Waheed Shabbir, Susan Tzotzos, Minela Bedak, Mohammad Aufy, Anita Willam, Martin Kraihammer, Alexander Holzner, Hendrik Fischer, Helmut Pietschmann, Bernhard Fischer, Rudolf Lucas and Rosa Lemmens-Gruber
Pulmonary edema is a life threatening condition, and a frequent complication of acute respiratory distress syndrome (ARDS), characterized by loss of epithelial sodium channel (ENaC) function and cell surface expression in diseased epithelia. We synthesized a synthetic peptide AP301, which mimics the lectin-like domain of TNF. AP301 has recently completed phase II clinical trials and is being developed as therapy for treatment of pulmonary edema in ARDS patients. We have previously shown that the proper glycosylation of H441 and A549 cells is crucial for its interaction with AP301 and initiates uptake of the peptide and interaction with the carboxy terminal domain of ENaC in H441. In this study we identify a glycosylation-dependent mechanism that preserves ENaC expression and function. Single- and multi-N-glycosylation site mutations were generated in α (N232, 293,312,397,511Q) - and δ (N166, 211,384Q)-subunits. Therefore, αL576X and αN232, 293,312,397,511Q, L576X mutants were generated by deletion of the carboxy terminal of wild type and quintuple (N232, 293,312,397,511Q) mutant α-hENaC. These constructs were co-expressed in HEK-293 cells with βγ-hENaC. In α (N232, 293,312,397,511Q), L576Xβγ-hENaC, AP301 activation, measured in the whole cell patch clamp mode, was abolished, and in αL576Xβγ-hENaC, it was attenuated. Taken together, our findings delineate an AP301 N-glycan dependent interaction leading to normalization of both sodium and fluid absorption in edematous alveoli to non-edematous levels. Futhermore, AP301 could also be a therapeutic options of other diseases where ENaC function is reduced due to some pathophysiological alterations.
Acknowledgment W.S. received financial support by APEPTICO GmbH.