|
|
|
|||||||
|
|||||||||
(Received for publication, October 31, 1995; and in revised form, November 20, 1995) From the
Leukotriene B
Leukotriene B
Total RNA was
prepared from the porcine kidney by a cesium-trifluoroacetic acid
method(24) . Poly(A) The conditions of
polymerase chain reaction were as follows: denaturation at 94 °C
for 1 min, annealing at 50 °C for 2 min, and elongation at 72
°C for 3 min. After 5 cycles, the annealing temperature was changed
to 55 °C. After 30 cycles of polymerase chain reaction, the
products were ethanol-precipitated and separated on an 1% agarose gel,
and 4 different bands were recovered from the gel using a QIAGEN gel
purification kit. Each band was ligated into a T-vector (Promega) by a
T An oligo(dT)-primed
Each mutagenetic primer (10
ng) and a selection primer (10 ng, Aat II/EcoRV,
5`-GTGCCACCTGATATCTAAGAAACC-3`) were annealed simultaneously to 10 ng
of pGEX-LTB12DH, and the first strand was synthesized with 4 units of
T The V
Figure 1:
The cDNA and deduced amino acid
sequences of porcine LTB
Figure 2:
Amino acid alignment of LTB
In addition, LTB
Figure 3:
Northern blotting of human tissues. Human
multiple tissue Northern blots (2 µg of poly(A) RNA/each lane,
Clonetech) were hybridized with a
[
All the
mutant proteins were detected as 62-kDa bands by Coomassie Brilliant
Blue G-staining,
Figure 4:
Expression of the wild type and mutant
enzymes as GST fusion proteins. A, purified recombinant
proteins (1 µg/lane) were separated on a SDS-PAGE gel (7.5%) and
stained with Coomassie Brilliant Blue G. The molecular weight marker (M.M.) contained phosphorylase b (94,000), albumin
(67,000), ovalbumin (43,000), and carbonic anhydrase (30,000). B, the same gel was blotted with an anti-LTB
Figure 5:
LTB
LTB The metabolism of LTB There is another
group of LTB In the present study, we cloned a cDNA for the porcine
LTB The tissue distribution of mRNA of
human enzyme corresponds well to the distribution of the enzyme
activities studied in porcine tissues (22) , with the highest
expression in the kidney and liver, followed by colon and small
intestine (Fig. 3). It is important to note that the mRNA is not
expressed in the human leukocytes where the LTB
Figure 6:
Amino
acid alignment of NAD
To determine which amino
acids are required for the enzyme activity, a site-directed mutagenesis
was carried out. Ala Fig. 5summarizes six experiments from three different
purifications. M3 (G152V), M4 (G155V), M6 (G166V), and M7 (A149V,
A150V, G152V, G155V, and G159V) mutants lost most of the enzyme
activity. M2 (A150V) mutant exhibited a full enzyme activity, whereas
about half and 90% of the activities were lost in M1 (A149V) and M8
(A149E) mutants, respectively. The K There is a proline-rich
motif that is conserved among three species in the C-terminal half of
LTB In
conclusion, LTB
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s) D49386 [GenBank]and D49387[GenBank].
Volume 271,
Number 5,
Issue of February 2, 1996 pp. 2844-2850
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
12-Hydroxydehydrogenase (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
12-hydroxydehydrogenase catalyzes the
conversion of leukotriene B into its biologically less
active metabolite, 12-oxo-leukotriene B. This is an initial
and key step of metabolic inactivation of leukotriene B in
various tissues other than leukocytes. Here we report the cDNA cloning
for porcine and human enzymes from kidney cDNA libraries. A full-length
cDNA of the porcine enzyme contains an open reading frame consisting of
987 base pairs, corresponding to 329 amino acids. The human enzyme
showed a 97.1% homology with the porcine enzyme. Northern blotting of
human tissues revealed its high expression in the kidney, liver, and
intestine but not in leukocytes. The porcine enzyme was expressed as a
glutathione S-transferase fusion protein in Escherichia
coli, which exhibited similar characteristics with the native
enzyme. Because the enzymes have a homology, in part, with
NAD(P)-dependent alcohol dehydrogenases, a
site-directed mutagenesis study was carried out. We found that three
glycines at 152, 155, and 166 have crucial roles in the enzyme
activity, possibly by producing an NADP
binding
pocket.
(LTB) (
)is a
potent chemotactic and proinflammatory factor produced in various
tissues(1, 2, 3, 4) . Arachidonic
acid, released from the cell membrane by various stimuli, is converted
to 5-hydroperoxyeicosatetraenoic acid and LTA by
5-lipoxygenase(5, 6, 7, 8) .
LTB is biosynthesized from LTA by the action of
LTA hydrolase(9, 10, 11, 12, 13) .
In human polymorphonuclear leukocytes, LTB is converted to
20-hydroxy-LTB by a cytochrome P-450 LTB
and further to
20-carboxy-LTB
(14, 15, 16, 17) .
The cDNA of cytochrome P-450 LTB was cloned, and the
mRNA is detected only in human leukocytes(18, 19) .
