ABCMdb

PMID: 24142515

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., [PubMed]

Sentences

No. Mutations Sentence Comment

47 ABCA3 p.Asn140Gln ABCA3 p.Asn124Gln ABCA3 p.Asn53Gln ABCA3 p.Asn945Gln 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=. Login to comment 122 ABCA3 p.Asn140Gln ABCA3 p.Asn124Gln 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. Login to comment 139 ABCA3 p.Asn140Gln ABCA3 p.Asn124Gln 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. Login to comment 153 ABCA3 p.Asn140Gln ABCA3 p.Asn124Gln ABCA3 p.Asn53Gln 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). Login to comment 179 ABCA3 p.Asn140Gln ABCA3 p.Asn124Gln Furthermore, the N124Q, N140Q, and N124Qaf9;N140Q substitutions all resulted in reduced total ABCA3 protein expression. Login to comment 190 ABCA3 p.Asn140Gln ABCA3 p.Asn124Gln 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. Login to comment 228 ABCA3 p.Leu101Pro Firstly, expression of the leucine-to-proline mutation at residue 101 (L101P) results in ER-retained, unprocessed ABCA3 product that induces the unfolded proteins response and ER stress (10, 37, 56), suggesting that this mutation causes gross misfolding of ABCA3. Login to comment 229 ABCA3 p.Arg43Leu ABCA3 p.Pro147Leu In contrast, a second group of lung disease-associated mutations that are proximal to N-glycan sites and/or that could profoundly alter the structural makeup of the loop such as P147L (23) and R43L (6, 20) may adversely influence glycosylation and subsequent ABCA3 biosynthesis. Login to comment 231 ABCA3 p.Asp123Asn ABCA3 p.Asp123Asn Specifically, mutation of aspartate to asparagine at residue 123 (D123N) has also been associated with familial ILD with fibrotic lung remodeling. Login to comment 233 ABCA3 p.Asn140His ABCA3 p.Asn124Ser Finally, ABCA3 sequence variants carrying a N124S (rs142977595) or N140H (rs45447801) substitution naturally occur in the human population [Human Amino Acid Missense Variant Server Database (humsavar) (http://decrypthon.igbmc. Login to comment 236 ABCA3 p.Asn140Gln ABCA3 p.Asn124Gln 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. Login to comment 247 ABCA3 p.Asn945Gln A: representative confocal images of A549 cells 24 h following plasmid introduction of EGFP-tagged to either WT or a mutant (N945Q) ABCA3 isoform showing predominant localization of both isoforms within CD63-positive LROs. Login to comment 248 ABCA3 p.Asn945Gln Bar, 5 òe;m. B: representative anti-GFP immunoblots (from 2 separate experiments) of whole cell lysates of A549 cells NT or transfected with either WT or N945Q mutant EGFP-tagged ABCA3 cDNAs showing similar band electrophoretic mobility and protein expression levels. Login to comment