Abstract
ObjectiveRecently 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.MethodsFragments 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.ResultsAll 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.ConclusionsCharacterization 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.
Highlights
Histamine mediates many biological processes including inflammation, gastric acid secretion, neuromodulation, and regulation of immune function by binding and activating four different G-protein-coupled receptors [1, 2]
Whereas a polyclonal antibody produced earlier against porcine diamine oxidase (DAO) [14] bound to all four constructs expressed by pGEX-huDAO01-04, the five monoclonal human DAO antibodies bound only to the fusion proteins expressed by pGEX-huDAO02 and pGEX-huDAO04 (Fig. 2a), indicating that their binding sites are located on peptide R190-F435 expressed by pGEX-huDAO02
Bacterial lysates containing considerable amounts of the respective fusion proteins (Fig. 1c) were separated by SDS polyacrylamide gel electrophoresis and blotted onto polyvinylidene fluoride (PVDF) membranes to test the binding of the antibodies
Summary
Histamine mediates many biological processes including inflammation, gastric acid secretion, neuromodulation, and regulation of immune function by binding and activating four different G-protein-coupled receptors [1, 2]. Histamine is synthesized by decarboxylation of the amino acid L-histidine, catalysed by the enzyme histidine decarboxylase (HDC, EC 4.1.1.22) [3, 4]. Histamine can be inactivated either by methylation of the imidazole ring nitrogen, catalysed by histamine N-methyltransferase (HNMT, EC 2.1.1.8) or by oxidative deamination of the primary amino group, catalysed by diamine oxidase (DAO, EC 1.4.3.22) [4,5,6]. DAO uses molecular oxygen to oxidize the primary amino group of histamine forming imidazole acetaldehyde, ammonia, and hydrogen peroxide [4,
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