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Posts Tagged ‘polyunsaturated fatty acid (PUFA)’


Insight into Blood Brain Barrier

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Gateway to The Brain

This image shows the structural model of critical transporter, Mfsd2a. Source: Duke-NUS Medical School
This image shows the structural model of critical transporter, Mfsd2a. Source: Duke-NUS Medical School.  http://www.dddmag.com/sites/dddmag.com/files/rd1604_brain.jpg

Scientists from Duke-NUS Medical School (Duke-NUS) have derived a structural model of a transporter at the blood-brain barrier called Mfsd2a. This is the first molecular model of this critical transporter, and could prove important for the development of therapeutic agents that need to be delivered to the brain — across the blood-brain barrier. In future, this could help treat neurological disorders such as glioblastoma.

Currently, there are limitations to drug delivery to the brain as it is tightly protected by the blood-brain barrier. The blood-brain barrier is a protective barrier that separates the circulating blood from the central nervous system which can prevent the entry of certain toxins and drugs to the brain. This restricts the treatment of many brain diseases. However, as a transporter at the blood-brain barrier, Mfsd2a is a potential conduit for drug delivery directly to the brain, thus bypassing the barrier.

In this study, recently published in the Journal of Biological Chemistry, first author Duke-NUS MD/PhD student Debra Quek and senior author Professor David Silver used molecular modeling and biochemical analyses of altered Mfsd2a transporters to derive a structural model of human Mfsd2a. Importantly, the work identifies new binding features of the transporter, providing insight into the transport mechanism of Mfsd2a.

“Our study provides the first glimpse into what Mfsd2a looks like and how it might transport essential lipids across the blood-brain barrier,” said Ms Quek. “It also facilitates a structure-guided search and design of scaffolds for drug delivery to the brain via Mfsd2a, or of drugs that can be directly transported by Mfsd2a.”

Currently this information is being used by Duke-NUS researchers to design novel therapeutic agents for direct drug delivery across the blood brain barrier for the treatment of neurological diseases. This initiative by the Centre for Technology and Development (CTeD) at Duke-NUS, is one of many collaborative research efforts aimed at translating Duke-NUS’ research findings into tangible commercial and therapeutic applications for patients.

Ms Quek plans to further validate her findings by purifying the Mfsd2a protein in order to further dissect how it functions as a transporter.

 

J Biol Chem. 2016 Mar 4. pii: jbc.M116.721035. [Epub ahead of print]
Structural insights into the transport mechanism of the human sodium-dependent lysophosphatidylcholine transporter Mfsd2a.

Major Facilitator Superfamily Domain containing 2A (Mfsd2a) was recently characterized as a sodium-dependent lysophosphatidylcholine (LPC) transporter expressed at the blood-brain barrier endothelium. It is the primary route for importation of docosohexaenoic acid and other long-chain fatty acids into foetal and adult brain, and is essential for mouse and human brain growth and function. Remarkably, Mfsd2a is the first identified MFS family member that uniquely transports lipids, implying that Mfsd2a harbours unique structural features and transport mechanism. Here, we present three 3D structural models of human Mfsd2a derived by homology modelling using MelB- and LacY-based crystal structures, and refined by biochemical analysis. All models revealed 12 transmembrane helices and connecting loops, and represented the partially outward-open, outward-partially occluded, and inward-open states of the transport cycle. In addition to a conserved sodium-binding site, three unique structural features were identified: A phosphate headgroup binding site, a hydrophobic cleft to accommodate a hydrophobic hydrocarbon tail, and three sets of ionic locks that stabilize the outward-open conformation. Ligand docking studies and biochemical assays identified Lys436 as a key residue for transport. It is seen forming a salt bridge with the negative charge on the phosphate headgroup. Importantly, Mfsd2a transported structurally related acylcarnitines but not a lysolipid without a negative charge, demonstrating the necessity of a negative charged headgroup interaction with Lys436 for transport. These findings support a novel transport mechanism by which LPCs are flipped within the transporter cavity by pivoting about Lys436 leading to net transport from the outer to the inner leaflet of the plasma membrane.

