sections were stained with anti-CSE N-terminal antibody (B, D, E, G–J, L)orwith
pre-immune serum as negative controls (A, C, F, K). BC, Bowman’s capsule; CV, central vein; GL, glomerulus; IHA, interlobular hepatic artery; ICT, interlobular connective tissue; IPV, interlobular
portal vein; RT, renal tubules. Scale bars, 1 mm (and 0.1 mm in E, H, I and J).
several rat and mouse tissue extracts (Figure 5B). The estimated
molecular masses of rat and mouse CSE are both 43.6 kDa (Fig-
ure 2), and match with the observed band mass in this study
(Figure 5B) and another [48] but not with the one (approx.
40 kDa) detected by other groups [12,49]. The reason for this
is unknown but anti-rat CSE C-terminal antibody also detected
the 44 kDa proteins as major bands in liver and kidney of both
rats and mice (results not shown). A faint level of expression
of the CSE transcript, protein and enzymic activity was observed
in the small intestine and stomach of both rodents. Previous studies
have shown that CSE is expressed in rat vascular smooth-muscle
cells [7] and, thus, the CSE gene may be expressed in tissues
of smooth-muscle cell lineages. Hydrogen sulphide produced by
CSE from cyst(e)ine [1–3] may regulate the relaxation of the
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2004 Biochemical Society
122 I. Ishii and others
smooth-muscle cells [5–7]. The 2 kb transcripts were also detected
in rat brain, mouse brain, heart, lung and adipose tissue, all of
which were not accompanied by expression of CSE protein and
activity (Figure 5). Whether or not they are the CSE transcripts
is unknown, although some previous reports suggested low levels
of CSE expression in rat brain [13,14].
Developmental expression of CSE in liver was previously
evaluated in human [10,45] and rat [14] but not in mouse. In
rat liver, the CSE activity was detected at very low levels around
E16, which was followed by gradual increases until it peaked just
after birth [14]. Thereafter, it remained constant in adulthood [14].
In contrast, the CSE activity in mouse liver increased significantly
after birth to its peak at P21, and thereafter remained constant in
adulthood (Figure 6); the h epatic CSE activity at P21 was 3.4-fold
higher than that at E18.5 in mice (Figure 6), but only 1.4-fold in
rats [14]. In mouse kidney, the CSE activity increased until the
peak at P21 and then decreased to half of the peak level in
adulthood (Figure 6). Considering that the hepatic and kidney CSE
activities in adult rats were both 2.5-fold higher than those in adult
mice (Figure 5C), CSE may play more important roles in mice
than in rats during the early postnatal period. A low level of the
CSE activity was detected at E12.5 in liver and kidney, which was
not accompanied by CSE protein expression (Figure 6), but the
reason for this is unclear. Except for CSE, propargylglycine has
been shown to inhibit some enzymes such as aspartate and alanine
aminotransferases [3]. Whether or not these enzymes could meta-
bolize β-chloro-
L-alanine to pyruvate in our assay system is
unknown. Interestingly, the hepatic rat CSE activity increased
during lactation with the peak at 2 weeks after delivery [49] and
the inhibition of CSE by propargylglycine administration was
followed by the significant decrease in lactation associated with
apoptosis of lactating mammary gland [50,51]. Although it is still
unknown whether hepatic CSE activity is also high in lactating
mice, the adequate supply of cysteine through lactation might
be more important for the newborn of mice than for those of
rats.
Finally, immunohistochemistry revealed enriched CSE expres-
sion in liver as early as E15.5 (Figure 7B), which is consistent
with our Western-blot results (Figure 6). The CSE expression was
much more evident in adult liver (Figures 7D and 7E). The CSE
in adult kidney was localized to the cortex rather than medulla,
especially to the renal tubule in the inner cortex. This is generally
consistent with the previous results obtained for rats by House
et al. [52]; they fractionated rat kidney and detected an enriched
distribution of the CSE activity in the inner cortex or cortical
tubule fractions. Kidney is a major locus for the removal and
subsequent metabolism of plasma homocysteine that is an inter-
mediate amino acid in the transsulphuration as well as an endo-
genous substrate of CBS. An increased level of plasma homo-
cysteine is a potential risk factor for cardiovascular diseases
such as atherosclerosis and thrombosis. The CSE, in combination
with the CBS (that is localized to the outer cortex [52]), may
influence the renal clearance of homocysteine.
In conclusion, we have cloned and characterized mouse CSE
cDNA and gene, generated specific anti-CSE antibodies and
examined its tissue distribution and developmental expression in
mice. This study has demonstrated that (i) CSE is a funda-
mental enzyme conserved through evolution; (ii) CSE core pro-
moter is located within the 5
-flanking region proximal (− 357 bp)
to the transcriptional start site; (iii) CSE is expressed mainly in
liver and kidney, and, at much lower levels, in small intestine
and stomach; (iv) CSE expression in liver and kidney is develop-
mentally regulated; and (v) CSE is highly expressed in adult hepat-
ocytes and kidney cortical tubuli. Such information is essential in
understanding physiological roles of CSE/transsulphuration, and
also provides useful information to generate and analyse CSE-
deficient mice as an animal model of cystathioninaemia.
We thank K. Saito, E. Yoshida, M. Kawabata, and other animal care staff at the Division of
Laboratory Animal Resources, NCNP. This work was supported in part by a grant-in-aid for
Young Scientists (No. 15790066) from the Ministry of Education, Culture, Sports, Science
and Technology (Japan), research grants from Uehara Memorial Foundation, Yamanouchi
Foundation for Research on Metabolic Disorders, ONO Medical Research Foundation,
Naito Foundation, Tokyo Biochemical Research Foundation (to I. I.) and National Institute
of Neuroscience (NCNP, Japan to H. K.).
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Received 13 February 2004/22 March 2004; accepted 23 March 2004
Published as BJ Immediate Publication 23 March 2004, DOI 10.1042/BJ20040243
c
2004 Biochemical Society
