Gene, 90 (1990) 293-297 Elsevier

293

GENE 03544

Cloning and expression of a c D N A encoding mouse kidney D-amino acid oxidase (Flavoprotein; recombinant DNA; cDNA library; phage ~ vectors; nucleotide sequence; amino acid sequence; plasmid vector; tac promoter; lac repressor gene)

Masazumi Tada, Kiyoshi Fukui, Kyoko Momoi and Yoshihiro Miyake Department of Biochemistry. National Cardiovascular Center Research Institute, Suita. Osaka 56~ (Japan) Received by H. Yoshikawa: 27 December 1989 " Revised: 2 January 1990 Accepted: 3 January 1990

SUMMARY

A cDNA encoding D-amino acid oxidase (DAO; EC 1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme. Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5'-terminal untranslated region of 68 nt, an open reading frame of 1035 nt that encodes 345 amino acids (aa), and a 3'-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence, ATTAAA. The deduced aa sequence showed 77 and 78 ~o aa identity with the porcine and human enzymes, respectively. Two catalytically important aa residues, Tyr 228 and His3°7, ofthe porcine enzyme, were both conserved in these three species. RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver. Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence ofthe mouse DAO cDNA downstream from the tac promoter ofplasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-/~-D-thiogalactopyranoside. Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme. Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E. coli cells.

INTRODUCTION

D-Amino acid oxidase (DAO; EC 1.4.3.3) is a flavoprotein associated with FAD, as the prosthetic group, which catalyzes the oxidative deamination of D-aa. Biological Correspondenceto: Dr. Y. Miyake, Department of Biochemistry,National Cardiovascular Center Research Institute, 5-7-1, Fujishiro-dai, Suita, Osaka 565 (Japan) Tel. 06-833-5012~ext. 2511; Fax 06-833-9865. Abbreviations: aa, amino acid(s); bp, base pair(s); cDNA, DNA complementary to RNA; DAO, D-amino acid oxidase; DAO, gene encoding DAO; FAD, flavin adenine dinucleotide; IPTG, isopropyl-p-D-thiogalaco topyranoside; kb, kilobase(s) or 1000bp; nt, nucleotide(s); ORF, open reading frame; SD, Shine and Dalgarno; SSC, 0.15 M NaCl/0.015 M Na3' citrate pH 7.6. 0378-11191901503.50© 1990ElsevierSciencePublishersB.V.(BiomedicalDivision)

studies of this enzyme have been performed extensively to obtain clues as to the physiological role, because D-aa do not appear to be involved in normal mammalian metabolism. However, no results have been obtained as yet. Recently, we showed that porcine DAO was synthesized on free polysomes as the mature enzyme without proteolytic processing (Fukui et al., 1986). We also isolated cDNA clones coding for the porcine (Fukui et al., 1987) and human (Momoi et al., 1988) kidney DAOs, and investigated the structure-function relationships of these enzymes (Watanabe et al., 1988; 1989a). In this study, to address the questions as to the biological sign'.'-.~canceof and genetic mechanism underlying the mutations and variations of this enzyme, we isolated DAO cDNA from a BALB/c mouse kidney cDNA library. Tissue-specific expression of DAO

294

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Fig, 1, Restriction endonuclease map and sequencing strategy for the cDNA clone, pMDAOiS. The blackened box represents the coding region for DAd; open boxes represent the 5'- and 3'-untranslated regions. The direction and extent of the sequence determination are indicated by horizontal arrows. The solid line at the bottom indicates an AccII-AcclI (1051-bp) fragment used as a probe for Northern-blot analysis.

mRNA among mouse tissues was examined and, moreover, we attempted to produce active mouse D A d in E. coll.

EXPERIMENTAL AND DISCUSSION

(a) Isolation of eDNA clones encoding mouse kidney DAd To isolate eDNA clones for mouse kidney DAd, 1 x 106 phage plaques of a mouse kidney ~.gt10 eDNA library were screened with a 1.4-kb SalI-PvulI fragment of the porcine kidney DAO eDNA clone (Fukui et el,, 1987) as a probe. On successive screening under low stringency washing conditions (1 × SSC, 42°C), 20 positive plaques were obtained from the library. Three eDNA clones containing an appropriately sized insert (1.8 kb) were subcloned and then subjected to further restriction mapping analysis. (b) Analysis of nt sequence and deduced aa sequence One of these clones, designated as pMDAO 15, was subjected to sequencing analysis (Fig. 1). The entire nt sequence and the deduced aa sequence of pMDAOI5 are shown in Fig. 2. The first Met codon is located at nt 69-71, followed by an ORF encoding a protein of 345 aa with a calculated Mr of 38 699. The coding region is followed by a 544=nt 3'-untranslated sequence which contains a polyadenylation signal, ATTAAA, 20 nt upstream from the poly(A) tract. The aa residues of mouse D A d are 77 and 78~0 identical with those of the porcine and human enzymes, respectively. The N-terminal sequence comprising the first 30 aa of the mouse protein was, like in the cases of the porcine and human DAOs, highly hydrophobic, and contains a sequence characteristic of a FAD binding site, GIy-X-Gly=X-X-GIy (Schulz et el., 1982). The aa residues Tyr22s and His 3°7, which are considered to be closely related to the catalytic function based on the results of studies involving site=directed mutagenesis (Watanabe etal., 1989a), were both conserved in these species.

