Human Histamine N-methyltransferase Research Articles

Histamine N-Methyltransferase in the Brain

Brain histamine is a neurotransmitter and regulates diverse physiological functions. Previous studies have shown the involvement of histamine depletion in several neurological disorders, indicating the importance of drug development targeting the brain histamine system. Histamine N-methyltransferase (HNMT) is a histamine-metabolising enzyme expressed in the brain. Although pharmacological studies using HNMT inhibitors have been conducted to reveal the direct involvement of HNMT in brain functions, HNMT inhibitors with high specificity and sufficient blood–brain barrier permeability have not been available until now. Recently, we have phenotyped Hnmt-deficient mice to elucidate the importance of HNMT in the central nervous system. Hnmt disruption resulted in a robust increase in brain histamine concentration, demonstrating the essential role of HNMT in the brain histamine system. Clinical studies have suggested that single nucleotide polymorphisms of the human HNMT gene are associated with several brain disorders such as Parkinson’s disease and attention deficit hyperactivity disorder. Postmortem studies also have indicated that HNMT expression is altered in human brain diseases. These findings emphasise that an increase in brain histamine levels by novel HNMT inhibitors could contribute to the improvement of brain disorders.

Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies

ObjectiveRecently, we characterized mouse monoclonal antibodies that allow the specific and sensitive detection of human histamine N-methyltransferase (HNMT). To understand differences in binding characteristics and recognition of enzyme variants we mapped the antibody binding sites.MethodsFragments of human HNMT were expressed as glutathione S-transferase fusion proteins that were used for testing antibody binding on immunoblots. Combined information from species cross-reactivity, sequence comparison, protein structure, and binding site prediction software were used to localize the epitope recognized by each antibody.ResultsAll eight monoclonal HNMT antibodies bound to linear epitopes in the C-terminal domain of the 292 amino acid protein. Of the five antibodies cross-reacting with HNMT from other species, one bound region L182–T223, three region M224–E261, and one region L262–A292. All three antibodies recognising only human HNMT bound the C-terminal region L262–A292 that contains residues present only in the human protein.ConclusionsOur HNMT monoclonal antibodies bind in three different regions of the protein and those binding the same putative epitope exhibit similar binding characteristics and species cross-reactivity. Antibodies binding non-overlapping epitopes will facilitate analyses of all clinically relevant variants described for HNMT.

Leucine 208 in human histamine N-methyltransferase emerges as a hotspot for protein stability rationalizing the role of the L208P variant in intellectual disability

The degradation of histamine catalyzed by the SAM-dependent histamine N-methyltransferase (HNMT) is critically important for the maintenance of neurological processes. Recently, two mutations in the encoding human gene were reported to give rise to dysfunctional protein variants (G60D and L208P) leading to intellectual disability. In the present study, we have expressed eight L208 variants with either apolar (L208F and L208V), polar (L208N and L208T) or charged (L208D, L208H, L208K and L208R) amino acids to define the impact of side chain variations on protein structure and function. We found that the variants L208N, L208T, L208D and L208H were severely compromised in their stability. The other four variants were obtained in lower amounts in the order wild-type HNMT>L208F=L208V>L208K=L208R. Biochemical characterization of the two variants L208F and L208V exhibited similar Michaelis-Menten parameters for SAM and histamine while the enzymatic activity was reduced to 21% and 48%, respectively. A substantial loss of enzymatic activity and binding affinity for histamine was seen for the L208K and L208R variants. Similarly the thermal stability for the latter variants was reduced by 8 and 13°C, respectively. These findings demonstrate that position 208 is extremely sensitive to side chain variations and even conservative replacements affect enzymatic function. Molecular dynamics simulations showed that amino acid replacements in position 208 perturb the helical character and disrupt interactions with the adjacent β-strand, which is involved in the binding and correct positioning of histamine. This finding rationalizes the gradual loss of enzymatic activity observed in the L208 variants.

Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies

ObjectiveThe lack of suitable antibodies for the histamine inactivating enzyme histamine N-methyltransferase (HMT) has so far prevented the direct analysis of HMT proteins in man and other mammals.MethodsA series of monoclonal antibodies was produced by immunizing mice with human and porcine HMT expressed in vitro. Antibodies were characterized by immunoblotting and immunohistochemical staining.ResultsSix different monoclonal antibodies specific for human HMT and four different monoclonal antibodies specific for porcine HMT were obtained that can detect HMT with up to tenfold greater sensitivity than the most sensitive enzymatic assays currently available. Using these antibodies allowed us to confirm the expression and cellular localization of HMT in various human and porcine tissues, where the presence of the enzyme had previously been deduced from activity measurement and HMT mRNA analysis. Immunohistochemical staining of human and porcine tissue sections clearly showed that HMT is a cytosolic protein, which is localized in specific cells of most mammalian tissues.ConclusionsThe new monoclonal antibodies not only allow a comprehensive quantitative evaluation of the expression of HMT at the cellular level in man and other mammals but will also facilitate sensitive analyses of disease-associated alterations of this protein.

Influence of iodinated contrast media on the activities of histamine inactivating enzymes diamine oxidase and histamine N-methyltransferase in vitro

BackgroundIodinated contrast media can cause pseudoallergic reactions associated with histamine release in significant numbers of patients. To clarify whether these adverse reactions may be aggravated by a compromised histamine catabolism we asked if radiographic contrast agents in vitro inhibit the histamine inactivating enzymes diamine oxidase (DAO) and histamine N-methyltransferase (HMT). MethodsNine iodinated contrast agents were tested in vitro. Following pre-incubation of purified porcine kidney DAO and recombinant human HMT with 0.1–10mM of the respective contrast medium (H2O and specific inhibitors of DAO and HMT as controls) enzyme activities were determined by using radiometric micro assays. ResultsNone of the contrast media irrespective of their structure showed significant inhibition of the activities of DAO and HMT. Pre-incubation of the enzymes with specific inhibitors led to complete inhibition of the respective enzymatic activity. ConclusionsThe iodinated contrast media tested in vitro did not exhibit inhibition of histamine converting enzymes at physiologically relevant concentrations. However due to the in vitro character of this study these results do not directly reflect the in vivo situation.

Erratum: Involvement of Human Histamine N-Methyltransferase Gene Polymorphisms in Susceptibility to Atopic Dermatitis in Korean Children

Erratum: Involvement of Human Histamine N-Methyltransferase Gene Polymorphisms in Susceptibility to Atopic Dermatitis in Korean Children

Involvement of Human Histamine N-methyltransferase Gene Polymorphisms in Susceptibility to Atopic Dermatitis in Korean Children

Histamine N-methyltransferase (HNMT) catalyzes one of two major metabolic pathways for histamine. Histamine is one of the mediators for pruritus of atopic dermatitis. The aim of this study was to evaluate the role of HNMT polymorphisms in children with atopic dermatitis. We genotyped 763 children for allelic determinants at four polymorphic sites, which were -465T>C, -413C>T, 314C>T and 939A>G in the HNMT gene, and the functional effect of the 939A>G polymorphism was analyzed. The genotyping was screened using the TaqMan fluorogenic 5’ nuclease assay (ABI, Foster City, CA, USA). Among these 763 children, 520 had eczema and 542 had atopy. Distributions of the genotype and allele frequencies of HNMT 314C>T polymorphism were significantly associated with non-atopic eczema (P = .004) and those of HNMT 939A>G polymorphism were significantly associated with eczema in atopy groups (P = .048). However, those of HNMT 654T>C and 413C>T polymorphisms were not. In addition, subjects with the homozygous AA or heterozygous AG of the 939A>G polymorphism showed significantly higher IgE levels than those with the homozygous GG genotype (P = .009). In U937 cells, the variant genotype reporter construct showed significantly higher mRNA stability (P < .001) and HNMT enzyme activity (P < .001) than the common genotype. Polymorphisms in the HNMT gene appear to confer susceptibility to atopic dermatitis in Korean children.

