ABCA3 p.Asn140Gln
| Predicted by SNAP2: | A: D (53%), C: D (53%), D: N (66%), E: N (66%), F: D (66%), G: N (61%), H: D (75%), I: D (80%), K: N (66%), L: D (63%), M: D (85%), P: N (53%), Q: N (66%), R: N (66%), S: N (82%), T: N (82%), V: D (53%), W: D (80%), Y: D (63%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: N, P: N, Q: N, R: N, S: N, T: N, 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.Asn140Gln 24142515:47:436
status: NEW122 Since data obtained thus far suggested that glycosylation takes place at N124 and N140, but not at N53, we next generated a double mutant construct containing both N124Q and N140Q mutations.
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ABCA3 p.Asn140Gln 24142515:122:174
status: NEW139 M.W., molecular weight. double mutant (N124Q and N140Q) was distributed in both calnexin (ER)- and CD63-positive compartments, suggestive of alterations in anterograde trafficking of ABCA3 caused by the absence of glycosylation at residues 124 and 140.
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ABCA3 p.Asn140Gln 24142515:139:50
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.Asn140Gln 24142515:153:211
status: NEW179 Furthermore, the N124Q, N140Q, and N124Qaf9;N140Q substitutions all resulted in reduced total ABCA3 protein expression.
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ABCA3 p.Asn140Gln 24142515:179:24
status: NEW190 Second, the immunoblot bands of single and double mutants of N124Q and N140Q supports the notion that the prominent changes in molecular weights (compared with WT ABCA3) are likely due to the absence of large sugar moiety and not due to the replacement of a single amino acid.
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ABCA3 p.Asn140Gln 24142515:190:71
status: NEW236 As Fig. 5 shows, the level of expression of single glycosylation mutants (N124Q; N140Q) are highly susceptible to MG132 treatment, indicating that as many as half of the glycosylation-deficient isoforms are susceptible to proteasomal degradation.
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ABCA3 p.Asn140Gln 24142515:236:81
status: NEW