Inhibition of Ceramide Synthase

In the current model for how FB₁ inhibits ceramide synthase, it has been proposed¹ that the aminopentol backbone competes for binding of the sphingoid base substrate whereas the tricarballylic acid side-chain interferes with utilization of the co-substrate fatty acyl-CoA, as can be envisioned by overlaying the structures in Figures 1A and B. Some of the evidence that supports this model is: 1) the potency of inhibition by FB₁ depends on the concentrations of both sphingoid bases and fatty acyl-CoA^6,7; 2) removal of the tricarballylic acids diminishes the potency of ceramide synthase inhibition^6,8 and, 3) upon removing the tricarballylic acids, AP₁ becomes a substrate for acylation (whereas FB₁ is not acylated).⁶ As will be discussed later, this acylation may explain why aminopentols remain toxic to animals despite the lower potency of these compounds as ceramide synthase inhibitors.

When added to mammalian cells in culture, FB₁ is usually effective in blocking the formation of ceramide, SM and glucosylceramide, and more complex sphingolipids, measured by incorporation of radiolabelled serine⁷ or of mass^5,9 (see below). Blockage of complex sphingolipid formation must be confirmed for each case because some organisms, such as yeast,¹⁰ take up fumonisins poorly unless treated specially and there are cases where other enzymmatic reactions, such as reversal of ceramidase,^11,12 can provide an alternative pathway for ceramide synthesis. It is also advisable to check the effectiveness of fumonisin stock solutions occasionally because potency is sometimes lost during storage, presumably due to hydrolysis of the ester-linked tricarballylic acid(s).

Analysis of Sphingolipids in Fumonisin-Treated Cells

Conventional methods for analysis of sphingolipids are sufficient to determine if FB₁ is blocking de novo synthesis. Among these, the most convenient are measurement of the incorporation of radiolabelled serine or palmitic acid into sphingolipids^13,14 or elevation of sphinganine (by HPLC with a fluorescence detector).^15,16 In addition to the relative convenience of the latter, it serves to remind the investigator that the elevation in sphinganine can be dramatic, as high as several nmol/10⁶ cells, amounts that are high enough to affect signaling pathways, such as the inhibition of protein kinase C.¹⁷ These conventional assays are labor intensive, provide little information about structural variants within each class (for example, the different molecular subspecies of ceramides), and are not very efficient at extracting and detecting polar compounds such as the sphingoid base 1-phosphates.

Analysis of Sphingolipids by Electrospray Tandem Mass Spectrometry

Heretofore, mass spectrometry has been used primarily for structural elucidation of sphingolipids (reviewed in ref. 18); however, the development of electrospray ionization (ESI) provides the ability to generate intact molecular ions of polar biomolecules directly from solution (i.e., the eluate from HPLC columns) and to perform quantitative analysis. Equally important has been the availability of a method to quantify different species (multiple reaction monitoring, illustrated in Fig. 3) and the commercial availability of internal standards for sphingoid bases (C20-sphingosine and -sphinganine), sphingoid base 1-phosphates (the C17 homologs), ceramides, sphingomyelins, glucosylceramide and lactosylceramide (all prepared as the C12-fatty acid homologs) (Avanti Polar Lipids, Alabaster, AL). With these capabilities, a step-by-step procedure has been developed for the extraction, analysis, and quantitation of all of these categories of sphingolipids (as well as lysosphingolipids and N-methylsphingoid bases) using HPLC electrospray tandem mass spectrometry.⁵ Its sensitivity allows analysis of all of these compounds in samples as small as a petri dish of cells in culture.

Figure 3

Illustration of the advantage of using multiple reaction monitoring (MRM) in the quantitative analysis of sphingolipids. Data from ref. .

Effects of Fumonisin B₁ on Sphingolipid Metabolism

Analyses of sphingolipid metabolism using diverse methods, most of which have now been confirmed by mass spectrometry, reveal interesting changes in the amounts of sphingolipids in cells. Pathways of sphingolipid metabolism that participate in the response to fumonisins are shown in Figure 2.

Figure 2

Pathway for sphingoid base biosynthesis with illustration of the side reactions (phosphorylation and acetylation) that can increase when sphinganine and sphingosine are elevated by (dihydro)ceramide synthase inhibition by fumonisins.

