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Mapping of the binding sites of human diamine oxidase (DAO) monoclonal antibodies.

ABSTRACT: 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.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.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.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.

SUBMITTER: Schwelberger HG

PROVIDER: S-EPMC5807474 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature

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Mapping of the binding sites of human diamine oxidase (DAO) monoclonal antibodies.

Schwelberger Hubert G HG Feurle Johannes J Houen Gunnar G

Inflammation research : official journal of the European Histamine Research Society ... [et al.] 20171121 3

<h4>Objective</h4>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.<h4>Methods</h4>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, sequen ...[more]

PMID: 29164268

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Project description:Unlike in flowering plants, the detailed roles of the enzymes in the polyamine (PA) pathway in conifers are poorly known. We explored the sequence conservation of the PA biosynthetic genes and diamine oxidase (DAO) in conifers and flowering plants to reveal the potential functional diversification of the enzymes between the plant lineages. The expression of the genes showing different selective constraints was studied in Scots pine zygotic embryogenesis and early seedling development. We found that the arginine decarboxylase pathway is strongly preferred in putrescine production in the Scots pine as well as generally in conifers and that the reduced use of ornithine decarboxylase (ODC) has led to relaxed purifying selection in ODC genes. Thermospermine synthase (ACL5) genes evolve under strong purifying selection in conifers and the DAO gene is also highly conserved in pines. In developing Scots pine seeds, the expression of both ACL5 and DAO increased as embryogenesis proceeded. Strong ACL5 expression was present in the procambial cells of the embryo and in the megagametophyte cells destined to die via morphologically necrotic cell death. Thus, the high sequence conservation of ACL5 genes in conifers may indicate the necessity of ACL5 for both embryogenesis and vascular development. Moreover, the result suggests the involvement of ACL5 in morphologically necrotic cell death and supports the view of the genetic regulation of necrosis in Scots pine embryogenesis and in plant development. DAO transcripts were located close to the cell walls and between the walls of adjacent cells in Scots pine zygotic embryos and in the roots of young seedlings. We propose that DAO, in addition to the role in Put oxidation for providing H2O2 during the cell-wall structural processes, may also participate in cell-to-cell communication at the mRNA level. To conclude, our findings indicate that the PA pathway of Scots pines possesses several special functional characteristics which differ from those of flowering plants.

Dataset Information

Mapping of the binding sites of human diamine oxidase (DAO) monoclonal antibodies.

Publications

Mapping of the binding sites of human diamine oxidase (DAO) monoclonal antibodies.

Similar Datasets

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