LTB
is also produced in tissues other than
leukocytes(20, 21) . We reported an alternative
pathway for LTB in various porcine tissues and purified a
cytosolic LTB 12-hydroxydehydrogenase from the porcine
kidney(22) . This enzyme converts LTB to
12-oxo-LTB in the presence of NADP.
12-Oxo-LTB
is at least 100 times less potent than LTB in increasing intracellular calcium concentrations in human
leukocytes(22) . However, the molecular structure of the enzyme
as well as its tissue distribution have not been known. Here we report
the primary structures of porcine and human LTB 12-hydroxydehydrogenases and the putative
NADP-binding domain. We clearly showed that the enzyme
is expressed in the kidney, liver, and various tissues but not in
leukocytes. Thus, this enzyme represents one of the major pathways of
the metabolic inactivation of LTB
in tissues other than
leukocytes.
Materials
LTB was kindly donated by
Ono Pharmaceutical Company (Osaka). EDTA-Na
,
dithiothreitol, pepstatin-A, and phenylmethylsulfonyl fluoride were
purchased from Wako Pure Chemicals (Osaka). NADP was
obtained from Sigma.
N-terminal and Internal Amino Acid Sequences of LTB
The porcine LTB 12-Hydroxydehydrogenase 12-hydroxydehydrogenase was purified as described
previously(22) . The purified enzyme (100 µg) was digested
with 5 µg of trypsin in 100 mM Tris-HCl, pH 8.5, at 37
°C for 8 h. Digested fragments were purified by reversed phase HPLC
using a Pharmacia Smart System(TM) equipped with a µRPC
C/C
column (2.1 
100 mm). The digested
enzyme was injected onto a µRPC C/C column
previously equilibrated with 0.1% trifluoroacetic acid in water and
eluted by a linear gradient to 80% acetonitrile with 0.1%
trifluoroacetic acid for 3.8 ml at a flow rate of 100 µl/min. The
eluted peptide fragments were monitored at 215 nm, and 31 fractions
were collected. LTB 12-hydroxydehydrogenase (5 µg) and
six of 31 peptide fragments (Fractions 8, 19, 24, 25, 27, and 37) were
loaded on polyvinlidene difluoride membranes with Prospin(TM)
(Perkin Elmer) and sequenced by Edman degradation using an automated
protein sequencer PPSQ-10 (Shimadzu, Kyoto). SWISS PROT protein data
base was used to search for homologous proteins using a BLAST
program(23) .cDNA Cloning of Porcine LTB
Degenerative reverse
transcriptase-polymerase chain reaction using mixed oligonucleotide
primers was performed to obtain a partial cDNA fragment for screening
of the library. Mixed oligonucleotide primers were designed according
to the amino acid sequences of N-terminal and Fraction 19. Each primer
was synthesized by Sawaday Technology (Tokyo), and the sequences of
sense and antisense primers were
5`-GTGCGCGCCAAGTCCTGGACCCTGAA(A/C)AA(A/C)CA(T/C)TT(T/C) GT-3`
(corresponding to N-terminal, 38 mers) and
5`-GCGGGCCACCTGCTC(A/G/C/T)CCCATCATCAT(A/G)TC-3` (corresponding to the
peptide of Fraction 19, 30 mers), respectively. 12-Hydroxydehydrogenase RNA was purified using
Oligotex(TM)-dT30 Super (Roche Japan, Tokyo) according to the
manufacturer's manual. An oligo(dT)
(Pharmacia)-primed cDNA was synthesized from 1 µg of
poly(A)
RNA by an Maloney murine leukemia virus
reverse transcriptase (Life Technologies Inc.).
DNA ligase, and the resulting constructs were transformed
into Escherichia coli strain JM 109 (Competent
high(TM), TOYOBO, Tokyo). Plasmids were purified by an alkaline
lysis method and sequenced with an ABI automated DNA sequencer 373A
(Perkin Elmer). A band of 220 base pairs encoded the 5` end of the cDNA
and was used as a probe to screen the library. Zap-II(TM) (Stratagene) porcine kidney cDNA library was
constructed from 4 µg of poly(A)
RNA with
Superscript II(TM) Choice System (Life Technologies Inc.) according
to the manufacturer's manual. The library yielded 1.6
10
independent clones. Full-length cDNA clones were
obtained by a plaque hybridization method. 1.0 10 clones were transferred to 10 sheets of Hybond N filters, and then the filters were alkaline-denatured and fixed
by baking at 80 °C for 2 h. The insert cDNA was digested out from
the vector, randomly labeled by [
P]dCTP using a
Multiprime Labeling System (Amersham Corp.), and used as a probe for
hybridization. After hybridization in Rapidhybri solution (Amersham
Corp.) at 65 °C for 8 h, each filter was washed extensively three
times in 0.1
SSC, 0.1% SDS at 65 °C for 20 min. Three
rounds of screening gave three positive clones named pBDH 9, 14, and
15. Each clone was excised in vivo into a pBluescript II
SK(-) phagemid by ExAssist helper phage (Stratagene), mapped
using various restriction enzymes, and sequenced as described
previously. All the clones showed the same restriction patterns, and
sequencing confirmed that these three clones code for full-length cDNAs
of LTB 12-hydroxydehydrogenase. Ten deletion mutants were
prepared by exonuclease III from pBDH 15, and both strands were
sequenced. In addition, six internal sequencing primers were
synthesized, and the sequences were confirmed.cDNA Cloning of Human LTB
Human cDNAs of LTB 12-Hydroxydehydrogenase 12-hydroxydehydrogenase were isolated from a human kidney gt
11 cDNA library (Clonetech) by a cross-hybridization method with a
porcine full-length cDNA (pBDH 15) as a probe. 6
10
clones were transferred to Biodyne nylon membranes (Pall) and
hybridized at 55 °C for 12 h with a
[P]dCTP-labeled full-length porcine cDNA (pDBH
15). Each filter was washed three times in 2
SSC, 0.1% SDS at
55 °C for 10 min. Three rounds of screening gave two phage clones
named hBDH4 and 8, which were then purified, digested by EcoRI, subcloned into a pBluescript II SK(+) vector, and
sequenced. Homology search was performed against the GenBank, EMBL, and
SCOP (structural classification of proteins) data bases using a BLAST
program(23) . The three-dimensional data of crystallized
proteins were obtained from the SCOP data base and analyzed using a
RASMOL program(25) . An open reading frame and the deduced
amino acid sequence were determined by a Genetyx Mac(TM) 6.0.2.
