ABCC5 functions as a transporter of glutamate conjugates and analogs

The ubiquitous efflux transporter ATP-binding cassette sub-family C member 5 (ABCC5) is present at high levels in the blood-brain barrier, neurons and glia, but its in vivo substrates and function are not known. Untargeted metabolomic screens revealed that Abcc5-/- mice accumulate endogenous glutamate conjugates and analogs in several tissues, but brain in particular. The abundant neurotransmitter N-acetylaspartylglutamate (NAAG), for example, was over 2-fold higher in Abcc5-/- brain. In line with ABCC5-mediated transport, the metabolites that accumulated in Abcc5-/- tissues were depleted in cultured cells that overexpressed human ABCC5. Using membrane vesicles, we show that ABCC5 not only transports the metabolites detected in our screen, but also a wide range of peptides containing a C-terminal glutamate. Glutamate conjugates are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter in the brain. We found that ABCC5 also transports exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid and N-methyl-D-aspartate (NMDA) and the therapeutic glutamate analog ZJ43. Taken together, we have identified ABCC5 as a general glutamate conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins and drugs. A set of 5 wildtype brains was compared to a set of 5 Abcc5-knockout mouse brains普遍存在的外流转运蛋白ATP结合盒C亚家族成员5（ABCC5）在血脑屏障、神经元与神经胶质细胞中高表达，但其体内底物与生理功能尚未明确。非靶向代谢组学筛选显示，Abcc5基因敲除小鼠的多种组织中会蓄积内源性谷氨酸结合物及其类似物，尤以脑组织最为显著。例如，丰度较高的神经递质N-乙酰天冬氨酰谷氨酸（NAAG）在Abcc5基因敲除小鼠脑组织中的含量较野生型高出2倍以上。与ABCC5介导的转运功能一致，在过表达人源ABCC5的培养细胞中，原本在Abcc5基因敲除组织中蓄积的代谢物含量显著降低。利用膜囊泡实验，我们证实ABCC5不仅能够转运筛选中发现的上述代谢物，还可识别并转运多种带有C端谷氨酸的肽类物质。谷氨酸结合物具有重要生理相关性，因为它们可影响脑内主要兴奋性神经递质谷氨酸的功能。我们进一步发现，ABCC5还能转运外源性谷氨酸类似物，包括经典的兴奋性毒性神经毒素红藻氨酸、软骨藻酸与N-甲基-D-天冬氨酸（NMDA），以及治疗用谷氨酸类似物ZJ43。综上，本研究鉴定ABCC5为一类广谱谷氨酸结合物及类似物转运蛋白，可影响内源性代谢物、神经毒素与治疗药物的体内处置。本研究将5份野生型小鼠脑组织与5份Abcc5基因敲除小鼠脑组织进行了对照分析。