Molecular cloning and characterization of rat liver catechol-O-methyltransferase

Gene, 93 (1990) 241-247 Elsevier

241

GENE 03628

Molecular cloning and characterization of rat liver catechol-O-methyltransferase (Methylating enzyme; ,I. gtll expression screening; recombinant DNA; genomic library; nucleotide sequencing; ILNA analysis)

Mar|o Salminen', Kenneth LundstrOma, Carola Tilgmann b, Raija Savolainen', Nisse Kalkkinen b and Ismo Ulmanen" a Orion Corporation, Laboratory of Molecular Genetics. Hebinla" (Finland) and b Institute of Biotechnology, University of Hebinld, Hebinki (Finland) Tel. 358(0)4346051 Received by J.K.C. Knowles: 9 December 1989 Revised: 13 February 1990 Accepted: 18 April 1990

SUMMARY

The coding sequence of rat liver catechoI-O-methyl-transferase (COMT; EC 2.1.1.6) was determined from rat cDNA and genomic libraries were screened with DNA probes and specific antiserum. The open reading frame consisted of 663 nucleotides coding for a 221-amino acid (aa) polypeptide with a deduced Mr of 24 747. No obvious hydrophobic signal sequence, membrane-spanning domains, or potential N-glycosylation sites were found in this sequence. The identity of the clone and the accuracy of the sequence was verified by direct aa sequencing of the tryptic peptides derived from the purified rat liver enzyme. Primer extension analysis showed that the transcription start point of the rat liver COMT mRNA was 450 bp upstream from the translation start codon. A putative polyadenylation signal (AT]'AAA) was found in the 3'-noncoding region. The predicted size of the COMT transcript was 1.8-2.0 kb, which could be confirmed from Northern hybridization ~malyses of the isolated rat liver mRNA. One polypeptide of 25 kDa, could be immunoprecipitated with anti-COMT ~ntibody from in vitro translation of rat liver mRNA. Employing the DNA blot analysis only one COMT-encoding gene was found in the rat genome.

INTRODUCTION

The enzyme COMT (EC 2.1.1.6) catalyses the transfer of a methyl group from S-adenosyl-L-methionine to one of the phenolic hydroxyl groups of a catechol substrate (Axelrod and Tomchick, 1958). In mammals this O-methylation !

Correspondence to: Dr. M. Salminen, Orion Corporation, Genetic Engi-, 'neering Laboratory, Valimotie 7, 00380 Helsinki (Finland) Tel. 358(0)4346051; Fax 358(0)5653164. Abbreviations: aa, amino acid(s); ATCC, American Type Culture Collection; bp, base pair(s); eDNA, DNA complementary to mRNA; COMT, catechoI-O-methyltransferase; COMT, gene encoding COMT; DTT, dithiothreitol; kb, kilobase(s) or 1000bp; MB-COMT, membrane-bound COMT; nt, nuclcotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PAGE, polyacrylamide-gei electrophoresis; s-COMT, ~soluble COMT; SDS, sodium dodecyl sulfate; TPCK, N-tosyI-L -phenylalanyl chloromethane; tsp, transcription start point(s). 0378-1119190/$03.50© 1990Elsevier Science Publishers B.V. (Biomedical Division)

reaction is important in the enzymatic inactivation of catechol hormones, catecholamine neurotransmitters and many neuroactive catechol drugs such as L-DOPA, ~methyl DOPA and isoprenaline (Ball et al., 1972; Guldberg and Marsden, 1975). To increase the efficiency of these drugs, synthetic COMT-inhibitors have been developed (Guldberg and Marsden, 1975; Lind6n etal., 1988; MannistO and Kaakkola, 1989). The COMT enzyme is present in many mammalian tissues and the highest activities have been reported in liver, kidney, gut, uterus and placenta (Axelrod et al., 1959; Guldberg and Marsden, 1975; Inoue et al., 1977; Nissinen et al., 1988). The enzyme exists mainly in two forms in rat and human tissues. Most of the COMT activity in the tissues corresponds to a soluble, cytoplasmic enzyme (s-COMT, 23 kDa). In addition, varying amounts (0-30~o) of membrane associated COMT activity (MB-COMT,

