Mfsd2a utilizes a flippase mechanism to mediate omega-3 fatty acid lysolipid transport.
Mfsd2a utilizes a flippase mechanism to mediate omega-3 fatty acid lysolipid transport.
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Mfsd2a利用翻转酶机制介导omega-3脂肪酸溶脂转运。
DOI：
10.1073/pnas.2215290120
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发表时间：
2023-03-07
影响因子：
11.1
通讯作者：
Silver, David L.
中科院分区：
文献类型：
--
作者：
Chua, Geok-Lin;Tan, Bryan C.;Loke, Randy Y. J.;He, Menglan;Chin, Cheen-Fei;Wong, Bernice H.;Kuk, Alvin C. Y.;Ding, Mei;Wenk, Markus R.;Guan, Lan;Torta, Federico;Silver, David L.
Mfsd 2a是一种在血脑屏障表达的转运蛋白，介导大脑从血液中获得必需脂肪酸的主要途径。Mfsd 2a在血液循环中转运结合为溶血磷脂酰胆碱（LPC）的脂肪酸。Mfsd 2a功能缺失突变的人表现为严重的小头畸形，表明LPC转运在脑发育中的关键作用。Mfsd 2a转运的模型假定Mfsd 2a充当LPC“翻转酶”，当其从质膜的外小叶到内小叶移动通过转运蛋白时使LPC反转。本研究通过建立体外生化分析方法，直接证明了Mfsd 2a具有“翻转酶”活性，从而明确了LPC转运入脑的生化机制。含有主要易化因子超家族结构域2a（Mfsd 2a）是一种钠依赖性溶血磷脂酰胆碱（LPC）转运蛋白，在血脑屏障上表达，构成大脑获得ω-3脂肪酸（如二十二碳六烯酸）的主要途径。人类Mfsd 2a缺乏导致严重的小头畸形，强调了Mfsd 2a转运LPC对大脑发育的重要性。生化研究和最近的冷冻电镜（cryo-EM）结构的Mfsd 2a结合LPC表明，Mfsd 2a运输LPC通过一个交替的访问机制之间的面向外和面向内的构象状态，其中LPC反转之间的运输膜的外部和内部小叶。然而，Mfsd 2a翻转酶活性的直接生物化学证据尚未得到证实，并且不了解Mfsd 2a如何以钠依赖性方式逆转膜的外小叶和内小叶之间的LPC。在这里，我们建立了一个独特的体外试验，使用重组Mfsd 2a在脂质体中重建，利用Mfsd 2a的能力，运输溶血磷脂酰丝氨酸（LPS）与小分子LPS结合荧光团，允许监测的LPS头基从外部到内部脂质体膜的方向翻转。使用该测定，我们证明了Mfsd 2a以钠依赖性方式将LPS从膜双层的外小叶翻转到内小叶。此外，使用cryo-EM结构作为指导连同诱变和基于细胞的运输测定，我们确定了Mfsd 2a活性的重要氨基酸残基，可能构成底物相互作用域。这些研究提供了直接的生化证据，Mfsd 2a功能作为一个溶解脂质翻转酶。
Mfsd2a is a transporter expressed at the blood–brain barrier that mediates the major pathway by which the brain acquires essential fatty acids from blood. Mfsd2a transports fatty acids conjugated as lysophosphatidylcholine (LPC) in blood circulation. Humans with loss of function mutations in Mfsd2a present with severe microcephaly, indicating a critical role of LPC transport in brain development. A model for Mfsd2a transport posits that Mfsd2a acts as a LPC “flippase,” inverting LPC as it moves through the transporter from the outer to inner leaflet of the plasma membrane. By establishing an in vitro biochemical assay, this study provides the direct proof that Mfsd2a has “flippase” activity, thus defining the biochemical mechanism of how LPC is transported into the brain. Major Facilitator Superfamily Domain containing 2a (Mfsd2a) is a sodium-dependent lysophosphatidylcholine (LPC) transporter expressed at the blood–brain barrier that constitutes the main pathway by which the brain obtains omega-3 fatty acids, such as docosahexanoic acid. Mfsd2a deficiency in humans results in severe microcephaly, underscoring the importance of LPC transport by Mfsd2a for brain development. Biochemical studies and recent cryo-electron microscopy (cryo-EM) structures of Mfsd2a bound to LPC suggest that Mfsd2a transports LPC via an alternating access mechanism between outward-facing and inward-facing conformational states in which the LPC inverts during transport between the outer and inner leaflet of a membrane. However, direct biochemical evidence of flippase activity by Mfsd2a has not been demonstrated and it is not understood how Mfsd2a could invert LPC between the outer and inner leaflet of the membrane in a sodium-dependent manner. Here, we established a unique in vitro assay using recombinant Mfsd2a reconstituted in liposomes that exploits the ability of Mfsd2a to transport lysophosphatidylserine (LPS) coupled with a small molecule LPS binding fluorophore that allowed for monitoring of directional flipping of the LPS headgroup from the outer to the inner liposome membrane. Using this assay, we demonstrate that Mfsd2a flips LPS from the outer to the inner leaflet of a membrane bilayer in a sodium-dependent manner. Furthermore, using cryo-EM structures as guides together with mutagenesis and a cell-based transport assay, we identify amino acid residues important for Mfsd2a activity that likely constitute substrate interaction domains. These studies provide direct biochemical evidence that Mfsd2a functions as a lysolipid flippase.
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影响因子：
64.8
作者：
Nguyen, Long N.;Ma, Dongliang;Silver, David L.
通讯作者：
Silver, David L.
影响因子：
2.2
作者：
Harel, Tamar;Quek, Debra Q. Y.;Edvardson, Shimon
通讯作者：
Edvardson, Shimon
影响因子：
4.8
作者：
Lin, Yibin;Deepak, R. N. V. Krishna;Zheng, Lei
通讯作者：
Zheng, Lei
DOI：
10.1073/pnas.2210353119
发表时间：
2022-10-04
影响因子：
11.1
作者：
通讯作者：
--
影响因子：
16.8
作者：
Martinez-Molledo, Maria;Nji, Emmanuel;Reyes, Nicolas
通讯作者：
Reyes, Nicolas