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

Abstract

ObjectiveRecently, 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.MethodsFragments 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.ResultsAll 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.ConclusionsOur 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.