software (Software Development, Tokyo, Japan).Northern Blot Analysis
Human multiple tissue
Northern blots (2 µg of poly(A) RNA/each lane,
Clonetech) were hybridized with a
[
P]dCTP-labeled full-length human LTB
12-hydroxydehydrogenase cDNA (hBDH 4) or a human 
-actin cDNA
for 3 h in Rapidhybri solution (Amersham Corp.). The membranes were
washed for 15 min once in 3 SSC, 0.1% SDS and for 20 min twice
in 0.1 SSC, 0.1% SDS at 65 °C. Autoradiogram was subjected
to a Bas-2000 system analyzer (Fuji Film, Tokyo, Japan).Expression of LTB
The porcine cDNA insert was digested out
from pBDH 15 by EcoRI and subcloned into a Pharmacia pGEX-1
expression vector (pGEX-LTB12DH). An E. coli strain
JM-109(TM) (TOYOBO) was transformed by heat shock, and then the
recombinant protein was induced with 0.1 mM isopropyl-1-thio- 12-Hydroxydehydrogenase as
a GST Fusion Protein-D-galactoside. The procedure was
basically as described in the manufacturer's manual, except that
the protein was induced at 20 °C overnight with 0.1 mM isopropyl-1-thio--D-galactoside. E. coli was collected, resuspended in PBS(-) containing 2 mM EDTA-Na, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 0.2 µg/ml pepstatin-A, 2
µg/ml leupeptine, and disrupted by sonication. The sonicates were
centrifuged for 10 min at 10,000 g, and 200 µl
(for 1 liter of E. coli culture) of GSH-sepharose (Pharmacia)
was added to the supernatant. After washing with PBS(-), the
protein was eluted in 50 mM Tris-HCl, pH 8.0, containing 10
mM GSH, and the purity was checked by SDS-PAGE. The 7.5%
polyacrylamide gel was stained with Coomassie Brilliant Blue G, and the
quantity of the recombinant protein was measured by scanning with
bovine serum albumin as a standard. The enzyme activity of the
recombinant protein was measured as described previously(22) .Peptide Antibody against LTB
A peptide (ESLEETLKKASPEG,
corresponding to the amino acid residues 197-210 of the porcine
enzyme) was synthesized as a multiple antigen peptide (Fmoc
MAP-peptide, 8-Branch, Applied Biosystems). An aliquot of 0.5 mg of the
peptide was emulsified with an equal volume of Freund's complete
adjuvant and injected into 3-9-month-old female New Zealand white
rabbits. After three immunizations at one-month intervals, blood
samples were collected and the serum was obtained by centrifugation.
The anti-serum was purified by affinity chromatography. The recombinant
LTB 12-Hydroxydehydrogenase 12-hydroxydehydrogenase (1 mg) was coupled to 0.5 g of
Epoxy-activated Sepharose (Pharmacia) in the coupling buffer (50 mM sodium-bicarbonate buffer, pH 9.0) at 25 °C for 10 h. The
Sepharose was loaded on a Poly-Prep Chromatography Column (Bio-Rad).