 

Brain and eye contain membrane phospholipids that are enriched in the omega-3 fatty acid docosohexaenoic acid (DHA). It is widely accepted that DHA is important for brain and eye function and brain development (1,2), although mechanisms for DHA function in these tissues are not well defined.   The mechanism by which DHA and other conditionally essential and essential fatty acids cross the blood-brain barrier (BBB) has been a long-standing mystery. Recently, we identified Major Facilitator Superfamily Domain containing 2a (Mfsd2a, aka NLS1) as the primary transporter by which the brain obtains DHA. Importantly, Mfsd2a does not transport unesterified DHA, but transports DHA in the chemical form of lysophosphatidylcholine (LPC) that are synthesized by the liver and circulate largely on albumin (3). This is consistent with biochemical evidence that the brain does not transport unesterified fatty acids (4) and that LPC is the preferred carrier of DHA to the brain (5,6).   Mfsd2a is a sodium-dependent transporter that is part of the Major Facilitator Superfamily (MFS) of proteins. Members of this family with elucidated structures have 12 transmembrane domains composed of two evolutionarily duplicated 6 transmembrane units (7). Transporting an LPC is a unique feature of Mfsd2a, since most members of this family transport water-soluble and minimally polar substrates such as sugars (GLUT, MelB, LacY), and amino acids (TAT1). Mfsd2a transport is not limited to LPCs containing DHA, as it can transport LPCs containing a variety of fatty acyl chains, with higher specificity for LPCs with unsaturated fatty acyl chains with a minimum chain length of 14 carbons (6,8). Crystal structures have been solved for more than a dozen members of the MFS family, with more than 19 structures, including that of Melibiose permease (MelB) of S. typhimurium (9), Lactose permease (LacY) of Escherichia coli (10), glycerol-3-phosphate transporter of E. coli (11) and the mammalian glucose transporters 1, 3, and 5 (GLUT1, GLUT3, GLUT5) (12-14). A common transport mechanism has emerged from both biochemical and structural analyses of MFSs, in which they transport via a rocker-switch, alternating access mechanism (7,15). In the rocker-switch model, rigid-body relative motion of the N- and C-termini domains renders the substrate-binding site alternatively accessible from either side of the membrane.

Mfsd2a is highly expressed at the bloodbrain barrier in both mouse and human (6,16). Mfsd2a deficient mice (KO) have significantly reduced brain DHA as a result of a 90% reduction in brain uptake of LPC containing DHA as well as other LPCs. The most prominent phenotype of Mfsd2a KO mice is microcephaly, and KO mice additionally exhibit motor dysfunction, and behavioral disorders including anxiety and memory and learning deficits (6). In line with the mouse KO phenotypes, human patients with partially or completely inactivating mutations in Mfsd2a presented with severe microcephaly, intellectual disability, and motor dysfunction (8,16). Plasma LPCs are significantly elevated in both KO mice and human patients with Mfsd2a mutations, consistent with reduced uptake at the blood-brain barrier. Taken together, these findings demonstrate that LPCs are essential for normal brain development and function in mouse and humans.

The fact that Mfsd2a transports a lysolipid, a non-canonical substrate for an MFS protein, might indicate unique structure features and a novel transport mechanism. However, no structural information or mechanism of transport of Mfsd2a is known. Human Mfsd2a is composed of 530 amino acids, with two glycosylation sites at Asn217 and Asn227. Mfsd2a is evolutionarily conserved from teleost fish to humans. Although not a functional ortholog of bacterial MFS transporters, Mfsd2a shares 25% and 26% amino acid sequence identity with S. typhimurium MelB (9,17), and LacY from E. coli (10), respectively. Given the high conservation of the MFS fold, the use of homology modeling to gain insight into the structure of S. typhimurium MelB, for example, has proven to be highly accurate and largely consistent with subsequent X-ray crystal data (9,18). Here, we take advantage of two recently derived high resolution X-ray crystal structures of S. typhimurium MelB (9), and a high resolution X-ray crystal structure of LacY (10) to generate three predictive structural models of human Mfsd2a. These models reveal three unique regions critical for function – an LPC headgroup binding site, a hydrophobic cleft occupied by the LPC fatty acyl tail, and three sets of ionic locks. These structural features indicate a novel mechanism of transport for LPCs.

Mfsd2a is a sodium-dependent lysophosphatidylcholine transporter essential for human brain growth and function (40). Mfsd2a is the only known MFS member or secondary transporter that transports a lipid. In line with its unique function, the current study has identified three unique structural features based on a combination of homology structural modeling and biochemical analysis – (1) a unique headgroup binding site and (2) a hydrophobic cleft for acyl chain binding, and (4) 3 sets of ionic locks that stabilize the outward open conformation. Drawing together these findings with studies of the mechanism of transport of other MFS family members, we propose the following alternatingaccess mechanism for LPC transport (Fig. 6). In the first steps, LPC inserts itself into the outer leaflet of the membrane and diffuses laterally into the transporter’s hydrophobic cleft. As Mfsd2a undergoes conformational changes from the outward open to the inward open conformation, the zwitterionic headgroup is inverted from the outer membrane leaflet to the inner membrane leaflet along a translocation pathway within the transporter, interacting with specific polar and charged residues lining the path. Since LPCs are hydrophobic phospholipids, it is unlikely that they will partition out of the transporter into the aqueous environment of the cytoplasm. We propose that the “flipped” LPC exits the transporter laterally into the membrane environment of the inner leaflet. This model of LPC flipping requires further biochemical proof. Of particular interest is the visualization of the interaction of the negatively charged phosphate headgroup of LPC with Lys436 that is maintained in both outward and inward open conformations. The sidechain of Lys436 is seen to be pointing in the upward direction in the outward open conformation, but pointing downward into the translocation cleft in the inward open conformation. These findings suggest that the Lys436 acts as a tether to push or pivot the headgroup down into the translocation cavity while the N- and C-termini of Mfsd2a rock and switch from outward to inward open.

