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Glycosylation‐dependent activation of ENaC by the TNF lectin like domain derived peptide AP301

Pulmonary edema is a life threatening condition, and a frequent complication of acute lung injury, characterized by loss of epithelial sodium channel (ENaC) function and cell surface expression in diseased epithelia. AP301 peptide, a mimic of the lectin‐like domain of TNF, is being developed as a therapy for pulmonary edema, having recently completed phase 2a clinical trials. 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 carboxyterminal domain of α‐ENaC in H441 cells. 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.