Inhibition of Cellular Uptake of Folate by Blocking Synthesis of the Membrane Folate Receptor

The folate receptor, bound to the plasma membrane through a glycosylphosphatidylinositol anchor, requires both sphingolipids and cholesterol in the membrane for full activity. In recent studies, treatment of cells in culture with the mycotoxin fumonisin B,, which inhibits sphingolipid synthesis, virtually abolished uptake of 5-methyltetrahydrofolate.

The compounds of the folate family, consisting of folate and its derivatives, are vitamins involved as coenzymes in reactions of one-carbon metabolism. They are essential for the biosynthesis of thymidine and purines and for the conversion of homocysteine to methionine, among other reactions.

cells by two different mechanisms: one uses a transmem- brane transporter protein called “reduced folate carrier,” with high affinity for reduced folates’ and the other in- volves a membrane-anchored receptor protein called “mem- brane folate receptor” (MFR) with high affinity for both folic acid and reduced folates.‘ The MFR is important for tissues that require high folate levels, such as kidney, placenta, and breast. Once inside the cells, the folate is retained in the cytoplasm by polyglutamation.*

The MFR has three distinguishing characteristics: (1) it is attached to the plasma membrane through a covalent link to a glycosylphosphatidylinositol (GPI) anchor: (2) it is located in a membrane domain that is rich in ~phingolipids~ and chole~terol,~ and (3) it can be isolated through its insolubility in nonionic detergents, such as Triton X-100 (TX100).’*6 Because of this insolubility, the suggestion was made that the MFR is located in caveolae, since the caveolae, which are invaginations of the plasma membrane, have domains that are insoluble in nonionic detergents. Morphologic and other investigations by immunolabeling appeared to show that GPI-anchored pro- teins, such as MFR, are concentrated in caveolae.’ More recent studies, however, demonstrate that GPI-linked pro- teins, including the MFR, are actually difhsely distrib- uted throughout the plasma membrane and cluster in caveolae only after cross-linking in the course of their isolation by antibody binding.* Uptake of folate by MFR, therefore, appears not to take place in caveolae, but rather

Folate is supplied by the diet and is transported i k

This review was prepared by George Wolf, D.Phil., Department of Nutritional Sciences, University of California, Berkeley, CA 94720-31 04, USA. Reprint requests are to be addressed to Nutrition Reviews, 71 1 Washington St., Boston, MA 021 11, U.S.A.

by the classical endocytosis process. The MFR on the plasma membrane is bound and folate is internalized by forming vesicles that are pinched off and enter the cell. After unloading the folate, the MFR is recycled back to the plasma membrane.’

Parton and Simonsq suggest that the insolubility of GPI-anchored proteins in nonionic detergents is caused by their close association with sphingolipids and choles- terol in specific plasma membrane domains. It is possible that the sphingolipids, particularly glycosphingolipids, interact through intermolecular hydrogen bonds with the GPI anchor of the membrane protein and thus localize it in particular domains of the plasma membrane.IO The local- ization, transport, and targeting of sphingolipids and GPI- anchored proteins generally appear to be linked.” Thus, inhibition of sphingolipid synthesis interferes with the transport of GPI-anchored proteins in yeast and animal epithelial cells to their correct membrane position.’*

A recent report by Stevens and Tang13 describes the effect of depleting intestinal cells (Caco-2 cells derived from a human colon adenocarcinoma) of sphingosine by treatment with the mycotoxin fumonisin B, (FB,), which inhibits sphingosine N-acyltransferase (ceramide syn- thase) and thereby blocks sphingolipid b i o s y n t h e ~ i s . ~ ~ ~ ~ ~ Treatment of these cells with FBI (27.7 pM) for 2 days resulted in a lowered sphingolipid level (about 20%), both in whole cells and in the TX100-insoluble fraction that contained the MFR. Because the MFR domain is enriched in cholesterol as well as spingolipid, the cells were treated with lovastatin, a cholesterol synthesis inhibitor. It caused a similar (about 20%) decline in the cells’ level of choles- terol.

Uptake into the cells of the folate derivative 5- methyltetrahydrofolate, which increased linearly with time in untreated cells, was virtually abolished by FBI and also by lovastatin. The FBI inhibition was concentration- and time-dependent over 2 days. The addition of ceramide to the FBI-treated cells reversed the inhibition of folate up- take. The authors” state that they cannot be sure whether this indicated the loss of mature sphingolipids or of ceramide itself.

The following results were obtained in control experi- ments. Treatment of cells with FBI for 1 hour (instead of 2 days) did not inhibit folate uptake, indicating that the in- hibition was not caused by direct toxicity of the FBI. Furthermore, FBI treatment had no effect on the facilitated transport into the cells of deoxyglucose or on the uptake of transferrin by endocytosis.

The authorsI3 next showed that about 80% of the total cell folate-binding capacity was localized in the

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sphingolipidcholesterol-rich TX 1 00-insoluble fraction of untreated cells. Treatment with FBI (to reduce sphin- golipid) or lovastatin (to reduce cholesterol) caused a time- and concentration-dependent decrease of about 50% in folate binding. Interestingly, 80% of the remaining folate- binding activity was still in the TX100-insoluble fraction. The authors state:I3 “Therefore, changes in the sphin- golipid or cholesterol levels appear to decrease the total amount of folate receptor in the cells, but do not change its enrichment in the TX100-insoluble domain.” A near- perfect correlation (r=0.967) was found between the amount of the folate receptor and the ability to transport folate into the cytoplasm of the cells. The authorsI3 con- cluded that the inhibition of the uptake of folate was caused by theposs of the receptor rather than by its redis- tribution within the membrane.

In summary, the authorsI3 showed that the MFR, which is attached to the plasma membrane by a GPI an- chor, is critically dependent on the sphingosine and cho- lesterol levels within the membrane. It appears that the GPI anchor requires a sphingolipicUcholestero1-enriched domain, possibly to target the receptor to a particular lo- cus on the membrane. Loss of either sphingolipid or cho- lesterol resulted in decreased receptor activity, although not by redistribution of the receptor.

Stevens and Tang’s reportI3 is important because it links FBI, a common contaminant of corn, to the cellular uptake of folate. They draw attention to the finding of fetal abnormalities observed in mice given FB,.I6 In preg- nant women, the risk of fetal neural tube defects is known to be associated with dietary folate def5ciency.I’ Epide- miologic studies18 have found that increased rates of neu- ral tube defects among Hispanics were clustered at certain periods in certain counties in Texas. Stevens and Tang considered the possibility that corn may have been con- taminated with mycotoxins at that time (1989) in Texas. FBI contamination, through inhibition of folate uptake, may be a risk factor in some birth defects. Recent find- ingslg of the correlation between increased risk of heart disease and elevated blood levels of homocysteine asso- ciated with folate depletion have focused attention on other human health problems that could be caused by food contaminated with FBI.

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Chang WJ, Rothberg KG, Kamen BA, Anderson RGW. Lowering of the cholesterol content of MA1 04 cells inhibits receptor-mediated transport of folate. J Cell Biol 1993;118:63-9 Hanada K, Nishijima M, Akamatsu V, Pagano RE. Both sphingolipids and cholesterol participate in the detergent-insolubility of alkaline phosphatase, a GPI- anchored protein in mammalian membranes. J Biol Chem 1995;270:6254-60 Anderson RGW. Plasmalemmal caveolae and GPI- anchored membrane proteins. Curr Opin Cell Biol

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