ABCMdb

PMID: 18682497

Chang XB, Mengos A, Hou YX, Cui L, Jensen TJ, Aleksandrov A, Riordan JR, Gentzsch M
Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator.
J Cell Sci. 2008 Sep 1;121(Pt 17):2814-23. Epub 2008 Aug 5., 2008-09-01 [PubMed]

Sentences

No. Mutations Sentence Comment

20 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp Mutation of native N-linked glycosylation sites (N894D/N900D) and treatment of cells with tunicamycin resulted in expression of unglycosylated CFTR (Fig. 2A). Login to comment 25 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp Inhibition of interactions with ER lectin chaperones does not affect localization of wild-type or ΔF508 CFTR To analyze whether the N-linked oligosaccharides are involved in retention of ΔF508 CFTR at the ER, we expressed ΔF508 with mutations at both N-glycosylation sites (ΔF508 N894D/N900D) (Fig. 3A). Login to comment 26 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp Similar to non-glycosylated wild-type CFTR, ΔF508 N894D/N900D did not interact with calnexin (Fig. 3B). Login to comment 28 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp At physiological temperature (37°C), ΔF508 localized only to the ER regardless of whether or not it was glycosylated (ΔF508 or ΔF508 N894D/N900D), as observed by immunolocalization of permeabilized cells (Fig. 3C). Login to comment 29 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp Staining of the surface CFTR of intact cells with an antibody recognizing an external epitope confirmed that unglycosylated ΔF508 N894D/N900D was not present at the plasma membrane (Fig. 3D). Login to comment 31 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp When cells were incubated at 27°C, both glycosylated ΔF508 and unglycosylated ΔF508 N894D/N900D could be detected at the cell surface (Fig. 3D). Login to comment 34 ABCC7 p.Asn900Asp ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ABCC7 p.Asn894Asp Cells expressing unglycosylated CFTR N894D/N900D showed a robust chloride efflux response to cAMP-elevating stimuli, whereas ΔF508 N894D/N900D did not display any activity, reflecting the absence of the channel protein at the plasma membrane. Login to comment 36 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp At 37°C, the wild-type protein was observed primarily in its native location in the apical membrane regardless of its glycosylation state, whereas neither glycosylated ΔF508 nor unglycosylated ΔF508 N894D/N900D CFTR was able to reach the apical membrane and was instead localized intracellulary (Fig. 3F). Login to comment 50 ABCC7 p.Asn894Asp The single-chain N894D species primarily formed a single band of mobility intermediate between the unglycosylated and wild-type Fig. 1. Login to comment 56 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp However, the other single-site variant, N900D, generated two strong bands, one with a similar mobility to that of N894D, probably representing the presence of the other single chain, and another more rapidly migrating band, most likely representative of a core rather than complex oligosaccharide chain at this site. Login to comment 57 ABCC7 p.Asn900Asp ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ABCC7 p.Asn894Asp The identity of the core-glycosylated forms of N894D and N900D was confirmed by digestion with endoglycosidase H, which selectively deglycosylates unprocessed core-glycosylated proteins and thus converted CFTR to a band with the same mobility as the N894D/N900D double mutant (Fig. 5B). Login to comment 59 ABCC7 p.Asn900Asp However, it appears that the nascent chain with only the N894 site available (N900D), progresses from the core to complex glycosylated state inefficiently. Login to comment 62 ABCC7 p.Asn900Asp ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ABCC7 p.Asn894Asp ABCC7 p.Asn894Asp The oligosaccharide at the N900 site was efficiently attached (N894D Journal of Cell Science 121 (17) in Fig. 5A) and was relatively refractory to removal by the PNGase (Fig. 5C, panel N894D), whereas that at the other site was rapidly removed to yield the unglycosylated species (Fig. 5C, compare panel N900D with N894D/N900D). Login to comment 63 ABCC7 p.Asn894Asp Interestingly, the wild-type protein (Fig. 5C, panel CFTR) behaved similarly to N894D, strongly suggesting that its two chains were differentially sensitive in the same way as when present individually. Login to comment 67 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp Although unglycosylated N894D/N900D can mature conformationally and reach the cell surface where it functions (Figs 2 and 3), it clearly turns over much more rapidly than the wild-type protein (Fig. 6A). Login to comment 68 ABCC7 p.Asn894Asp By contrast, the single-chain species N894D appeared to mature as effectively as the wild-type protein, with its two chains. Login to comment 69 ABCC7 p.Asn900Asp As had been suggested by the western blots (Fig. 5A), the N900D species matured much less effectively than the wild- Fig. 2. Login to comment 71 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp (A) Western blot of CFTR and unglycosylated CFTR created either by mutation of the glycosylation sites (N894D/N900D) or by expressing CFTR in tunicamycin-treated cells (+ Tunicamycin). Login to comment 72 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp CFTR and CFTR N894D/N900D variants were stably (left panel) or transiently (right panel) expressed in BHK-21 cells. Login to comment 75 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp (B) CFTR N894D/N900D does not interact with calnexin. Login to comment 80 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp Single-channel measurements were performed using membrane vesicles prepared from stably transfected BHK-CFTR, BHK- N894D/N900D cells or from cells transiently transfected with CFTR that were treated with tunicamycin. Login to comment 82 ABCC7 p.Asn894Asp type or N894D proteins, and the mature product appeared to turn over faster than wild-type CFTR. Login to comment 87 ABCC7 p.Asn894Asp The N894D species also turned over at a very similar rate to the wild-type protein in the BFA-exposed cells (Fig. 