©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
cDNA Cloning, Expression, and Mutagenesis Study of Leukotriene B 12-Hydroxydehydrogenase (*)

(Received for publication, October 31, 1995; and in revised form, November 20, 1995)

Takehiko Yokomizo Yoko Ogawa Naonori Uozumi Kazuhiko Kume Takashi Izumi Takao Shimizu (§)

From the Department of Biochemistry, Faculty of Medicine, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Leukotriene B(4) 12-hydroxydehydrogenase catalyzes the conversion of leukotriene B(4) into its biologically less active metabolite, 12-oxo-leukotriene B(4). This is an initial and key step of metabolic inactivation of leukotriene B(4) 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.


INTRODUCTION

Leukotriene B(4) (LTB(4)) (^1)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(4) by 5-lipoxygenase(5, 6, 7, 8) . LTB(4) is biosynthesized from LTA(4) by the action of LTA(4) hydrolase(9, 10, 11, 12, 13) . In human polymorphonuclear leukocytes, LTB(4) is converted to 20-hydroxy-LTB(4) by a cytochrome P-450 LTB(4) and further to 20-carboxy-LTB(4)(14, 15, 16, 17) . The cDNA of cytochrome P-450 LTB(4) was cloned, and the mRNA is detected only in human leukocytes(18, 19) . LTB(4) is also produced in tissues other than leukocytes(20, 21) . We reported an alternative pathway for LTB(4) in various porcine tissues and purified a cytosolic LTB(4) 12-hydroxydehydrogenase from the porcine kidney(22) . This enzyme converts LTB(4) to 12-oxo-LTB(4) in the presence of NADP. 12-Oxo-LTB(4) is at least 100 times less potent than LTB(4) 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(4) 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(4) in tissues other than leukocytes.


EXPERIMENTAL PROCEDURES

Materials

LTB(4) was kindly donated by Ono Pharmaceutical Company (Osaka). EDTA-Na(2), 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(4) 12-Hydroxydehydrogenase

The porcine LTB(4) 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(2)/C(18) column (2.1 times 100 mm). The digested enzyme was injected onto a µRPC C(2)/C(18) 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(4) 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(4) 12-Hydroxydehydrogenase

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.

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(4) 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 times 10^6 independent clones. Full-length cDNA clones were obtained by a plaque hybridization method. 1.0 times 10^6 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 times 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(4) 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(4) 12-Hydroxydehydrogenase

Human cDNAs of LTB(4) 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 times 10^5 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 times 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(4) 12-hydroxydehydrogenase cDNA (hBDH 4) or a human beta-actin cDNA for 3 h in Rapidhybri solution (Amersham Corp.). The membranes were washed for 15 min once in 3 times SSC, 0.1% SDS and for 20 min twice in 0.1 times SSC, 0.1% SDS at 65 °C. Autoradiogram was subjected to a Bas-2000 system analyzer (Fuji Film, Tokyo, Japan).

Expression of LTB(4) 12-Hydroxydehydrogenase as a GST Fusion Protein

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-beta-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-beta-D-galactoside. E. coli was collected, resuspended in PBS(-) containing 2 mM EDTA-Na(2), 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 times 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(4) 12-Hydroxydehydrogenase

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(4) 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 alpha2 antibody hereafter.

Site-directed Mutagenesis of the Putative NADP-binding Domain

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`.

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(4) DNA polymerase and 6 units of T(4) 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 5times 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 alpha2 antibody (200 times dilution) or rabbit anti-GST antibody (Pharmacia) as the first antibody and visualized using an Amersham ECL system.

The V(max) and K(m) values against LTB(4) and NADP were determined as described previously (22) six times in three independent experiments.


RESULTS

cDNA Cloning of Porcine and Human LTB(4) 12-Hydroxydehydrogenase

Screening of 1.0 times 10^6 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(4) 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 times 10^5 clones) with pBDH 15 gave two independent clones, hBDH 4 and 8, which were identical. The primary structures of porcine and human LTB(4) 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(4) 12-hydroxydehydrogenase. pBDH 15 contains an open reading frame of 987 base pairs and coded for 329 amino acids. The calculated M(r) 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(4) 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).


Figure 1: The cDNA and deduced amino acid sequences of porcine LTB(4) 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(4) 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. bullet indicates amino acids that are identical in two species.



In addition, LTB(4) 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(4) 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(4) 12-hydroxydehydrogenase (see below).