47 Involvement of Human Histamine N-Methyltransferase Gene Polymorphisms in Susceptibility to Atopic Dermatitis in Korean Children

BackgroundHistamine N-methyltransferase (HNMT) catalyzes one of 2 major metabolic pathways for histamine. Histamine is one of the mediators for pruritus of atopic dermatitis. The aim of this study was to evaluate the role of HNMT polymorphisms in children with atopic dermatitis.MethodsWe genotyped 763 children for allelic determinants at 4 polymorphic sites, which were −465T>C, −413C>T, 314C>T and 939A>G in the HNMT gene, and the functional effect of the 939A>G polymorphism was analyzed. The genotyping was screened using the TaqMan fluorogenic 5' nuclease assay (ABI, Foster City, CA, USA).ResultsAmong these 763 children, 520 had eczema and 542 had atopy. Distributions of the genotype and allele frequencies of HNMT 314C>T polymorphism were significantly associated with non-atopic eczema (P = 0.004) and those of HNMT 939A>G polymorphism were significantly associated with eczema in atopy groups (P = 0.048). However, those of HNMT 654T>C and 413C>T polymorphisms were not. In addition, subjects with the homozygous AA or heterozygous AG of the 939A>G polymorphism showed significantly higher IgE levels than those with the homozygous GG genotype (P = 0.009). In U937 cells, the variant genotype reporter construct showed significantly higher mRNA stability (P < 0.001) and HNMT enzyme activity (P < 0.001) than the common genotype.ConclusionsPolymorphisms in the HNMT gene appear to confer susceptibility to atopic dermatitis in Korean children.

Involvement of Human Histamine N-Methyltransferase Gene Polymorphisms in Susceptibility to Atopic Dermatitis in Korean Children

PurposeHistamine N-methyltransferase (HNMT) catalyzes one of two major histamine metabolic pathways. Histamine is a mediator of pruritus in atopic dermatitis (AD). The aim of this study was to evaluate the association between HNMT polymorphisms and AD in children.MethodsWe genotyped 763 Korean children for allelic determinants at four polymorphic sites in the HNMT gene: -465T>C, -413C>T, 314C>T, and 939A>G. Genotyping was performed using a TaqMan fluorogenic 5' nuclease assay. The functional effect of the 939A>G polymorphism was analyzed.ResultsOf the 763 children, 520 had eczema and 542 had atopy. Distributions of the genotype and allele frequencies of the HNMT 314C>T polymorphism were significantly associated with non-atopic eczema (P=0.004), and those of HNMT 939A>G were significantly associated with eczema in the atopy groups (P=0.048). Frequency distributions of HNMT -465T>C and -413C>T were not associated with eczema. Subjects who were AA homozygous or AG heterozygous for 939A>G showed significantly higher immunoglobulin E levels than subjects who were GG homozygous (P=0.009). In U937 cells, the variant genotype reporter construct had significantly higher mRNA stability (P<0.001) and HNMT enzyme activity (P<0.001) than the common genotype.ConclusionsPolymorphisms in HNMT appear to confer susceptibility to AD in Korean children.

The Histamine N-Methyltransferase T105I Polymorphism Affects Active Site Structure and Dynamics

Histamine N-methyltransferase (HNMT) is the primary enzyme responsible for inactivating histamine in the mammalian brain. The human HNMT gene contains a common threonine-isoleucine polymorphism at residue 105, distal from the active site. The 105I variant has decreased activity and lower protein levels than the 105T protein. Crystal structures of both variants have been determined but reveal little regarding how the T105I polymorphism affects activity. We performed molecular dynamics simulations for both 105T and 105I at 37 degrees C to explore the structural and dynamic consequences of the polymorphism. The simulations indicate that replacing Thr with the larger Ile residue leads to greater burial of residue 105 and heightened intramolecular interactions between residue 105 and residues within helix alpha3 and strand beta3. This altered, tighter packing is translated to the active site, resulting in the reorientation of several cosubstrate-binding residues. The simulations also show that the hydrophobic histamine-binding domain in both proteins undergoes a large-scale breathing motion that exposes key catalytic residues and lowers the hydrophobicity of the substrate-binding site.