6B,C). Login to comment 89 ABCC7 p.Asn900Asp Again, in the BFA-treated cells, the N900D species was distinctly different from the others, turning over much more rapidly (Fig. 6B,C). Login to comment 90 ABCC7 p.Asn900Asp Thus, N900D CFTR modified with an N-linked oligosaccharide chain only at the N894 site is Fig. 3. Login to comment 91 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ΔF508 N894D/N900D cannot escape ER quality control. Login to comment 92 ABCC7 p.Asn900Asp ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ABCC7 p.Asn894Asp (A) Western blot of CFTR, CFTR N894D/N900D, ΔF508 and ΔF508 N894D/N900D expressed in BHK-21 cells. Login to comment 94 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp (B) ΔF508 N894D/N900D does not interact with calnexin. Login to comment 95 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ΔF508 N894D/N900D was immunoprecipitated by incubation with anti-CFTR antibody 596 crosslinked to Dynabeads. Login to comment 110 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp This was further confirmed by the application of the proteasomal inhibitor ALLN, which significantly stabilized CFTR N900D, but neither CFTR N894D nor the double mutant (supplementary material Fig. S4). Login to comment 112 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp We found that N894D interacted more strongly with calnexin than did N900D (Fig. 7A), but more of the latter was found in association with EDEM (Fig. 7B). Login to comment 113 ABCC7 p.Asn894Asp The oligosaccharide at position 900 might mediate prolonged or preferential association with calnexin, as reflected in the decreased binding of the N894D mutant to EDEM but unaffected interaction with calnexin as compared with wild-type CFTR. Login to comment 114 ABCC7 p.Asn900Asp By contrast, the oligosaccharide at position 894, which seems to direct the single mutant primarily into a degradative pathway at the ER, might support EDEM interaction in the N900D mutant, which is similar to that of the wild-type protein, whereas binding to calnexin is reduced. Login to comment 181 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp The CFTR variant with a single oligosaccharide at position 894 (N900D) was much more susceptible to deglycosylation by PNGase, whereas the variant with glycosylation at position 900 (N894D) was surprisingly resistant to this treatment. Login to comment 183 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp This suggests that N900D CFTR, with a glycan at position 894, might be a much better substrate for this pathway than the N894D variant. Login to comment 185 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp With just the oligosaccharide chain at position 900 (N894D), turnover was similar to that of the wild-type protein; however, with an oligosaccharide attached only at position 894 (N900D), CFTR maturation was very inefficient. Login to comment 187 ABCC7 p.Asn900Asp ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ABCC7 p.Asn894Asp The conclusion that N900D CFTR is a better substrate for ERAD than N894D CFTR was further strengthened by the preferential interaction of N900D CFTR with EDEM, which was similar to that of the wild-type protein, but which was reduced in the N894D mutant (Fig. 7), and by the observation Fig. 7. Login to comment 188 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp Interaction of N894D and N900D single-chain mutants with calnexin and EDEM. Login to comment 189 ABCC7 p.Asn900Asp ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ABCC7 p.Asn894Asp (A) Cell lysates from BHK-21 cells stably expressing CFTR, CFTR N894D, CFTR N900D or CFTR N894D/N900D were subjected to immunoprecipitation by incubation with anti-CFTR antibody 596 crosslinked to Dynabeads (IP: CFTR, as indicated on the left). Login to comment 192 ABCC7 p.Asn900Asp ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ABCC7 p.Asn894Asp (B) CFTR was immunoprecipitated from cells that were stably expressing CFTR, CFTR N894D, CFTR N900D or CFTR N894D/N900D and that were also transiently expressing HA-tagged EDEM, by incubation with anti-CFTR monoclonal antibody 596 crosslinked to Dynabeads. EDEM was detected by western blotting using monoclonal antibody HA11 and CFTR using antibody 596. Login to comment 202 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp The carbohydrate at position 900 in CFTR N894D supports the route to maturation and progress to the Golgi, whereas the oligosaccharide at asparagine residue 894 in N900D promotes degradation. Login to comment 203 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp that the proteasomal inhibitor ALNN stabilized N900D CFTR, but not N894D CFTR (supplementary material Fig. S4). Login to comment 204 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp It seems likely that the double mutant behaves in the ER like CFTR N894D rather than like CFTR N900D, because the presence of the oligosaccharide at N894 supports the ERAD pathway, whereas the absence of this oligosaccharide allows the double glycosylation mutant to exit the ER and proceed to the Golgi and plasma membrane. Login to comment 207 ABCC7 p.Asn900Asp We cannot rule out the possibility that the lack of glycans might affect the conformation of CFTR and that the N900D mutation is accompanied by a significant degree of misfolding that makes it a better ERAD substrate. Login to comment 219 ABCC7 p.Asn900Asp ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ABCC7 p.Asn894Asp Materials and Methods Mutagenesis and stable expression of CFTR Replacement of asparagine residues N894 and N900 with aspartate residues was achieved by replacing a CFTR HpaI-DraIII cDNA fragment (bp 2463 to 3328) in pNUT CFTR with a counterpart generated by PCR coding for N894D, N900D or N894D/N900D, to produce CFTRs with only one or no glycosylation site (Chang et al., 1994). Login to comment 224 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp The well-differentiated cultures were then transduced with adenoviral vectors as described previously (Coyne et al., 2000) to express Extope-CFTR and Extope-ΔF508 with intact or mutated glycosylation sites (N894D/N900D). Login to comment