Genomics 61, 285297 (1999) Article ID geno.1999.5960, available online at http://www.idealibrary.

com on

Human Indolethylamine N-Methyltransferase: cDNA Cloning and Expression, Gene Cloning, and Chromosomal Localization
Michael A. Thompson,* Eunpyo Moon, , Ung-Jin Kim, Jingping Xu, Michael J. Siciliano, and Richard M. Weinshilboum* ,1
*Department of Pharmacology, Mayo Medical School/Mayo Clinic/Mayo Foundation, Rochester, Minnesota 55905; Division of Biology, California Institute of Technology, Pasadena, California 91125; Department of Biological Sciences, Ajou University, Suwon, Korea; and Department of Molecular Genetics, University of Texas, M.D. Anderson Cancer Center, Houston, Texas 77030
Received May 6, 1999; accepted July 6, 1999

Indolethylamine N-methyltransferase (INMT) catalyzes the N-methylation of tryptamine and structurally related compounds. We recently cloned and characterized the rabbit INMT cDNA and gene as a step toward cloning the cDNA and gene for this enzyme in humans. We have now used a PCR-based approach to clone a human INMT cDNA that had a 792-bp open reading frame that encoded a 263-amino-acid protein 88% identical in sequence to rabbit INMT. Northern blot analysis of 35 tissues showed that a 2.7-kb INMT mRNA species was expressed in most tissues. When the cDNA was expressed in COS-1 cells, the recombinant enzyme catalyzed the methylation of tryptamine with an apparent K m value of 2.9 mM. The human cDNA was then used to clone the human INMT gene from a human genomic BAC library. The gene was 5471 bp in length, consisted of three exons, and was structurally similar to the rabbit INMT gene as well as genes for nicotinamide N-methyltransferase and phenylethanolamine N-methyltransferase in several species. All INMT exonintron splice junctions conformed to the GT-AG rule, and no canonical TATA or CAAT sequences were present within the 5 -anking region of the gene. Human INMT mapped to chromosome 7p15.2p15.3 on the basis of both PCR analysis and uorescence in situ hybridization. Finally, two possible single nucleotide polymorphisms were identied within exon 3, both of which altered the encoded amino acid. The cloning and expression of a human INMT cDNA, as well as the cloning, structural characterization, and mapping of its gene represent steps

toward future studies of the function and regulation of this methyltransferase enzyme in humans. 1999
Academic Press

INTRODUCTION

Supported in part by NIH Grants RO1 GM28157 (R.M.W.), RO1 GM35720 (R.M.W.) and RO1 CA34936 (M.J.S.) as well as DOE Grant DEFC03-97ER62299 (U.J.K.). Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. (human INMT cDNA) AF128846 and AF128847 and (human INMT gene) AF128848. 1 To whom correspondence and reprint requests should be addressed. Telephone: (507) 284-2246. Fax: (507) 284-9111. E-mail: weinshilboum.richard@mayo.edu.

Methylation is an important reaction in the metabolism of many drugs, other xenobiotics, and endogenous molecules (Weinshilboum, 1989; Weinshilboum et al., 1999). The methylation of tryptamine and structurally related compounds by a cytosolic S-adenosyl-Lmethionine (AdoMet) 2-dependent methyltransferase was described in the rabbit lung nearly 40 years ago (Axelrod, 1961). Interest in this activity initially focused on its possible participation in the pathophysiology of neuropsychiatric disease (Axelrod, 1961). Since N,N-dimethyltryptamine is a hallucinogen (Szara, 1956; Axelrod, 1961; Strassman et al., 1994), it seemed possible that the in vivo N-methylation of tryptamine and structurally related indoleamines might be involved in the etiology of schizophrenia or other psychiatric illnesses. However, this area of research proved to be controversialin part because of difculty in consistently detecting tryptamine N-methylation in human tissues. Therefore, we recently cloned the cDNA and gene for rabbit INMT (Thompson and Weinshilboum, 1998). Recombinant rabbit INMT expressed in COS-1 cells catalyzed the methylation of tryptamine, 5-hydroxytryptamine (serotonin), structurally related indoleamines, and -carbolines, and it was inhibited by N,N-dimethyltryptamine, a product of the methylation reaction (Thompson and Weinshilboum, 1998).

Abbreviations used: AdoHcy, S-adenosyl-L-homocysteine; AdoMet, S-adenosyl-L-methionine; BAC, bacterial articial chromosome; DEAE, diethylaminoethyl; DMEM, Dulbeccos modied Eagles medium; DMSO, dimethyl sulfoxide; EST, expressed sequence tag; FCS, fetal calf serum; ORF, open reading frame; PAC, P1 articial chromosome; UTR, untranslated region.

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FIG. 1. Human INMT cDNA cloning strategy. The full- length INMT cDNA is depicted at the top of the diagram. The solid box represents the ORF, and open boxes represent 5 - and 3 -UTRs. RI, RII, and RIII represent the locations of signature amino acid sequences for methyltransferase enzymes. The dashed line represents anchor sequence. Boldface numbers in parentheses on the left represent sequential PCRs that were used to clone the cDNA. The tissue from which template cDNA was obtained is indicated next to the reaction. See text for details.