Depletion of More Complex Sphingolipids

Fumonisins can completely block synthesis of new sphingolipids and severely deplete the total mass of cellular sphingolipids.^1,7,20,21 As an example of the findings from mass spectrometric analysis of the effects of FB₁ on cellular amounts of SM, glucosylceramide and ceramide in NIH3T3 cells are shown in Figure 4. These changes can disrupt cell functions dependent on complex sphingolipids; for example, FB₁ treatment of intestinal cells in culture blocks folate uptake because the folate transporter is a glycosylphosphatidylinositol-anchored protein, which typically require sphingolipids and cholesterol to function normally.²²

Figure 4

Amounts of ceramide (Cer), sphingomyelin (SM) and glucosylceramide (GlcCer) inIH 3T3 cells incubated for 24 h with (or without) 50 mM FB₁ and analyzed by electrospray tanden mass spectrometry.

Production of Sphingoid Base 1-Phosphates and Down-Stream Metabolites (e.g., Ethanolamine Phosphate)

Sphingoid bases are catabolized by phosphorylation and lytic cleavage to a fatty aldehyde and ethanolamine phosphate (see Fig. 2). FB₁ has been shown to increase the amounts of cellular sphingoid base 1-phosphates^4,5 (see also Fig. 5) as well as to increase the amount of sphingolipid-derived ethanolamine phosphate that is incorporated into phosphatidylethanolamine.⁴

Figure 5

Amounts of sphinganine (Sa), sphingosine (So) and sphinganine 1-phosphate (SaP) in NIH 3T3 cells incubated for 24 h with (or without) 50 mM FB₁ and analyzed by electrospray tandem mass spectrometry.

Additional Findings with Aminopentols and Other Sphingoid-Base Analogs

Animals (including humans) are naturally exposed to the fumonisin aminopentols because they are formed during the treatment of corn with lye, a common practice in the preparation of flour for tortillas; furthermore, the intestinal microflora of primates can hydrolyze FB₁ to AP₁. Although AP₁ is a weak inhibitor of ceramide synthase, it is acylated to form derivatives such as the one shown in Figure 6 (N-palmitoyl-AP₁ or PAP₁).⁶ These acylated aminopentols are more potent inhibitors of ceramide synthase and are as, or more, cytotoxic than FB₁.⁶

Figure 6

Acylation of AP₁ and some of the properties of the original aminopentol and this metabolite.

Structural differences between the aminopentols and sphinganine include the absence of a hydroxyl group at the 1-position, the presence of an additional hydroxyl at position 5, and a threo- versus erythro-stereochemistry at positions 2 and 3. When the toxicities of naturally occurring fumonisin aminopentols (e.g., AP₁) are compared to sphinganine with cells in culture, AP₁ is less toxic. This may be due to the lower hydrophobicity of the aminopentol because when 1-deoxy-sphinganine analogs have been synthesized and compared (K. Desai et al, manuscript in preparation), they are approximately 10-fold more toxic than sphinganine. These types of compounds offer promise as probes for the mechanisms of action of both fumonosins and sphingoid bases.

Conclusions

It should be clear from this overview that perturbation of one aspect of sphingolipid metabolism has ramifications for other pathways. Of particular interest is the finding that fumonisins can increase not only sphinganine growth inhibitory and pro-apoptotic compound but also sphinganine 1-phosphate, a mitogenic and anti-apoptotic sphingolipid. This may account for the puzzling research literature for fumonisins in which they have been studied by toxicologists for their toxicity, but have been recently used by other researchers to protect cells against apoptosis (indeed, some companies market FB₁ as an inhibitor of apoptosis). This may also have an in vivo correlate since fumonisins are toxic in vivo but also carcinogenic for liver, kidney and some other organs.

The possible mechanisms of action of fumonisins are probably quite complex, involving reductions in complex sphingolipids, elevations in sphingoid bases (sometimes counterbalanced with sphingoid base 1-phosphates), and formation of other interesting compounds such as the C2-(dihydro)ceramides and acylated aminopentols. One way to resolve some of these discrepancies is to use other inhibitors of sphingolipid biosynthesis, such as ISP1 or myriosin.³² Nonetheless, this complexity probably explains why these mycotoxins show such a wide spectrum of pathologies including neurotoxicity, pulmonary toxicity, liver and renal toxicity (and carcinogenicity) inter alia.^1,2

Acknowledgments

Work from the lab of the corresponding author was supported by grants from the NIH (GM46368 and ES09204). We are also grateful to the many valuable collaborators and particularly Ron Riley and colleagues at the USDA laboratories in Athens, GA, who have contributed to findings discussed in this review.

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