ABCMdb

ABCC7 p.Asn894Asp

None has been submitted yet.

Publications

PMID: 18682497 [PubMed] Chang XB et al: "Role of N-linked oligosaccharides in the biosynthetic processing of the cystic fibrosis membrane conductance regulator."

No. Sentence Comment

20 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 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 Similar to non-glycosylated wild-type CFTR, ΔF508 N894D/N900D did not interact with calnexin (Fig. 3B). Login to comment
28 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 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 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 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 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 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 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 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
62 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 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 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 By contrast, the single-chain species N894D appeared to mature as effectively as the wild-type protein, with its two chains. Login to comment
71 (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 CFTR and CFTR N894D/N900D variants were stably (left panel) or transiently (right panel) expressed in BHK-21 cells. Login to comment
75 (B) CFTR N894D/N900D does not interact with calnexin. Login to comment
80 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 type or N894D proteins, and the mature product appeared to turn over faster than wild-type CFTR. Login to comment
87 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
91 ΔF508 N894D/N900D cannot escape ER quality control. Login to comment
92 (A) Western blot of CFTR, CFTR N894D/N900D, ΔF508 and ΔF508 N894D/N900D expressed in BHK-21 cells. Login to comment
94 (B) ΔF508 N894D/N900D does not interact with calnexin. Login to comment
95 ΔF508 N894D/N900D was immunoprecipitated by incubation with anti-CFTR antibody 596 crosslinked to Dynabeads. Login to comment
110 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 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 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
181 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 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 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 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 Interaction of N894D and N900D single-chain mutants with calnexin and EDEM. Login to comment
189 (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 (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 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 that the proteasomal inhibitor ALNN stabilized N900D CFTR, but not N894D CFTR (supplementary material Fig. S4). Login to comment
204 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
219 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 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

PMID: 19307599 [PubMed] Glozman R et al: "N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic."

No. Sentence Comment

24 Results Cell surface expression of glycosylation-deficient CFTR is severely reduced CFTR glycosylation was abolished by mutagenesis of the two consensus Asn-linked N-glycosylation sites (Kornfeld and Kornfeld, 1985) individually (N894D and N900D) or together (2D-CFTR) in CFTR. Login to comment
26 [ID]FIG1[/ID] The endoglycosidase (endo) H and F sensitivity of N894D- and N900D-CFTR verified that both glycan chains underwent complex glycosylation, whereas the 2D-CFTR failed to obtain N-glycans (Fig. 1 B). Login to comment
27 Indirect immunostaining of the extracellular 3HA tag in nonpermeabilized cells revealed that N894D-, N900D-, and 2D-CFTR were expressed at the cell surface, as proved by colocalization with Alexa Fluor 594-conjugated wheat germ agglutinine (WGA), a plasma membrane marker (Fig. 1 C). Login to comment
28 Quantitative analysis of CFTR cell surface density by anti-HA antibody (Ab) binding to the 3HA tag revealed that the N894D-, N900D-, and 2D-CFTR expression level was decreased by ~37%, ~63%, and ~87%, respectively, relative to wild-type (wt) CFTR, which is in line with the immunostaining results (Fig. 1 D). Login to comment
54 The wt CFTR folding efficiency (34.0 ± 5.6%) was reduced to 15.6 ± 0.5% and 13.8 ± 1.2% in N894D- and N900D-CFTR, respectively (Fig. 2 A). Login to comment
92 The transport-competent native 2D-CFTR has threefold accelerated (t1/2 = ~4 h), whereas the N894D- and N900D-CFTR have approximately twofold faster metabolic turnover rates than the wt channel (t1/2 = ~12 h; Fig. 4, A and B). Login to comment
303 The N894D-/N900D-CFTR (2D-CFTR) was generated using the N894D-CFTR as a template and the N900D mutagenic primers. Login to comment
306 Cell culture and transfection Stably transfected BHK cell lines expressing wt, N894D, N900D, 2D-, 2A-, and 2Q- or ΔF508-CFTR-2D as well as N900D+T908N- and 2D+T908N-CFTR variants were generated as described previously (Du et al., 2005). Login to comment

PMID: 20008117 [PubMed] Cholon DM et al: "Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures."

No. Sentence Comment

268 A: Western blot of lysates of HAE cells adenovirally expressing CFTR N894D N900D, in which asparagine residues N894 and N900 are replaced with aspartate, confirmed lack of glycosylation. Login to comment
269 B: primary HAE cultures were adenovirally transduced to express wild-type CFTR and a nonglycosylated variant (N894D N900D) of CFTR. Login to comment
272 Time course experiments were performed on 3 different HAE cultures; 1 representative example is shown. D: immunofluorescence labeling of sodium-potassium-chloride cotransporter isoform 1 (NKCC1) confirms that internalized CFTR N894D N900D is transported to basolateral compartments. Login to comment
273 HAE cultures were adenovirally transduced to express CFTR N894D N900D. Login to comment
266 A: Western blot of lysates of HAE cells adenovirally expressing CFTR N894D N900D, in which asparagine residues N894 and N900 are replaced with aspartate, confirmed lack of glycosylation. Login to comment
267 B: primary HAE cultures were adenovirally transduced to express wild-type CFTR and a nonglycosylated variant (N894D N900D) of CFTR. Login to comment
270 Time course experiments were performed on 3 different HAE cultures; 1 representative example is shown. D: immunofluorescence labeling of sodium-potassium-chloride cotransporter isoform 1 (NKCC1) confirms that internalized CFTR N894D N900D is transported to basolateral compartments. Login to comment
271 HAE cultures were adenovirally transduced to express CFTR N894D N900D. Login to comment

