N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic

Abstract

N-glycosylation, a common cotranslational modification, is thought to be critical for plasma membrane expression of glycoproteins by enhancing protein folding, trafficking, and stability through targeting them to the ER folding cycles via lectin-like chaperones. In this study, we show that N-glycans, specifically core glycans, enhance the productive folding and conformational stability of a polytopic membrane protein, the cystic fibrosis transmembrane conductance regulator (CFTR), independently of lectin-like chaperones. Defective N-glycosylation reduces cell surface expression by impairing both early secretory and endocytic traffic of CFTR. Conformational destabilization of the glycan-deficient CFTR induces ubiquitination, leading to rapid elimination from the cell surface. Ubiquitinated CFTR is directed to lysosomal degradation instead of endocytic recycling in early endosomes mediated by ubiquitin-binding endosomal sorting complex required for transport (ESCRT) adaptors Hrs (hepatocyte growth factor–regulated tyrosine kinase substrate) and TSG101. These results suggest that cotranslational N-glycosylation can exert a chaperone-independent profolding change in the energetic of CFTR in vivo as well as outline a paradigm for the peripheral trafficking defect of membrane proteins with impaired glycosylation.

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

“…The CFTR EL4-FAP construct did not produce a distinct mature band, which suggests that the efficiency of its glycosylation was impaired. The fourth extracellular loop of CFTR is the locus of two asparagine residues (N894 and N900) that acquire N-linked glycosylation and other modifications; therefore, careful consideration was taken to preserve these residues, and the consensus glycosylation sequences (NXS/T) surrounding them, in the FAP fusion construct (29,30). The observed incomplete glycosylation could be due to reduced glycan modification or partial oligosaccharide modification at these sites, which may result from impaired glycosylation enzyme recognition and/or accessibility at these sites when the FAP tag is present.…”

“…Because CFTR had been found to tolerate these modifications while retaining functional activity, we chose this location to create an FAP-CFTR chimeric protein. The CFTR EL4-FAP chimera was generated by inserting the FAP in the EL4 between the two asparagine residues required for the posttranslational glycosylation of CFTR (29,30).…”

Comparative Processing and Function of Human and Ferret Cystic Fibrosis Transmembrane Conductance Regulator

“…This glycosylation aids in the proper folding, enhances stability of the protein, and serves as a convenient method for studying protein processing (17)(18)(19). On an electrophoretic gel there are three potential CFTR bands (band A, B, and C) that can be seen (18,20).…”

GABRB3 Mutation, G32R, Associated with Childhood Absence Epilepsy Alters α1β3γ2L γ-Aminobutyric Acid Type A (GABAA) Receptor Expression and Channel Gating

“…N-Linked glycosylation serves several functions in the biogenesis of multimeric proteins. First, addition of glycans facilitates monomer folding and multimer assembly, thus preventing aggregation and degradation of newly synthesized subunits (20,21). Furthermore, glycan conjugation may favor assembly of certain subunits, thereby determining subunit stoichiometry (22).…”