The mutant human CFTR protein ΔF508, which is present in 90% of cystic fibrosis patients, has some residual chloride channel activity (Dalemans et al., 1991
; Drumm et al., 1991
). However, it does not mature and traffic to the cell surface but is retained at the ER and eliminated by the proteasome (Jensen et al., 1995
; Ward et al., 1995
). Therefore, the mechanisms resulting in the different fates of the wild-type and mutant proteins are subject to intense investigation.
Although an increasing number of proteins, including components of the Hsp70 and Hsp90 chaperone complexes, have been identified that interact with ΔF508 CFTR at the ER and affect its processing to some extent, the detailed mechanism of its ER retention is not completely understood (Amaral and Kunzelmann, 2007
; Riordan, 2008
; Wang et al., 2006
). It has been repeatedly proposed that calnexin might be a target for developing a therapeutic approach to rescue processing of ΔF508 CFTR and therefore it is important to clarify whether or not ΔF508 CFTR is retained by this lectin.
Numerous membrane glycoproteins have been shown to interact with calnexin, but in many cases it has not been clearly demonstrated that calnexin is a primary determinant of ER retention or export (Dickson et al., 2002
; Halaban et al., 2000
; Lu et al., 2003
; Morello et al., 2001
; Nagaya et al., 1999
; Rigot et al., 1999
). We show in this study that the failure to export the ΔF508 CFTR from the ER is independent of N-glycosylation and ER lectin interactions. The mutant protein was not able to reach the cell surface when interactions with ER-lectin chaperones were inhibited either by removal of N-linked glycosylation sites or by pharmacological inhibition (Figs and ). Another very recent study confirmed, using calnexin knockout mice, that calnexin is not necessary for ER retention of ΔF508 CFTR (Okiyoneda et al., 2008
). This is in contrast to some other membrane glycoproteins, such as the major histocompatibility complex class I molecules and influenza hemagglutinin, which are retained by calnexin when not correctly assembled (Brothers et al., 2006
; Jackson et al., 1994
; Molinari et al., 2004
; Rajagopalan and Brenner, 1994
; Zhang et al., 1997
A lack of N-linked oligosaccharides also did not affect sorting of wild-type CFTR to the plasma or apical membrane. Unlike CFTR, the N-glycans of some other membrane proteins, such as the β-subunits of the Na,K-ATPase or the gastric H,K-ATPase, carry apical sorting information (Vagin et al., 2007
). Other examples of membrane proteins for which N-linked glycosylation is required for efficient trafficking to the cell surface are shaker K+
channels (de Souza and Simon, 2002
), the glutamate receptor (Standley and Baudry, 2000
), NKCC2 (SLC12A1) (Paredes et al., 2006
) and aquaporin 2 (Hendriks et al., 2004
We found that complete removal of N-linked glycosylation at both sites of CFTR did however influence turnover of the mature CFTR protein in post-ER compartments. The non-glycosylated double mutant turned over rapidly but behaved like wild-type CFTR in the presence of brefeldin A, which blocks transport from the ER to the Golgi apparatus (). Farinha and Amaral (Farinha and Amaral, 2005
) observed a shortened half-life of CFTR when both N-glycosylated asparagines were replaced by alanines or glutamines, but did not test whether degradation occurred at the ER or in a later compartment. Our results clearly show that unglycosylated CFTR is degraded after the protein has exited the ER.
Rapid turnover and decreased stability in the absence of N-linked glycosylation have also been observed in other membrane proteins. Mutation of N-linked glycosylation sites of the human κ opioid receptor led to a threefold faster turnover than that of the wild-type protein (Li et al., 2007
). Similarly, another ABC transporter, P-glycoprotein (ABCB1), turns over three times faster when N-linked glycosylation is inhibited (Zhang et al., 2004
). This degradation apparently occurs as a consequence of increased ubiquitylation. Ubiquitylation of mature CFTR has been shown to promote its targeting to lysosomal degradation (Sharma et al., 2004
) and, therefore, a similar mechanism might contribute to the faster turnover of non-glycosylated CFTR.
