ABCMdb

PMID: 19307599

Glozman R, Okiyoneda T, Mulvihill CM, Rini JM, Barriere H, Lukacs GL
N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic.
J Cell Biol. 2009 Mar 23;184(6):847-62., 2009-03-23 [PubMed]

Sentences

No. Mutations Sentence Comment

24 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp 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 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp [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 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp 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 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp 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 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp 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 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp 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 263 ABCC7 p.Thr908Asn In accord, with the localized effect of the glycan chain, partial reversion of the expression and folding defect of the 900D- and 2D-CFTR was observed by introducing a validated glycosylation site (T908N) in the fourth extracellular loop (Hammerle et al., 2000; Fig. S5). Login to comment 303 ABCC7 p.Asn900Asp ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp ABCC7 p.Asn894Asp The N894D-/N900D-CFTR (2D-CFTR) was generated using the N894D-CFTR as a template and the N900D mutagenic primers. Login to comment 306 ABCC7 p.Asn900Asp ABCC7 p.Asn894Asp 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