Review
Cell-specific in vivo functions of glycosphingolipids: Lessons from genetic deletions of enzymes involved in glycosphingolipid synthesis

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Abstract

Glycosphingolipids (GSLs) are believed to be involved in many cellular events including trafficking, signaling and cellular interactions. Over the past decade considerable progress was made elucidating the function of GSLs by generating and exploring animal models with GSL-deficiency. Initial studies focused on exploring the role of complex sialic acid containing GSLs (gangliosides) in neuronal tissue. Although complex gangliosides were absent, surprisingly, the phenotype observed was rather mild. In subsequent studies, several mouse models with combinations of gene-deletions encoding GSL-synthesizing enzymes were developed. The results indicated that reduction of GSL-complexity correlated with severity of phenotypes. However, in these mice, accumulation of precursor GSLs or neobiosynthesized GSL-series seemed to partly compensate the loss of GSLs. Thus, UDP-glucose:ceramide glucosyltransferase (Ugcg), catalyzing the basic step of the glucosylceramide-based GSL-biosynthesis, was genetically disrupted. A total systemic deletion of Ugcg caused early embryonic lethality. Therefore, Ugcg was eliminated in a cell-specific manner using the cre/loxP-system. New insights into the cellular function of GSLs were gained. It was demonstrated that neurons require GSLs for differentiation and maintenance. In keratinocytes, preservation of the skin barrier depends on GSL synthesis and in enterocytes of the small intestine GSLs are involved in endocytosis and vesicular transport.

Introduction

GSLs are amphiphathic molecules and consist of a hydrophilic sugar part and a lipophilic ceramide anchor. With their ceramide anchor they are integrated in the outer leaflet of the plasma membrane of all eukaryotic cells. The ceramide contains a sphingoid base linked via amide bond to a fatty acid of different chain lengths. Synthesis of all sphingoid bases starts with the condensation of l-serine and an acyl-CoA. The fatty acid constitution can vary in its length, dependent on the cell type. To the ceramide, a phosphate group, a phosphorylcholine or a monosaccharide may be added to the hydroxyl group of the sphingoid base in position 1 resulting into ceramide-1-phosphate, sphingomyelin, and glycosphingolipids, respectively (Fig. 1). Glucosylceramide synthase-catalyzed addition of UDP-activated glucose to ceramide leads to glucosylceramide (GlcCer) on the cytoplasmic surface of the Golgi [1], [2], [3]. GlcCers may be elongated with additional carbohydrates resulting into a great variety of complex sugar moieties and GSL-series. Else, UDP-activated galactose may be added to ceramide on the luminal side of the ER [3]. Acidic glycosphingolipids such as sulfatides and gangliosides are formed by addition of sulfuric- or sialic acid residues. Whereas only comparatively few GSLs are derived from galactosylceramide (GalCer), hundreds of structurally different glycosphingolipids including gangliosides and higher sulfatides are based on the glucosylceramide core structure.

GSLs are constituents of eukaryotic cell membranes. A major portion is located exclusively on the outer leaflet of the cellular plasma membrane. GSLs influence cell adhesion [4], [5] and cell differentiation [6]. Intracellularly, glycosphingolipids may be important for protein and lipid trafficking [7], [8], [9]. GSLs have been described also to be located intracellularly associated with small vesicles, tubulovesicular structures, the surface of phase-dense lipid droplets, intermediate filaments of the cytoskeleton as well as with mitochondria [10]. Their subcellular localization varies in different cell types [10]. GSLs may concentrate in lipid rich domains [11], where they are supposed to participate in signaling events [12], [13], [14], [15], modify insulin- and EGF-receptor activities [16], [17], [18], [19] and modulate Notch ligand activity in Drosophila [20]. Specific GSLs function as binding ligands of bacterial toxins [21], [22], [23] and viruses [24], [25]. Moreover, they were described to be associated with or enriched in certain mammalian cancers [26], [27], [28]. Consequently, GSLs were used as target molecules in immunological approaches of tumour therapy [29], [30], [31], [32], [33].

GSLs are degraded in the lysosomes by soluble exo-glycosidases. A defect or mutation in genes encoding sphingolipid digesting enzymes or proteins supporting sphingolipid degradation leads to the accumulation of GSLs in lysosomal compartments. GSL storage then causes severe neurodegenerative and visceral diseases known as metachromatic leukodystrophy, GM1-gangliosidosis, Fabry-, Tay-Sachs-, Sandhoff-, Gaucher-, and Krabbe disease [34].

In the present report, we focus on the consequences from deletions of enzymes involved in GSL-synthesis. The predominant database to elucidate the functions of glycosphingolipids was obtained by in vitro studies in cell culture. To investigate whether or not those findings could be transferred to in vivo situations, great efforts were undertaken to generate animal models with various deletions of genes involved in the synthesis of glycosphingolipids.

