Expression of recombinant soluble and membrane-bound catechol O-methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain.

The rat and human recombinant soluble and membrane-bound catechol O-methyltransferase (S- and MB-COMT, respectively) were expressed using mammalian and baculovirus vectors. Low levels of rat and human S-COMT polypeptides were detected by immunoprecipitation in K-562 cell lines transfected with the S-COMT vectors. From K-562 cells transfected with the rat MB-COMT construct, both S- and MB-COMT recombinant proteins were detected by a rat COMT-specific anti-serum. Infection of lepidopteran Spodoptera frugiperda cells with recombinant S- or MB-COMT baculovirus constructs yielded high amounts of enzymically active and immunoreactive S- or MB-COMT proteins, respectively. Pulse/chase experiments with [35S]methionine-labelled insect cells infected with the MB-COMT baculovirus showed that the 30-kDa recombinant human MB-COMT polypeptide was not processed into the 25-kDa S-COMT form. Subcellular fractionations of insect cells, followed by immunoblotting with COMT antiserum, showed that recombinant S-COMT was found only in the soluble, cytoplasmic fraction, whereas MB-COMT resided both in soluble and membrane fractions. The recombinant MB-COMT sedimented in Percoll gradients at the density of 1.042 g/ml cosedimenting with the plasma-membrane marker. Fractionation and immunoblotting experiments on homogenized total rat brains indicated that the rat S-COMT (24 kDa) and some of the rat MB-COMT (28 kDa) was recovered in soluble fractions, whereas the microsomal material having COMT activity contained the MB-COMT polypeptide. The rat brain microsomal MB-COMT had a density of 1.042 g/ml in Percoll gradients, cosedimenting with the plasma-membrane and rough-endoplasmic-reticulum marker enzymes. The meta/para methylation ratio of dihydroxybenzoic-acid substrate by different recombinant and rat brain COMT-containing subcellular fractions was analysed.     

Purification methods of mammalian catechol-O-methyltransferases

The protein purification strategies used for obtaining homogeneous rat and human soluble catechol-O-methyltransferase (S-COTM) polypeptides are reviewed. Expression and purification of recombinant rat and human S-COMT in Escherichia coli and for human S-COMT in baculovirus-infected insect cells made it possible to elucidate the S-COMT polypeptides in more detail. The application of these purification methods has allowed the crystallization of the rat S-COMT protein and the analysis of the kinetic properties of the enzyme in great detail. The availability of the pure S-COMT protein together with the structural data has also greatly enhanced the development of more potent COMT inhibitors.     

Differential expression and activity of catechol-O-methyl transferase (COMT) in a generalist (Neotoma albigula) and juniper specialist (Neotoma stephensi) woodrat

Mammalian herbivores, particularly dietary specialists must have an efficient means to metabolize the high doses of plant secondary compounds they consume. We found previously that Neotoma stephensi, a juniper specialist, upregulated catechol-O-methyl transferase (COMT) mRNA almost seven fold in response to an ecologically relevant diet (70% juniper). To further investigate the relevance of this enzyme with respect to juniper metabolism, we compared the protein expression, activity and kinetics of the two forms of COMT, soluble (S-COMT) and membrane bound (MB-COMT), in the blood, kidneys and liver of N. stephensi on its natural juniper diet to that of N. stephensi fed an experimental diet of 70% juniper as well as a non-toxic control diet under laboratory conditions. In addition, we compared these results to that of Neotoma albigula, a generalist species, which consumes a diet of 25% juniper in the wild. The specialist consuming juniper under both field and laboratory conditions had increased S-COMT expression and activity in their livers and kidneys, and increased S-COMT activity in their blood compared to the specialist and generalist fed the control diet. The specialist showed expression and activity of S-COMT in their kidneys that was as high as or higher than that in their livers. The generalist had an elevated V_max for MB-COMT compared to the specialist that resulted in higher activity for MB-COMT than the specialist despite lower expression of MB-COMT in the generalist's livers and kidneys. This high activity MB-COMT may be in part responsible for differences in the behaviors of the generalist compared to the specialist. We conclude that S-COMT is important in the specialist's ability to consume high levels of juniper.     