By making use of our knowledge of rabbit INMT, we have now cloned the human INMT cDNA and gene as the next steps required to study the function of this enzyme in humans. The human INMT cDNA encoded a protein with an amino acid sequence 88% identical to that of rabbit INMT. Recombinant human INMT, like the rabbit enzyme, catalyzed the N-methylation of tryptamine. We also used the human cDNA to clone and structurally characterize the human INMT gene. INMT mapped to chromosome 7p15.2p15.3. Molecular characterization of the cDNA and gene for INMT in humans will now make it possible to study the function and regulation of this methyltransferase enzyme.
MATERIALS AND METHODS
Materials. [ 14C-CH 3]AdoMet (60 mCi/mmol) was obtained from Dupont NEN Products (Boston, MA), and [ - 32P]dCTP (3000 Ci/ mmol) was purchased from Amersham Life Science (Arlington Heights, IL). Ultrapure agarose, restriction enzymes, Superscript II reverse transcriptase, DMEM, and fetal calf serum were obtained from Gibco BRL (Gaithersburg, MD). TA cloning kits (pCR2.1 and

pCR3.1) were purchased from Invitrogen (San Diego, CA). AdoMet HCl, bovine serum albumin, and tryptamine HCl were obtained from Sigma Chemical Co. (St. Louis, MO). Wizard Miniprep and Maxiprep DNA purication systems were purchased from Promega (Madison, WI). A low-molecular-weight protein marker set was obtained from Bio-Rad (Hercules, CA). DEAESepharose CL-6B was purchased from Pharmacia LKB Biotechnology, Inc. (Piscataway, NJ). Human INMT cDNA cloning. The strategy used to clone a human INMT cDNA is depicted schematically in Fig. 1. As a rst step, the rabbit INMT amino acid sequence was used to design degenerate forward (DF4 and DF5) and reverse (DR4 and DR5) primers (Fig. 1, Step 1). The sequences of these and all other primers described subsequently are listed in Table 1. These degenerate primers were used to amplify a region of the human INMT cDNA using human skeletal muscle cDNA as template. This source of template cDNA was chosen on the basis of Northern blot analysis of mRNA from a series of human tissues that was performed with the rabbit INMT cDNA as a probe (data not shown). The amplication product from the initial reaction performed with degenerate primers was sequenced, and that sequence was used to design human INMT-specic primers. DNA sequencing was performed in the Mayo Clinic Molecular Biology Core Facility with an Applied Biosystems Model 377 DNA sequencer. All DNA sequences reported were veried by sequencing both strands. The template for subsequent PCR ampli-

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TABLE 1 Sequences of Primers Used to Clone and Study the Human INMT cDNA and Gene
Primer designation INMT degenerate primers DF4 DF5 DR4 DR5 INMT forward primers F1 F51 F455 F669 INMT reverse primers R240 R271 rabINMT R792 R792 R864 I1R77 Vector primers M13 For M13 Rev T7 Seq primer pCR3.1 REV 5 -RACE anchor primers dC 15-adaptor AP1 AP2 Primer location Primer sequence

Exon Exon Exon Exon Exon Exon Exon Exon

1 2 3 3 1 1 3 3

5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

-TAYTAYTCITTYCARTCIGGICC-3 -ATIGAYATIGGITCIGGICCIAC-3 -GCRCAYTCCATIGCIARIARIGT-3 -GTIACIARRTGICCICCIGGYTT-3 -ATGAAGGGTGGCTTCACTGGG-3 -CAGGGACTACTTGGCTACTTACTA-3 -CTGTGTTGCCTCTCGCCGAC-3 -GGCTGTCCTGGATGCTGGCTTTGA-3 -GTCTTGGAAGGAATCACAGGCAGC-3 -GGTTGCGGTCGGTAAAGTCGG-3 -TCAGGACCCCGGCTTCTTGC-3 -TCAGGGCCCAGGCTTCTTGCGA-3 -CCTCACCCCTCTCTAGCATACAG-3 -CCCCAGGACACCGTCTCCCCA-3 -TCCCAGTCACGACGT-3 -AACAGCTATGACCATG-3 -TAATACGACTCACTATAGGG-3 -TAGAAGGCACAGTCGAGG-3