Actions of tacrine and galanthamine on histamine-N-methytransferase

Histamine-synthesizing neurons in the brain may play an important role in cognition, and a histaminergic deficit has been found in Alzheimer's disease (AD). The AD medication tacrine was previously shown to inhibit some forms of rodent histamine-N-methyltransferase (HNMT), but the effects of AD drugs have not been investigated on human HNMT activity. Presently, the effects of tacrine and galanthamine (another AD medication) were studied on the activity of several forms of human and rat HNMT. Tacrine (0.01-10 microM) inhibited both human and rat HNMT activity in a concentration-dependent manner, but was less potent on both human embryonic kidney and recombinant human brain HNMT than on rat kidney HNMT (IC50 values were 0.46 and 0.70 microM vs. 0.29 microM, respectively). Galanthamine (up to 10 microM) did not influence the activity of rat kidney or human HNMT. Tacrine, but not galanthamine, may achieve brain levels sufficient to influence histamine metabolism in some patients treated for AD.

Characterization of a new mRNA species from the human histamine N-methyltransferase gene

Histamine N-methyltransferase (HNMT), a cytosolic histamine-metabolizing enzyme, is the only known product of the 50-kb human HNMT. Here, a detailed investigation of HNMT products revealed the existence of a new brain mRNA product of HNMT. This species, named HNMT-Short (HNMT-S), encodes a 126-amino-acid protein. Northern blot analysis detected HNMT-S mRNA (1.0 kb) in placenta, but not in several other human tissues. In addition, unlike the known HNMT cDNA, HNMT-S cDNA did not result in histamine-methylating activity after transfection into COS-7 cells. These studies show that HNMT-S is a new mRNA species and putative protein product from HNMT. The physiological role of HNMT-S remains to be investigated.

Genotype–phenotype correlation for histamine N-methyltransferase in a Chinese Han population

Background: Two potential single-nucleotide polymorphisms (SNP) (C314T and A595G) exist in the gene for human histamine N-methyltransferase (HNMT). Methods: A radiochemical microassay was used to measure the erythrocyte HNMT activities, whereas the polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) was employed to perform the genetic analysis in 247 unrelated Chinese Han subjects. Results: All subjects had detectable HNMT activity. The activity of HNMT was gender related (males>females, p<0.0001), with a 5.5-fold individual variation. The distribution of HNMT activity was compatible with a normal distribution. There were 28 heterozygotes for the variant T314 allele among the 247 subjects, whereas no A595G transition was observed. All heterozygotes for the T314 allele displayed an intermediate or low HNMT activity, with an average HNMT activity being 34.0% lower than those with wild-type genotype (623.1±136.0 vs. 944.8±249.3 U/ml red blood cells [RBC], p<0.0001). Conclusion: The C314T polymorphism was functionally important and contributes in part to phenotypic variance of HNMT activity in Chinese Han population. Additional unknown genetic or epigenetic factors should also play important roles in the regulation of HNMT activity.

Human histamine N-methyltransferase pharmacogenetics: gene resequencing, promoter characterization, and functional studies of a common 5′-flanking region single nucleotide polymorphism (SNP)

Histamine N-methyltransferase (HNMT) catalyzes one of two major metabolic pathways for histamine. The levels of HNMT activity and immunoreactive protein in human tissues are regulated primarily by inheritance. Previous studies of HNMT identified two common single nucleotide polymorphisms (SNPs), including a functionally significant nonsynonymous coding SNP (cSNP), (C314T, Thr105Ile), but that polymorphism did not explain all of the phenotypic variation. In the present study, a genotype-to-phenotype strategy was used to search for additional genetic factors that might contribute to the regulation of human HNMT activity. Specifically, we began by resequencing the human HNMT gene using 90 ethnically anonymous DNA samples from the Coriell Cell Repository and identified a total of eight SNPs, including the two that had been reported previously. No new nonsynonymous cSNPs were observed, but three of the six novel SNPs were located in the 5′-flanking region (5′-FR) of the gene—including a third common polymorphism with a frequency of 0.367 (36.7%). That observation directed our attention to possible genetic effects on HNMT transcription. As a first step in testing that possibility, we created and studied a series of reporter gene constructs for the initial 1 kb of the HNMT 5′-FR. The core promoter and possible regulatory regions were identified and verified by electrophoresis mobility shift assays. We then studied the possible functional implications of the new common HNMT 5′-FR SNP. However, on the basis of reporter gene studies, that SNP appeared to have little effect on transcription. Phenotype–genotype correlation analysis performed with 112 human kidney biopsy samples that had been phenotyped for their level of HNMT activity confirmed that the common 5′-FR SNP was not associated with the level of HNMT activity in vivo. In summary, this series of experiments resulted in the identification of several novel HNMT polymorphisms, identification of the HNMT core promoter, and a comprehensive functional genomic study of a common HNMT 5′-FR SNP. These results represent an additional step in the definition of molecular genetic mechanisms involved in the regulation of this important autacoid-metabolizing enzyme in humans.