242 RESULTS AND DISCUSSION

25 kDa) has been detected (Inscoe eL aL, 1965; Assicot and Bohuon, 1970; 1971; Borchardt et al., 1974; White and Wu, 1975; Tong and D'Iorio, 1977; Roth, 1980; Rivett et al., 1983; Grossmann et al., 1985; Heydorn et al., 1986; Nissinen ctal., 1988). MB-COMT has a higher activity to catechol substrates than s-COMT (Rivett et al., 1982; Rivett and Roth, 1982). Other, larger immunoreactive polypeptides have been demonstrated in Western blots by using different kinds of anti-s-COMT antibodies (Huh and Friedhof, 1979; Grossman et al., 1985). Only the s-COMT form could be immunologically detected in the in vitro translations of polysomal RNA from rat liver (Grossmann et al., 1989). Whether the different polypeptides represent separate gene products or posttranslational modifications of a single polypeptide or are due to unspecific antisera is, at present, unknown. Neither has the nature of membrane association of the MB-COMT been solved. The aim of the present work is to isolate and analyse COMT eDNA from rat liver and to get information on the primary structure of the protein. Further we wanted to study the transcripts and genomic organization of COMT.

8TOP

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- 100

(a) Identification of the C O M T clones and sequence analyses The isolated rat liver COMT eDNA 1349-bp clone contained a 622-bp ORF and a 727-bp 3'-untranslated region (Figs• 1 and 2). To conrwm the nt sequence of the eDNA, 22 tryptic peptides from the purified s-COMT enzyme (Fig. 3) were directly sequenced in a gas-pulsed liquid sequencer. These peptides covered accurately 204 out of 207 aa coded by ~he eDNA (underlined in Fig. 2). Only ! the deduced C-terminal Ser and a dipeptide AspJ4S-Arg14~ were not found among the trypti¢ pcptides. The peptide No. 3 showed an N-terrrdnal sequence Tyr-Val-Gln, which was absent in the aa sequence deduced from the eDNA clone. This indicated that the isolated eDNA clone was not complete. Because several attempts to obtain a full-length eDNA from the rat liver library failed, we~tecided to determine the total ORF by isolating and sequencing the 5'region of the rat COMT gene (Fig. 2). The COMT genomic clone revealed a 41-bp extension of

1340

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Fig. I. Partial restriction map and the sequencing strategy ofrat liver COMT. A rat liver eDNA library (Ciontech Laboratories Inc.) in ~stl ! was screened using E. colt YI090 as a host. The immunoscreening method (Young and Davis, 1983) was ped'ormed by using the ProtoBIot Immunoscreening System (Promega, M~dison, WI) and rabbit polyclonal antibody raised against a highly purified rat liver COMT enzyme (Tilgmann and Kalkkinen, 1990). Positive plaques were obtained at a frequency of 0.02%. The largest insert from the ten positive clones was subclonod for sequence determination. Rat genomic library, ,1.Charon4A (Clontech Laboratories Inc.), was screened with the filter hybridizationmethod of Benton and Davis (1977) using Hy~nd-N filters. A 209.bp EcoRI.Pstl fragment from the COMT eDNA clone was labelled (Multiprimer DNA labelling kit) with [0t.3"P]dCrP and used as a probe. The filters, labelling kit and 32p were from Amersham International. One positive clone was found from 0.5 x l0 s plaques scr,~ned. The upper part of the figure represents the restriction map where the thick line corresponds to the sequence deriv~l from the eDNA clone and thin line the sequence derived From the genomic clone, In the middle pan, the shaded box represents the rat liver COMT coding region and the open box the untranslated 3' region. The putative polyedenylation signal, poly(A), is shown in the sequence derived from the rat COMT genomic clone. SP6 and 1"7 promoter primers (Promega, Madison, Wl) and nine synthetic oligo primers were used for sequ~cing the eDNA clone. The arrows represent the direction and extent of the sequence data generated by each of the primers used•

243 -100 - 90 - 4S

(: CT6 TtqS TTG GCC CT(: (:T(: CTG CT(: TTG CGA CA(: CTG GG(: TGG GGC TTG GTG ACT ATT TTC T l ~ TTT GAG TA¢ GTO CTG CAG (:C~ GTC CA¢ AA(: ~ ATC

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GGC CAA ATC ATG GAT GCA G'TG ATT ¢GG GAG TAC AGC C(:C TC¢

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60

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226

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90

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135

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361

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180

GCG TAT GTG AGA GGG AG(: &OC AO(: TTC GAG TGC &CA ¢AC TA¢ AGC AIa TYr V J l Arg Gly 8or Sot 8or PAo G2u Cys Thr B18 Tyr S e t