Exon 2 Exon 2 Exon 3 Exon 3 Exon 3 Intron 1

5 -AAAAGATCTGTCGACCCCCCCCCCCCCCC-3 5 -CCATCCTAATACGACTCACTATAGGGC-3 5 -ACTCACTATAGGGCTCGAGCGGC-3

Note. D, degenerate; F, forward, R, reverse; and I, for intron. Numbers in primer designations indicate nucleotide positions, with 1 used to designate either the A in the cDNA translation initiation codon or the rst nucleotide in an intron. IUPAC codes are used to refer to bases listed in primer sequences. cations was either human skeletal muscle cDNA or human placental cDNA. The placental cDNA was synthesized from the corresponding poly(A) RNA (Clontech, Palo Alto, CA) with Superscript II reverse transcriptase or was purchased as human placental Marathon RACE-Ready cDNA (Clontech). Thermus aquaticus (Taq) DNA polymerase was used to perform these amplications in a PerkinElmer GeneAmp PCR System 2400 thermal cycler (Foster City, CA). The reason for using both placental and skeletal muscle cDNA to perform these experiments is discussed subsequently. Amplication products from all of these PCRs were either sequenced directly or sequenced after cloning into pCR2.1 Additional open reading frame (ORF) sequence was obtained by performing a reaction with the human INMT-specic primer F1 paired with rabbit INMT primer R792 (Fig. 1, Step 3), and the remainder of the human INMT cDNA sequence was obtained by performing 5 - and 3 -RACE with human INMTspecic primers paired with appropriate anchor primers (AP1, AP2, or a dC 15-adaptor primer) (Table 1 and Fig. 1). 5 -RACE was performed with an anchor generated by using terminal transferase to add guanine nucleotides to rst-strand cDNA (Fig 1, Steps 2 and 5). When 3 -RACE was performed with human skeletal muscle cDNA as template, the reaction was unsuccessful. Therefore, 3 -RACE and 5 -RACE were also performed with human placental Marathon RACE-Ready cDNA (Clontech) (Fig. 1, Steps 4 and 5). Finally, the entire ORF and a portion of the 3 -untranslated region (UTR) were amplied in a single reaction (Fig. 1, Step 6) with placental cDNA as template to conrm the ORF sequence that had been obtained during the individual reactions. Human INMT Northern blot analysis. Human multiple tissue Northern blots (Clontech) were used to perform Northern analyses. Each lane contained approximately 2 g of poly(A) RNA, and the probe consisted of nucleotides 1 to 767 of the human INMT cDNA that had been radioactively labeled with [ - 32P]dCTP by random priming performed with the Oligolabeling kit (Pharmacia). The Northern blots were then stripped and probed with human -actin cDNA as a control for mRNA loading. Human INMT COS-1 cell expression. The human INMT cDNA ORF was amplied with Taq DNA polymerase and primers F1 and R792. The 792-bp amplication product obtained during this reaction was cloned into the eukaryotic expression vector pCR3.1, and the insert in this expression construct was sequenced on both strands to ensure that alterations in sequence had not been introduced during the amplication. Four micrograms of the expression construct was then used to transfect COS-1 cells with the DEAE dextran technique (McCutchan and Pagano, 1968) as described elsewhere (Aksoy et al., 1994). The cells were harvested, and a cytosol preparation was prepared as described previously (Rini et al., 1989). These preparations were used to study recombinant human INMT enzymatic activity. Recombinant human INMT was partially puried by DEAE Sepharose CL-6B anion-exchange chromatography using a modication of the procedure described by Thompson and Weinshilboum (1998). Specically, 5-ml fractions were collected from a 9.5 cm 1.6 cm column with a total volume of 76 ml at a ow rate of 2 ml/min. Twelve fractions were collected with only running buffer (1 mM EDTA in 50 mM Tris, pH 7.3), followed by a gradient from 0.1 to 0.4 M KCl in the running buffer. INMT enzyme assay. A radiochemical assay was used to measure INMT activity (Thompson and Weinshilboum, 1998). The assay utilized [ 14C-CH 3]AdoMet as methyl donor and tryptamine as the methyl acceptor substrate. The formation of 14C-methylated tryptamine was determined after incubation for 60 min at 37C in a 200- l volume that contained, unless otherwise stated, 50 mM Tris HCl, pH 8.5; 34 M [ 14C-CH 3]AdoMet (24 Ci/ mol); 250 g/ml bovine serum albumin; and 8 mM tryptamine. The reaction was terminated by the addition of 0.5 ml of 0.5 M potassium borate, pH 10, followed by extraction of the methylated product into 5 ml of 3%

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FIG. 2. Human INMT Northern blot analysis. Human multiple tissue Northern blots (2 g of poly(A) RNA/lane, Clontech) were probed with the human INMT cDNA ORF (A and C). The same blots were also probed with human -actin cDNA as a control (B and D). Exposure times are indicated beneath the blots.

