(Received for publication, October 31, 1995; and in revised form, November 20, 1995)
From the
Leukotriene B 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.
Leukotriene B (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.
Total RNA was
prepared from the porcine kidney by a cesium-trifluoroacetic acid
method(24) . Poly(A) 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.).
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 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.
An oligo(dT)-primed
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.
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 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.
The V and K
values against LTB
and
NADP
were determined as described previously (22) six times in three independent experiments.
Figure 1:
The cDNA and deduced amino acid
sequences of porcine LTB 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.
Figure 2:
Amino acid alignment of LTB 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.
In addition, LTB 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).
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
[P]dCTP-labeled hBDH4 or a human
-actin
cDNA. kb, kilobases.
All the
mutant proteins were detected as 62-kDa bands by Coomassie Brilliant
Blue G-staining, 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).
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 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.
Figure 5:
LTB 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.
LTB 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.
The metabolism of LTB 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) .
There is another
group of LTB 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.
In the present study, we cloned a cDNA for the porcine
LTB 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.
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 -oxidation pathway is
present.
LTB 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).
Figure 6:
Amino
acid alignment of NAD/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.
To determine which amino
acids are required for the enzyme activity, a site-directed mutagenesis
was carried out. Ala, 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.
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 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.
There is a proline-rich
motif that is conserved among three species in the C-terminal half of
LTB 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.
In
conclusion, LTB 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.
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].