After washing with 5 ml of the coupling buffer, 5 ml of the blocking
buffer (50 mM Tris-HCl, 0.1 M ethanolamine, pH 8.0)
was added to block the unbound resin. After washing with 10 ml of
water, 10 ml of the elution buffer (0.1 M glycine-HCl, pH
2.5), and 10 ml of the wash buffer (20 mM Tris-HCl, 1 M NaCl, 1% Triton X-100, pH 7.5), the column was equilibrated with
PBS(-). 2 ml of anti-serum was applied on the column and allowed
to stand at 25 °C for 1 h. After washing the column with 10 ml of
PBS(-), 30 ml of the wash buffer, 30 ml of PBS (-), 30 ml
of 0.15 M NaCl, the antibody was eluted in 2 ml of the elution
buffer. The eluate was immediately neutralized with 100 µl of 1 M Tris-HCl, pH 8.0. The concentration of the purified antibody
was 236 µg/ml. The affinity-purified antibody is termed 
2
antibody hereafter.Site-directed Mutagenesis of the Putative
NADP
A mutagenesis study was
performed by an oligonucleotide-derived mutagenesis method (26) using a Transformer(TM) site-directed mutagenesis kit
(Clonetech). The mutagenetic primers were designed as follows: M1
(A149V), 5`-GATGGTTAATGTCGCAGCAGGGG-3`; M2 (A150V),
5`-GTTAATGCGGTAGCAGGGGCC-3`; M3 (G152V), 5`-GCGGCAGCAGTCGCCGTGGGCTC-3`;
M4 (G155V) 5`-GGGGCCGTGGTCTCTGTCGTG-3`; M5 (G159V),
5`-CTCTGTCGTGGTCCAGATCGCGAAG-3`; M6 (G166V),
5`-CGAAGCTCAAGGTCTGCAAAGTTG-3`; M7 (A149V, A150V, G152V, G155V, G159V),
5`-GATGGTTAATGTCGTAGCAGTCGCCGTGGTCTCTGTCGTGGTCCAGATCGCGAAG-3`; and M8
(A149E), 5`-GATGGTTAATGCGGCAGCAGGGG-3`.-binding Domain
DNA polymerase and 6 units of T DNA ligase in
30 µl at 37 °C for 2 h. AatII (20 units) was added to
selectively linearize the parental DNA. 40 µl of the
electrocompetent BMH71-18 mutS strain (Clonetech, CA)
was transformed with 2 µl of 5 diluted reaction mixture
using a Gene Pulser Unit (Bio-Rad). The condition of electroporation
was 1.8 kV, 25 microfarad, 100 . After shaking the culture in 10
ml of TB medium overnight, the plasmids were recovered by an alkaline
lysis method, and 100 ng of plasmids were digested with AatII
(10 units) again. JM 109 cells were transformed with 10 ng of
digested plasmids by heat shock, and colonies were isolated. Each
mutated plasmid was sequenced entirely to check for unexpected
mutations. The mutant proteins were purified as GST fusion proteins as
described previously. Purified proteins (1 µg/lane) were separated
on a 7.5% SDS-PAGE gel and transferred to a Hybond ECL membrane
(Amersham Corp.). It was blotted with
2 antibody (200
dilution) or rabbit anti-GST antibody (Pharmacia) as the first antibody
and visualized using an Amersham ECL system.
and K
values against LTB and
NADP were determined as described previously (22) six times in three independent experiments.
cDNA Cloning of Porcine and Human LTB
Screening of 1.0 12-Hydroxydehydrogenase 10 porcine clones with the probe coding for the 5` end gave three
independent positive clones, pBDH 9, 14, and 15. Three clones were
excised in vivo into pBluescript II SK(-) and mapped
using several restriction enzymes. All the inserts gave an identical
restriction map, and DNA sequencing confirmed that these three clones
coded for full-length cDNAs of LTB 12-hydroxydehydrogenase.
pBDH 15 was further sequenced by deletion with exonuclease III and 6
internal sequencing primers. Screening of a human kidney cDNA library
(6 10 clones) with pBDH 15 gave two independent
clones, hBDH 4 and 8, which were identical. The primary structures of
porcine and human LTB 12-hydroxydehydrogenases are shown in Fig. 1and 2. The deduced amino acid sequences of the porcine
enzyme contained all the amino acid sequences of seven peptide
fragments obtained from the native porcine kidney enzyme (Fig. 1). The cDNA of pBDH 15 contained a polyadenylation signal
after the stop codon (Fig. 1), showing that it codes for a
full-length LTB 12-hydroxydehydrogenase. pBDH 15 contains
an open reading frame of 987 base pairs and coded for 329 amino acids.
The calculated M
of the porcine enzyme is 35,761,
a value similar to that of the native enzyme(22) . Because hBDH
4 and 8 lack the stop codon, the human enzyme seems to have additional
amino acids in the C-terminal. Several trials to acquire the
full-length clones using rapid amplification of cDNA ends were
unsuccessful. The identity between the porcine and human enzymes was
83.5% at the amino acid level and 84.7% at the nucleotide level. Amino
acid homology was 97.1%. Both porcine and human enzymes showed a high
homology (94.5 and 96.1%, respectively) with a previously reported
rabbit protein, AdRab-F, which is expressed only in adult rabbit small
intestine but not in the baby(27) . Function of the AdRab-F
protein has not been documented(27) , but it seems to be a
rabbit homologue of LTB 12-hydroxydehydrogenase judging
from the high homology. These three proteins contain a proline-rich
motif (250-257 residues) in the C-terminal half ( Fig. 1and Fig. 2).
12-hydroxydehydrogenase. The underlined letters indicate peptide sequences from the
purified porcine kidney enzyme. F followed by a number indicates the
fraction number of peptide fragments digested by Lys-C and separated by
HPLC (see ``Experimental Procedures''). The underlined and bold letters (residues 149-166) show the
putative NADP-binding domain. The bold lowercase
letters show the polyadenylation
signal.
