RESUMEN
OBJECTIVE: Recently we characterized five mouse monoclonal antibodies that allow the specific and sensitive detection of human diamine oxidase (DAO). To understand differences in binding characteristics and recognition of enzyme variants, we mapped the antibody binding sites. METHODS: Fragments of human DAO 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 and binding site-prediction software were used to localize the epitope recognized by each antibody. RESULTS: All five monoclonal DAO antibodies bound to linear epitopes between the N3 and enzymatic domains of the 732 amino acid protein. The binding sites could be mapped onto amino acid regions V262-E278 and P279-R288, respectively, which exhibit considerable sequence variation in mammals explaining the fact that the human DAO antibodies do not cross-react with DAO from other species. The antibodies efficiently bind only denatured human DAO but not the native protein. CONCLUSIONS: Characterization of the binding sites of the DAO antibodies revealed that the antibodies bind two adjacent epitopes and exhibit similar binding characteristics and species cross-reactivity. As the epitopes do not overlap any of the amino acid substitutions described for clinically significant DAO gene polymorphisms, our antibodies will also be useful for analyses of the mutant DAO proteins.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/metabolismo , Anticuerpos Monoclonales/farmacología , Sitios de Unión , Amina Oxidasa (conteniendo Cobre)/genética , Amina Oxidasa (conteniendo Cobre)/inmunología , ADN Complementario/genética , Epítopos , Escherichia coli/genética , Humanos , Plásmidos , Unión ProteicaRESUMEN
OBJECTIVE: Recently, 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. METHODS: Fragments 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. RESULTS: All 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. CONCLUSIONS: Our 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.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Histamina N-Metiltransferasa/metabolismo , Anticuerpos Monoclonales/química , Sitios de Unión , Epítopos/química , Epítopos/metabolismo , Histamina N-Metiltransferasa/química , Histamina N-Metiltransferasa/genética , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
OBJECTIVE: The 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. METHODS: A 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. RESULTS: Six 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. CONCLUSIONS: The 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.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Histamina N-Metiltransferasa/inmunología , Histamina N-Metiltransferasa/metabolismo , Adulto , Animales , Femenino , Glutatión Transferasa/genética , Histamina N-Metiltransferasa/genética , Humanos , Riñón/metabolismo , Masculino , Ratones Endogámicos BALB C , Persona de Mediana Edad , Proteínas Recombinantes de Fusión/inmunología , Porcinos , Adulto JovenRESUMEN
Diamine oxidase (DAO) was purified to homogeneity from human seminal plasma by consecutive chromatographic fractionation on heparin-sepharose, phenyl-sepharose, CIM-QA, and Superdex 200. Human seminal plasma DAO behaves electrophoretically similar to DAO proteins from other human tissues and has very similar enzymatic properties with histamine and aliphatic diamines being the preferred substrates as well as significant conversion of polyamines. The cellular source and functional importance of DAO in human semen remain to be determined.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/metabolismo , Semen/enzimología , Adulto , Humanos , Masculino , Persona de Mediana Edad , Poliaminas , Sefarosa/análogos & derivadosRESUMEN
Diamine oxidase (DAO) oxidatively deaminates histamine and other diamines. Due to the lack of antibodies for human DAO, many findings on this enzyme had not been confirmed in man. Therefore, we produced a series of monoclonal antibodies by immunizing mice with human DAO protein fragments expressed in vitro. Five different monoclonal antibodies specific for human DAO were obtained that do not recognize any other human protein and can detect DAO with 100-fold greater sensitivity than the most sensitive enzymatic assays currently available. Using these antibodies allowed confirming the expression and cellular localization of DAO in various human tissues such as kidney, intestine and placenta where the presence of the enzyme had previously been deduced from activity measurement and DAO mRNA analysis. Due to the high sensitivity of the novel monoclonal antibodies, DAO was also detected at sites that previously evaded unequivocal proof of DAO enzymatic activity such as the urine. On the other hand, with these antibodies it was possible to show that DAO is normally not present in human liver and blood serum. The new monoclonal antibodies not only allow a comprehensive quantitative evaluation of the expression of DAO at the cellular level in man but will also facilitate sensitive analyses of disease-associated alterations of this enzyme.
Asunto(s)
Amina Oxidasa (conteniendo Cobre)/inmunología , Amina Oxidasa (conteniendo Cobre)/metabolismo , Anticuerpos Monoclonales/metabolismo , Amina Oxidasa (conteniendo Cobre)/química , Amina Oxidasa (conteniendo Cobre)/genética , Animales , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Intestinos/enzimología , Riñón/enzimología , Ratones , Ratones Endogámicos BALB C , Fragmentos de Péptidos/inmunología , Placenta/enzimología , Embarazo , Suero/enzimología , PorcinosRESUMEN
RATIONALE AND OBJECTIVES: The observation that the intravenous application of gadolinium-based contrast media can lead to nephrogenic systemic fibrosis (NSF) has raised interest in their interactions with pathways. In this context, histamine is a focus because of its stimulating effect on fibrogenesis. In humans, histamine can be inactivated either by diamine oxidase (DAO) or by histamine N-methyltransferase (HMT), and numerous drugs are known to inhibit these enzymes. Therefore, it was the aim of this study to investigate whether magnetic resonance imaging contrast agents have an inhibitory effect on the enzymatic activities of DAO and HMT. MATERIALS AND METHODS: Seven gadolinium-based (gadoterate meglumine, gadoteridol, gadobutrol, gadobenate dimeglumine, gadopentetate dimeglumine, gadoxetate disodium, gadodiamide) and one manganese-containing (mangafodipir) contrast agents were tested in vitro. Following the preincubation of purified DAO and HMT with 0.1 to 10 mmol/L of the respective contrast medium, enzyme activities were determined using radiometric microassays. Enzyme activities measured in the absence of contrast agents and after preincubation with specific inhibitors of DAO and HMT, respectively, served as controls. RESULTS: The gadolinium-containing and manganese-containing contrast media tested did not show significant inhibition of the activities of DAO and HMT. No significant difference was observed between ionic and nonionic or between cyclic and linear gadolinium compounds. Preincubation of the enzymes with specific inhibitors led to complete inhibition of the respective enzymatic activity. CONCLUSION: The gadolinium-containing and manganese-based magnetic resonance imaging contrast media tested did not exhibit significant inhibition of histamine-inactivating enzymes at physiologically relevant concentrations.