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

Hubert G Schwelberger,Johannes Feurle,Gunnar Houen

doi:10.1007/s00011-017-1086-7

Hubert G Schwelberger, Johannes Feurle + Show 1 more

Open Access

https://doi.org/10.1007/s00011-017-1086-7

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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.

Highlights

Histamine binds and activates four different G-proteincoupled receptors and thereby mediates many biological processes including inflammation, gastric acid secretion, neuromodulation, and regulation of immune function [1, 2]
Fragments of human histamine N-methyltransferase (HNMT) were expressed as glutathione S-transferase fusion proteins that were used for testing antibody binding on immunoblots
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

Summary

Introduction

Histamine binds and activates four different G-proteincoupled receptors and thereby mediates many biological processes including inflammation, gastric acid secretion, neuromodulation, and regulation of immune function [1, 2]. Histamine can be inactivated either by oxidative deamination of the primary amino group, catalyzed by diamine oxidase (DAO, EC 1.4.3.22), or by methylation of the imidazole ring, catalyzed by histamine N-methyltransferase (HNMT, EC 2.1.1.8) [4,5,6]. Human HNMT is a small monomeric protein of 33 kDa consisting of a single polypeptide chain of 292 amino acid residues and catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to the secondary amino group of the imidazole ring of histamine forming Nsmethylhistamine [6]. HNMT is a cytosolic protein that is responsible for the inactivation of intracellular histamine, which is either synthesized in the cell or taken up from the extracellular space after binding to one of its receptors present on the cell surface or by plasma membrane transporters [2, 4]

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