12-hydroxydehydrogenases and AdRab-F protein. The human sequence
is considered to be partial. The hypothetical AdRab-F protein (27) was also aligned as rabbit. The asterisk indicates amino acids that are identical among three species.
indicates amino acids that are identical in two
species.
12-hydroxydehydrogenases have a weak homology with
NAD/NADP
-dependent short chain
alcohol dehydrogenases (28, 29) and a
-crystallin(30) , identity being 30-35%. Especially,
a fragment from 149 to 166 of the porcine LTB
12-hydroxydehydrogenase has a relatively high homology (50%)
with these dehydrogenases. Because this domain is considered to be a
NAD
/NADP
-binding domain in these
dehydrogenases(28, 29) , a mutagenesis study was
carried out to determine the putative NADP
-binding
domain of LTB
12-hydroxydehydrogenase (see below).Northern Blot Analysis
Fig. 3shows the
tissue distribution of mRNA of LTB 12-hydroxydehydrogenase
in human tissues. The mRNA is expressed most abundantly in the kidney
and liver, followed by small intestine and colon. It was absent in
human leukocytes. The distribution of mRNA matches the tissue
distribution of the enzyme activities studied in various porcine
tissues(22) .
P]dCTP-labeled hBDH4 or a human
-actin
cDNA. kb, kilobases.
Expression of LTB
The recombinant porcine LTB 12-Hydroxydehydrogenase as
a GST Fusion Protein 12-hydroxydehydrogenase was overexpressed as a GST fusion protein
in the E. coli system. When the transformed E. coli was cultured at 37 °C, most of the recombinant protein was
precipitated by centrifugation at 10,000 g, possibly
existing in bacterial inclusion bodies. By decreasing the culture
temperature to 20 °C, good yields of a soluble protein were
obtained. The recombinant protein was purified by affinity column
chromatography using a GSH-sepharose column. A typical yield was 3 mg
of protein from 1 liter of bacterial culture, and the purity was around
70% judging from SDS-PAGE. The purified GST fusion protein exhibited
characteristics similar to the native enzyme, with a V value of forming 6 nmol of 12-oxo-LTB/min/mg fusion
protein. The K values of the recombinant enzyme
were 20 µM against LTB and 10 µM against NADP. After digesting out GST from the
fusion protein with thrombin, the specific activity of the enzyme
remained unchanged. GST only had no apparent enzyme activity (date not
shown). These results indicate that the cDNA of pBDH 15 codes for
LTB
12-hydroxydehydrogenase.Site-directed Mutagenesis of the Putative
NADP
A computer-assisted
homology analysis revealed that the fragment 149-166 of LTB-binding Domain
12-hydroxydehydrogenase was homologous to the
NAD/NADP
-binding domain of other
short chain alcohol dehydrogenases(28, 29) . To
determine whether this domain is essential for binding to
NADP
and for the enzyme activity, a site-directed
mutagenesis study was done. Ala
, Ala
,
Gly
, Gly
, Gly
, or Gly
was converted to Val or Glu, and the mutant proteins were
expressed in E. coli as GST fusion proteins. The recombinant
proteins were purified with a GSH-sepharose column and quantified on
Coomassie Brilliant Blue G-stained SDS-PAGE gels, and the enzyme
activities were measured. The K
and V values were determined by changing the
concentrations of LTB and NADP.
2 antibody raised against the porcine LTB 12-hydroxydehydrogenase (Fig. 4), and anti-GST antibody
(data not shown). Among them, M6 and M7 were unstable, showing smaller
bands (Fig. 4). All the mutants had decreased V values, and the remaining activities varied among the mutants (Fig. 5). M2 (A150V) had an almost full (91% of wild type)
enzyme activity, M1 (A149V) showed 56% activity, and M5 (G159V) 40%
activity. M3 (G152V, 0%), M4 (G155V, 1%), M6 (G166V, 1%), and M7
(A149V, A150V, G152V, G155V, and G159V, 2%) lost most of the enzyme
activity. M8 (A149E) showed a 9% activity against the wild type. There
were no significant differences between the wild type and the mutant
enzymes in terms of K values against LTB and NADP (data not shown).
12-hydroxydehydrogenases antibody (2) and visualized using
an ECL system (Amersham Corp.). WT, the wild type enzyme; M1, A149V; M2, A150V; M3, G152V; M4, G155V; M5, G159V; M6, G166V; M7, A149V, A150V, G152V, G155V, and G159V; and M8,
A149E.
12-hydroxydehydrogenase
activities in the mutants. Relative activities are shown in percentages
with the wild type as 100%. The mean of six different experiments (closed columns) ± S.D. is shown. WT, the wild type
enzyme; M1, A149V; M2, A150V; M3, G152V; M4, G155V; M5, G159V; M6, G166V; M7, A149V, A150V, G152V, G155V, and G159V; and M8,
A149E.
is a potent lipid mediator that activates
leukocytes to migrate from vessels, to generate superoxide anions, and
to release lysosomal enzymes(3) . This potent mediator is
produced in various tissues like the kidney (21, 31, 32) or skin (33, 34, 35) under pathophysiological
conditions. has been intensively
studied in leukocytes. Human polymorphonuclear leukocytes convert
LTB into 20-hydroxy-LTB by a microsomal
NADPH-dependent cytochrome P-450
LTB(14, 36, 37, 38, 39, 40, 41) .
