DNA AND CELL BIOLOGY Volume 10, Number 3, 1991 Mary Ann Liebert, Inc., Publishers Pp. 181-189

Cloning and Characterization of Human Placental Catechol-O-Methyltransferase cDNA KENNETH

LUNDSTRÖM,

MARJO SALMINEN,* ANU JALANKO, RAIJA SAVOLAINEN, and ISMO ULMANEN

ABSTRACT

Catechol-O-methyltransferase (COMT) cDNA clones were isolated from a human placental cDNA library using synthetic oligonucleotides as probes. AH four positive clones isolated contained an open reading frame,, which potentially coded for a 24.4-kD polypeptide, presumably corresponding to the cytoplasmic

form of the COMT (S-COMT). In addition to the S-COMT sequences, two of the clones carried extensions in the 5' end, which potentially coded for a 50-amino-acid peptide extending the S-COMT reading frame. This sequence contained a stretch of signal sequence-like hydrophobic amino acids in its amino terminus. The deduced human COMT polypeptide had 80% similarity with the previously characterized rat COMT. Expression of one of the cDNA clones in human K-562 cells resulted in cell clones with 3- to 10-fold increased COMT activity. Cell-free translation of transcripts synthesized in vitro from one of the short cDNAs yielded a 26-kD product, similar in size to human S-COMT. Translation of transcripts from one of the long cDNAs gave 30-kD and 26-kD polypeptides, suggesting translation initiation from two different AUG initiation codons. The 30-kD protein, but not the 25-kI) protein, associated with microsomal membranes in translation lysates. A potential polyadenylation signal AATTAA was detected in the 3' ends of two of the clones 265 nucleotides downstream from the COMT translation termination codon. RNA blotting on human placental RNA revealed a 1.5-kb-long COMT-specific transcript. DNA analysis suggested that human, as well as rat, canine and monkey cells have one gene for COMT.

INTRODUCTION

(COMT, 2.1.1.6) Catechol-O-methyltransferase major responsible

EC is one of the three for the enzymes inactivation of catechol hormones, catecholamine neurotransmitters, and many catechol drugs such as l-DOPA, amethyl DOPA, and isoprenaline (Ball et ai, 1972; Guldberg and Marsden, 1975). COMT catalyses the transfer of a methyl group from S-adenosyl-Lmethionine (SAM) to a hydroxyl group of the catechol moiety (Axelrod and Tom-

chick, 1958). COMT activity has been detected in many mammalian tissues, including liver, brain, kidney, gut, uterus, and placenta (Axelrod et ai, 1959; Iisalo and Castren, 1967; Guldberg and Marsden, 1975; Inoue et ai, 1977; Rivett et ai, 1983; Barnea et ai, 1988; Nissinen et ai, 1988). COMT may function as a physiological barrier for cate-

cholamines (Kaplan et ai, 1981). In human placenta, COMT has an important role in inactivating circulating catecholamines (Castren and Saarikoski, 1974; Saarikoski, 1972; Nandakumaran et ai, 1983; Barnea et ai, 1988). Decreased COMT activity in placenta has been detected in pregnancies associated with toxemia and chronic hypertension (Barnea et ai, 1988). Although COMT is predominanty a soluble cytosolic enzyme (S-COMT), a membrane-bound (MB-COMT) activity has been described (White and Wu, 1975; Roth, 1978). MB-COMT has a higher affinity for catechol substrates than S-COMT (Rivett et ai, 1982; Rivett and Roth, 1982). MB-COMT is tightly associated with membranes, and can be released only by solubilization of the membranes by detergent. Even if this could indicate that MB-COMT is an

integral membrane protein, Roth (1984), the mechanism

as suggested by Jeffery and of the membrane association

Orion Corporation, Laboratory of Molecular Genetics, Valimotie 7, 00380 Helsinki, Finland. •Present address: INSERM U129, 24 rue du Faubourg Saint-Jacques 75674 Paris Cedex 14, France.

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et ai, 1977; Tabor and Richardson, 1987). DNA sequence editing, analysis of functional protein domains, and homology comparisons to

chain-termination method (Sanger

of the MB-form is still unknown. S-COMT has been purified from the human placenta (Gugler et ai, 1970; Darmenton et ai, 1976; Tilgmann and Kalkkinen, 1991) and from rat liver (Tilgmann and Kalkkinen, 1990). Recently, the primary structure of the 24.7-kD rat SCOMT was deduced from the DNA sequences of rat liver cDNA and the rat genomic DNA for COMT (Salminen et ai, 1990). Here we report the isolation, expression, and sequence analysis of human placental cDNAs, which define the primary structure of human S-COMT. A comparison of the human and rat S-COMT amino acid sequences reveals 80% similarity between the two species. In vitro transcription translation experiments of the cDNAs produced two COMT polypeptides, one corresponding to S-COMT and the other having a hydrophobic amino terminal extension to the S-COMT sequence. The implications of these observations to the production of two COMT-forms (MBand S-COMT) in vivo from one single gene is discussed.

performed using the PC Gene Inc./Genofit SA, Switsoftware (IntelliGenetics computer zerland). The search for sequence homologies was done in protein (SwissProt) and DNA (EMBL) data banks. rat COMT sequences were

Expression of cDNA

COMT enzyme assay

A human placental cDNA library (Clontech Laboratories Inc., CA) in bacteriophage Xgtll was screened using synthetic oligonucleotides (Lathe, 1985). The two oligonucleotides used (5'-TGCAAGCTTGCGCTGCTCCTTTGTGTCACCC-3' and GA-31) were designed from the amino acid sequence obtained from sequencing tryptic peptides of purified human placental COMT enzyme (Tilgmann and Kalkkinen, 1991) and from the previously known rat COMT cDNA sequence (Salminen et ai, 1990). COMT-positive clones were subcloned into the pGEM-7Zf(+) vector (Promega, Madison WI) prior to sequencing by the dideoxynucleotide

COMT-activity was measured from the cell lysates as described (Schultz et ai, 1989), using reversed-phase HPLC chromatography by monitoring at 254 nm. Briefly, 4 x 106 cells were washed once with phosphate-buffered saline, lysed in 100 ¿d of 1% Nonidet P-40, 0.4 M NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.02% sodium azide by incubating at 0°C for 30 min and centrifuged for 10 min at 12,000 x g and 4°C. The lysates were incubated for 30

5'-GA

Cloning and characterization of human placental catechol-O-methyltransferase cDNA.

Catechol-O-methyltransferase (COMT) cDNA clones were isolated from a human placental cDNA library using synthetic oligonucleotides as probes. All four...
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