Catechol-O-methyltransferase: effects of the val108met polymorphism on protein turnover in human cells

A single nucleotide polymorphism in the human COMT (catechol-O-methyltransferase) gene has been associated with increased risk for breast cancer and several CNS diseases and disorders. The G to A polymorphism causes a valine (val) to methionine (met) substitution at codon 108 soluble - (S)/158 membrane - (MB)-COMT, generating alleles encoding high and low-activity forms of the enzyme, COMT H and COMT L, respectively. Tissues and cells with a COMT LL genotype have decreased COMT activity compared to COMT HH cells. Previously, we reported that the decreased activity was due to decreased amounts of S-COMT L protein in human hepatocytes. In this study, we investigated the role of S-COMT protein synthesis and turnover as determinates of reduced COMT protein in COMT LL compared to COMT HH cells. No association between S-COMT protein synthesis and COMT genotype was detected. Using a pulse-chase protocol, the half-life of S-COMT H was determined to be 4.7 days, which was considerably longer than expected from the half-lives of other phase 2 enzyme proteins. The half-life of S-COMT L compared to S-COMT H protein was significantly shorter at 3.0 days, but the difference was affected by the medium used during the chase period. These results suggest that increased turnover may contribute to reduced COMT activity in cells and tissues from COMT LL individuals. Subtle differences appear to be able to affect the stability of the S-COMT L protein, and this may contribute to the differences observed in epidemiological studies on the association of this polymorphism with breast cancer risk.     

Orientation and cellular distribution of membrane-bound catechol-O-methyltransferase in cortical neurons: implications for drug development

Catechol-O-methyltransferase (COMT) is a key enzyme for inactivation and metabolism of catechols, including dopamine, norepinephrine, caffeine, and estrogens. It plays an important role in cognition, arousal, pain sensitivity, and stress reactivity in humans and in animal models. The human COMT gene is associated with a diverse spectrum of human behaviors and diseases from cognition and psychiatric disorders to chronic pain and cancer. There are two major forms of COMT proteins, membrane-bound (MB) COMT and soluble (S) COMT. MB-COMT is the main form in the brain. The cellular distribution of MB-COMT in cortical neurons remains unclear and the orientation of MB-COMT on the cellular membrane is controversial. In this study, we demonstrate that MB-COMT is located in the cell body and in axons and dendrites of rat cortical neurons. Analyses of MB-COMT orientation with computer simulation, flow cytometry and a cell surface enzyme assay reveal that the C-terminal catalytic domain of MB-COMT is in the extracellular space, which suggests that MB-COMT can inactivate synaptic and extrasynaptic dopamine on the surface of presynaptic and postsynaptic neurons. Finally, we show that the COMT inhibitor tolcapone induces cell death via the mechanism of apoptosis, and its cytotoxicity is dependent on dosage and correlated with COMT Val/Met genotypes in human lymphoblastoid cells. These results suggest that MB-COMT specific inhibitors can be developed and that tolcapone may be less hazardous at low doses and in specific genetic backgrounds.     

Immunoaffinity purification and partial amino acid sequence analysis of catechol-O-methyltransferase from pig liver

Monoclonal antibodies (mAbs) against the soluble form (S-COMT) of catechol-O-methyltransferase (COMT, EC 2.1.1.6) were produced using a purified preparation of the enzyme from pig liver as antigen. The selected monoclonal antibodies recognized the enzyme with different capacities. One of them (Co60-1B/7) showed a significant cross reaction with S-COMT from rat and human liver. A protein band of 23 kDa was recognized by the mAbs on Western blots of the soluble fraction of pig liver. The mAbs were also able to recognize the membrane-bound form of the enzyme, which was found to be mainly localized in the microsomal fraction of pig and rat liver as well as of the human hepatoma cell line Hep G2. The protein bands detected in microsomes had a molecular mass of 26 kDa in pig and rat liver and displayed a slightly higher molecular mass (29 kDa) in the Hep G2 cell line. A single step method for the immunoaffinity purification of pig liver S-COMT was developed by using a Sepharose 4B column to which the mAb Co54-5F/8 was covalently coupled. Acid elution conditions were optimized to obtain the enzyme in active form with a good yield. SDS-PAGE analysis of the purified preparation revealed a single protein band with a molecular mass of 23 kDa with 154-fold enrichment in enzyme activity over the starting material. Since the N-terminus was blocked, purified enzyme preparations were cleaved with trypsin. Two fragments of 22 and 33 amino acids in length could be sequenced by Edman degradation.     