20-Hydroxy-LTB
is further metabolized to
20-carboxy-LTB(42) . 20-Hydroxy- and
20-carboxy-LTB was 10-30 times less active in
neutrophil chemotaxis(43, 44) . metabolites. Porcine leukocytes converts
LTB to 10,11-dihydro-LTB,
10,11-dihydro-12-oxo-LTB, and
10,11-dihydro-12-epi-LTB(37, 45, 46, 47) .
Wainwright and Powell (48) extensively studied the mechanism
and found that LTB is first converted to 12-oxo-LTB by a microsomal NAD-dependent
12-hydroxydehydrogenase and then to 10,11-dihydro-12-oxo-LTB
by a cytosolic NADH-dependent 10,11-reductase in porcine
polymorphonuclear leukocytes(48) . We purified
LTB-specific 12-hydroxydehydrogenase from porcine kidney (22) and found that it was different in nature from the porcine
polymorphonuclear leukocyte enzyme (48) . The purified kidney
enzyme also converts LTB to 12-oxo-LTB, but it
is a cytosolic enzyme and utilizes NADP as a
cofactor(22) . A similar conversion of LTB
was
reported in the human lung(49) , kidney(50) ,
keratinocytes(51) , and the guinea pig kidney and liver. ()The enzyme purified from the porcine kidney is a monomeric
protein with an M of 35,000 and an isoelectric
point over 9.5. It specifically recognizes the
12(R)-hydroxy-moiety of LTB, thus it was named
LTB 12-hydroxydehydrogenase(22) . The product,
12-oxo-LTB, was at least 100 times less potent in
increasing the intracellular calcium concentration in human leukocytes (22) . Recently, the method of the chemical synthesis of
12-oxo-LTB was established(52) , thus enabling us
to determine the biological activity and precise metabolism of this
compound. 12-hydroxydehydrogenase by screening a kidney cDNA
library using a probe obtained from its partial amino acid sequences of
the purified enzyme (Fig. 1). Porcine LTB 12-hydroxydehydrogenase cDNA contained an open reading frame of
987 base pairs and coded for 329 amino acids. The deduced amino acid
sequence contained all the sequences from Lys-C-digested peptide
fragments (Fig. 1). The calculated M of the
porcine enzyme is 35,761, which agrees well with that of the native
enzyme. In addition, we obtained a cDNA of the human enzyme by
cross-hybridization with the porcine cDNA. The primary structures of
the porcine and human enzymes are similar, with an amino acid homology
of 97.1%. Both enzymes were highly homologous (94.5 and 96.1%) with a
rabbit AdRab-F hypothetical protein (Fig. 2), the mRNA of which
was expressed only in the adult rabbit and not in the
baby(27) . The function of AdRab-F protein has not been
reported, but it seems to be a rabbit homologue of LTB 12-hydroxydehydrogenase. Further studies are required to
determine the developmental change of the expression of LTB 12-hydroxydehydrogenase.-oxidation pathway is
present.
12-hydroxydehydrogenases were homologous
with other NAD/NADP
-dependent short
chain alcohol dehydrogenases. Although the total homology was 35% or
less, there was a relatively highly homologous domain (Fig. 6).
Among these homologous proteins, three enzymes were crystallized, and
the structures were well
studied(53, 54, 55) . Crystal structure
analyses revealed that this domain forms a compact
-sheet--helix--sheet structure and was determined to
form a NAD/NADP
-binding domain (Fig. 6). An acidic residue adjacent to this domain is supposed
to bind to the 2` and 3` hydroxyl groups of the adenine ribose of
NAD
/NADP
(29) . In addition,
mutagenesis studies of the other dehydrogenases indicate that the
GXGXX(G/A)XXXGXXXXXXG consensus
sequence is important to maintain a close contact between the coenzyme
and the enzyme by forming an
-helix
structure(29, 56) . By changing two Gly in this domain
of NAD-dependent pyruvate dehydrogenase to Ala, the
enzyme activity was decreased(57) . This domain is highly
conserved in the porcine and human LTB
12-hydroxydehydrogenases and AdRab-F hypothetical protein, and
the consensus sequence is AAXGXXGXXXGXXXXXXG
( Fig. 2and Fig. 6).
