ABCMdb

PMID: 11443282

Hammerle MM, Aleksandrov AA, Chang XB, Riordan JR
A novel CFTR disease-associated mutation causes addition of an extra N-linked oligosaccharide.
Glycoconj J. 2000 Nov;17(11):807-13., [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Thr908Asn This substitution (T908N) creates a consensus sequence (N X S=T) for addition of an N-linked oligosaccharide chain near the C-terminal end of EL4. Login to comment 5 ABCC7 p.Thr908Asn However, the T908N site is used, even though it is within four residues of the predicted membrane interface and the oligosaccharide chain added binds calnexin, a resident chaperone of the ER membrane. Login to comment 13 ABCC7 p.Thr908Asn However, while analyzing the functional impact of missense mutations in the putative extracytoplasmic loops (ELs) of the protein [7], we realized that one (T908N) created a consensus sequence (N X S=T) for N-glycosylation. Login to comment 20 ABCC7 p.Thr908Asn BHK cells stably expressing T908N-CFTR were similarly established. Login to comment 23 ABCC7 p.Thr908Asn ABCC7 p.Asn900Asp ABCC7 p.Thr910Asn Tel.: 480-301-6206; Fax: 480-301-7017; E-mail: riordan@mayo.edu N900D, T908N or T910N to produce a CFTR with different numbers or no consensus site for glycosylation on extracytoplasmic loop 4. Login to comment 46 ABCC7 p.Thr908Asn Single channel records Microsomal vesicles were isolated from BHK cells expressing wildtype and T908N CFTR and phosphorylated with protein kinase A [17]. Login to comment 52 ABCC7 p.Thr908Asn Reconstruction and expression of the T908N variant in BHK cells resulted in the synthesis of a protein which ran with reduced mobility in SDS-PAGE (Figure 1B) consistent with the possibility that this additional N-glycosylation site had been used. Login to comment 54 ABCC7 p.Thr908Asn The sialidase treatment increased the mobility of both the wild-type and the T908N variant substantially, indicating that the oligosaccharide chains are highly sialylated. Login to comment 55 ABCC7 p.Thr908Asn After N-glycosidase F treatment the mobility is increased further and the deglycosylated form of both the wild-type and T908N migrate to the same position. Login to comment 57 ABCC7 p.Thr908Asn The difference in mobility between wild-type and T908N was entirely eliminated after cells were grown in presence of the inhibitor. Login to comment 60 ABCC7 p.Thr908Asn (A) Sketch of CFTR topology indicating position of T908N relative to the membrane and native glycosylation sites at N894 and N900. Login to comment 61 ABCC7 p.Thr908Asn (B) Cells stably expressing wild type and T908N-CFTR were lysed in NP-40 lysis buffer (see Materials and methods). Login to comment 65 ABCC7 p.Thr908Asn Cells expressing wild-type and T908N-CFTR were pulse labeled and chased as described in Methods. oligosaccharyl transferase and is an acceptor of an N-linked oligosaccharide chain. Login to comment 69 ABCC7 p.Thr908Asn Conversion of precursor to product occurs with low efficiency ($ 30%) but this also is similar with wild-type and the T908N variant. Login to comment 70 ABCC7 p.Thr908Asn Hence the additional oligosaccharide in T908N does not have a profound effect on the biosynthetic processing of CFTR. Login to comment 89 ABCC7 p.Thr908Asn As already noted in Figure 1 the T908N variant in the wild-type background has substantially decreased mobility. Login to comment 90 ABCC7 p.Thr910Asn However, the mobility of the T910N variant also is somewhat slower compared to wild-type suggesting that it also can serve as oligosaccharyl transferase acceptor. Login to comment 98 ABCC7 p.Thr908Asn T908N reduces the rate of 36 Cl7 efflux and alters both the single channel gating kinetics and conductance of CFTR. Login to comment 108 ABCC7 p.Thr908Asn As shown in Figure 1B, removal of sialic acid from complex glycosylated CFTR results in a higher mobility of wild-type and T908N. Login to comment 114 ABCC7 p.Thr908Asn Similar amounts of calnexin were detected in CFTR immunoprecipitates containing wild-type and T908N, and 894N and 900N alone whereas much less was detected when single oligosaccharide chains were present at 908N and especially at 910N (Figure 3). Login to comment 116 ABCC7 p.Thr908Asn To assess how the presence of a third oligosaccharide chain influences CFTR function, the rate of 36 Cl7 efflux from cells expressing the T908N variant was compared with wild-type and was found to be reduced about 50% (Figure 4A). Login to comment 119 ABCC7 p.Thr908Asn Thus both measures of the T908N CFTR chloride channel indicate that while still active, it is substantially altered from the wild-type. Login to comment 120 ABCC7 p.Thr908Asn Discussion Although all types of mutations have been detected in the CFTR gene of patients with cystic fibrosis, T908N is the only example of a single residue substitution that introduces a consensus site for glycosylation. Login to comment