HNMT Human, Active

What is the molecular structure of human HNMT?

Human HNMT is a monomeric protein of 33 kDa consisting of a single polypeptide chain of 292 amino acid residues. It has a characteristic two-domain structure with the larger N-terminal domain containing a classical methyltransferase fold with an S-adenosyl-L-methionine (SAM) binding motif. The protein is encoded by the HNMT gene located on chromosome 2q22.1 and organized into six exons . The enzyme exhibits high substrate specificity for histamine and is inhibited by its reaction products as well as by SH-group reagents like p-chloromercuribenzoate and N-ethylmaleimide, and antimalarial drugs including quinacrine and amodiaquine .

How does HNMT function in histamine metabolism?

HNMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the secondary amino group of the imidazole ring of histamine, forming N-τ-methylhistamine . As a cytosolic protein, HNMT is responsible for the inactivation of intracellular histamine, which is either synthesized within the cell or taken up from the extracellular space after binding to cell surface receptors or via plasma membrane transporters . This represents one of two major histamine metabolic pathways in humans, making HNMT crucial for regulating histamine-mediated responses in tissues.

What methods are available for measuring HNMT enzyme activity?

HNMT activity can be measured by quantifying the formation of N-τ-methylhistamine using radioactively labeled substrates. A standard methodology involves incubating cell lysates containing HNMT with S-adenosyl-L-[methyl-14C]methionine as a methyl donor, followed by measuring the radioactivity of formed N-τ-methylhistamine using a liquid scintillation counter (such as the LS-3801 from Beckman Instruments) . Enzyme activity is typically expressed as the amount of N-τ-methylhistamine formed per hour of incubation at 37°C, and results should be normalized to total protein concentration for accurate comparisons across samples .

What monoclonal antibodies are available for human HNMT detection?

Several mouse monoclonal antibodies have been developed for the specific and sensitive detection of human HNMT. These include antibody clones HYB372-04/-05/-06/-07/-08/-09 (resulting from immunization with human HNMT) and clones HYB373-02/-03 (resulting from immunization with porcine HNMT but cross-reacting with human HNMT) . These antibodies exhibit different binding characteristics and species cross-reactivity, making them valuable tools for various experimental applications including immunoblotting, immunoprecipitation, and immunohistochemical analyses .

How can epitope mapping be performed for HNMT antibodies?

Mapping the binding sites of anti-HNMT antibodies involves a multi-faceted approach combining several techniques:

• Expression of HNMT fragments as glutathione S-transferase (GST) fusion proteins in bacterial systems

• Testing antibody binding to these fragments using immunoblotting

• Analyzing species cross-reactivity across human, porcine, rat, and mouse HNMT

• Performing sequence comparison using tools such as the NCBI constrained-based Multiple Alignment Tool

• Generating antigenicity plots with prediction tools like BepiPred Linear Epitope Prediction Tool

• Correlating findings with structural information using software such as NCBI Cn3D

This comprehensive approach allows precise localization of epitopes recognized by different antibodies, which is essential for selecting appropriate antibodies for specific research applications.

What are the antibody binding regions on the HNMT protein?

Studies have shown that all eight monoclonal HNMT antibodies described in the literature bind to linear epitopes in the C-terminal domain of the 292 amino acid protein . Specifically:

The C-terminal region L262–A292 contains residues present only in the human protein, explaining why antibodies binding to this region are specific to human HNMT .

What are the major polymorphisms in the human HNMT gene?

Several significant polymorphisms have been identified in the human HNMT gene, with three common single nucleotide polymorphisms (SNPs): -463T>C (also referred to as -465T>C in some studies), 314C>T, and 939A>G . These polymorphisms have varying effects on enzyme function and have been associated with different clinical phenotypes. Additional polymorphisms include -413C>T and 1097A>T, though these are less extensively characterized in the literature .

How do HNMT polymorphisms affect enzyme activity?

Different HNMT polymorphisms have distinct functional consequences:

The 314T allele consistently shows association with reduced HNMT activity across studies, while the 939G allele demonstrates enhanced mRNA stability and consequently increased enzyme activity .

What experimental approaches verify the functional effects of the 939A>G polymorphism?

The functional impact of the 939A>G polymorphism has been experimentally verified through:

• mRNA stability analysis: After treating U937 cells transfected with either 939A or 939G reporter constructs with actinomycin D (10 μg/mL), the 939G-fused reporter gene mRNA showed significantly greater stability than the 939A-fused variant (p<0.001) .

• Enzyme activity measurements: In transfected U937 cells, the 939G construct demonstrated significantly higher HNMT enzyme activity compared to the 939A construct (p<0.001), as measured by N-τ-methylhistamine formation .

These findings provide strong experimental evidence for the molecular mechanism by which this polymorphism influences HNMT function.

How are HNMT polymorphisms associated with atopic dermatitis?

Research has demonstrated significant associations between HNMT polymorphisms and atopic dermatitis (AD):

These findings suggest that different HNMT polymorphisms may contribute to distinct subtypes of eczema (atopic versus non-atopic), potentially through different pathophysiological mechanisms .

How do HNMT genotypes affect immunoglobulin E levels?

Total IgE levels vary significantly based on HNMT 939A>G genotypes:

• In children with atopy, subjects homozygous for GG at position 939 showed significantly lower serum total IgE levels compared to combined AA homozygous and AG heterozygous subjects (p=0.009) .