/NADP
-binding
domains of LTB
12-hydroxydehydrogenases and other
homologous proteins. The amino acid sequences of the porcine and human
LTB 12-hydroxydehydrogenases (LTB12DH) are aligned with
rabbit AdRab-F protein (27) and other homologous proteins. CRZ (MOUSE), mouse -crystallin(30) ; ADH
(YEAST), Saccharomyces cerevisiae alcohol dehydrogenase
1(61) ; FAS (RAT), rat fatty acid
synthase(62) ; PKS (S. hygro.), Streptomyces
hygroscopicus polyketide synthase(63) ; QOR (E.
coli), E. coli quinone oxidoreductase (54) (SCOP
entry 1qor); HDC (S. hydro.), Streptomyces hydrogenans 3-
, 20--hydroxysteroid dehydrogenase (53) (SCOP
entry 2hsd); and ADH (HORSE), horse alcohol dehydrogenase (55) (SCOP entry 2ohs). The bold letters show amino
acids identical with the porcine LTB 12-hydroxydehydrogenase. The underlined letters show
amino acids that form -helix structures, and the letters in the shaded boxes are -helix structures derived from the
crystal structure analyses of three proteins (QOR, HDC, and ADH). Three Gly in the open boxes (152, 155, and 166) play important roles in porcine LTB 12-hydroxydehydrogenases activity and are well conserved among
these proteins shown in this figure.
, Ala
,
Gly
, Gly
, Gly
, and
Gly
were changed into Val, which has a longer side chain
than Ala and Gly, or to Glu, which is negatively charged, and the
enzyme activities were measured. M6 (G166V) and M7 (A149V, A150V,
G152V, G155V, and G159V) mutants readily cleaved into shorter peptides,
as shown in Fig. 4. The K
values against
LTB and NADP of the recombinant wild type
enzyme were 20 µM and 10 µM, respectively.
Because these values of the native enzyme purified from the porcine
kidney were 10 µM and 1 µM(22) ,
respectively, the recombinant protein may contain a slight change in
the three-dimensional structure. To exclude the influence of the enzyme
instability and degradation, the quantities of mutant proteins were
standarized on Coomassie Brilliant Blue G-stained SDS-PAGE gels (Fig. 4). The enzyme activities of mutant proteins were measured
as the relative activities toward the wild type enzyme.
values for
NADP of M1, M5, and M8 mutants were not significantly
different from that of the recombinant wild type enzyme, although the V
values were all decreased. Similar results
were obtained from the mutagenesis study in the short-chain alcohol
dehydrogenase(58) . These results suggest that the longer side
chain of Val may inhibit the NADP binding in G152V,
G155V, and G166V mutants. Because the enzyme activity remains partially
in A149V and G159V mutants, the conformational change of the binding
pocket might be moderate in these mutants. In contrast, by changing
Ala
to Glu, most of the enzyme activity was lost,
possibly due to the negative charge of Glu. These results indicate that
Gly
, Gly
, and Gly
of
LTB
12-hydroxydehydrogenase are essential for the enzyme
activity, probably by forming an NADP binding pocket (Fig. 6). As seen in Fig. 6, these three Gly are well
conserved among LTB
12-hydroxydehydrogenases and other
homologous proteins, suggesting that these Gly are important.
Ala and Gly
seem to play some roles in the
enzyme activity but are not essential. Ala
seems to have
only a little role in the enzyme activity.
12-hydroxydehydrogenase (Fig. 2, 250-257
residues). The proline-rich motif was reported to play crucial roles by
binding src homology 3 (SH3) domains in the signal
transduction system of tyrosin-kinase type receptors(59) .
Recently, the binding of proline-rich domains to SH3 domain was
reported to be involved in the translocation and activation of
5-lipoxygenase(60) , which catalyzes the initial step of
biosynthesis of leukotrienes. The role of the proline-rich domain of
LTB 12-hydroxydehydrogenase remains to be clarified. 12-hydroxydehydrogenase cDNAs were isolated
from the porcine and human kidney, and their primary structures were
identified. Northern blotting revealed that the mRNA was expressed in
the kidney, liver, small intestine, and colon but not in leukocytes. By
a site-directed mutagenesis study, we found that three Gly residues at
152, 155, and 166 play important roles in the enzyme activity. The
acquisition of the cDNA and the antibody paves the way for the further
analysis of the cellular localization and the biological significance
of the enzyme under various physiological and pathological conditions.
),
5(S),12(R)-dihydroxy-6,14cis-8,10-trans-eicosatetraenoic
acid; LTA,
5(S)-trans-5,6-oxide-7,9trans-11,14-cis-eicosatetraenoic
acid; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance
liquid chromatography; PBS(-), phosphate-buffered saline without
calcium; GST, glutathione S-transferase.)