As has been reported in previous studies, we did not find the chloride channel function of CFTR to be affected by lack of N-glycosylation (Chang et al., 1994
; Gregory et al., 1991
; Morris et al., 1993
). Analogous to CFTR, the related ABC transporter P-glycoprotein functions without attached oligosaccharides (Gribar et al., 2000
; Kramer et al., 1995
; Schinkel et al., 1993
; Urbatsch et al., 2001
). Moreover, it has recently been shown that N-linked glycosylation is not essential for expression, transport activity, or trafficking of another human ABC protein, ABCG2 (Diop and Hrycyna, 2005
; Mohrmann et al., 2005
We found that the individual CFTR glycosylation mutants, with just one oligosaccharide chain attached, behaved differently to each other in several respects. 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. PNGase is a cytoplasmic enzyme that catalyzes deglycosylation of proteins during ERAD, before they are degraded by the proteasome, and it has been shown that this N-glycanase acts specifically on denatured and misfolded proteins (Hirsch et al., 2003
; Hirsch et al., 2004
; Joshi et al., 2005
; Suzuki and Lennarz, 2003
). This suggests that N900D CFTR, with a glycan at position 894, might be a much better substrate for this pathway than the N894D variant.
Pulse-chase labeling experiments demonstrated that the individual oligosaccharides do affect the fate of the wild-type protein at the ER differently. 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. When transport from the ER was inhibited by brefeldin A, the CFTR mutant with an oligosaccharide attached to position 894 was very unstable and was rapidly removed by ERAD (). 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 (), and by the observation that the proteasomal inhibitor ALNN stabilized N900D CFTR, but not N894D CFTR (supplementary material Fig. S4
). 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.
The concept that each glycan of CFTR is capable of directing the processing of the protein at the ER in a different way fits extraordinarily well with the results obtained in this study. We suggest a model in which the oligosaccharide at position N894 accelerates ERAD, whereas the oligosaccharide attached to position 900 promotes maturation and allows the protein to progress to the Golgi ().
Fig. 8 Individual N-linked oligosaccharides promote different fates in the processing of CFTR. Immediately after addition of the core glycan to the nascent polypeptide chain, the outermost of the three glucose residues is removed by glucosidase I. Subsequently, (more ...)
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. However, this does not seem a very likely scenario because the double mutant, which lacks both oligosaccharide chains, is stable at the ER, similar to wild-type CFTR.
Interestingly, distinct functions of N-glycans have been observed in the processing of other glycoproteins. Hebert et al. (Hebert et al., 1997
) reported that the number and location of glycans on influenza hemagglutinin determined its folding and association with calnexin. In another study, two out of eight N-glycosylation sites were shown to be the major determinants for efficient apical sorting of the transmembrane protein endolyn (CD164) (Potter et al., 2004
). In case of the gastric H,K-ATPase β-subunit, just one out of four N-glycosylation sites affected ER-to-Golgi trafficking and enhanced endocytosis from the apical membrane (Vagin et al., 2004
). In yeast, it has been observed that the N-linked oligosaccharides of CPY have different impacts on the processing of the protein and the most C-terminal of the four N-linked oligosaccharides is specifically required for ERAD (Kostova and Wolf, 2005
; Spear and Ng, 2005
The exact, molecular details of the means by which the individual glycans affect the processing of CFTR are not yet apparent, but CFTR-interacting lectins, including calnexin and EDEM, might act upon the variously glycosylated forms differentially (). The interactions of N-linked carbohydrates of CFTR at the ER might not be limited to these two chaperones, but could also involve lectin receptors such as ERGIC53 (LMAN1), VIP36 (LMAN2), VIPL (LMAN2L) or erlectin, which have all been shown to facilitate trafficking of certain glycoproteins in the early secretory pathway (Appenzeller-Herzog and Hauri, 2006
; Appenzeller et al., 1999
; Cruciat et al., 2006
; Neve et al., 2003
; Shimada et al., 2003
In conclusion, the two N-linked oligosaccharides are not responsible for retention of ΔF508 CFTR at the ER, but they strongly influence turnover of the mature wild-type protein in post-ER compartments. In addition, it is notable that each individual oligosaccharide chain directs ER processing of CFTR differently. This might be due either to different oligosaccharide structures at the two sites, or to differences in the accessibility of the N-glycans at their separate locations in the polypeptide chain.