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FIG. 3. Recombinant human INMT Western blot analysis. Two different alleles for human INMT were transiently expressed in COS-1 cells, and 100,000g supernatant preparations were used to perform Western blot analysis after SDSPAGE. INMT was detected with an antibody directed against the C-terminus of the protein. isoamyl alcohol in toluene. After centrifugation, 3.5 ml of the organic phase was added to 5 ml of BioSafe II (Research Products International Corp., Mount Prospect, IL) prior to determination of the level of radioactivity. Protein concentrations were measured with the dye binding assay of Bradford (1976) with bovine serum albumin as a standard. Human INMT Western analysis. The amino acid sequence of the protein encoded by human INMT cDNA was analyzed for hydrophilicity, surface probability, and antigenic index by the Mayo Research Resource Protein Core Facility using the sequence analysis software package of the Genetics Computer Group (Madison, WI) before portions of the sequence were selected to use in the generation of antibodies. Peptides corresponding to human INMT amino acids 124 (N-terminal antibody) and 237263 (C-terminal antibody), with an additional cysteine residue added at the C-terminus for peptide 124 and at the N-terminus for peptide 237263, were synthesized on the basis of those analyses. The sequences of these peptides were compared with those in the GenBank nonredundant and human EST databases and were not found to be identical to the sequences of any known proteins. The synthetic peptides were then conjugated to keyhole limpet hemocyanin and were used to generate rabbit polyclonal antibodies (Cocalico Biologicals, Inc., Reamstown, PA). The rabbit antiserum was then used to perform Western blot analysis with cytosolic proteins that had been subjected to SDSPAGE (Towbin et al., 1979). Bound antibody was detected by use of the ECL Western blotting system (Amersham) as described in detail elsewhere (Aksoy et al., 1993). Human INMT gene cloning. Nucleotides 1 to 767 of the human INMT cDNA were used to probe an arrayed human genomic DNA bacterial articial chromosome (BAC) library (fourfold coverage of the human genome). Taking into account the presence of introns, the cDNA probe was fragmented by AluI digestion prior to random hexamer labeling. As a result of using this probe, seven positive clones were obtained. These clones were further conrmed by Southern hybridization, and all of them contained a 7.5-kb HindIII fragment corresponding to the cDNA probe. DNA from BAC clone 2018A05 was puried by use of the Qiagen Plasmid Maxiprep kit (Qiagen, Inc., Chatsworth, CA), and the structure of the gene was determined by using primers designed on the basis of the human INMT cDNA sequence to sequence directly BAC DNA with BigDye terminator chemistry. Human INMT chromosomal localization. The chromosomal location of human INMT was determined by performing the PCR with

template DNA from NIGMS Human/Rodent Somatic Cell Hybrid Panels 1 and 2 (Coriell Institute for Medical Research, Camden, NJ). Exonbased primer F51 and intron-based primer I1R77 were used to perform these reactions. Those experiments served to localize the gene to human chromosome 7. Sublocalization was then accomplished by the use of two-color uorescence in situ hybridization (FISH) performed with metaphase preparations of normal human lymphoblastoid cells (Marlton et al., 1995). Digoxigenin-labeled BAC 2018A05 and biotin-labeled chromosome 7 DNA were used as probes to perform these experiments. The chromosome 7 probe was generated by inter-Alu PCR (Liu et al., 1993) performed with a somatic cell hybrid monochromosomal for human chromosome 7 (cell line 1HL11, Coriell Institute). The biotinlabeled probe was visualized by using FITC-avidin (green uorescence) and anti-digoxigen antibody obtained from Oncor (Gaithersburg, MD). The digoxigenin-labeled probe was detected with anti-digoxigenin antibody (Oncor) labeled with rhodamine (red uorescence) and rabbit anti-sheep antibody as suggested by the manufacturer. The slides were counterstained with propidium iodide/antifade or DAPI/antifade and were examined with an Olympus uorescence microscope equipped with red- and green-pass lters. No electronic amplication or correction of the signal was employed. Data analysis. The University of Wisconsin Genetics Computer Group (GCG) software package Version 9.1 (Devereux et al., 1984) was used to analyze DNA and protein sequences. The TFSITES transcription factor database, Release 7.5 (Ghosh, 1990), was used to identify sequence motifs that might be involved in transcription initiation or regulation. Apparent K m values were calculated by the method of Wilkinson (1961) with a computer program written by Cleland (1963).

RESULTS

Human INMT cDNA Cloning A PCR-based strategy was used to clone a human INMT cDNA as described under Materials and Methods. The human cDNA had a 792-bp ORF that encoded a 263-amino-acid protein 88% identical to the sequence of rabbit INMT with a calculated M r value of 29,000. The initial ATG in the human cDNA was present within a translation initiation consensus sequence (Kozak, 1987). Direct sequencing of a 5 -RACE PCR product obtained with skeletal muscle cDNA as template showed that the 5 -UTR was 16 bp in length. That length was conrmed by the analysis of seven 5 -RACE clones, three of which had 5 -UTRs that were 16 bp, three that were 14 bp, and one that was 13 bp in
TABLE 2 Apparent K m Values for Recombinant Human and Rabbit INMT
Recombinant enzyme source Rabbit Human Human Codon 205 Met Val Met Codon 219 Glu Glu Gly Tryptamine apparent K m (mM) 0.27 2.92 2.92 0.05 0.07 0.24 AdoMet apparent K m ( M) 22 28 23 5 5 3

Note. All values are expressed as means SEM for triplicate determinations. Apparent K m values for AdoMet were determined with 8 mM tryptamine as the methyl acceptor substrate for the human enzyme. Apparent K m values for the rabbit enzyme are those reported by Thompson and Weinshilboum (1998).