Two Polymorphic Forms of Human Histamine Methyltransferase: Structural, Thermal, and Kinetic Comparisons

Background: Histamine plays important biological roles in cell-to-cell communication; it is a mediator in allergic responses, a regulator of gastric acid secretion, a messenger in bronchial asthma, and a neurotransmitter in the central nervous system. Histamine acts by binding to histamine receptors, and its local action is terminated primarily by methylation. Human histamine N-methyltransferase (HNMT) has a common polymorphism at residue 105 that correlates with the high- (Thr) and low- (Ile) activity phenotypes. Results: Two ternary structures of human HNMT have been determined: the Thr105 variant complexed with its substrate histamine and reaction product AdoHcy and the Ile105 variant complexed with an inhibitor (quinacrine) and AdoHcy. Our steady-state kinetic data indicate that the recombinant Ile105 variant shows 1.8- and 1.3-fold increases in the apparent K M for AdoMet and histamine, respectively, and slightly (16%) but consistently lower specific activity as compared to that of the Thr105 variant. These differences hold over a temperature range of 25°C–45°C in vitro. Only at a temperature of 50°C or higher is the Ile105 variant more thermolabile than the Thr105 enzyme. Conclusions: HNMT has a 2 domain structure including a consensus AdoMet binding domain, where the residue 105 is located on the surface, consistent with the kinetic data that the polymorphism does not affect overall protein stability at physiological temperatures but lowers K M values for AdoMet and histamine. The interactions between HNMT and quinacrine provide the first structural insights into a large group of pharmacologic HNMT inhibitors and their mechanisms of inhibition.

Theoretical 3D Model of Histamine N-Methyltransferase: Insights into the Effects of a Genetic Polymorphism on Enzymatic Activity and Thermal Stability

Histamine N-methyltransferase (HNMT) catalyzes the N-methylation of histamine in mammals. The experimentally determined HNMT three-dimensional (3D) structure is not available. However, there is a common genetic polymorphism for human HNMT (Thr105Ile) that reduces enzymatic activity and is a risk factor for asthma. To obtain insights into mechanisms responsible for the effects of that polymorphism on enzymatic activity and thermal stability, we predicted the 3D structure of HNMT using the threading method and molecular dynamics simulations in water. Herein, we report a theoretical 3D model of human HNMT which reveals that polymorphic residue Thr105Ile is located in the turn between a beta strand and an alpha helix on the protein surface away from the active site of HNMT. Ile105 energetically destabilizes folded HNMT because of its low Chou–Fasman score for forming a turn conformation and the exposure of its hydrophobic side chain to aqueous solution. It thus promotes the formation of misfolded proteins that are prone to the clearance by proteasomes. This information explains, for the first time, how genetic polymorphisms can cause enhanced protein degradation and why the thermal stability of allozyme Ile105 is lower than that of Thr105. It also supports the hypothesis that the experimental observation of a significantly lower level of HNMT enzymatic activity for allozyme Ile105 than that with Thr105 is due to a decreased concentration of allozyme Ile105, but not an alternation of the active-site topology of HNMT caused by the difference at residue 105.

Guinea Pig Histamine N-Methyltransferase: cDNA Cloning and mRNA Distribution

We report here the isolation of histamine N-methyltransferase (HMT) cDNA from the guinea pig brain by the polymerase chain reaction on the basis of nucleotide sequences of rat and human counterparts. Guinea pig HMT consists of 292 amino acids, with homologies of 75.6% and 79.1% to rat and human HMT, respectively. Northern blotting analysis indicated that the 1.6-kb guinea pig HMT transcript was expressed at various levels in different tissues at the following relative abundance: jejunum, brain > lung, spleen, stomach > liver, kidney. HMT mRNA localized throughout the jejunum, and it was mainly expressed in epithelial cells and in Auerbach’s plexus.

Human HistamineN-Methyltransferase Gene: Structural Characterization and Chromosomal Localization

Histamine N-methyltransferase (HNMT) catalyzes the Nτ-methylation of histamine. The level of HNMT activity in human red blood cells is controlled by a common genetic polymorphism. We previously cloned and expressed a cDNA for human kidney HNMT, and we have now determined the structural organization of the human HNMT gene as a step toward studies of the genetic regulation of levels of HNMT activity in human tissue. Structural characterization of the HNMT gene was performed by use of a polymerase chain reaction (PCR)-based strategy. The gene was approximately 34 kb in length and contained 6 exons. All exon–intron splice junction sequences conformed to the “GT-AG” rule. The longest transcript isolated after 5′-rapid amplification of cDNA ends indicated that transcription initiation occurred 252 nucleotides 5′-upstream from the cDNA translation initiation codon.HNMTmapped to human chromosome 2. Structural characterization of the gene for HNMT will make it possible to study molecular genetic mechanisms involved in the regulation of this important enzyme in humans.

Localization of histamine N-methyltransferase messenger RNA in human nasal mucosa

Background: Histamine is metabolized mainly by histamine N-methyltransferase (HMT) to N τ -methylhistamine in human nasal mucosa. Human HMT cDNA has been cloned and expressed in COS cells. The purpose of this study was to determine the localization of HMT mRNA in human nasal mucosa. Methods: The fragment (nucleotide residues 430-1055) of human HMT cDNA was subcloned in a Bluescript vector (Stratagene, La Jolla, Calif.), and HMT sense and anti-sense RNA probes were made with T 7 and T 3 RNA polymerases. In situ hybridization with digoxigenin-labeled RNA probes was performed on surgical specimens of human nasal turbinates. Results: HMT mRNA was localized in cells in the epithelium and submucosa, and densely in endothelial cells of vessels. No HMT mRNA was identified in the submucosal glands. The presence of HMT mRNA was confirmed by Northern blot analysis, and HMT activities were also detected in nasal mucosa. Conclusion: Our study indicates that endothelium expresses HMT mRNA, whereas cells in the epithelium and submucosa, which remain unidentified, are an additional source of HMT mRNA. (J A LLERGY C LIN I MMUNOL 1995;95:96-102.)

Structure and function of human histamine N-methyltransferase: critical enzyme in histamine metabolism in airway

In mammals, histamine is inactivated principally by two enzymes: histamine N-methyltransferase (HMT; EC 2.1.1.8) and diamine oxidase (DAO; EC 1.4.3.6.). The cDNA clone of human HMT (hHMT) has been isolated from a cDNA library of human kidney and its nucleotide, and deduced amino acid sequences have been determined. One clone, phHMT-1, containing an insert of 1.4 kb, was confirmed to encode HMT by transient expression of HMT activity in COS cells. hHMT consists of 292 amino acid residues [relative molecular weight (M(r)) = 33,279] and shares 82% identity with that of rat HMT. Northern blot analysis with hHMT cDNA probe revealed that 1.6-kb HMT mRNA transcript was expressed in the lung, nasal polyps, and kidney. HMT activity was measured in human trachea and bronchi. In addition, the contractile response of isolated human bronchi to histamine was potentiated in the presence of an HMT inhibitor, SKF 91488, but a DAO inhibitor, aminoguanidine, was without effect. These results suggest that HMT plays an important role in degrading histamine and in regulating the airway response to histamine. Therefore, the level of HMT gene expression in human airway may be one of the critical factors determining the airway responsiveness to histamine. In situ chromosomal hybridization demonstrated that human HMT gene was localized in chromosome 1 p32.