O-methylation of (+)-(R)- and (−)-(S)-6,7-dihydroxy-1-methyl-1,2,3,4-tetra-hydroisoquinoline (salsolinol) in the presence of pig brain catechol-o-methyltransferase

(+)-(R)- and (−)-(S)-salsolinol, dopamine-derived tetrahydroisoquinolines, were tested as substrates of pig brain soluble and membrane-bound catechol-O-methyltransferase (COMT) and as inhibitors of O-methylation of dopamine by soluble COMT in vitro. Methylation products were separated by high-performance liquid chromatography with electrochemical detection. Quantification of the products showed that O-methylation of (+)-(R)-salsolinol by soluble COMT afforded the 7-O-methylated product salsoline preferentially, whereas (−)-(S)-salsolinol yielded almost equivalent amounts of the 6- and 7-methyl ethers. Unlike O-methylation by soluble COMT, 7-O/6-O-methylation ratio produced by membrane-bound COMT varied with (+)-(R)-salsolinol concentration. As to the O-methylation of dopamine by soluble COMT, comparable competitive inhibition was observed with both (+)-(R)- and (−)-(S)-salsolinol. Chirality 9:367–372, 1997. © 1997 Wiley-Liss, Inc.     

Catechol-O-methyltransferase and aromatic L-amino acid decarboxylase activities in human gastrointestinal tissues

Human gastrointestinal samples from the corpus, antrum, bulbus, jejunum and ileum were assayed for soluble and membrane-bound catechol-O-methyltransferase (COMT) and aromatic L-amino acid decarboxylase (AADC) activity in vitro. The mean soluble COMT activities with 3,4-dihydroxybenzoic acid (DBA) and 3,4-dihydroxyphenylalanine (L-DOPA) as substrate were 70-242 and 70-174 pmol/min mg, respectively. The membrane-bound COMT activities ranged from 33 to 60 pmol/min mg in the different parts of the intestine. The AADC activities, measured with L-DOPA as the substrate, increased from 114 pmol/min mg in the corpus to 3488 pmol/min mg in the jejunum. The affinity of the soluble COMT was approximately 20 times higher for DBA (Km 15-19 microM) than for L-DOPA (Km 300-600 microM). The Km-values for L-DOPA of AADC and COMT were of the same order of magnitude. The specific COMT inhibitors, nitecapone and OR-611, effectively inhibited in vitro the human intestinal COMT activity. Nanomolar concentrations caused 50% inhibition with both DBA and L-DOPA as substrate.     

A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane

We cloned and expressed a new organic anion transporting polypeptide (OATP), termed human OATP2, (OATP-C, LST-1; symbol SLC21A6), involved in the uptake of various lipophilic anions into human liver. The cDNA encoding OATP2 comprised 2073 base pairs, corresponding to a protein of 691 amino acids, which were 44% identical to the known human OATP. An antibody directed against the carboxy terminus localized OATP2 to the basolateral membrane of human hepatocytes. Northern blot analysis indicated a strong expression of OATP2 only in human liver. Transport mediated by recombinant OATP2 and its localization were studied in stably transfected Madin-Darby canine kidney strain II (MDCKII) and HEK293 cells. Confocal microscopy localized recombinant OATP2 protein to the lateral membrane of MDCKII cells. Substrates included 17beta-glucuronosyl estradiol, monoglucuronosyl bilirubin, dehydroepiandrosterone sulfate, and cholyltaurine. 17beta-Glucuronosyl estradiol was a preferred substrate, with a Michaelis-Menten constant value of 8.2 microM; its uptake was Na(+) independent and was inhibited by sulfobromophthalein, with a inhibition constant value of 44 nM. Our results indicate that OATP2 is important for the uptake of organic anions, including bilirubin conjugates and sulfobromophthalein, in human liver.     