• In children with eczema, the GG genotype was similarly associated with lower total IgE levels (p=0.011), and when comparing AA versus combined AG/GG genotypes, the latter demonstrated significantly lower IgE levels (p=0.007) .

These findings suggest that the 939G allele may have a protective effect against excessive IgE production, potentially influencing the severity of allergic responses.

What cell models are appropriate for studying HNMT function?

U937 cells (human monocytic cell line) have been successfully employed as a cellular model for studying HNMT function and the effects of genetic polymorphisms . These cells can be transfected with various HNMT constructs, including:

• pHNMT CDS-3'-UTR constructs for enzyme activity measurements

• pEGFP-HNMT 3'-UTR constructs for mRNA stability assays

The use of a consistent cell model allows for controlled comparison of different HNMT variants without the confounding variables present in primary human samples .

What considerations are important when designing genotyping studies for HNMT polymorphisms?

When designing studies investigating HNMT polymorphisms, researchers should consider:

• Appropriate genotyping methods: TaqMan fluorogenic 5' nuclease assay has been validated for HNMT polymorphism detection .

• Sample preparation: Genomic DNA should be extracted using standardized methods (e.g., FlexiGene DNA kit) from whole blood samples .

• Statistical approaches:

  • Check for Hardy-Weinberg equilibrium for genotype frequencies

  • Use logistic regression with appropriate models (co-dominant, dominant, and recessive)

  • Control for covariates such as age and gender

  • Apply appropriate corrections for multiple comparisons (e.g., Bonferroni's correction)

• Sample size calculation: Ensure adequate statistical power to detect associations, particularly for polymorphisms with lower minor allele frequencies.

How can immunoprecipitation be optimized for HNMT studies?

For optimal immunoprecipitation of HNMT:

• Incubate tissue lysates containing comparable HNMT activity with varying concentrations of monoclonal HNMT antibodies for 16 hours at 4°C.

• Follow with incubation with Protein A-Sepharose for 1 hour at 4°C.

• Separate immunoprecipitates by centrifugation at 6700× g, 4°C for 1 minute.

• Wash three times with TBST and solubilize in SDS sample buffer.

• Analyze HNMT presence in both precipitate and supernatant by immunoblotting.

• Determine HNMT activity in the supernatant using radioactive methylation assays .

This protocol allows for both qualitative detection of the protein and quantitative assessment of the immunoprecipitation efficiency.

How can researchers investigate the relationship between HNMT polymorphisms and drug responses?

Investigating HNMT polymorphism effects on drug responses requires a multi-faceted approach:

• In vitro enzyme inhibition studies: Test whether HNMT variants show differential sensitivity to inhibitors like antimalarial drugs (quinacrine and amodiaquine) or SH-group reagents (p-chloromercuribenzoate and N-ethylmaleimide) .

• Cell-based pharmacological studies: Transfect cells with different HNMT variants and assess how they respond to potential HNMT inhibitors or activators.

• Genotype-phenotype correlation studies: In clinical cohorts, correlate HNMT genotypes with treatment responses to medications that might be affected by histamine metabolism.

• Pharmacokinetic analyses: Investigate whether HNMT polymorphisms affect the metabolism and clearance of drugs that interact with histamine pathways.

The methodological approach should include appropriate controls and statistical analyses to account for confounding variables.

What strategies can be used to study tissue-specific expression and regulation of HNMT?

To investigate tissue-specific HNMT expression and regulation:

• Immunohistochemical analysis: Use well-characterized monoclonal antibodies that bind to different epitopes to detect HNMT in various human tissues .

• Quantitative PCR: Measure HNMT mRNA levels across different tissues and correlate with enzyme activity to identify potential post-transcriptional regulation.

• Promoter analysis: Investigate how polymorphisms in the promoter region (-465T>C, -413C>T) affect transcriptional regulation in different cell types.

• Epigenetic studies: Analyze DNA methylation and histone modifications in the HNMT gene locus across various tissues to identify epigenetic mechanisms of tissue-specific expression.

Each approach should include appropriate controls and validation steps to ensure reliable results.

How might high-throughput screening be applied to identify novel HNMT modulators?

Development of high-throughput screening for HNMT modulators could involve:

• Establishing a fluorescence-based or luminescence-based assay for HNMT activity that can be miniaturized for microplate formats.

• Creating stably transfected cell lines expressing wild-type or variant HNMT for cell-based screening approaches.

• Developing computational models based on the HNMT structure to perform in silico screening of compound libraries.

• Validating hit compounds using secondary assays including radioactive enzymatic assays and cell-based functional studies.

Such approaches could identify both inhibitors and activators with potential therapeutic applications in histamine-related disorders.

What are promising approaches for studying the interaction between HNMT and environmental factors?

To investigate HNMT-environment interactions:

• Exposure studies in cellular models: Expose cells expressing different HNMT variants to environmental factors (allergens, pollutants, dietary components) and measure effects on enzyme expression and activity.

• Epigenetic analyses: Investigate how environmental exposures might influence epigenetic regulation of the HNMT gene.

• Animal models with humanized HNMT: Develop transgenic animals expressing human HNMT variants to study environmental interactions in vivo.

• Population studies with extensive environmental data collection: Correlate HNMT genotypes with environmental exposure data and clinical outcomes in large cohorts.

These approaches would provide insights into how environmental factors might interact with genetic predisposition to influence histamine-related disorders.