We are grateful to Dr. H. Toh (Kyushu Industrial
College) and Dr. M. Miyano (Japan Tobacco Inc.) for discussion and to
M. Ohara for pertinent comments.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Porte, E. Valencia, E. A. Yakovtseva, E. Borras, N. Shafqat, J. E. Debreczeny, A. C. W. Pike, U. Oppermann, J. Farres, I. Fita, et al. Three-dimensional Structure and Enzymatic Function of Proapoptotic Human p53-inducible Quinone Oxidoreductase PIG3 J. Biol. Chem., June 19, 2009; 284(25): 17194 - 17205. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. Buczynski, D. S. Dumlao, and E. A. Dennis Thematic Review Series: Proteomics. An integrated omics analysis of eicosanoid biology J. Lipid Res., June 1, 2009; 50(6): 1015 - 1038. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W.-L. Chou, L.-M. Chuang, C.-C. Chou, A. H.-J. Wang, J. A. Lawson, G. A. FitzGerald, and Z.-F. Chang Identification of a Novel Prostaglandin Reductase Reveals the Involvement of Prostaglandin E2 Catabolism in Regulation of Peroxisome Proliferator-activated Receptor {gamma} Activation J. Biol. Chem., June 22, 2007; 282(25): 18162 - 18172. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Christmas, K. Tolentino, V. Primo, K. Z. Berry, R. C. Murphy, M. Chen, D. M. Lee, and R. J. Soberman Cytochrome P-450 4F18 Is the Leukotriene B4 {omega}-1/{omega}-2 Hydroxylase in Mouse Polymorphonuclear Leukocytes: IDENTIFICATION AS THE FUNCTIONAL ORTHOLOGUE OF HUMAN POLYMORPHONUCLEAR LEUKOCYTE CYP4F3A IN THE DOWN-REGULATION OF RESPONSES TO LTB4 J. Biol. Chem., March 17, 2006; 281(11): 7189 - 7196. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Hori, T. Yokomizo, H. Ago, M. Sugahara, G. Ueno, M. Yamamoto, T. Kumasaka, T. Shimizu, and M. Miyano Structural Basis of Leukotriene B4 12-Hydroxydehydrogenase/15-Oxo-prostaglandin 13-Reductase Catalytic Mechanism and a Possible Src Homology 3 Domain Binding Loop J. Biol. Chem., May 21, 2004; 279(21): 22615 - 22623. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Dick, X. Yu, and T. W. Kensler NADPH Alkenal/One Oxidoreductase Activity Determines Sensitivity of Cancer Cells to the Chemotherapeutic Alkylating Agent Irofulven Clin. Cancer Res., February 15, 2004; 10(4): 1492 - 1499. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hong, K. Gronert, P. R. Devchand, R.-L. Moussignac, and C. N. Serhan Novel Docosatrienes and 17S-Resolvins Generated from Docosahexaenoic Acid in Murine Brain, Human Blood, and Glial Cells. AUTACOIDS IN ANTI-INFLAMMATION J. Biol. Chem., April 18, 2003; 278(17): 14677 - 14687. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Brink, S.-E. Dahlen, J. Drazen, J. F. Evans, D. W. P. Hay, S. Nicosia, C. N. Serhan, T. Shimizu, and T. Yokomizo International Union of Pharmacology XXXVII. Nomenclature for Leukotriene and Lipoxin Receptors Pharmacol. Rev., March 1, 2003; 55(1): 195 - 227. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.'i. Mano, Y. Torii, S.-i. Hayashi, K. Takimoto, K. Matsui, K. Nakamura, D. Inze, E. Babiychuk, S. Kushnir, and K. Asada The NADPH:Quinone Oxidoreductase P1-{zeta}-crystallin in Arabidopsis Catalyzes the {alpha},{beta}-Hydrogenation of 2-Alkenals: Detoxication of the Lipid Peroxide-Derived Reactive Aldehydes Plant Cell Physiol., December 15, 2002; 43(12): 1445 - 1455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Kayo, D. B. Allison, R. Weindruch, and T. A. Prolla Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys PNAS, April 12, 2001; (2001) 81061898. [Abstract] [Full Text]
|
|
|
|
 |
|
 T. Yokomizo, K. Kato, K. Terawaki, T. Izumi, and T. Shimizu A Second Leukotriene B4 Receptor, BLT2: A New Therapeutic Target in Inflammation and Immunological Disorders J. Exp. Med., August 8, 2000; 192(3): 421 - 432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. YOKOMIZO, K. MASUDA, K. KATO, A. TODA, T. IZUMI, and T. SHIMIZU Leukotriene B4 Receptor . Cloning and Intracellular Signaling Am. J. Respir. Crit. Care Med., February 1, 2000; 161(2): S51 - 55. [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Drazen, E. Israel, and P. M. O'Byrne Treatment of Asthma with Drugs Modifying the Leukotriene Pathway N. Engl. J. Med., January 21, 1999; 340(3): 197 - 206. [Full Text] [PDF]
|
|
|
|
|
|
C. B. Clish, B. D. Levy, N. Chiang, H.-H. Tai, and C. N. Serhan Oxidoreductases in Lipoxin A4 Metabolic Inactivation. A NOVEL ROLE FOR 15-OXOPROSTAGLANDIN 13-REDUCTASE/LEUKOTRIENE B4 12-HYDROXYDEHYDROGENASE IN INFLAMMATION J. Biol. Chem., August 11, 2000; 275(33): 25372 - 25380. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Dick, M.-K. Kwak, T. R. Sutter, and T. W. Kensler Antioxidative Function and Substrate Specificity of NAD(P)H- dependent Alkenal/one Oxidoreductase. A NEW ROLE FOR LEUKOTRIENE B4 12-HYDROXYDEHYDROGENASE/15-OXOPROSTAGLANDIN 13-REDUCTASE J. Biol. Chem., October 26, 2001; 276(44): 40803 - 40810. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Kayo, D. B. Allison, R. Weindruch, and T. A. Prolla Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys PNAS, April 24, 2001; 98(9): 5093 - 5098. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |