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Mapping of the binding sites of human histamine N-methyltransferase (HNMT) monoclonal antibodies

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: Frag...

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Detalles Bibliográficos
Autores principales: Schwelberger, Hubert G., Feurle, Johannes, Houen, Gunnar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer International Publishing 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633628/
https://www.ncbi.nlm.nih.gov/pubmed/28791419
http://dx.doi.org/10.1007/s00011-017-1086-7
Descripción
Sumario: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 L(182)–T(223), three region M(224)–E(261), and one region L(262)–A(292). All three antibodies recognising only human HNMT bound the C-terminal region L(262)–A(292) 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.