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  • Title: Evidence for stabilization of aquaporin-2 folding mutants by N-linked glycosylation in endoplasmic reticulum.
    Author: Buck TM, Eledge J, Skach WR.
    Journal: Am J Physiol Cell Physiol; 2004 Nov; 287(5):C1292-9. PubMed ID: 15253895.
    Abstract:
    Aquaporin-2 (AQP2) is the vasopressin-sensitive water channel that regulates water reabsorption in the distal nephron collecting duct. Inherited AQP2 mutations that disrupt folding lead to nephrogenic diabetes insipidus (NDI) by targeting newly synthesized protein for degradation in the endoplasmic reticulum (ER). During synthesis, a subset of wild-type (WT) AQP2 is covalently modified by N-linked glycosylation at residue Asn123. To investigate the affect of glycosylation, we expressed WT AQP2 and four NDI-related mutants in Xenopus laevis oocytes and compared stability of glycosylated and nonglycosylated 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 nonglycosylated counterparts. For nonglycosylated isoforms, the half-life of WT AQP2 was significantly greater (>48 h) than that of 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 (approximately 25 h), 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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