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. 2015 Dec 18;290(51):30429-40.
doi: 10.1074/jbc.M115.692103. Epub 2015 Oct 29.

ATP-binding Cassette Subfamily C Member 5 (ABCC5) Functions as an Efflux Transporter of Glutamate Conjugates and Analogs (V体育官网入口)

Robert S Jansen  1 , Sunny Mahakena  1 , Marcel de Haas  1 , Piet Borst  1 , Koen van de Wetering  2
Affiliations

ATP-binding Cassette Subfamily C Member 5 (ABCC5) Functions as an Efflux Transporter of Glutamate Conjugates and Analogs

Robert S Jansen et al. J Biol Chem. .

Abstract (VSports app下载)

The ubiquitous efflux transporter ABCC5 (ATP-binding cassette subfamily C member 5) is present at high levels in the blood-brain barrier, neurons, and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens, we show that Abcc5(-/-) mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular VSports手机版. The abundant neurotransmitter N-acetylaspartylglutamate was 2. 4-fold higher in Abcc5(-/-) brain. The metabolites that accumulated in Abcc5(-/-) tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay, ABCC5 also transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid, and NMDA; the therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. After CO2 asphyxiation, several immediate early genes were expressed at lower levels in Abcc5(-/-) brains than in wild type brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins, and drugs. .

Keywords: MRP5; blood-brain barrier; brain; drug transport; glutamate; neurotoxin; neurotransmitter transport. V体育安卓版.

