Abstract
In a previous study we observed that long term (5 days) incubation with fumonisin B1 (FB1), an inhibitor of acylation of sphingoid long chain bases to (dihydro)ceramide, resulted in morphological and biochemical changes in 3T3 fibroblasts (Meivar-Levy, I., Sabanay, H., Bershadsky, A. D., and Futerman, A. H. (1997) J. Biol. Chem. 272, 1558-1564). Among these were changes in the profile of synthesis of sphingolipids (SLs) and glycosphingolipids (GSLs). Whereas [3H]globotriaosylceramide ([3H]Gb3) comprised 1.9% of the total [3H]SLs and [3H]GSLs synthesized in control cells, it comprised 16. 5% in FB1-treated cells. We now demonstrate by in vitro analysis that inhibition of ceramide synthesis by FB1 for 5 days results in up-regulation of the activities of three enzymes in the pathway of Gb3 synthesis, namely glucosylceramide, lactosylceramide, and Gb3 synthases; up-regulation is due to an increase in Vmax, with no change in Km values toward lipid substrates. Moreover, molecular analysis (reverse transcriptase-polymerase chain reaction) of glucosylceramide synthase indicated that this enzyme is up-regulated at the transcriptional level. No changes in either the Vmax or Km values of sphingomyelin or of GM3 synthase were detected after FB1 treatment. Analysis of SL and GSL synthesis in cultured cells using [4,5-3H]sphinganine as a metabolic precursor demonstrated that at low substrate concentrations, Gb3 synthesis is favored over GM3 synthesis and glucosylceramide synthesis is favored over sphingomyelin synthesis, whereas the opposite is true at high substrate concentrations. These data demonstrate that GSL synthesis and in particular Gb3 synthesis are tightly regulated in fibroblasts, presumably so as to maintain constant levels of Gb3 on the cell surface.
Highlights
Sphingolipids (SLs)1 and glycosphingolipids (GSLs) are ubiquitous and essential components of eukaryotic cell membranes [1]
Using [4,53H]sphinganine as a precursor of SL and GSL synthesis, we previously observed that residual levels of synthesis of each lipid differed after 5 days incubation with fumonisin B1 (FB1) (20 M) [13], and unexpectedly, Gb3 synthesis was not inhibited to any extent
We have systematically determined the activity of the five enzymes responsible for the synthesis of each lipid by in vitro analyses and analyzed whether differences in Km values between the different enzymes might account for the change in the profile of SL and GSL synthesis
Summary
FB1 was from the Division of Food Science and Technology (CSIR, Pretoria, South Africa). D-Sphinganine and lactosylceramide (LacCer) were from Matreya (Pleasant Gap, PA). N-Hexanoic acid [1-14C]n-hydroxysuccinimide ester (55 mCi/mmol) was from American Radioactive Chemicals (St. Louis, MO). CMP[14C]sialic acid (294 mCi/mmol) and UDP[U-14C]Gal (261 mCi/mmol) were from Amersham International plc (Amersham, UK). TitanTM one tube RT-PCR system was from Boehringer Mannheim. Silica Gel 60 plates were from Merck. Ultima Gold was from Packard (Meriden, CT). Other chemicals were from Sigma, and solvents (analytical grade) were from Bio-Lab Laboratories Ltd. Other chemicals were from Sigma, and solvents (analytical grade) were from Bio-Lab Laboratories Ltd. (Jerusalem, Israel)
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