Expression and characterization of rat soluble catechol-O-methyltransferase fusion protein

Rat soluble catechol-O-methyltransferase cDNA was cloned into the pCAL-n-FLAG vector and expressed in Escherichia coli as a fusion protein with a calmodulin-binding peptide tag. The recombinant protein, comprising up to 30% of the total protein in the soluble fraction of E. coli, was purified by calmodulin affinity chromatography and gel filtration. Up to 16 mg of pure recombinant enzyme was recovered per liter of culture. Recombinant catechol-O-methyltransferase, in the bacterial soluble fraction, exhibited the same affinity for adrenaline as rat liver soluble catechol-O-methyltransferase (K(m) 428 [246, 609] microM and 531 [330, 732] microM, respectively), as well as the same affinity for the methyl donor, S-adenosyl-l-methionine (K(m) 27 [9, 45] microM and 38 [21, 55] microM, respectively). In addition, both the recombinant and the liver enzymes displayed the same sensitivity to the inhibitor 3,5-dinitrocatechol (IC(50) 132 [44, 397] nM and 74 [38, 143] nM, respectively), and both had the same catalytic number, respectively, 10.1 +/- 1.5 min(-1) and 8.3 +/- 0.3 min(-1). The purified recombinant enzyme also displayed the same affinity for the substrate as the purified rat liver catechol-O-methyltransferase (K(m) 336 [75, 597] microM and 439 [168, 711] microM, respectively) as well as the same inhibitor sensitivity (IC(50) 44 [19, 101] nM and 61 [33, 111] nM, respectively). This recombinant form of catechol-O-methyltransferase is kinetically identical to the rat liver enzyme. This system provides an easy and quick way of obtaining large amounts of soluble catechol-O-methyltransferase for both pharmacological and structural studies.     

TIG-Fc真核表达载体的构建及其对自然杀伤细胞功能的影响

T细胞免疫球蛋白和免疫受体酪氨酸抑制性基序结构域\[T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain,TIGIT\]是一种新型的免疫抑制性受体,在机体的免疫调节网络中扮演着重要角色。为了进一步探究TIGIT对自然杀伤（natural killer,NK）细胞的免疫调节功能,构建了融合蛋白TIGIT胞外段（即TIGIT第1~135位氨基酸,以保留功能结构域IgV区,此胞外段简称为TIG）-人免疫球蛋白G3（immunoglobulin G3,IgG3）Fc段的真核表达载体,并对TIG-Fc融合蛋白的表达及其对NK-92细胞功能的影响进行了初步研究。利用分子生物学方法,将携带人TIG与人IgG3 Fc段的基因融合,然后插入真核表达载体pcDNA3.1(-)中,以构建重组质粒pcDNA3.1(-)-TIG-Fc。随后将重组质粒转染至人胚肾细胞HEK-293T中,通过流式细胞仪和Western blot检测TIG-Fc融合蛋白在293T细胞中的表达;再将重组质粒转染至NK-92细胞中,通过WST-1检测TIG-Fc融合蛋白对NK-92细胞增殖的影响,并利用ELISA法检测TIG-Fc融合蛋白对NK-92细胞分泌IFN-γ的水平的影响。结果成功构建了人TIG与人IgG3 Fc融合表达的真核表达载体,且TIG-Fc融合蛋白的表达能够显著抑制NK-92细胞的增殖和分泌IFN-γ的能力（P<0.05）,为后期TIGIT的功能研究奠定了重要基础。     

The purification and characterization of recombinant human renin expressed in the human kidney cell line 293

