Human diamine oxidase undergoes transcytosis and colocalizes with the plasma membrane monoamine transporter indicating intracellular histamine degradation

Secreted human diamine oxidase (hDAO) is the only enzyme capable of extracellular histamine inactivation. Histamine is the key mediator released at high concentrations from activated mast cells causing various unpleasant to life-threatening symptoms. Recombinant hDAO is internalized into various cell types. However, rhDAO-containing intracellular vesicles do not colocalize with the lysosomes indicating that rhDAO is unlikely targeted for direct degradation. After internalization rhDAO was rapidly exocytosed without loss of enzymatic activity and re-internalized. Total internal reflection fluorescence microscopy revealed that rhDAO undergoes transcytosis in endothelial cells. The number of transcytosis events was dose-dependent and saturable indicating a specific, receptor-mediated uptake mechanism. Addition of unlabeled DAO, heparin and heparin-binding motif mutated DAO significantly reduced the number of transcytosis events. After intravenous injection of rhDAO into mice it was detected in vesicles not only within but also below the endothelial cell layer of the thoracic aorta. Localization of hDAO within extracellular vesicles was not detected in biological fluids. In various cell types intracellular vesicular rhDAO colocalized with plasma membrane monoamine transporter (PMAT), but not with the organic cationic transporters 1-3. Histamine is described to be a substrate for all four transporters. Cytoplasmic intravesicular localization of DAO has been detected for many decades but no definite physiological function could be assigned. Newly discovered transcytosis might transport DAO into the interstitial fluid compartment with widespread tissue distribution, especially during pregnancy. Vesicular colocalization of DAO with the histamine transporter PMAT indicates intracellular histamine degradation within these poorly characterized vesicles.