Human ABCG2 belongs to the ATP-binding cassette (ABC) transporter family and plays an important role in various biological reactions, such as xenobiotic elimination and homeostasis of protoporphyrin. We previously reported that ABCG2 exists in the plasma membrane as a homodimer bound via a disulfide bond at Cys-603. In the present study, we examined the importance of an intramolecular disulfide bond for stability of the ABCG2 protein.Substitution of either Cys-592 or Cys-608 located in the extracellular loop to glycine resulted in a significant decrease in protein levels of ABCG2 when expressed in Flp-In-293 cells. Interestingly, the protein levels of those ABCG2 variants were remarkably enhanced by treatment with the proteasome inhibitor MG132. Concomitantly, increases in ubiquitinated forms of those variant proteins were detected by immunoprecipitation. In contrast, neither the protein level nor the ubiquitinated state of the ABCG2 wild-type (WT) was affected by MG132 treatment. Ubiquitin-mediated protein degradation is suggested to be involved in degradation of misfolded ABCG2 proteins lacking the intramolecular disulfide bond. On the other hand, the protein level of ABCG2 WT increased more than 4-fold when cells were treated with bafilomycin A 1 , which inhibits lysosomal degradation, whereas the C592G or C608G variant was little affected by the same treatment. These results strongly suggest that two distinct pathways exist for protein degradation of ABCG2 WT and mutants lacking the intramolecular disulfide bond. Namely, the WT ABCG2 is degraded in lysosomes, and the misfolded ABCG2 lacking intramolecular disulfide bond undergoes ubiquitin-mediated protein degradation in proteasomes.

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.

Since porphyrins are regarded as endogenous substrates for the ATP-binding cassette (ABC) transporter ABCG2, it is hypothesized that functional impairment owing to genetic polymorphisms or inhibition of ABCG2 by drugs may result in a disruption of cellular porphyrin homeostasis. In the present study, we expressed ABCG2 genetic variants, i.e., V12M, Q141K, S441N, and F489L, as well as the wild type (WT) in Flp-In-293 cells to examine the hypothesis. Cells expressing S441N and F489L variants exhibited high levels of both cellularly accumulated pheophorbide a and photosensitivity, when those cells were incubated with pheophorbide a and irradiated with visible light. To further elucidate the significance of ABCG2 in cellular porphyrin homeostasis, we observed cellular accumulation and compartmentation of porphyrin and pheophorbide a by means of a new fluorescence microscopy technology, and found that accumulation of porphyrin and pheophorbide a in the cytoplasm compartment was maintained at low levels in Flp-In-293 cells expressing ABCG2 WT, V12M, or Q141K. When ABCG2 was inhibited by imatinib or novobiocin, however, those cells became sensitive to light. Based on these results, it is strongly suggested that certain genetic polymorphisms and/or inhibition of ABCG2 by drugs can enhance the potential risk of photosensitivity.

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Tetrahydrobiopterin (BH4) is a cofactor for tyrosine hydroxylase that is essential for the biosynthesis of dopamine. Parkinson's disease (PD) is characterized by a progressive degeneration of nigrostriatal dopaminergic neurons, and biomarkers reflecting the degree of neurodegeneration are important not only for basic research but also for clinical diagnosis and the treatment of the disease. Although the total neopterin and biopterin levels in the cerebrospinal fluids (CSF) of the patients with PD were reported, alterations in the composition of reduced and oxidized forms of pteridine compounds have not been examined. In this study, we first examined the time-dependent alterations in BH4 and other reduced pteridine compounds in the CSF of an MPTP-treated monkey as a primate PD model. We found that the CSF levels of BH4 and dihydroneopterin, an intermittent metabolite of BH4-biosynthesis, altered inversely with progression of neurodegeneration, whereas those of dihydrobiopterin and neopterin were relatively low and constant. Next, we assayed the amounts of reduced pteridine compounds in the CSF of 36 pre-symptomatic LRRK2-mutation (N1437H or G2019S) carriers (LRRK2-carrier), 13 patients with PD symptoms (LRRK2-PD), 46 patients with sporadic PD (sPD), and 26 non-PD individuals. The BH4 levels were significantly lower in both the LRRK2-PD and sPD patients, and the LRRK2-carriers exhibited higher BH4 levels compared with the sPD patients. The total neopterin levels in the CSF of the LRRK2-PD were significantly higher than those in the sPD and non-PD individuals, which indicated greater inflammatory responses in the brains of LRRK2-PD patients. The present results suggest that detailed analyses of pteridine levels in the CSF might be useful for understanding the pathophysiology of familial PD and for monitoring PD progression.