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"V体育官网" Figures

FIGURE 1.
FIGURE 1.
MS2 fragmentation spectra of unknowns and available reference standards resulted in the identification of most of the accumulating metabolites. The neutral loss of 79.957 in the spectrum of C11H18N2O5S indicates the presence of a sulfate moiety. The spectrum of putative BCG2 contains fragments (m/z 128.035 and 240.051) that are identical to that of BCG, strengthening its putative identification. The peak at m/z 196.919 in the spectrum of Asp-Gly-Glu is a nonspecific background peak that we commonly observe in low abundant MS2 spectra. The unknowns with m/z 421.0557 and 456.1745 did not yield any database hits, nor could we accurately determine the elemental composition because of the relatively high mass and low abundance.
FIGURE 2.
FIGURE 2.
Chemical structures of glutamate conjugates and analogs found to be transported by ABCC5. A, in vivo substrates found through comparative metabolomics of wild type and Abcc5−/− mouse tissues. B, additional ABCC5 substrates identified through in vitro vesicular transport studies. MTX transport was shown by Wielinga et al. (14). Glutamate moieties are highlighted in red, whereas aspartate moieties are highlighted in blue.
FIGURE 3.
FIGURE 3.
Nine metabolites accumulated in Abcc5−/− tissues. Tissues of wild type and Abcc5−/− mice were analyzed using untargeted LC-MS metabolomics, after which the data sets were compared using the XCMS online platform. Only metabolites that were significantly (p < 0.05) and over 2-fold increased in at least two Abcc5−/− tissues are shown here. The elemental composition could be determined for a single unknown, whereas two other unknowns could not be identified beyond their mass over charge ratio (m/z). The metabolite concentrations in brains were relatively high and showed clear ABCC5 dependence. The data are presented on a log scale as means plus S.D. (n = 5 per genotype). AU, arbitrary units. p values were calculated using the Student's t test. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 4.
FIGURE 4.
The metabolites detected in our screen strongly accumulated in Abcc5−/− brain. Tissues of wild type and Abcc5−/− mice were analyzed using untargeted LC-MS metabolomics, after which the data sets were compared using the XCMS online platform. Of the nine metabolites that were significantly (p < 0.05) and over 2-fold increased in at least two Abcc5−/− tissues, eight accumulated in Abcc5−/− brain. Two unknowns could not be identified beyond their mass over charge ratio (m/z). The data are presented as means plus S.D. (n = 5 per genotype). AU, arbitrary units.
FIGURE 5.
FIGURE 5.
Metabolites that accumulate in Abcc5−/− organs in vivo are also effluxed from HEK293 cells by human ABCC5 in vitro. HEK293 parental cells and HEK293 cells stably overexpressing RIMKLA or RIMKLB in combination with ABCC5 (depicted in gray) or GFP (depicted in black) were cultured for 3 days. Cell lysate (L) and culture medium (M) were analyzed using untargeted LC-MS metabolomics. The data are presented as means plus S.D. (n = 3). AU, arbitrary units; ND, not detected.
FIGURE 6.
FIGURE 6.
Vesicular transport assays show that ABCC5 transports glutamate conjugates and analogs. Inside-out membrane vesicles without (control) or with ABCC5 (ABCC5) were incubated (10 min, 37 °C) with test substrates (100 μm) in the presence and absence of 5 mm ATP. The relative amount of test substrate in the vesicles was determined using LC-MS. The data are presented as means plus S.D. (n = 3). AU, arbitrary units; ND, not detected.
FIGURE 7.
FIGURE 7.
Vesicular transport of an N-acetylated dipeptide library shows that ABCC5 is a general C-terminal glutamate dipeptide transporter. Inside-out membrane vesicles with or without ABCC5 were incubated (10 min, 37 °C) with a library of 20 N-acetylated, C-terminal glutamate dipeptides (∼10 μm per peptide) in the presence and absence of 5 mm ATP. The relative amount of each peptide in the vesicles was determined using LC-MS and normalized on the peptide signal in the original peptide library. Poor LC-MS sensitivity for (Ac)Lys-Glu resulted in a low signal in the input library and undetectable levels in vesicles. We were unable to discriminate (Ac)Ile-Glu and (Ac)Leu-Glu because of their identical mass. Hence the uptakes of both of these dipeptides are labeled (Ac)(iso)Leu-Glu. The data are presented as means plus S.D. (n = 4). AU, arbitrary units.
FIGURE 8.
FIGURE 8.
NAAG and ZJ43 are transported into inside-out membrane vesicles by ABCC5. A and B, 100 μm NAAG (A) or 100 μm ZJ43 (B) was incubated with control vesicles (squares) and ABCC5-containing vesicles (circles) at 37 °C, in the presence (solid line) and absence (dashed line) of 5 mm ATP. At the indicated time points, samples containing 75 μg of protein were taken. After washing over a filter, the vesicular content was analyzed by LC-MS (n = 3). C and D, various concentrations of NAAG (C) or ZJ43 (D) were incubated with control vesicles and ABCC5-containing vesicles for 10 min at 37 °C, in the presence of 5 mm ATP (n = 4). ABCC5-dependent transport was calculated and fitted to Michaelis-Menten kinetics (solid line) using GraphPad Prism. The data are presented as means ± S.E. because rare outliers resulted in a high S.D. that required a scale on which the data were not interpretable. AU, arbitrary units.
FIGURE 9.
FIGURE 9.
Expression levels of several immediate early genes are down-regulated in Abcc5−/− mouse brains. mRNA of whole brains collected from wild type and Abcc5−/− mice was sequenced (n = 5 single brains/group), as described under “Experimental Procedures.”
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References

    1. Borst P., de Wolf C., and van de Wetering K. (2007) Multidrug resistance-associated proteins 3, 4, and 5. Pflügers Arch. 453, 661–673 - PubMed
    1. Dean M., and Allikmets R. (2001) Complete characterization of the human ABC gene family. J. Bioenerg. Biomembr. 33, 475–479 - PubMed
    1. Belinsky M. G., Bain L. J., Balsara B. B., Testa J. R., and Kruh G. D. (1998) Characterization of MOAT-C and MOAT-D, new members of the MRP/cMOAT subfamily of transporter proteins. J. Natl. Cancer Inst. 90, 1735–1741 - PubMed
    1. Kool M., de Haas M., Scheffer G. L., Scheper R. J., van Eijk M. J., Juijn J. A., Baas F., and Borst P. (1997) Analysis of expression of cMOAT (MRP2), MRP3, MRP4, and MRP5, homologues of the multidrug resistance-associated protein gene (MRP1), in human cancer cell lines. Cancer Res. 57, 3537–3547 - PubMed
    1. McAleer M. A., Breen M. A., White N. L., and Matthews N. (1999) pABC11 (also known as MOAT-C and MRP5), a member of the ABC family of proteins, has anion transporter activity but does not confer multidrug resistance when overexpressed in human embryonic kidney 293 cells. J. Biol. Chem. 274, 23541–23548 - PubMed

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