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TCA TA(: CTG GAG TA(: ATG AAA GTT GTA GAC GGC TTG GAG AAG GCA fief TFr Leu Glu 5~r lU~ r y e v a l ,v82 Asp Gly r e u a l u r y s AI_.A

210

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676 131 766 811 8S6 901 946 991 1036 1081 1136 1171 1316

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1306

TOG GOC TTG TCC TCG GGG OCA CAT TTT ~ GGT AGT NkC CCT ~ JUW CAC CT& GG? A(:A CT& GAT CAC AOC TT& &5~ CTC TOC ACC ¢Gk OkO &TC 'nC &CO 000 TTT CAO TOT G0G AT(: ACT AGO CAT TOT AT(: ACC AGT TAG O©C CAO GAG ATA &CA OCk GAg OAA GTC AGC A / ~ C ~ TJtA ATO TCC AGA ¢0C ~ OCT CAO AGA CAG TAC TOT CAC TTA CTO TCC ¢Ck OCA 0CT CAC &CC AC~ T~'~ CkT ~ &kO AkT GAG TCL CAk OCT T/C CA6 OTA TOO TOO TOC ACG CCT GTC AT(: CCC AGT AT(: CkO 0/W 0CA OkO OCA '/CO OkO GAC AOC CkO TTC AO¢ GCC A0C ¢TO GTC TAC ATA AGT GAG TTC ~ GGC TA(: GTA. GAA ATA CAT TTT TAA .s..s..s AGT TAT AAC CTT OOO GTT GGG GAT TTA OCT CAG TOG TAG AG(: GAT TO(: C~A GC

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Fig. 2. The nt sequence ofthe rat liver COMT and the deduced aa sequence. The inserts from the eDNA and genomic clones in the pGem-7Zf( + )-vector (Promega, Msdison, WI) were sequenced as described (Sanger et al., 1977; Holmes and Quigiey, 1981; Tabor and Richardson, 1987; Wang, 1988). Nucleotide sequence editing and the analysis of functional protein domains was performed using the PC Gene computer software (IntelliGenetics lnc./Genofit SA, Switzerland). The search for sequence homologies was done in both protein (SwissProt Release 1 !.0, June 1989) and DNA (EMBI. Release 20.0, August 89) data banks. The nt in the figure are numbered on the left and the aa on the right margin. The nine methionine residues are shown in bold-face letters in the aa sequence. The nt from 42-1340 and 3' (a) sequence are derived from the rat liver eDNA clone, and the nt from -100-41 and the 3' (b) sequence are derived from the rat genomic clone. The doubly underlined sequence is the putative polyadenylation signal. The underlined an have been verified by sequencing the tryptic peptides. For the sequence analysis the peptides were dissolved in 30 ~tl of 20% trifiuoroacetic acid before loading on a Polybrene-treated (2 rag/30/~1 H20) glassfibre filter (Whatman GF/C). Edman degradations were carried out in a gas-pulsed liquid phase sequencer (Kalkkinen and Tilgmann, 1988) equipped with an on-line phenylthiohydantoin aa analyzer (corresponding to the Applied Biosystems 120A). Sequencing was performed using the Applied Biosystems 03RPTH program.

244 21 7

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Fig. 3. Reversed-phase chromatography of the tryptic peptides from the rat liver COMT enzyme• The COMT enzyme was purified from rat liver as described in detail by Tilgmann and Kalkkinen (1990). The pure enzyme was alkylated with 4-vinylpyridine (Friedman et el., 1970; Fullmer, 1984), desalted by reversed-phase chromatography and treated with 4% w/w TPCK-trypsin in 0.1 M ammonium bicarbonate for 8 h. Tryptic pep,ides were separated on a 0.46 × 10cm Vydac 218TPB$ column using a linear gradient of acetonitrile (0-60 % in 90 min) in 0. 1% trifluoroacetic acid. For elution a linear gradient of acetonitrile (0-60% in 90rain) in 0.1% trifluoroacetic acid was used at a flow I ml/min. Chromatography was monitored at 218 nm and pep,ides collected manually and dried in a vacuum centrifuge and stored dry at -20°C prior to the sequence analysis. The numbers refer to the peptides subjected for sequence analysis. X-axis, elution time (min); Y-axis, absorbance units/full scale (AUFS).