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FIG. 4. Human INMT gene nucleotide and deduced amino acid sequences. Intron, anking region sequences, and possible SNPs are shown in lowercase, while exon sequences are shown in uppercase and are boxed. The deduced amino acid sequence of the encoded protein is shown in single-letter code beneath the nucleotide sequence. Changes in amino acids encoded by codons containing possible SNPs are indicated in parentheses. Methyltransferase enzyme signature sequences for regions I, II, and III are underlined, and a polyadenylation signal is shown in boldface type. A possible Sp1 site within the 5 -anking region is boxed.

length. 5 -RACE was also performed with human placental cDNA, and a 16-bp 5 -UTR was also found in that tissue both by directly sequencing the PCR amplication product and by analyzing 11 clones, one of which had a 17-bp 5 -UTR, seven were 16 bp, and three

were 14 bp in length. The 5 -UTR of the rabbit lung INMT cDNA was 15 bp in length (Thompson and Weinshilboum, 1998). 3 -RACE performed with human placental cDNA revealed a 3 -UTR length of 1758 bp. We also obtained preliminary evidence for the possible

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FIG. 4Continued

presence of single nucleotide polymorphisms (SNPs) within the cDNA ORF based on cDNA data, information obtained during our subsequent gene cloning studies, and the sequence of a genome survey sequence clone that we were able to identify as INMT after we had cloned the human cDNA. Specically, nucleotide 613 could be either A or G, resulting in amino acid 205 being either Met or Val, respectively, while nucleotide 656 could be either G or A, resulting in amino acid 219 being either Gly or Glu, respectively. These SNPs were

observed in the combinations 613A/656G (gene and cDNA clone), 613G/656A (cDNA clone), and 613A/656A (genome survey sequence clone AQ182801 and PAC clone DJ1131617, GenBank Accession No. AC006022). Obviously, the existence and frequencies of these possible alleles for human INMT will have to be conrmed in the course of future, larger population studies. GenBank accession numbers for the human INMT cDNA are AF128846 (613G/656A) and AF128847 (613A/ 656G).

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FIG. 5. INMT uorescence in situ hybridization on human chromosome 7. The arrow indicates the location of the centromere. See text for details.

approximately 29,000 as determined by SDSPAGE (Fig. 3). This recombinant protein was detected by antibodies directed against both the N- and the Ctermini of human INMT. Both of these recombinant proteins, after partial purication by DEAE anion-exchange chromatography, were then tested for their ability to catalyze the methylation of tryptamine, the prototypic substrate for this enzyme in rabbit lung (Axelrod, 1961; Thompson and Weinshilboum, 1998). Mock- transfected COS-1 cells failed to demonstrate detectable INMT activity, but preparations from cells transfected with cDNAs that encoded both recombinant human allozymes catalyzed the methylation of tryptamine. When eight concentrations of tryptamine that varied from 0.03 to 4.0 mM were tested, apparent K m values of both recombinant proteins for tryptamine were identical (Table 2). However, those K m values were approximately an order of magnitude higher than that of recombinant rabbit INMT (Table 2). Apparent K m values for AdoMet were very similar for both human INMT allozymes and rabbit INMT when ve concentrations of AdoMet that varied from 0.75 to 12 M were studied (Table 2). Human INMT Gene Cloning The human INMT cDNA ORF was used as a probe to screen a human genomic DNA BAC library with fourfold coverage of the human genome. Seven positive clones were identied. One of those clones was sequenced and found to contain the entire human gene. The structure of the human INMT gene was similar to that of the rabbit INMT gene. The human gene was 5471 bp in length and consisted of three exons with an initial 1426-bp intron and a second intron that was 1479 bp in length (Fig. 4). All exon intron splice junction sequences conformed to the GT-AG rule (Mount, 1982). The 5 -anking region of the gene did not contain a canonical TATA box. However, a possible initiator (Inr) sequence was present at the site of transcription as determined by 5 RACE. Specically, the human gene contained a sequence (GGC/ACATTT) at the 5 -anking/5 -UTR interface that was 67% identical to the canonical Inr motif (Smale and Baltimore, 1989). An Sp1 motif was also located approximately 220 bp upstream from the site of transcription initiation (Fig. 4). The GenBank accession number for the human INMT gene is AF128848. After we had completed the cloning and characterization of both the human INMT cDNA and the gene, the sequence of a PAC clone (PAC clone DJ1131617, GenBank Accession No. AC006022) was deposited in the database that was identied as containing a human thioether S-methyltransferaselike gene. That gene was human INMT, but it differed from our gene sequence at cDNA nucleotide 761 (G 3 T, resulting in Cys254 3 Phe).

