Molecular mechanism of omega-3 fatty acid transport into the brain

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ABSTRACT Major Facilitator Superfamily Domain containing 2A (MFSD2A) is a 58 kDa integral membrane protein that is highly expressed within endothelial cells of the blood-brain and blood-retinal barriers where it mediates uptake of the ω-3 fatty acid docosahexaenoic acid (DHA) into the brain and eyes respectively. DHA comprises up to 20% of total brain lipids. Despite this, the brain cannot synthesise DHA de novo and MFSD2A-mediated transport is the primary route by which the brain acquires DHA. Mouse MFSD2A KO models exhibit severe brain DHA deficiency and generalized microcephaly, and human patients with homozygous loss-of-function mutations in MFSD2A present with severe microcephaly and developmental delay. Previous cell-based studies have demonstrated that MFSD2A transports DHA in the form of lysophosphatidylcholine (LPCDHA), but not unesterified fatty acid, in a Na+-dependent manner. Similar to all other MFS family members, MFSD2A has twelve transmembrane domains but unlike most MFS proteins that transport water soluble compounds, MFSD2A transports lysolipids, raising the possibility of a unique transport mechanism. It has been hypothesized that MFSD2A transports mono-acyls chain lipids across membranes by flipping the LPC between membrane leaflets, acting essentially as a “flippase”. Understanding MFSD2A- mediated transporter has the potential to aid drug delivery to the brain. Proof of concept studies have indicated that small molecules covalently linked to LPC can be transported by MFSD2A, potentially providing a new platform for drug delivery across the blood brain barrier – a major bottleneck in neurotherapeutic development. Currently, there are no known atomic resolution structures of MFSD2A and a limited understanding of its molecular transport mechanism. This has hindered our understanding of who DHA is delivered into brain and precludes structure-based LPC-prodrug design for neurotherapeutic intervention. Here we present the first structure of MFSD2A, in an apo inward-open conformation - in complex with a Fab to increase the size of and introduce features to the imaged particles – determined using single particle cryo electron microscopy to 3.45 Å resolution. In collaboration with, Dr. David Silver at DUKE-NUS (Singapore), a pioneer in the MSFD2A physiology field, we have developed biochemical assays to probe structure-based functional hypotheses for MSFD2A in cells, solution, and proteoliposomes. We are proposing to determine the structures of MSFD2A in complex with its ligand and in an outward-facing conformation, and to use our newly developed biochemical assays to probe and fully understand the molecular features of these structures. The results generated from this application will elucidate the molecular-mechanism behind MSFD2A-mediated LPCDHA transport and will provide information regarding how DHA enters the brain. Furthermore, this research has the potential to lay the foundation for the rational design of neurotherapeutics that “hijack” MFSD2A for delivery across the blood brain barrier.