ABCA3 p.Asn53Gln
| Predicted by SNAP2: | A: D (59%), C: D (59%), D: N (61%), E: N (61%), F: D (66%), G: N (57%), H: N (61%), I: D (63%), K: N (66%), L: D (66%), M: D (63%), P: D (59%), Q: N (57%), R: N (61%), S: N (72%), T: N (72%), V: D (59%), W: D (85%), Y: D (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, P: N, Q: N, R: N, S: N, T: D, V: D, W: D, Y: D, |
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[hide] Disruption of N-linked glycosylation promotes prot... Am J Physiol Lung Cell Mol Physiol. 2013 Dec;305(12):L970-80. doi: 10.1152/ajplung.00184.2013. Epub 2013 Oct 18. Beers MF, Zhao M, Tomer Y, Russo SJ, Zhang P, Gonzales LW, Guttentag SH, Mulugeta S
Disruption of N-linked glycosylation promotes proteasomal degradation of the human ATP-binding cassette transporter ABCA3.
Am J Physiol Lung Cell Mol Physiol. 2013 Dec;305(12):L970-80. doi: 10.1152/ajplung.00184.2013. Epub 2013 Oct 18., [PMID:24142515]
Abstract [show]
The lipid transport protein, ABCA3, expressed in alveolar type 2 (AT2) cells, is critical for surfactant homeostasis. The first luminal loop of ABCA3 contains three putative N-linked glycosylation sites at residues 53, 124, and 140. A common cotranslational modification, N-linked glycosylation, is critical for the proper expression of glycoproteins by enhancing folding, trafficking, and stability through augmentation of the endoplasmic reticulum (ER) folding cycle. To understand its role in ABCA3 biosynthesis, we utilized EGFP-tagged fusion constructs with either wild-type or mutant ABCA3 cDNAs that contained glutamine for asparagine substitutions at the putative glycosylation motifs. In A549 cells, inhibition of glycosylation by tunicamycin increased the electrophoretic mobility (Mr) and reduced the expression level of wild-type ABCA3 in a dose-dependent manner. Fluorescence imaging of transiently transfected A549 or primary human AT2 cells showed that although single motif mutants exhibited a vesicular distribution pattern similar to wild-type ABCA3, mutation of N124 and N140 residues resulted in a shift toward an ER-predominant distribution. By immunoblotting, the N53 mutation exhibited no effect on either the Mr or ABCA3 expression level. In contrast, substitutions at N124 or N140, as well a N124/N140 double mutation, resulted in increased electrophoretic mobility indicative of a glycosylation deficiency accompanied by reduced overall expression levels. Diminished steady-state levels of glycan-deficient ABCA3 isoforms were rescued by treatment with the proteasome inhibitor MG132. These results suggest that cotranslational N-linked glycosylation at N124 and N140 is critical for ABCA3 stability, and its disruption results in protein destabilization and proteasomal degradation.
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No. Sentence Comment
47 The primers [primer nucleotide sequence is obtained from the National Center for Biotechnology (NCBI) of human ABCA3, data accession number NM_001089] generated for these mutant constructs are as follows: for N53Q: forward, 5=-tcggaaaatgtgccccaggccaccatctacccg-3=, reverse, 5=-cgggtagatggtggcctggggcacattttccga-3=; for N124Q: forward, 5=-ctacattaggtacgaccagtgctcgtccagcgtgc-3=, reverse, 5=-gcacgctggacgag- cactggtcgtacctaatgtag-3=; for N140Q, forward, 5=-tcgagcaccccttccagca- cagcaaggagcc-3=, reverse, 5=-ggctccttgctgtgctggaaggggtgctcga-3=, and for N945Q: forward, 5=-ccctcctggccatccagtactcctcggagct-3=, reverse, 5=- agctccgaggagtactggatggccaggaggg-3=.
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ABCA3 p.Asn53Gln 24142515:47:209
status: NEW153 As was observed in epithelial cell lines, immunoblot analysis of primary human AT2 cells revealed increased electrophoretic mobility of the primary translation products of single and double mutants of N124Q and N140Q but not the single mutant of N53Q (Fig. 7A).
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ABCA3 p.Asn53Gln 24142515:153:246
status: NEW