the eDNA ORF starting with a putative start codon (Figs. 1 and 2). As the N-terminal end of the purified COMT most likely is blocked (Tilgmann and Kalkkinen, 1990), we could not directly show that this ATG is used in initiation. However, indirect evidence supports that assumption. The sequence at this site (ATCATGG) resembled closely the optimal consensus .qequence (ACCATGG) for translation initiation by euka,~jotie ribosomes (Kozak, 1986). The deduced Mr of s-COMT is 24 747 starting from this ATG, which is ve;y close to the value of 25 kDa for the highly purified s-COMT determined by" SDS-PAGE (Tilgmann ~ d Kalkkinen, 1990), but is somewhat higher than that previously reported (23kD~) for the rat s-COMT (Grossman etal., 1985; Heydorn eta]., 1986). In the genomic sequence, the ORF continues upstream from the putative ATG start codon up to -250 having three ~dditional ATG codons (not shown). No pep.ides correspond. ing to the upstream sequences were found in the tryptic digest of rat COMT, suggesting that these ATGs are not used in translation initiation. This is further supported by the low homology of these sequence contexts with the optimal initiation sites. At present we have no information on

the possible splicing pattern of the 5'-noncoding region. Thus we do not know, whether the upstream sequences containing these ATGs actually occur in the COMT transcripts. The sequence analysis of the polypeptide derived from the eDNA did not reveal any regions characteristic of integral membrane proteins. This polypeptide has no hydrophobic signal sequence, membrane-spanning domains or N-glycosylation sites. Thus, by the methods used, we could only fred clones representing the s-COMT. The homology searches of the COMT ORF nt or aa sequence in protein or DNA data banks did not reveal any significant similarity to known sequences. The 3'-untranslated region of the eDNA has no consensus polyadenylation signals (Fig. 2a). The comparison of the eDNA and genomic clones revealed a 50 bp discrepancy between their 3'-untranslated regions (Fig. 2, a and b). It can be assumed that the foreign sequence in the eDNA clone most likely is a cloning artefact created during the eDNA preparation. Alternatively, it can be due to splicing. However, introns have rarely been found in the 3'-untranslated region of mammalian genes (Breathnach and Chambon, 1981) and further, the putative donor site does not follow the GT-AG rule (Mount, 1982). Examining the genomic sequence (Fig. 2b) corresponding to the 3'-end of the eDNA, one possible polyadenylation signal (ATTAAA) can be found (doubly underlined in Fig. 2b). This variant polyadenylation signal occurs in 12% of 3'-terminal sequences of eukaryotic mRNAs (Wickens and Stephenson, 1984). .(b) RNA analysis and in vitro translation

COMT mRNA from rat liver was resolved in agarose ~gels as a 1,8-2,0-kb band (Fig. 4). The in vitro translation ~ f rat liver mRNA in rabbit reticulocyte lysate produced a polypeptide of about 25 kDa, which could be precipitated • with the ~ti-COMT antiseram (Fig. 4). No active enzyme was detected in these lysates, which can be due to the uncorrect formation ofthe S-S bonds between the four Cys residues found in the COMT sequence (Til~pnann and Kalkkinen, 1990). However, the COMT activity increases considerably in mammalian cells when transfected with an expression vector containing the complete rat liver COMT ORF r e , on (A. Jalanko, personal communication). Primer extension of rat liver mRNA suggested that there is one major transcript 5'-end 450 nt upstream from the translation start codon (Fig. 5). Based on this the length of the COMT transcript is 1.8 kb, which corresponds well to the size of the COMT mRNAs observed in RNA blots. In addition, primer extension revealed two minor 5'-ends for COMT transcripts in rat liver, about 500 and 300nt upstream from the ATG (Fig. 5). Whether this heterogeneity is actually due to utilization of different tsp or to

245

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0 -14.3 Fig. 4. Northern hybridization and in vitro translation ofmRNA from rat liver. Total RNA was isolated and purified from rat liver (Chirgwin et al., 1979; GilPin et al., 1974) and poly(A)+mRNA prepared by HybondT M -mAP. 10pg of poly(A)+RNA from rat liver was analyzed in a 1% agarose-2.2 M formaldehyde gel, b|otted onto a Hybond-N membrane and hybridized with a 209-bp EcoRI-Pstl [32P]DNA fragment (Maniatis et al., 1982) from the coding region of the COMT eDNA clone (lane I). Two pg of rat liver poly(A) + mRNA was translated in vitro using retieulocyte lysate (Promega, Madison, Wl) in the presence of L-[35S]methionine. For immunopreoipitation, the lysates (50/~1) were diluted with 400/zl of NET buffer (1% NP-40/400 mM NaCI/50 mM Tris. HCi pH 8.0/5 mM EDTA) containing 10/zg/ml of aprotinin (Boehringer-Mannheim) and 0.2% SDS. Preimmune- (lane ~) or anti-COMT polyelonal rabbit (lane 3) serum (Tilgmann and Kalkkinen, 1990) was added with Protein A-Sepharose (Pharmaeia-LKB). The samples were mixed for 2 h at +220C and the precipitates subjected to 0.1% SDS-10% PAGE (Laemmli, 1970).