PMID: 21571517 [PubMed] Okiyoneda T et al: "Protein quality control at the plasma membrane."

No. Sentence Comment

31 Structural destabilization of the Pma1 and Gap1 transmembrane domains in strains defective of sphingoid base synthesis could be mechanistically similar to the farnesol-induced 484 Membranes and organelles Table 1 Peripheral protein QC substrates Membrane protein Mutation/condition Degron PM stability Related disease Reference Mammalian bile salt export pump (BSEP) E297G (Cy) D482G (Cy) 2-3 Ub # progressive familial intrahepatic cholestasis type 2 (PFIC2) [36] CFTR rDF508 (Cy) poly/multimono Ub # cystic fibrosis (CF) [44 ,27 ] D70 (Cy truncation) poly/multimono Ub # [27 ] N894D, N900D (Ex) poly/multimono Ub # [22] Na/H exchanger (NHE6) D255-256 (TM) poly/multimono Ub # Angelman syndrome [35] MLC1 multiple (TM or Cy) ND # megalencephalic leukoencephalopathy with subcortical cysts (MLC) [66] HERG low K+ Ub # type 2 long QT syndrome [37] LDL receptor high salt or low pH (Ex) ND # hypercholesterolemia [16] Dopamine D4.4 receptor M345T (TM) poly/multimono Ub # attention deficit hyperactivity disorder [28 ] Vasopressin V2 receptor W164S (TM) poly/multimono Ub # nephrogenic diabetes insipidus [28 ] alpha-2A adrenergic receptor D79N (TM) ND # cardiovascular diseases [14] N422D (TM) ND # [14] Di3loop (Cy) ND # [69] CD4tl-lm L57C (Cy) poly/multimono Ub # model protein [28 ] H+ /K+ -ATPase b subunit N99Q, N130Q, N161Q, N222Q (Ex) ND # gastric, autoimmune diseases [18] k opioid receptor N25/39Q (Ex) ND # pain control, neuronal phenotypes [19] D opioid receptor N18Q/N33Q (Ex) ND # pain control, neuronal phenotypes [20] GLUT1 N45Y, Q or D (ex) ND # GLUT1 deficiency syndrome [70] EGFR L858R (Cy), exon 19 deletion (Cy) poly/multimono Ub # cancer suspectibility [71] ErbB2 Hsp90 inhibition poly/multimono Ub # breast cancer [72] TGFBR2 Hsp90 inhibition poly/multimono Ub # tumor suspectibility [73] Yeast Pma1 Icb1-100 poly/multimono Ub # NA [29] Pma1-7 poly/multimono Ub # NA [7] Pma1-10 poly/multimono Ub # NA [52] Gap1 absence of sphingolipids poly/multimono Ub # NA [9] Abbreviations: Cy, cytosolic; Ex, extracellular; TM, transmembrane; Ub, ubiquitin; ND, not determined; #, decreasing stability; CFTR, cystic fibrosis transmembrane conductance regulator; HERG, human ether-a` -go-go related gene; LDL, low-density lipoprotein; GLUT, glucose transporter; EGFR, epidermal growth factor receptor; ErbbB2, v-erb-b2 erythroblastic leukemia viral oncogene homolog 2; TGFBR2, transforming growth factor (TGF)- beta type II; Pma1, H(+)-ATPase; Gap1, general amino acid permease. Login to comment

PMID: 17113596 [PubMed] Cui L et al: "Domain interdependence in the biosynthetic assembly of CFTR."

No. Sentence Comment

211 Complex N-glycosylation of EL1 only, was confirmed when EL4 glycosylation sites were mutated14 (substitutions N894D/N900D), and of both EL1 and EL4 when sites were present in both loops. Login to comment
209 Complex N-glycosylation of EL1 only, was confirmed when EL4 glycosylation sites were mutated14 (substitutions N894D/N900D), and of both EL1 and EL4 when sites were present in both loops. Login to comment

PMID: 7518437 [PubMed] Chang XB et al: "Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion."

No. Sentence Comment

22 A fragment inwild- type pNUT-CFTR (21, 22) was replaced by a PCR fragment (between HpaI site atnucleotide 2463 andDraIII site atnucleotide 3328)coding for the N894D and N900D changes to produce aCFTR with no consensus glycosylation sites on extracytoplasmic loops (designated ELO,Fig. Login to comment