Human INMT Northern Analysis Northern blot analysis of 35 human tissues performed with the human INMT cDNA as a probe demonstrated the expression in most tissues of a transcript approximately 2.7 kb in length (Fig. 2), a length compatible with a 16-bp 5 -UTR, a 792-bp ORF, and a 1758-bp 3 -UTRplus a poly(A) tract. mRNA expression appeared to be highest in the thyroid, in adrenal gland, and in both adult and fetal lung. Very low or absent expression was seen in the adult brain, spleen, thymus, peripheral blood leukocytes, and bone marrow, as well as fetal brain, liver, and kidney (Fig. 2). It was of interest that, even though this mRNA was not expressed in either total brain preparations or tissue obtained from specic sublocalizations within the brain, it was well expressed in the spinal cord. The possible functional implications of that observation should be explored in the future. In the rabbit, Northern blot analysis had demonstrated that, in the three tissues studied, INMT mRNA was present in the rank order of lung liver brain (Thompson and Weinshilboum, 1998). Human INMT COS-1 Cell Expression The ORFs encoded by two potential human INMT alleles, those that encoded Val205-Glu219 and Met205-Gly219, were cloned into the eukaryotic expression vector pCR3.1, and those expression constructs were used to transfect COS-1 cells. Western analysis demonstrated that the transfected cells expressed recombinant human INMT with an M r value of

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FIG. 6. Small molecule methyltransferase gene family amino acid sequence alignment. Amino acid sequences are shown for members of the INMT small molecular methyltransferase enzyme family, including human (h)INMT (this report), rabbit (rab)INMT (Thompson and Weinshilboum, 1998), mouse (m)TEMT (Warner et al., 1995), hNNMT (Aksoy et al., 1994), mNNMT (Yan et al., 1997), and PNMT from four different species (Baetge et al., 1986; Batter et al., 1988; Morita et al., 1992; Suh et al., 1994), with bovine abbreviated b. Amino acid sequences were analyzed by the use of the PILEUP program, Version 8.0 (Feng and Doolittle, 1987) from the GCG package (Devereux et al., 1984). Amino acids that are conserved in seven of the eight proteins are shown as white type against a black background and in capital letters on the consensus line, and amino acids conserved in all proteins except PNMT are shown against a gray background and as lowercase letters on the consensus line. Regions I, II, and III are boxed. Changes in amino acid sequence resulting from the presence of possible SNPs in hINMT are shown in parentheses above the hINMT sequence.

Human INMT Chromosomal Localization The chromosomal location of human INMT was determined by performing the PCR with INMT genespecic primers F51 and I1R77 and with NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panels

1 and 2 (Coriell Institute for Medical Research). Those studies indicated that INMT was located on human chromosome 7. The gene was then mapped to 7p15.2p15.3 by FISH analysis. Specically, red uorescing BAC hybridization was specic to the border of the R-band at 7p15.3 and the G-band at 7p15.2

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FIG. 6Continued

in all 10 metaphases analyzed, placing INMT at 7p15.2p15.3 (Fig. 5). The mapping of the human INMT gene to 7p was supported by the fact that the PAC clone referred to earlier mapped to 7p14 p15.1. Comparison with Other Methyltransferase Enzymes and Genes The amino acid sequence encoded by INMT was compared with those of other known cytosolic small molecule methyltransferase enzymes. Human INMT was 88, 58, 53, 53, 39, 38, 36, and 36% identical with the amino acid sequences of rabbit INMT (Thompson and Weinshilboum, 1998), mouse thioether methyltransferase (TEMT) (Warner et al., 1995), human and mouse nicotinamide methyltransferase (NNMT) (Aksoy et al., 1994; Yan et al., 1997), and bovine, human, mouse, and rat phenylethanolamine N-methyltransferase (PNMT) (Baetge et al., 1986; Batter et al., 1988; Morita et al.,

1992; Suh et al., 1994), respectively. A NCBI protein BLAST (blastp) search performed with the human INMT peptide sequence and the Caenorhabditis elegans nonredundant (nr) database revealed homology to three hypothetical C. elegans proteins (Accession Nos. 283554, 4008393, and 365682). The genes encoding those hypothetical proteins had been identied by use of the Genender program (P. Green, University of Washington). These three proteins were from 20 to 28% identical in amino acid sequence with human INMT. However, none of these three hypothetical proteins was present in EST clones in the C. elegans database. Ingrosso et al. (1989) have reported that enzymes that utilize AdoMet as a cosubstrate often contained three regions of high sequence homology, which they designated regions I, II, and III. Others subsequently extended this analysis (Gomi et al., 1992; Wu et al.,

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TABLE 3 Conservation of Splice Junction Locations and Sequences in a Family of Related Methyltransferase Genes
Exon I hINMT Exon II Exon III