Interestingly, Lys436 is orthologous to the residue Lys377 in the melibiose transporter of S. typhimurium. Based on the S. typhimurium MelB crystal structure, Lys377 has been predicted to be involved in binding melibiose, and in forming a hydrogen bond with Tyr120, likely separating the sodium binding site from the central hydrophilic cavity (9). In a recent molecular dynamic simulation of E. coli MelB, Lys377 was noted to interact differently with residues involved in the sodium binding site (Asp55, Asp59, and Asp124) in the presence or absence of a sodium ion, and thought to be critical for the spatial organization of the sodium binding site (41). Similarly, in our refined models of Mfsd2a, Lys436 is localized in close proximity to the sodium-binding site residue, Asp93, and the central translocation pathway where it has been identified by docking studies to interact with the charged headgroup of LPC. We hypothesize that Lys436 may shuttle between the two binding sites, communicating and coordinating the occupancy status of the two sites. Interestingly, there is a distinct mobility shift in Mfsd2a bands on SDS-PAGE between wild-type Mfsd2a and the L-3 mutant (R498E, R499E, R500E, K503E, K504E) (Fig. 5I) that is not seen when each of the residues are mutated individually (Fig. S1). These findings are consistent with a conformational change in the L-3 mutant. Given that the L-3 ionic lock is visualized in the outward partially occluded model, we hypothesize that the loss of the L-3 ionic lock results in Mfsd2a being trapped in an energetically more favorable inward open conformation, resulting in the loss of transport function (Fig. 5H).

Patients with the partially inactivating mutation p.(S399L) exhibited significant increases specifically in plasma LPCs having monounsaturated (18:1 – 92%, p=0.004) and polyunsaturated LPCs (18:2, 20:4, 20:3 – 254%, p=0.002; 117%, p=0.007, and 238%, p=0.002), but not in the most abundant LPCs – saturated LPCs (C16:0, C18:0) (8). This is consistent with a greater specificity of Mfsd2a for LPCs with unsaturated fatty acyl chains (6)…A possible explanation for this acyl chain specificity is related to the mobility of the acyl tail in the membrane. It is known that phospholipids with unsaturated fatty acyl chains disrupt the packing of the bilayer, resulting in greater lateral membrane fluidity (42). Therefore, one possible mechanism for LPC specificity is that LPCs with unsaturated fatty acyl chains have greater lateral mobility in the membrane, increasing the Ka for interacting with the transport cleft of Mfsd2a.

Another important structural feature of the physiological ligand, LPC, is a minimum acyl chain length of 14 carbons is required for transport by Mfsd2a. A possible explanation for this requirement is that the hydrocarbon chain must extend beyond the cleft, protruding into the hydrophobic milieu of the phospholipid bilayer core. This interaction of the fatty acyl tail with the acyl chains of the membrane bilayer may provide a hydrophobic force strong enough to pull the molecule through and out of the transporter as the LPC headgroup partitions into the inner leaflet of the membrane. A similar scenario is seen in the Sec translocon where a hydrophobic transmembrane domain of a protein partitions laterally from the Sec61p complex channel into the lipid bilayer (43,44). This proposal that the omega carbon of the fatty acyl chain sticks out of the Mfsd2a pocket is consistent with the observation that Mfsd2a can transport nitrobenzoxadiazole (NBD) or Topfluor when these moieties are attached to the omega carbon of the LPC fatty acyl tail [1].

Other known transmembrane phospholipid transporters include flippases, floppases, and scramblases. Flippases and floppases utilize ATP to drive the uphill transport of aminophospholipids from the outer to the inner leaflet, and specific substrates from the inner to the outer leaflet, respectively (45-47). Scramblases are less well understood, facilitating transport of substrates in either direction down concentration gradients upon activation. While the substrates are similar, several differences make comparisons between Mfsd2a and phospholipid transporters of limited relevance. First, the shapes of the substrates differ in shape and size – lysophospholipids are smaller and conical while phospholipids are cylindrical. Second, unlike flippases and floppases, Mfsd2a is a secondary transporter, utilizing a sodium electrochemical gradient to drive the transport of lysophospholipids from one leaflet to the other. Third, the overall structure of MFS members is different from P4- ATPases and ABC transporters. Consequently, the mechanism of action between Mfsd2a and flippases such as P4-ATPases and ABC transporters, or floppases is expected to differ.