some other reason, such as alternative splicing, remains to be evaluated. It will also be interesting to investigate if liver COMT transcripts ofdifferent lengths have any relationship to the enzyme forms described in the literature. (e) DNA analysis Analysis of genomic DNA from two rat cell lines by Southern blotting showed that only one HindIll or EcoRl restriction enzyme fragment is hybridized with the COMT eDNA probe from the 5'-region of the coding sequence (Fig. 6). When EcoRl, EcoRV, Hindlll and Xbal digests of these DNAs were probed with a eDNA fragment covering the entire coding region one (EcoRl and Hindlll) or two (EcoRV and Xbal) hybridizing fragments were detected (not shown). This strongly suggests that rat cells have only one COMT gene coding for the s-COMT. Thus the different forms ofthe COMT enzyme can not be products of separate genes but they rather result from alternative processing of transcripts and/or from some posttranslational modifications of the COMT polypeptide. The cloning of rat COMT eDNA and gene presented here provides valuable tools to study further the expression and the biosynthesis of COMT and to solve the open question of different forms of the enzyme. It also gives

300-

--0

Fig. 5. Localization of the 5' end of the COMT-specific transcripts by primer extension. A 18-mer oligo primer (5'-AGGCTTTGCATTCTGCAG-Y) from the $'-part ofthe coding region (nt 40-57 in Fig. 2), was labelled with 32p using T4-polynucleotide kinase. Ten ng of the primer was heated at +95°C for $min and annealed with 25/48 of poly(A)* RNA (lane I) or total (lane 2) rat liver RNA in a buffer consisting of 0.4M NaCI/40mM piperazine-Nfl'-bis(2-ethane-sulfonie acid) pH 6.4/1 mM EDTA/80% (v/v) formamide. The annealing was done by cooling the samples for 2.5 h to room temperature. The annealed primers were extended with avian myeloblastosis virus reverse transcriptase (30 units) for I h at + 42°C in 50 mM Tris. HCI pH 8.3/50 mM KCI/10mM MgCI2/2 mM DTT/60 units of RNasin and 0.5 mM of deoxynucleotides. The extended products were analyzed in a 8 M urea-6% polyacrylamide gel and autoradiographed.

1234 kb

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Fig. 6. Southern hybridization of DNAs isolated from rat cell lines L6J ! [lanes I and 3 (Ringertz et al., 1978) obtained from Dr. Wahrmann, lnstitut de Pathologie et Biologic Cellulaires et Mol6culaires, Paris, France] and XC (lanes 2 and 4; ATCC CCL165). Isolated DNA from these cells (Maniatis et al., 1982) was digested with EcoRi (lanes I and 2) or Hindlll (lanes 3 and 4) restriction enzymes. 20/=g of each digested DNA was electrophoresed on a 0.8% agarose gel and transferred to a Hybond-N nylon membrane (Amersham International). Hybridization of the membrane was performed with the same 209-bp COMT-specific probe as above or with a probe covering the entire coding region. Hindilldigested DNA from the bacteriophage ~. was used as Mr markers.

~6 possibilities to examine the structure of the enzyme and to def'me its functionally important regions. This would obviously allow rational design of improved inhibitors of the enzyme.

ACKNOWLEDGEMENTS We are grater'el to Dr. Christophe Roos and Kai Korpela for their indispensable aid in the computer analyses. We wish to thank Mia Bengtstr0m and Maritta Putkiranta for the expert synthesis of oligos, Seija Jarvenpaa for technical assistance and Dr. Eero Castr~n for providing material. We are most grateful to J o h a n Peranen and to Drs. Pentti Pohto, Inge-Britt Lind6n, Ha n s S0derlund and Arja Kallio for the advice and valuable comments during the preparation of the manuscript.

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