ABCMdb

ABCG5 p.Asn591Gln

Predicted by SNAP2:	A: D (63%), C: D (53%), D: D (63%), E: D (66%), F: D (75%), G: D (59%), H: D (71%), I: D (71%), K: D (71%), L: D (71%), M: D (75%), P: D (75%), Q: D (53%), R: D (71%), S: N (66%), T: N (61%), V: D (66%), W: D (85%), Y: D (75%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, P: D, Q: D, R: D, S: N, T: N, V: D, W: D, Y: D,

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Publications
[hide] Inhibition of post-translational N-glycosylation b... Sci Rep. 2014 Mar 3;4:4258. doi: 10.1038/srep04258. Suzuki S, Shuto T, Sato T, Kaneko M, Takada T, Suico MA, Cyr DM, Suzuki H, Kai H
Inhibition of post-translational N-glycosylation by HRD1 that controls the fate of ABCG5/8 transporter.
Sci Rep. 2014 Mar 3;4:4258. doi: 10.1038/srep04258., [PMID:24584735]

Abstract [show]
N-glycosylation of proteins in endoplasmic reticulum is critical for protein quality control. We showed here a post-translational N-glycosylation affected by the HRD1 E3 ubiquitin ligase. Both WT- and E3-defective C329S-HRD1 decreased the level of high mannose form of ABCG8, a protein that heterodimerizes with ABCG5 to control sterol balance. Meanwhile, HRD1 increased the non-glycosylated ABCG8 regardless of its E3 activity, thereby suppressing full maturation of ABCG5/8 transporter. Pulse chase and mutational analysis indicated that HRD1 inhibits STT3B-dependent post-translational N-glycosylation of ABCG8. Whereas, HRD1 had only slight effect on the N-glycosylation status of ABCG5; rather it accelerated ABCG5 degradation in an E3 activity-dependent manner. Finally, RMA1, another E3 ubiquitin ligase, accelerated the degradation of both ABCG5 and ABCG8 via E3 activity-dependent manner. HRD1 and RMA1 may therefore be negative regulators of disease-associated transporter ABCG5/ABCG8. The findings also highlight the unexpected E3 activity-independent role of HRD1 in the regulation of N-glycosylation.

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Sentences [show]
No. Sentence Comment
50 Single substitution of glutamine for asparagine at position 584 (N584Q) and at position 591 (N591Q) in human ABCG5 reduced the apparent molecular mass to a similar extent between two mutants (Figure 1b). Login to comment
51 Double mutation (N584Q/N591Q) further reduced the band size identical to those of EndoH- and PNGase F-treated ABCG5 proteins (Figure 1b,c; Supplementary Figure S4), indicating that human ABCG5 contains two N-linked glycans at positions 584 and 591. Login to comment
67 (b) Steady-state expression of monomeric Myc-tagged WT-, N584Q-, N591Q- and N584Q-/N591Q-ABCG5 was analyzed by immunoblotting. Login to comment
68 (c) Endo H (500 U) and PNGase F (500 U) sensitivity of Myc-tagged WT-, N584Q-, N591Q- and N584Q-/N591Q-ABCG5 proteins shown in (b). Login to comment
83 (f-i) Pulse chase (pulse labeled for 10 min) of HEK293 cells expressing Myc-tagged WT-, N584Q- and N591Q-ABCG5 (f). Login to comment
84 Relative quantity of each glycosylated form of WT-ABCG5 (g), N591Q-ABCG5 (h) and N584Q-ABCG5 (i) within all ABCG5 was quantified. Login to comment
91 To further characterize the glycosylation timing of each Asn residues (N584 and N591) of ABCG5 protein, mutational analysis using N584Q- and N591Q-ABCG5 constructs were performed. Login to comment
92 Notably, Non-G forms of both N584Q and N591Q ABCG5 proteins were abundantly expressed (Figure 2f,h,i: 70.1% vs. 54.6%), indicating that both Asn residues (N584 and N591) have the potential to be post-translationally glycosylated although the tendency seems to be different among residues. Login to comment
93 Consistently, pulse-chase analysis using N584Q and N591Q ABCG5 revealed that the expression level of glycosylated forms of N584Q and N591Q ABCG5 are gradually increased during the chase period (Figure 2f,h,i: 45.4% to 72.6% for N584Q-ABCG5; 29.9% to 71.9% for N591Q-ABCG5). Login to comment
94 Because of the high expression of Non-G form of N591Q ABCG5 at 0-min chase (Figure 2f,h 70.1%) and of the time-dependent increase of Mono-G form of ABCG5 (Figure 2f,h), we can deduce that most of the N584 residue of ABCG5 is post-translationally glycosylated, while N591 residue is both co- and post-translationally glycosylated. Login to comment
302 Glycosylation-defective mutants N584Q-, N591Q-, N584Q/N591Q-ABCG5, N619Q-ABCG8, and E3-defective mutants C291S/C329S-HRD1, H260Q CHIP and C42S-RMA1 were generated using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to previous reports12,17,32-34 . Login to comment