...TTCGGCCCTG]gtgagc...cacag[GAGGCCTCCAA...GAAGGAAACAG]gtaggg...cgcag[CGGCCGATGGGAG... F G P G G L Q E G N S G R W E rabINMT ...TTCGGCCCTG]gtgagc...cacag[GCGGCCTCCAA...GAAGGGAACAG]gtaggt...tgcag[TGGCCGGTGGCAG... F G P G G L Q E G N S G R W Q hNNMT ...TTCTGCCTAG]gtagag...tccag[ACGGTGTGAAG...GAAGGGAACAG]gtagag...ttcag[AGTCAAGGGTCCA... F C L D G V K E G N R V K G P mNNMT ...TTCTGCCTGG]gtaagt...cccag[GTGCTGTAAAG...GAAGGCAACAG]gtagag...ttcag[AATGAAGGGACCT... F C L G A V K E G N R M K G P hPNMT ...TTCGCCACCG]gtgagc...cccag[GTGAAGTGTCC...GAGGGCAAGGG]gtaagg...cacag[GGAATGCTGGCAG... F A T G E V S E G K G E C W Q bPNMT ...TTCGCCACCG]gtgagc...tccag[GTGAGGTGTCT...GAGGGCAAGGG]gtaagg...aacag[GGAATCCTGGCAG... F A T G E V S E G K G E S W Q rPNMT ...TTTGCCACCG]gtgagc...accag[GTGAGGTGTCT...GAGGACAAGGG]gtgaga...tacag[AGAGTCCTGGCAG... F A T G E V S E D K G E S W Q mPNMT ...TTTGCTACCG]gtgagc...accag[GTGAGGTGTCG...GAGGACAAGGG]gtgaga...tacag[TGAGTCCTGGCAG... F A T G E V S E D K G E S W Q Donor consensus sequence: A 62G 77 G 100T 100A 60A 74G 84T 50 Acceptor consensus sequence: Y nNC 78A 100G 100 G 55

Note. Abbreviations for enzyme names are those used in the legend to Fig. 6. Exon and encoded amino acid sequences are shown in uppercase and intron sequences are in lowercase. Splice donor and acceptor consensus sequences are those described by Padgett et al. (1986).

1992; Fujioka, 1992; Kagan and Clarke, 1994; Niewmirzycka and Clarke, 1999), and in this paper we have chosen to use the nomenclature of Kagan and Clarke (1994). Djordjevic and Stock (1997) have suggested that these regions might reect the common origin of these enzymes rather than a necessary requirement for AdoMet binding. Human INMT contained areas with homology to regions I, II, and III, which were located between amino acids 59 and 67, 155 and 162, and 186 and 195, respectively (Fig. 6). The degree of sequence conservation within region II was not as great as was that for regions I and III in this particular family of methyltransferase enzymes (Fig. 6). The close relationship of these proteins based on amino acid sequence identity was supported by a comparison of the structures of the genes that encoded the proteins. Although the gene for mouse TEMT has not been cloned, gene structures have been reported for rabbit INMT (Thompson and Weinshilboum, 1998), for NNMT in two species (Aksoy et al., 1994; Yan et al., 1998), and PNMT in four species (Baetge et al., 1988; Batter et al., 1988; Morita et al., 1992; Suh et al., 1994). All of those genes have three exons with a central exon that is 208 bp in length. Furthermore, exonintron splice junctions interrupt codons at the same position in all of these genes (Table 3).
DISCUSSION

Methylation is an important pathway in the biotransformation of both exogenous and endogenous molecules (Weinshilboum, 1989; Weinshilboum et al., 1999). Tryptamine N-methylating activity has been detected in a variety of tissues and species (Axelrod, 1961, 1962; Saavedra and Axelrod, 1972; Mandel et al.,

1972; Narasimhachari et al., 1972; Saavedra et al., 1973; Wyatt et al., 1973; Mandell et al., 1974; Bhikharidas et al., 1975; Raisanen and Karkkainen, 1978; Porta et al., 1979; Kennedy et al., 1995), but this activity has been best characterized biochemically in the rabbit lung. Therefore, a cDNA and gene encoding a rabbit tryptamine N-methyltransferase, INMT, were recently cloned and characterized (Thompson and Weinshilboum, 1998). In the present study, we set out to clone the cDNA and gene for the human orthologue of this rabbit gene as steps toward understanding the function and regulation of INMT in humans. A PCRbased approach was used to clone a human INMT cDNA with a 792-bp ORF that encoded a 263-aminoacid protein with a predicted M r value of 29,000. Northern blot analysis indicated that an INMT mRNA transcript was expressed in many human tissues but not in brain (Fig. 3). Therefore, even though interest in INMT was originally related to its possible role in neurotransmitter metabolism or hallucinogen biosynthesis in vivo (Axelrod, 1961), our Northern blot results make that possibility less likely. Expression of two human INMT allozymes showed that both catalyzed the methylation of tryptamine with similar apparent K m values for both the methyl acceptor and the donor cosubstrates (Table 2)thus conrming functionally that this cDNA encoded the human orthologue of rabbit INMT. We also cloned and structurally characterized the human INMT gene, a gene that mapped to human chromosome 7p15.2p15.3 by FISH analysis (Fig. 5). The structure of INMT was very similar to those of genes for a family of related small molecule methyltransferase enzymes. INMT is clearly a member of this growing familya family that currently also includes TEMT, NNMT, and PNMT. That conclusion is

