Identification of a receptor for human diamine oxidase

The human protein diamine oxidase (hDAO) is important for histamine inactivation. Histamine is a small substance, which contributes to symptoms during allergic reactions like nausea, headache, runny nose, and in more severe cases low blood pressure and fortunately rarely fatal hypersensitivity reactions. Medications blocking histamine are not powerful enough to prevent severe symptom development. Human DAO might be used as a new treatment option for histamine-mediated symptoms, but there are too many unknowns about DAO. We therefore produced hDAO in animal cells. When the protein was injected into animals it rapidly disappeared from the circulation. The carbohydrates on the surface of DAO are not involved in the fast removal of the protein from the plasma of animals. Our preliminary experiments show that hDAO is taken up by human endothelial cells. This is likely mediated by a so far unknown specific docking protein, a so called receptor, on the surface of these cells. In this project we will investigate, which other cells are able to show uptake of hDAO. Then we will characterize the interaction between the docking station and the protein in different cell types. Identification of the receptor will be performed by testing all human genes expressed in receptor- positive cells in receptor-negative cells. Repeated rounds of testing for receptor-positive cells should eventually result in the isolation of the receptor DNA. Finally, we will examine the mechanism by which hDAO enters cells and determine its fate inside cells using different microscopy techniques. This will be the first study, which investigates the clearance mechanism of hDAO. The underlying mechanism could also apply to other proteins. Isolation of the docking protein for DAO will also allow us to better understand, how the human body inactivates histamine. The generated knowledge will be essential for the development of recombinant hDAO as a new, old treatment approach for human diseases with excess histamine. Diamine oxidase purified from porcine kidney tissue was used for the treatment of various conditions more than 80 years ago, but in the 21st century recombinant human proteins are much more suitable for human usage. Successful completion of this project will move us closer to reach this important goal.

The human protein diamine oxidase (hDAO) is important for histamine inactivation. Histamine is a small substance, which contributes to symptoms during allergic reactions like nausea, headache, runny nose, and in more severe cases low blood pressure and fortunately rarely fatal hypersensitivity reactions. Medications blocking histamine are not powerful enough to prevent severe symptom development. Human DAO might be used as a new treatment option for histamine mediated symptoms. We therefore produced hDAO in animal cells. When the protein was injected into rats and mice it rapidly disappeared from the circulation, which is highly unfavorable for a pharmaceutical. The carbohydrates on the surface of DAO are not involved in the fast removal of the protein from the plasma of animals, but hDAO is taken up by various cell types endothelial cells in particular. We found that DAO binds to carbohydrate structures on the cell surface, so-called heparan sulfate proteoglycans (HSPGs). After altering the HSPG-binding site of DAO the half-life in the blood of animals increased significantly. However, HSGPs did not seem to be the only interaction partners for DAO but our data pointed to another docking site, a so-called receptor, on the cell surface. We were able to identify neuropilin 1 (NRP1) as a potential candidate receptor for DAO binding and cellular uptake. In addition, we found that unlike many other proteins DAO is not degraded after entering the cells but that is passes through and exits them via a mechanism named transcytosis. In this study we have generated important knowledge that will be essential for the development of recombinant hDAO as a new, "old" treatment approach for human diseases with excess histamine. Diamine oxidase purified from porcine kidney tissue was used for the treatment of various conditions more than 80 years ago, but in the 21st century recombinant human proteins are much more suitable for human usage. The successful completion of this project has moved us closer to reach this important goal.