Section snippets

Genetic inhibition of GSL-series synthesis

In mammals, enzyme-catalyzed linkage of UDP-activated glucose to ceramide results in glucosylceramide. Addition of beta-galactose to GlcCer leads to lactosylceramide (LacCer). Elongation of LacCer with further sugars results in GSL series (Fig. 1[a–d]). Hence, the addition of alpha-galactose, N-acetylglucosamine or N-acetylgalactosamine correlates with synthesis of GSLs of the globo-, lacto-, or ganglio-series, respectively. During embryonic development, a switch from globo- and lacto-series

Glycosphingolipids and brain function

Nervous tissue contains high concentrations of glycosphingolipids, in particular complex gangliosides [52] as compared to organs of mesodermal- and endodermal origin [53]. Only intestinal epithelial cells reach abundant GSL concentrations comparable to the brain. For decades, researchers in the glycosphingolipid field claimed that gangliosides may play a critical role during development and maturation of the central- (CNS) and peripheral nervous systems (PNS). Ganglioside GM3 and GD3 constitute

Glycosphingolipids and skin function

Land dwelling animals need a water impermeable skin to prevent dehydration. This barrier is believed to be generated by interplay of proteins of the cornified envelope with lipids. Glucosylceramides (GlcCers) are the GSLs mostly synthesized by keratinocytes. GlcCers constitute approximately 4% of the total epidermal lipid mass [118]. Epidermal GlcCers consists of a unique fatty acid composition in their ceramide anchor. Besides a fatty acid chain length of 16–26 carbon atoms common to many

Glycosphingolipids and liver function

The liver is the central organ for lipid, amino acid, and carbohydrate metabolism. GSLs, as membrane constituents were thought to be one mechanism to actively trigger membrane lipid transporters located in the hepatocyte canalicular membrane [136] to regulate cholesterol- and bile acid homeostasis [137], [138]. Pharmacologic GSL reduction by inhibitors for Ugcg suggested that GSLs might be involved in the development of liver steatosis. [139], [140]. Glucosylceramide reduction of approximately

Glycosphingolipids and intestinal function

The intestine is the principal organ for digestion and absorption of nutrients. The surface of intestinal villi is covered by columnar epithelial cells, enterocytes, each containing apically thousands of microvilli, thereby enormously expanding the surface area for absorption of nutrients (Fig. 5A). GSLs were found to be major components of enterocytes [142] and therefore thought to be involved in several cellular processes such as epithelial polarization [143] and differentiation [144].

Glycosphingolipids and adipocyte function

Investigations of adipose tissue of Lepob mice indicated enhanced GSL concentration as compared to fat tissue of lean control mice [151]. On the other hand, adipogenesis was significantly improved in mice in which the GSL content was reduced by inhibitors of Ugcg [139], [140]. Those results implicated GSLs as important mediators in fat metabolism. As the Ugcg-inhibitors have been orally applied to mice, their effect might not have been restricted to the adipose tissue. Therefore, tissue-

Cerebroside sulfotransferase deficiency and renal function

Sulfatides have been described to be ligands of L-selectin [153]. In addition, they were discussed to exert modulatory functions in blood coagulation [154], [155], [156] and to act as cofactors of basolateral Na+-K+-ATPase activity by binding K+ or by facilitating the membrane relocalization of the enzyme [157], [158], [159], [160]. The kidney as well as the brain contains relevant amounts of sulfatides. In contrast to sulfatide expression in the brain predominantly restricted to the simple

Lowering renal GSL concentrations ameliorates polycystic kidney disease

Adult polycystic kidney disease (PKD) is an autosomal dominant hereditary disease affecting approximately 0.2% of the population. The progressive formation of fluid-filled cysts with compression of adjacent renal tissue which atrophies and shows fibrosis leads to end-stage renal disease. No therapy for PKD is currently available. Recent investigations suggested that cyst formation might be influenced by glycolipid-synthesis [162]. GlcCer and ganglioside GM3 levels were found to be increased in

Conclusions and perspectives

In the past 20 years efforts have been undertaken to develop animal models in C. elegans, Drosophila, and mice with deletions or silencing of glycosphingolipid synthesizing enzymes. Studies in those animal models provided new insights which cellular processes sphingolipids might influence. The in vitro and pharmacologic data from the last decades could be partially substantiated or partially disproven in vivo.

  • (I)

    In the brain, it could convincingly be demonstrated that GSLs play critical roles in

Acknowledgements

We cordially thank Herbert Wiegandt for critical remarks. We apologize to those whose research has not been cited. The field reviewed in this manuscript is expanding enormously. Accordingly, not all relevant references might be listed here.

Studies by us cited in this review have been founded by the DFG, SFB 938 to H.-J.G.

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