(c) Tissue distribution of mouse DAO mRNA Polyadenylated RNA was analyzed by Northern-blot analysis using the coding region probe (Fig. 1) to examine the tissue distribution ofthe mouse DAO mRNA. As shown in Fig. 3, a single intense signal was detected for the kidney at 2 kb, even after high-stringency washing. The 2-kb transcript was clearly observed in the brain as well, but with a weak hybridizing signal. The DAO mRNA was also detected in porcine brain, as previously reported (Fukui et el., 1988). The absence ofDAO mRNA in mouse tissues other than kidney and brain was in sharp contrast to the expression pattern observed in porcine tissues, where liver contains a relatively large amount of DAO mRNA (Fukui et el., 1988). It was reported that D A d activity is undetectable in the liver of mouse (Shack, 1943), but in a recent cytochemical study, the enzyme activity was detected in hepatocytes from a mouse fetus (Dabholkar, 1986). The difference in mRNA expression between mouse and porcine liver is unknown. However, an interesting possibility as to mRNA expression in fetal mouse liver is that the D A d activity in mouse liver disappears in the process of development of a fetus into an adult. Further investigation is required to solve the question of whether or not the enzyme or its mRNA exists in the liver and other tissues during early developmental stages. (d) Construction of an expression plasmid As an approach for enhancing the efficiency of the expression of exogenous mouse DAO eDNA in a prokaryotic cell, an expression plasmid was designed. First, a strong tac promoter was located upstream from the start codon of the cloned eDNA to obtain high expression efficiency. Second, the distance between the ribosome-binding site (SD sequence) and the start codon was shortened by the introduction of a unique restriction enzyme site in the SD sequence. A SphI-$cal fragment containing the tac promoter and rrnB terminators of pKK223-3, in which the endogenous BamHI site had been destroyed, was inserted into the SphI-SmaI site of pGEM3Zf( - ), Oligodeoxyribonucleotide-directed mutagenesis was performed to generate a unique BamHI site into the SD sequence, which yielded a mutant plasmid, pGEM223-3B. Third, a represser sufficiently capable of regulating the tac promoter, i.e., the lac represser (laclq), was introduced to avoid overproduction of the exogenous protein, that could be toxic for the host cell. The lacIq gene in pMJR1560 was excised as a Kpnl-Sacl fragment, following substitution of the Pstl site for a Sacl site through the use of the SacI linker. The iacl q

295 1 AC,CAGTCCTGCTGGAACCTGCACCCCAGG'J~rA~-~-i-i~L'TCCCGACACCTGGrACCAb'~GGC~GO]rG~G

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143

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GAT TAT GGC TAC GGC TGG TTC &AT &CA AGC CTC Ctvl' CTA GAG GGG /LAG AGC TAC CTG CCA TGG CTA ACt' GAG AGG Asp T y r G l y T y r G l y T r p Phe Ann T h r Ser Leta 1,eta Zeta Glta Gly Lys Ser T y r Zeta Pro T r p Zeta Thr Glta Arcj 126 130 135 140 145 150

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~ T ',CA TCC CAC C'JL'C TGA GGAC'L'CTAN~NI'CAC 11;t2 Asn Leu Phe Gly Lys l i e Leu Glu Glu Lys Lye Leu Sag Arg Leu P r o ' P r o ~er H18 Leu , t , AAC CTC 'lvl'c GGG AAA A'L~ CTA GAG GAA AAG AAG TTG TCC AGG ~ 326

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Fig. 2. The nt sequence o f e D N A ~ncoding mouse kidney D A O and deduced aa sequence. The nt are numbered in the 5' --, 3' direction, beginning with the first nt o f the e D N A insert. The M e t corresponding to the start codon A T G at nt 69-71 is numbered I. The steep codon is indicated by asterisks. The polyadenylation signal is boxed ( G e n B a n k accession number: M32299).

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(5 pg) from each of various tissues was subjected to Northern blotting with an Accll-Accll fragment (Fig. i) as a probe. Membrane washing was performed under relatively stringent conditions (0.1 x SSC, 55°C~ The size marker used was mouse rRNA. RNAs were prepared from the following tissues: brain 0ane !); heart (lane 2); kidney (lane 3); liver (lane 4); lung (lane 5); pancreas (lane 6); spleen (lane 7); submandibular gland (lane 8); testis (lane 9); and thymus (lane 10). Exposure for autoradiography was performed at - 8 0 ° C with an intensifying screen for three days.

296

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Fig. 4. Construction ofthe expression plasmid, pEQMDI. The blackened and hatched boxes represent the insert DNA of mouse DAO cDNA and the tac promoter containing the SD sequence, respectively. The open arrows represent the rrnB transcription terminators. The solid thin arrows represent the lacl q gene. The BamHI site (boxed by a solid line) was introduced into the promoter region of pKK223-3 by in vitro mutagenesis. The BamHI site (boxed by a dashed line) was introduced later into the 5'-untranslated region of pMDAOI5, also by in vitro mutagenesis.

