Signature Research Program in Cardiovascular and Metabolic Disorders, Duke-National University of Singapore Medical School, Singapore 169857, Singapore.
Singapore Lipidomics Incubator, Life Sciences Institute, National University of Singapore, Singapore 117456, Singapore.
Proc Natl Acad Sci U S A. 2023 Mar 7;120(10):e2215290120. doi: 10.1073/pnas.2215290120. Epub 2023 Feb 27.
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.
主要溶质载体家族 2 成员 A（Mfsd2a）是一种位于血脑屏障上的钠离子依赖性溶血磷脂酰胆碱（LPC）转运体，它构成了大脑获取ω-3 脂肪酸（如二十二碳六烯酸）的主要途径。人类 Mfsd2a 缺陷会导致严重的小头畸形，这突显了 Mfsd2a 对 LPC 转运对于大脑发育的重要性。生化研究和最近的 Mfsd2a 与 LPC 结合的冷冻电子显微镜（cryo-EM）结构表明，Mfsd2a 通过外向构象和内向构象之间的交替访问机制来转运 LPC，在此过程中，LPC 在穿过膜的内外叶之间转运时会反转。然而，尚未证明 Mfsd2a 的翻转酶活性的直接生化证据，也不了解 Mfsd2a 如何以钠离子依赖的方式在膜的内外叶之间翻转 LPC。在这里，我们使用重组 Mfsd2a 重建在脂质体中建立了一种独特的体外测定方法，该方法利用 Mfsd2a 转运溶血磷脂酰丝氨酸（LPS）的能力，同时结合一种小分子 LPS 结合荧光染料，该染料可用于监测 LPS 头部从外向脂质体膜翻转到内向脂质体膜。使用该测定法，我们证明 Mfsd2a 以钠离子依赖的方式将 LPS 从外膜翻转到内膜。此外，使用 cryo-EM 结构作为指导，结合突变和基于细胞的转运测定，我们确定了与 Mfsd2a 活性相关的重要氨基酸残基，这些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，证明 Mfsd2a 是一种溶血磷脂翻转酶。
