Disruption of N‐linked glycosylation enhances ubiquitin‐mediated proteasomal degradation of the human ATP‐binding cassette transporter ABCG2

Abstract

The human ATP-binding cassette (ABC) transporter, ABCG2 (BCRP ⁄ MXR ⁄ ABCP), is a plasma membrane protein containing intramolecular and intermolecular disulfide bonds and an N-linked glycan at Asn596. We have recently reported that the intramolecular disulfide bond is a critical checkpoint for determining the degradation fates of ABCG2. In the present study, we aimed to analyze quantitatively the impact of the N-linked glycan on the protein stability of ABCG2. For this purpose, we incorporated one single copy of ABCG2 cDNA into a designated site of genomic DNA in Flp-In-293 cells to stably express ABCG2 or its variant proteins. When ABCG2 wild type-expressing cells were incubated with various N-linked glycosylation inhibitors, tunicamycin profoundly suppressed the protein expression level of ABCG2 and, accordingly, reduced the ABCG2-mediated cellular resistance to the cancer chemotherapeutic SN-38. When Asn596 was converted to Gln596, the resulting variant protein was not glycosylated, and its protein level was about one-third of the wild type level in Flp-In-293 cells. Treatment with MG132, a proteasome inhibitor, increased the level of the variant protein. Immunoblotting with anti-ubiquitin IgG1k after immunoprecipitation of ABCG2 revealed that the N596Q protein was ubiquitinated at levels that were significantly enhanced by treatment with MG132. Immunofluorescence microscopy demonstrated that treatment with MG132 increased the level of ABCG2 N596Q protein both in intracellular compartments and in the plasma membrane. In conclusion, we propose that the N-linked glycan at Asn596 is important for stabilizing de novo-synthesized ABCG2 and that disruption of this linkage results in protein destabilization and enhanced ubiquitin-mediated proteasomal degradation.

Identification of Glycosylation Sites Essential for Surface Expression of the CaVα2δ1 Subunit and Modulation of the Cardiac CaV1.2 Channel Activity

“…The relative contribution of lectin chaperones, such as calnexin and calreticulin, to protein biogenesis and the nature of the key enzymes responsible for degradation within the ubiquitin-proteasome system of ERAD (68, 70 -72) also remain to be identified. Outstanding issues include the identification of the quality control networks that are affected by these specific glycosylation sites (73,74) and the potential cross-talk between ERAD and autophagy, the two major cellular degradative pathways (75). Finally, there is always the question of overexpressing cardiac proteins in a model cell to study translation events.…”

The ABC of glycosylation

“…Interestingly, glycosylation might regulate ABC transporter levels, as altered glycosylation of ABCG2 (also known as BCRP) results in increased degradation 8 . Indeed, N-linked glycans are thought to be crucial regulators of the stability of ABCG2 in the endoplasmic reticulum 8,9 . In fact, N-glycosylated wildtype ABCG2 is degraded in lysosomes, whereas misfolded mutant proteins have been shown to undergo ubiquitin-mediated degradation in the proteasome 10,11 .…”

Xanthines Down-Regulate the Drug Transporter ABCG2 and Reverse Multidrug Resistance

“…It has been reported previously that wild-type ABCG2 is primarily degraded through the lysosome-mediated pathway, although misfolded ABCG2 is degraded by ubiquitinmediated proteasomal machinery (Nakagawa et al, 2009). To determine whether caffeine or its analog, theophylline, enhanced ABCG2 lysosomal degradation, we examined the effect of lysosome inhibitors on caffeine-mediated ABCG2 down-regulation.…”

“…Mutated or misfolded newly synthesized ABCG2 protein is rapidly eliminated via ubiquitin-mediated proteasome degradation before reaching the membrane (Nakagawa et al, 2009). In contrast, membrane-associated wild-type ABCG2 is normally degraded through the lysosome (Wakabayashi et al, 2007).…”