DAO that reacted with the antiserum was the same as that of DAO observed in the mouse kidney extract (data not shown). Next, the catalytic activity of the recombinant mouse DAO synthesized in E. coil was examined by means of a spectrophotometric assay method with D-Ala as a substrate (Fukui et al., 1988). When 200 mM D-Ala was used, the DAO activity was detectable, and it was inhibited by the addition of 100 mM benzoate, a potent competitive inhibitor of DAO. Judging from the amount of the mouse DAO protein fraction on immunoblot analysis, the maximum activity of the mouse recombinant DAO is comparable to that of the porcine recombinant DAO (Watanabe et al., 198%) from kinetics analysis. These results suggest that the mouse DAO synthesized in IPTG-induced cells is a functional DAO. The availability of this expression system will greatly facilitate detailed studies on the enzymatic properties of mouse DAO. The appreciation and comparison of the catalytic properties of mouse DAO with those of the authentic porcine enzyme may provide some information on the catalytic function of DAO. Furthermore, through the use of the mouse eDNA clone, genetical analysis of mouse DAO can be carried out. These approaches would form a basis for understanding the biological significance of DAO.

ACKNOWLEDGEMENTS

fragment and the SphI.Kpnl fragment from pGEM223-3B were inserted into the Sphl-$acl site of pUC 18 to generate the expression vector, pUC223-3BQ (Fig. 4). In addition, the mouse cDNA was also modified to remove the 5'-untranslated sequence by introducing a BamHI site at the position of 10 nt upstream from the start codon. The sequence around the start codon of the mouse cDNA was found to be as follows; 5'-GTGATG-3'. Since the underlined triplet, G TG, may be used as a start codon instead of the authentic ATG codon in prokaryotes, the GTG sequence was converted to GGC by in vitro mutagenesis. Finally, the BamHI-EcoRV fragment of this modified mouse DAO cDNA was inserted into the BamHI-Smal site of the expression vector, pUC223-3BQ. In the resultant expression plasmid, pEQMD1, the distance between the SD sequence and the start codon was only 7 nt. (e) Synthesis of mouse kidney DAO in Escherichia coil Using the expression system described above, it was examined as to whether the mouse DAO protein is synthesized in E. cell under IPTG regulation. On analysis of the cell-free extract by immunoblottingusing anti-porcine kidney DAO antiserum, a single polypeptide band was observed for IPTG-induced cells harboring pEQMD 1. The molecular size of the immunoreactive recombinant mouse

This work was supported in part by a Research Grant for Cardiovascular Diseases (62A-1) from the Ministry of Health and Welfare of Japan, and Grants-in-Aid for Scientific Research (63570137 and 01770170) from the Ministry of Education, Science and Culture of Japan.

REFERENCES Dabholkar, A.S.: Ultrastructural localization of catalase and D-amino acid oxidase in 'normal' fetal mouse liver. Experientia 42 (1986) 144-147. Fukui, K., Momoi, K., Watanabe, F. and Miyake, Y.: Biosynthesis of porcine kidney D.amino acid oxidase. Biochem. Biophys. Res. Commun. 141 (1986) 1222-1228. Fukui, K., Watanabe, F., Shibata, T. and Miyake Y.: Molecular cloning and sequence analysis of eDNA encoding porcine kidney D-amino acid oxidase. Biochemistry 26 (1987) 3612-3618. Fukui, K., Momoi, K., Watanabe, F, and Miyake, Y.: In vivo and in vitro expression of porcine D-amino acid oxidase: in vitro system for the synthesis of a functional enzyme. Biochemistry 27 (1988) 6693-6697. Momoi, K., Fukui, K., Watanabe, F. and Miyake, Y.: Molecular cloning and sequence analysis of eDNA encoding human kidney D-amino acid oxidase. FEBS Lett. 238 (1988) 180-184. Schulz, G.E., Schirmer, R.H. and Pal, E.F.: FAD-binding site of glutathione reductase. J. Mol. Biol. 160 (1982) 287-308. Shack, J.: Cytochrome oxidase and D-amino acid oxidase in tumor tissue. J. Natl. Cancer Inst. 3 (1943) 389-396.

297 Watanabe, F., Fukui, K., Momoi, K. and Miyake, Y.: Effect of site-specific mutagenesis of tyrosine-55, methionine-i 10 and histidine-217 in porcine kidney D-amino acid oxidase on its catalytic function. FEBS I~tt. 238 (1988) 269-272. Watanabe, F., Fukui, K., Momoi, K. and Miyake, Y.: Site-specific mutagenesis of lysine-204, tyrosine-224, tyrosine-228, and histidine-307 of

porcine kidney D-amino acid oxidase and the impfications as to its catalytic function. J. Biochem. 105 (1989a) 1024-1029. Watanabe, F., Fukui, K., Momoi, K. and Miyake, Y.: Expression of normal and abnormal porcine kidney D-amino acid oxidases in Escherichia coli: purification and characterization of the enzymes. Biochem. Biophys. Res. Commun. 165 (1989b) 1422-1427.