Being expressed at the blood-brain barrier, Mfsd2a is a potential conduit for drug delivery to the brain. The blood-brain barrier is highly impermeable, protecting the brain from bloodderived molecules, pathogens, and toxins. However, its impermeability poses a challenge for pharmacological treatment of brain diseases. It has been predicted that 98% of small molecule drugs are excluded from the brain by the blood-brain barrier (48). Currently, most drugs used to treat brain diseases are lipid soluble small molecules with a molecular weight of less than 400 Da (49). A small number of drugs traverse the blood-brain barrier by carrier-mediated transport. An example of this is Levodopa, a treatment for Parkinson’s Disease, which is a precursor of the neurotransmitter dopamine. Levodopa is transported across the blood-brain barrier by the large neutral amino acid transporter, LAT1 (50). Our findings here provide a further refinement of understanding of the structure-activity relationship of LPCs to their transport, and educates the search and design of drugs that can be transported by Mfsd2a. Candidates for transport, whether as a drug itself or as a LPC scaffold, must have a zwitterionic headgroup, but not necessarily a phosphate, and a minimal threshold of hydrophobic character. As the binding pocket is several times larger than LPC, it is sterically feasible to attach a small molecule drug onto LPC or LPC-like scaffolds for delivery across the blood-brain barrier.

In summary, these studies represent a first structural model of human Mfsd2a based on homology modeling and biochemical interrogation. We expect that this model will serve as a foundation for the future development of X-ray crystal structures of the protein, which would provide further insight into the structure and function of this physiologically important transporter required for human brain growth and function.

REFERENCES

1. Salem, N., Jr., Litman, B., Kim, H. Y., and Gawrisch, K. (2001) Mechanisms of action of docosahexaenoic acid in the nervous system. Lipids 36, 945-959

2. Bazan, N. G. (2009) Neuroprotectin D1-mediated anti-inflammatory and survival signaling in stroke, retinal degenerations, and Alzheimer’s disease. Journal of lipid research 50 Suppl, S400- 405

3. Baisted, D. J., Robinson, B. S., and Vance, D. E. (1988) Albumin stimulates the release of lysophosphatidylcholine from cultured rat hepatocytes. The Biochemical journal 253, 693-701

4. Edmond, J., Higa, T. A., Korsak, R. A., Bergner, E. A., and Lee, W. N. (1998) Fatty acid transport and utilization for the developing brain. Journal of neurochemistry 70, 1227-1234

5. Lagarde, M., Bernoud, N., Brossard, N., Lemaitre-Delaunay, D., Thies, F., Croset, M., and Lecerf, J. (2001) Lysophosphatidylcholine as a preferred carrier form of docosahexaenoic acid to the brain. Journal of molecular neuroscience : MN 16, 201-204; discussion 215-221

6. Nguyen, L. N., Ma, D., Shui, G., Wong, P., Cazenave-Gassiot, A., Zhang, X., Wenk, M. R., Goh, E. L., and Silver, D. L. (2014) Mfsd2a is a transporter for the essential omega-3 fatty acid docosahexaenoic acid. Nature 509, 503-506

7. Law, C. J., Maloney, P. C., and Wang, D. N. (2008) Ins and outs of major facilitator superfamily antiporters. Annual review of microbiology 62, 289-305

8. Alakbarzade, V., Hameed, A., Quek, D. Q. Y., Chioza, B. A., Baple, E. L., Cazenave-Gassiot, A., Nguyen, L. N., Wenk, M. R., Ahmad, A. Q., Sreekantan-Nair, A., Weedon, M. N., Rich, P., Patton, M. A., Warner, T. T., Silver, D. L., and Crosby, A. H. (2015) A partially inactivating mutation in the sodium-dependent lysophosphatidylcholine transporter MFSD2A causes a non-lethal microcephaly syndrome. Nat Genet 47, 814-817

9. Ethayathulla, A. S., Yousef, M. S., Amin, A., Leblanc, G., Kaback, H. R., and Guan, L. (2014) Structure-based mechanism for Na(+)/melibiose symport by MelB. Nature communications 5, 3009

10. Guan, L., Mirza, O., Verner, G., Iwata, S., and Kaback, H. R. (2007) Structural determination of wild-type lactose permease. Proceedings of the National Academy of Sciences of the United States of America 104, 15294-15298

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