The cDNA encoding human preprorenin has been introduced into the adenovirus-transformed human kidney cell line 293. The recombinant 293 cells expressed and secreted prorenin; trypsin was used to activate the secreted prorenin to renin in vitro. The recombinant protein was purified to homogeneity by a single affinity chromatographic step. Using synthetic tetradecapeptide, the Km was 57.1 +/- 9.3 microM and the kcat was (7.48 +/- 1.57) x 10(3)/hr. Activation with trypsin resulted in a secondary cleavage between Arg53 and Leu54 generating a two chain form held together via a disulfide between Cys51 and Cys58. This secondary cleavage did not affect enzyme activity as determined by the ability of renin to degrade a synthetic tetradecapeptide substrate. Our paper demonstrates the potential for producing large quantities of renin from human kidney cells and also suggests that the use of trypsin, which has been widely used to convert prorenin to renin in vitro, causes a secondary cleavage in the renin peptide chain.     

Kinetic reaction mechanism for magnesium binding to membrane-bound and soluble catechol O-methyltransferase

Catechol O-methyltransferase (COMT, EC 2.1.1.6) from human brain occurs in both a membrane-bound (MB-COMT) and a soluble form (SOL-COMT). While these enzymes appear to be distinct molecular entities, both catalyze the O-methylation of catecholamines through an ordered reaction mechanism in which S-adenosylmethionine (SAM) is the leading substrate [Rivett, A. J., & Roth, J. A. (1982) Biochemistry 21, 1740-1742; Jeffery, D. R., & Roth, J. A. (1985) J. Neurochem. 44, 881-885]. Both MB-COMT and SOL-COMT require the presence of divalent cations for catalytic activity. This series of experiments provides evidence indicating that magnesium ions bind to both MB-COMT and SOL-COMT in a rapid equilibrium sequence prior to the addition of SAM. An equation is presented that predicts the qualitative results obtained in all kinetic experiments carried out with either MB-COMT or SOL-COMT.     

Increased catechol-O-methyltransferase activity and protein expression in OX-42-positive cells in the substantia nigra after lipopolysaccharide microinfusion

Activated microglial cells are found in the substantia nigra and the striatum of Parkinson's disease patients. These cells have been shown to express catechol-O-methyltransferase activity which may increase during pathological conditions. Lipopolysaccharides are potent activators of microglial cells. After paranigral lipopolysaccharide infusion to rats we observed intense microglial activation around the lesion area followed by a delayed injury in nigrostriatal pathway in 2 weeks. Simultaneously, catechol-O-methyltransferase activity in the substantia nigra was gradually increased up to 213%. In the Western blot the amount of soluble COMT and membrane bound COMT proteins were increased by 255% and 86%, respectively. Increased catechol-O-methyltransferase immunoreactivity was located primarily into the activated microglial cells in the lesion area. Interestingly, catechol-O-methyltransferase and OX-42 stained also intensively microglia/macrophage-like cells which surrounded the adjacent blood vessels. Inhibition of catechol-O-methyltransferase activity by tolcapone or entacapone did not increase lipopolysaccharide-induced neurotoxicity. We conclude that catechol-O-methyltransferase activity and protein expression were increased in the substantia nigra after inflammation induced by lipopolysaccharides. These changes in glial and perivascular catechol-O-methyltransferase activity may have clinical relevance for Parkinson's disease drug treatment due to increased metabolism of levodopa in the brain.     

大鼠p75NTR荧光真核表达载体的构建及其在HEK293细胞中的表达

目的:构建大鼠p75神经营养素受体(p75 neurotrophin receptor,p75NTR)cDNA序列的绿色荧光真核表达载体并鉴定其在人胚肾293(human embryo kidney 293,HEK293)细胞中的表达.方法:采用PCR方法从含野生型大鼠p75NTR的pDC316-RP75质粒中扩增目的片段,经EcoRⅠ和SaⅡ双酶切,定向克隆于pEGFP-N1质粒中,构建绿色荧光真核表达栽体pEGFP-N1-RP75,经酶切及测序鉴定后,通过脂质体转染HEK293细胞,激光共聚焦及免疫组织化学法鉴定大鼠p75NTR的表达.结果:重组质粒经酶切鉴定和序列分析证实含有大鼠p75NTR的编码序列,转染后经激光共聚焦显微镜及免疫组织化学染色观察表明重组质粒能够在HEK293细胞中表达出具有活性的大鼠p75NTR片段.结论:大鼠p75NTR绿色荧光真核表达栽体构建成功并可在HEK293细胞中表达,为进一步研究奠定了基础.     

