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1 Molecular Medicine, Oregon Health & Sciences University, Portland, Oregon, USA
* To whom correspondence should be addressed. E-mail: skachw{at}ohsu.edu.
Aquaporin-2 (AQP2) is the vasopressin sensitive water channel that regulates water reabsorbtion in the distal nephron collecting duct. Inherited AQP2 mutations that disrupt folding lead to nephrogenic diabetes insipidus by targeting newly synthesized protein for degradation in the endoplasmic reticulum (ER). During synthesis, a subset of WT AQP2 is covalently modified by N-linked glycosylation at residue Asn123. To investigate the affect of glycosylation, we expressed WT and four NDI-related mutants in Xenopus laevis oocytes and compared stability of glycosylated and non-glycosylated isoforms. In all constructs, approximately 15-20% of newly synthesized AQP2 was covalently modified by N-linked glycosylation. At steady state, however, core glycosylated WT protein was nearly undetectable, whereas all mutants were found predominantly in the glycosylated form (60-70%). Pulse chase metabolic labeling studies revealed that glycosylated isoforms of mutant AQP2 were significantly more stable than their non-glycosylated counterparts. For non-glycosylated isoforms, the half-life of WT AQP2 was significantly greater (>48 h) than mutant AQP2 (T126M 4.1± 1.0 h, A147T 4.2± 0.60 h, C181W 4.5± 0.50 h, R187C 6.8 ± 1.2 h). This is consistent with rapid turnover in the ER as previously reported. In contrast, the half-lives of mutant proteins containing N-linked glycans were similar to WT (T1/2=~25h), indicating that differences in steady state glycosylation profiles are caused by increased stability of glycosylated mutant proteins. These results suggest that addition of a single N-linked oligosaccharide moiety can partially compensate for ER folding defects induced by disease related mutations.
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