Abstract
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.
Highlights
Histamine is an important mediator of many biological processes including inflammation, gastric acid secretion, neuromodulation, and regulation of immune function acting through four different G-protein-coupled receptors [1, 2]
Six different monoclonal antibodies specific for human histamine N-methyltransferase (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
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
Summary
Histamine is an important mediator of many biological processes including inflammation, gastric acid secretion, neuromodulation, and regulation of immune function acting through four different G-protein-coupled receptors [1, 2]. The major routes of histamine inactivation in mammals are oxidative deamination of the primary amino group, catalyzed by diamine oxidase (DAO, EC 1.4.3.22), and methylation of the imidazole ring, catalyzed by histamine N-methyltransferase (HMT, EC 2.1.1.8) [4,5,6]. HMT 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 Nsmethylhistamine [6]. HMT appears to be a cytosolic protein that is responsible for the inactivation of intracellular histamine, which is either synthesized in the cell or taken up from the extracellular space after binding to one of its receptors present on the cell surface or by plasma membrane transporters [4]
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