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THOMPSON ET AL. and structure of the gene encoding bovine phenylethanolamine N-methyltransferase. J. Neurosci. Res. 19: 367376. Bhikharidas, B., Mann, L. R. B., and McLeod, W. R. R. (1975). Indolamine N-methyltransferase activity in human tissues. J. Neurochem. 24: 203205. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72: 248 254. Cleland, W. W. (1963). Computer programmes for processing enzyme kinetic data. Nature 198: 463 465. Devereux, J., Haeberli, P., and Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387395. Djordjevic, S., and Stock, A. M. (1997). Crystal structure of the chemotaxis receptor methyltransferase CheR suggests a conserved structural motif for binding S-adenosylmethionine. Structure 5: 545558. Feng, D.-F., and Doolittle, R. F. (1987). Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J. Mol. Evol. 25: 351360. Fujioka, M. (1992). Mammalian small molecule methyltransferases: Their structural and functional features. Int. J. Biochem. 24: 19171924. Ghosh, D. (1990). A relational database of transcription factors. Nucleic Acids Res. 18: 1749 1756. Gomi, T., Tanihara, K., Takayasu, D., and Fujioka, M. (1992). Rat guanideoacetate methyltransferase: Mutation of amino acids within a common sequence motif of mammalian methyltransferase does not affect catalytic activity but alters proteolytic susceptibility. Int. J. Biochem. 24: 1639 1649. Ingrosso, D., Fowler, A. V., Blebau, A. V., and Clarke, S. (1989). Sequence of the D-aspartyl/L-isoaspartyl protein methyltransferase from human erythrocytes. J. Biol. Chem. 264: 2013120139. Kagan, R. M., and Clarke, S. (1994). Widespread occurrence of three sequence motifs in diverse S-adenosylmethionine-dependent methyltransferases suggests a common structure for these enzymes. Arch. Biochem. Biophys. 310: 417 427. Kennedy, B., Bigby, T. D., and Ziegler, M. G. (1995). Nonadrenal epinephrine-forming enzymes in humans. Characteristics, distribution, regulation, and relationship to epinephrine levels. J. Clin. Invest. 95: 2896 2902. Kozak, M. (1987). An analysis of 5 -noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15: 8125 8148. Liu, P., Siciliano, J., Seong, D., Craig, J., Zhao, Y., de Jong, P. J., and Siciliano, M. J. (1993). Dual Alu-PCR primers and conditions for isolation of human chromosome painting probes from hybrid cells. Cancer Genet. Cytogenet. 65: 9399. Mandel, L. R., Ahn, H. S., VandenHeuvel, W. J. A., and Walker, R. W. (1972). Indoleamine-N-methyltransferase in human lung. Biochem. Pharmacol. 21: 11971200. Mandell, A. J., Knapp, S., and Hsu, L. L. (1974). Some factors in the regulation of central serotonergic synapses. Life Sci. 14: 117. Marlton, P., Claxton, D. F., Liu, P., Estey, E., Beran, M., Le Beau, M. M., Testa, J. R., Collins, F. S., Rowley, J. D., and Siciliano, M. J. (1995). Molecular characterization of 16p deletions associated with inversion 16 denes the critical fusion for leukemogenesis. Blood 85: 772779. McCutchan, J. H., and Pagano, J. S. (1968). Enhancement of the infectivity of simian virus 40 deoxyribonucleic acid with diethylaminodextran. J. Natl. Cancer Inst. 41: 351357. Morita, S., Kobayashi, K., Hidaka, H., and Nagatsu, T. (1992). Organization and complete nucleotide sequence of the gene encoding mouse phenylethanolamine N-methyltransferase. Mol. Brain Res. 13: 313319. Mount, S. M. (1982). A catalogue of splice junction sequences. Nucleic Acids Res. 10: 459 472.

supported both by comparisons of amino acid sequence and by the highly homologous gene structures, including conservation of the locations of splice junctions (Table 3). Whether other members of this emerging gene family remain to be discovered is currently unknown. The apparent K m value of recombinant human INMT for tryptamine of 2.9 mM can be compared to values of 1.2 mM (Mandel et al., 1972) and 0.43 mM (Raisanen and Karkkainen, 1978) that have been reported by others for human tryptamine N-methyltransferase activity in human lung preparations. However, the functional role of INMT in human tissue remains unclear. The fact that the apparent K m value of the human enzyme for tryptamine is approximately an order of magnitude higher than that of rabbit INMT (0.27 mM) (Thompson and Weinshilboum, 1998) makes it even less likely that tryptamine is an endogenous substrate for INMT in humans. However, the cloning and expression of a human INMT cDNA as well as the cloning and structural characterization of its gene represent important steps toward an eventual understanding of the function and regulation of this methyltransferase enzyme in humans.
ACKNOWLEDGMENTS
We thank Dr. Daniel McCormick in the Mayo Research Resource Protein Core for designing the peptides used to generate antibodies, Diane Otterness for performing the human multiple tissue Northern blot experiments, Thomas Wood for assistance with the transient expression experiment, and Luanne Wussow for assistance with the preparation of the manuscript.

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