Abstract. Glucosylceramide (GlcCer) is synthesized at the cytosolic surface of the Golgi complex while enzymes acting in late steps of glycosphingolipid biosynthesis have their active centers in the Golgi lumen. However, the topology of the "early" galactose-transferring enzymes is largely unknown. We used shortchain ceramides with either a 2-hydroxy fatty acid (HFA) or a normal fatty acid (NFA) to determine the topology of the galactosyltransferases involved in the formation of HFA-and NFA-galactosylceramide (GalCer), lactosylceramide (LacCer), and galabiosylceramide (GaECer).Although the HFA-GaICer synthesizing activity colocalized with an E R marker, the other enzyme activities fractionated at the Golgi density of a sucrose gradient. In cell homogenates and permeabilized cells, newly synthesized short-chain GlcCer and GalCer were accessible to serum albumin, whereas LacCer and Ga2Cer were protected. From this and from the results obtained after protease treatment, and after interfering with UDP-Gal import into the Golgi, we conclude that (a) GlcCer and NFA-GaICer are synthesized in the cytosolic leaflet, while LacCer and GaECer are synthesized in the lumenal leaflet of the Golgi. (b) HFA-GalCer is synthesized in the lumenal leaflet of the ER, but has rapid access to the cytosolic leaflet. (c) GlcCer, NFA-GaICer, and HFA-GaICer translocate from the cytosolic to the lumenal leaflet of the Golgi membrane. The transbilayer movement of GlcCer and NFA-GalCer in the Golgi complex is an absolute requirement for higher glycosphingolipid biosynthesis and for the cell surface expression of these monohexosyl sphingolipids.LYCOSPHINGOLIPIDS are universal membrane components of eukaryotic cells. They are enriched at the cell surface and in the lumen of endosomes and lysosomes (60). At the cell surface the glycosphingolipids not only exert functions in cell-cell interaction, cell-substrate interaction, and signal transduction, but are also used as receptors by bacteria, bacterial toxins, and viruses. Glycosphingolipids are a relatively minor constituent of most membranes, but a major component of myelin and of the apical plasma membrane of the polarized epithelial cells that line the gastrointestinal and urinary tract. In the latter tissues, glycosphingolipids play a structural role in rigidifying and protecting the cell surface. The apical plasma membrane of polarized epithelial cells is enriched in glycosphingolipids relative to the basolateral plasma membrane which contains less glycosphingolipids Please address all correspondence to K. Burger, Department of Cell Biology, Universiteit Utrecht, AZU H02.314, 3584 CX Utrecht, The Netherlands, Tel: 31 30 2506480. Fax: 31 30 2541797. E-mail: K.N.J.Burger @med.ruu.nl but more of the phospholipid phosphatidylcholine and the (sphingo)phospholipid sphingomyelin (SM). t Studies using epithelial cells in culture indicate that glycosphingolipids and phospholipids are sorted intracellularly before reaching the cell surface. Moreover, glycosphingolipids are thought to play a key role ...

Galactosylceramide (“galactocerebroside”; GalC) is a major glycolipid in the myelin sheath of the CNS and the PNS. The enzyme UDP‐galactose:ceramide galactosyltransferase (CGalT) catalyzes the final step of the synthesis of GalC: the transfer of galactose to ceramide. By a differential screening approach, we have isolated a cDNA, the sequence of which is identical to the recently isolated cDNA clones for CGalT. By northern analysis and in situ hybridization we demonstrated that CGalT mRNA is expressed at birth in oligodendrocytes and Schwann cells, an expression pattern corresponding to the onset of myelination. In addition to the high expression levels of CGalT in oligodendrocytes and Schwann cells, in situ hybridization also showed expression in subtypes of neurons in spinal cord, cerebellum, and brainstem in the adult CNS, but at a much lower level than in oligodendrocytes. Expression of CGalT in COS cells demonstrated that CGalT has a preference for hydroxyceramide as a substrate. CGalT‐expressing COS cells synthesize and transport GalC to their cell surface as shown by immunofluorescence and by lipid analysis of living cells. Our results suggested that the CGalT specifically uses hydroxyceramide for the synthesis of GalC and that separate (co)enzymes are not needed.

Galactosylceramide (GalCer) is the major glycolipid in brain. In order to characterize the activity of brain UDPgalactose: ceramide galactosyltransferase (CGalT), it has been stably expressed in CGalT-negative Chinese hamster ovary (CHO) cells. After fractionation of transfected cells, CHO-CGT, on sucrose gradients, the activity resides at the density of endoplasmic reticulum and not of Golgi. A lipid chromatogram from CHO-CGT cells revealed two new iodine-staining spots identified as GalCer, since they comigrate with GalCer standards, can be metabolically labelled with [3H]galactose, are recognized by anti-GalCer antibodies, and are resistant to alkaline hydrolysis. A third [3H]galactose lipid was identified as galactosyldiglyceride. In the homogenate CGalT displays a 25-fold preference for hydroxy fatty acid-containing ceramides. Remarkably, endogenous GalCer of transfected cells contains exclusively non-hydroxy fatty acids: fast atom bombardment and collision-induced dissociation mass spectrometric analysis revealed mainly C16:0 in the lower GalCer band on TLC and mainly C22:0 and C24:0 in the upper band. Our results suggest that CGalT galactosylates both hydroxy- and non-hydroxy fatty acid-containing ceramides and diglycerides, depending on their local availability. Thus, CGalT alone may be responsible for the synthesis of hydroxy- and non-hydroxy-GalCer, and galactosyldiglyceride in myelin.

Abstract. The high concentration of glycosphingolipids on the apical surface of epithelial cells may be generated by selective transport from their site of synthesis to the cell surface. Previously, we showed that canine kidney MDCK and human intestinal Caco-2 cells convetted a ceramide carrying the short fluorescent fatty acid Cr-NBD to glucosylceramide (GlcCer) and sphingomyelin (SM), and that GlcCer was preferentially transported to the apical surface as compared to SM. Here, we address the point that not all glycosphingolipid classes are apically enriched in epithelia. We show that a ceramide containing the 2-hydroxy fatty acid C60H was preferentially converted by MDCK and Caco-2 cells to galactosylceramide (GalCer) and its derivatives galabiosylceramide (Ga2Cer) and sulfatide (SGalCer) as compared to SM and GlcCer--all endogenous lipid classes of these cells. Transport to the apical and basolateral cell surface was monitored by a BSAdepletion assay. In MDCK cells, GalCer reached the cell surface with a two-to sixfold lower apical/basolateral polarity than GlcCer. Remarkably, in Caco-2 cells GalCer and GlcCer displayed the same apical/basolateral polarity, but it was sixfold lower for lipids with aCrOH chain than for C6-NBD lipids. Therefore, the sorting of a sphingolipid appears to depend on lipid structure and cell type. We propose that the different ratios of gluco-and galactosphingolipid synthesis in the various epithelial tissues govern lipid sorting in the membrane of the trans Golgi network by dictating the composition of the domains from where vesicles bud to the apical and basolateral cell surface.

In most cell types sphingomyelin is synthesized predominantly in the cis-medial compartments of the Golgi stacks whereas the contribution of the plasma membrane is much lower. The aim of this study was to assess the contribution of both compartments to the synthesis of sphingomyelin in myelinating cells. Therefore, oligodendroeytes from rat spinal cord were incubated in culture with fluorescently-or radiolabelled ceramides, and the effects of a block in the vesicular flow (monensin, brefeldin A, low temperature) on surface synthesis of sphingomyelin were evaluated. The results indicate that = 50% of the sphingomyelin synthase is present at the plasma and myelin membranes of oligodendrocytes.