Northern Blot Analysis

Fig. 3shows the tissue distribution of mRNA of LTB(4) 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) .


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 beta-actin cDNA. kb, kilobases.



Expression of LTB(4) 12-Hydroxydehydrogenase as a GST Fusion Protein

The recombinant porcine LTB(4) 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 times 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(max) value of forming 6 nmol of 12-oxo-LTB(4)/min/mg fusion protein. The K(m) values of the recombinant enzyme were 20 µM against LTB(4) 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(4) 12-hydroxydehydrogenase.

Site-directed Mutagenesis of the Putative NADP-binding Domain

A computer-assisted homology analysis revealed that the fragment 149-166 of LTB(4) 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(m) and V(max) values were determined by changing the concentrations of LTB(4) and NADP.

All the mutant proteins were detected as 62-kDa bands by Coomassie Brilliant Blue G-staining, alpha2 antibody raised against the porcine LTB(4) 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(max) 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(m) values against LTB(4) 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(4) 12-hydroxydehydrogenases antibody (alpha2) 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(4) 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.




DISCUSSION

LTB(4) 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(4) has been intensively studied in leukocytes. Human polymorphonuclear leukocytes convert LTB(4) into 20-hydroxy-LTB(4) by a microsomal NADPH-dependent cytochrome P-450 LTB(4)(14, 36, 37, 38, 39, 40, 41) . 20-Hydroxy-LTB(4) is further metabolized to 20-carboxy-LTB(4)(42) . 20-Hydroxy- and 20-carboxy-LTB(4) was 10-30 times less active in neutrophil chemotaxis(43, 44) .

There is another group of LTB(4) metabolites. Porcine leukocytes converts LTB(4) to 10,11-dihydro-LTB(4), 10,11-dihydro-12-oxo-LTB(4), and 10,11-dihydro-12-epi-LTB(4)(37, 45, 46, 47) . Wainwright and Powell (48) extensively studied the mechanism and found that LTB(4) is first converted to 12-oxo-LTB(4) by a microsomal NAD-dependent 12-hydroxydehydrogenase and then to 10,11-dihydro-12-oxo-LTB(4) by a cytosolic NADH-dependent 10,11-reductase in porcine polymorphonuclear leukocytes(48) . We purified LTB(4)-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(4) to 12-oxo-LTB(4), but it is a cytosolic enzyme and utilizes NADP as a cofactor(22) . A similar conversion of LTB(4) was reported in the human lung(49) , kidney(50) , keratinocytes(51) , and the guinea pig kidney and liver. (^2)The enzyme purified from the porcine kidney is a monomeric protein with an M(r) of 35,000 and an isoelectric point over 9.5. It specifically recognizes the 12(R)-hydroxy-moiety of LTB(4), thus it was named LTB(4) 12-hydroxydehydrogenase(22) . The product, 12-oxo-LTB(4), 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(4) 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(4) 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(4) 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(r) 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(4) 12-hydroxydehydrogenase. Further studies are required to determine the developmental change of the expression of LTB(4) 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(4) 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 beta-sheet-alpha-helix-beta-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 alpha-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(4) 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(4) 12-hydroxydehydrogenases and other homologous proteins. The amino acid sequences of the porcine and human LTB(4) 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-alpha, 20-beta-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(4) 12-hydroxydehydrogenase. The underlined letters show amino acids that form beta-helix structures, and the letters in the shaded boxes are alpha-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(4) 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(m) values against LTB(4) 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(m) values for NADP of M1, M5, and M8 mutants were not significantly different from that of the recombinant wild type enzyme, although the V(max) 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(4) 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(4) 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(4) 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(4) 12-hydroxydehydrogenase remains to be clarified.

In conclusion, LTB(4) 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.


FOOTNOTES

*
This work was supported in part by grants-in-aid from the Ministry of Education, Science, and Culture and the Ministry of Health and Welfare of Japan and by grants from the Yamanouchi Foundation for Metabolic Disorders and the Human Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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].

§
To whom correspondence should be addressed. Fax: 81-3-3813-8732; :tshimizu{at}m.u-tokyo.ac.jp.

(^1)
The abbreviations used are: LTB(4), 5(S),12(R)-dihydroxy-6,14cis-8,10-trans-eicosatetraenoic acid; LTA(4), 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.

(^2)
N. Uozumi and T. Yokomizo, unpublished data.


ACKNOWLEDGEMENTS

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.


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