Human extravillous trophoblast (EVT) invasion of the pregnant uterus constitutes a pivotal event for the establishment of the maternal-fetal interface. Compromised EVT function manifesting in inadequate arterial remodeling is associated with the severe pregnancy disorder early-onset preeclampsia (eoPE).

Recent studies suggest that EVTs invade the entire uterine vasculature including arteries, veins and lymphatics in the first trimester of pregnancy. We therefore hypothesized that EVT-derived factors accumulate in the circulation of pregnant women early in gestation and may serve to predict eoPE.

In contrast to published literature, we demonstrate that placenta-associated diamine oxidase(DAO) is not expressed by maternal decidual cells but solely by EVTs, especially when in close proximity to decidual vessels.

Cultures of primary EVTs express and secret large amounts of bioactive DAO.

ELISA measurements indicate a pregnancy-specific rise in maternal DAO plasma levels around gestational week (GW) 7 coinciding with vascular invasion of EVTs.

Strikingly, DAO levels from eoPE cases were significantly lower (40%) compared to controls in the first trimester of pregnancy but revealed no difference at mid gestation. Furthermore, DAO-containing pregnancy plasma rapidly inactivates pathophysiologically relevant histamine levels.

This study represents the first proof of concept suggesting EVT-specific signatures as diagnostic targets for the prediction of eoPE.

OBJECTIVE:

Recently we characterized five mouse monoclonal antibodies that allow the specific and sensitive detection of human diamine oxidase (DAO). To understand differences in binding characteristics and recognition of enzyme variants, we mapped the antibody binding sites.

METHODS:

Fragments of human DAO 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 and binding site-prediction software were used to localize the epitope recognized by each antibody.

RESULTS:

All five monoclonal DAO antibodies bound to linear epitopes between the N3 and enzymatic domains of the 732 amino acid protein. The binding sites could be mapped onto amino acid regions V²⁶²-E²⁷⁸ and P²⁷⁹-R²⁸⁸, respectively, which exhibit considerable sequence variation in mammals explaining the fact that the human DAO antibodies do not cross-react with DAO from other species. The antibodies efficiently bind only denatured human DAO but not the native protein.

CONCLUSIONS:

Characterization of the binding sites of the DAO antibodies revealed that the antibodies bind two adjacent epitopes and exhibit similar binding characteristics and species cross-reactivity. As the epitopes do not overlap any of the amino acid substitutions described for clinically significant DAO gene polymorphisms, our antibodies will also be useful for analyses of the mutant DAO proteins.