N-Glycanase 1 Regulates Aquaporins Independent of Its Enzymatic Activity

Patients with pathogenic mutations in NGLY1 cannot make tears (alacrima), have global developmental delay and liver dysfunction. Traditionally, N-glycanase 1 (NGLY1) cleaves intact N-glycans from misfolded, retrotranslocated glycoproteins prior to their proteasomal degradation. We found that Ngly1-null mouse embryonic fibroblasts (MEFs), NGLY1 knockout human cells, and patient fibroblasts are resistant to hypotonic lysis. Ngly1-deficient MEFs swell slower and have reduced aquaporin 1 (Aqp1) mRNA and protein expression. Ngly1 knockdown and overexpression confirmed that Ngly1 regulates Aqp1 and hypotonic cell lysis. Patient fibroblasts and NGLY1 KO cells show reduced AQP11 mRNA, supporting NGLY1 regulates expression of multiple AQPs across species. Complementing Ngly1-deficient cells with catalytically inactive NGLY1 (p.Cys309Ala) restored normal hypotonic lysis and Aqp1 protein. We show that transcription factor Creb1 regulates Aqp1 and that the Atf1/Creb1 signaling pathway is disrupted in Ngly1-deficient MEFs. These results identify a novel, non-enzymatic, regulatory function of NGLY1 in AQP transcription possibly related to alacrima.