Localization of Membrane-Bound Catechol-O-Methyltransferase

Abstract: Previous studies of the distribution of catechol-O-methyltransferase (COMT) have concentrated on the soluble enzyme activity. In this study the activity of the membrane-bound enzyme was determined in different brain regions and peripheral tissues of the rat. Membrane-bound COMT, like the soluble enzyme, has a general distribution with high levels in liver, kidney, and vas deferens. However, the ratio of membrane-bound to soluble activity varies almost 30-fold in different tissues, with the highest ratio in brain. Membrane-bound activity varies twofold within brain. In view of their different localization and kinetic properties, it seems likely that the two forms of COMT have different functions in vivo.     

Detoxication of structurally diverse polycyclic aromatic hydrocarbon (PAH) o-quinones by human recombinant catechol-O-methyltransferase (COMT) via O-methylation of PAH catechols

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Metabolic activation of intermediate PAH trans-dihydrodiols by aldo-keto reductases (AKRs) leads to the formation of electrophilic and redox-active o-quinones. We investigated whether O-methylation by human recombinant soluble catechol-O-methyltransferase (S-COMT) is a feasible detoxication step for a panel of structurally diverse PAH-catechols produced during the redox-cycling process. Classes of PAH non-K-region o-quinones (bay region, methylated bay region, and fjord region o-quinones) produced by AKRs were employed in the studies. PAH o-quinones were reduced to the corresponding catechols by dithiothreitol under anaerobic conditions and then further O-methylated by human S-COMT in the presence of S-[3H]adenosyl-l-methionine as a methyl group donor. The formation of the O-methylated catechols was detected by HPLC-UV coupled with in-line radiometric detection, and unlabeled products were also characterized by LC-MS/MS. Human S-COMT was able to catalyze O-methylation of all of the PAH-catechols and generated two isomeric metabolites in different proportions. LC-MS/MS showed that each isomer was a mono-O-methylated metabolite. 1H NMR was used to assign the predominant positional isomer of benzo[a]pyrene-7,8-catechol as the O-8-monomethylated catechol. The catalytic efficiency (k(cat)/K(m)) varied among different classes of PAH-catechols by 500-fold. The ability of S-COMT to produce two isomeric products from PAH-catechols was rationalized using the crystal structure of the enzyme. We provide evidence that O-8-monomethylated benzo[a]pyrene-7,8-catechol is formed in three different human lung cell lines. It is concluded that human S-COMT may play a critical role in the detoxication of PAH o-quinones generated by AKRs.     

Purification and Kinetic Mechanism of Human Brain Soluble Catechol-O-Methyltransferase

The soluble form of human brain catechol-O-methyltransferase (EC 2.1.1.6, COMT) has been purified approximately 4,000-fold from a 250,000 X g supernatant solution. The purified enzyme exhibits a molecular weight near 27,500 and a pI value equal to approximately pH 5.0. Initial velocity and product inhibition studies are consistent with an ordered reaction mechanism for soluble COMT. Tropolone, a dead-end inhibitor, exhibited a competitive pattern of inhibition when dopamine (DA) was the varied substrate and an uncompetitive pattern when S-adenosyl-L-methionine (SAM) was the varied substrate. These observations strongly suggest that the soluble form of COMT from human brain catalyzes the O-methylation of catecholamines via an ordered reaction mechanism in which SAM is the leading substrate. Since the membrane-bound form of COMT catalyzes the O-methylation of catecholamines through an identical reaction mechanism, these data provide further evidence that two forms of COMT, while being localized in distinct subcellular compartments, are quite similar in their molecular structure.