Abstract
This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DH-Cer), an intermediate in de novo sphingolipid biosynthesis. When 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl-DH-Cer (C6-NBD-DH-Cer) was incubated with HT29, NRK, BHK, or HL-60 cells, it was efficiently converted to dihydrosphingomyelin and dihydroglucosylceramide, and a number of other sphingolipids, with the nature of the products depending on the cell line. In addition, complex sphingolipids were formed that contained a desaturated (sphingosine) backbone, indicating that DH-Cer (and/or its metabolites) were substrates for the desaturase(s) that introduce the 4,5-trans double bond. Based on the kinetics and inhibitor studies, double bond addition did not appear to occur with the complex sphingolipids directly, but rather, during turnover and resynthesis. The conversion of C6-NBD-DH-Cer to more complex sphingolipids was highly stereoselective for the natural D,erythro isomer of C6-NBD-DH-Cer. Interestingly, the stereochemistry of the sphingoid base backbone also affected the localization of fluorescent sphingolipids: the D,erythro species appeared in the Golgi apparatus, whereas other stereo-isomers accumulated in the endoplasmic reticulum. In addition to C6-NBD-Cer and C6-NBD-DH-Cer, C6-NBD-4-D-hydroxy-DH-Cer gave rise to formation of complex sphingolipids and localized at the Golgi apparatus. These studies indicate that dihydroceramide is used as the initial backbone of complex (glyco)sphingolipids, perhaps to avoid build up of ceramide as an intermediate since this is such a potent bioactive compound. The stereoselectivity in transport and metabolism suggests that trafficking of ceramide is protein-directed rather than simply a consequence of vesicular membrane flow.
Highlights
This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DH-Cer), an intermediate in de novo sphingolipid biosynthesis
C6-NBD-DH-Cer Is Converted to Complex Sphingolipids, Both Saturated and Desaturated—C6-NBD-Cer, a short-chain fluorescent analog of ceramide, is a well established precursor forsphingolipid biosynthesis [17, 23, 30]
We examined the fate of a C6-NBD-analog of dihydroceramide (C6-NBD-DH-Cer, Fig. 2C), which should, in principle, serve as a precursor for the synthesis of C6-NBD-Cer via double bond introduction between C4 and C5 of the sphinganine backbone
Summary
This study utilized fluorescent analogs to characterize the intracellular transport and metabolism of dihydroceramide (DH-Cer), an intermediate in de novo sphingolipid biosynthesis. Little is known about introduction of the 4-trans double bond into the sphingoid base backbone during de novo biosynthesis of sphingolipids or about the fate of dihydroceramide (DH-Cer) of complex sphingolipids when they are turned over. The goal of this study was to clarify the metabolic pathways for conversion of DH-Cer to Cer and more complex sphingolipids, as well as their intracellular localization and transport For this purpose, we made use of a fluorescent analog of DH-Cer, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoyl-dihydroceramide (C6-NBD-DH-Cer) in analogy to C6-NBD-Cer [14, 15], which after insertion into the outer leaflet of the plasma membrane undergoes transbilayer movement and diffusion into the cell interior, where it integrates into the membranes of various organelles [16, 17]. It is not known how (C6-NBD-)Cer is translocated from the ER to the Golgi, whether by co-transport with proteins in vesicular shuttles or by (protein-mediated) transport through the cytosol
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