© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs219204. doi:10.1242/jcs.219204 REVIEW Glycosphingolipid metabolism in cell fate specification Domenico Russo1,*, Laura Capolupo1,2, Jaipreet Singh Loomba1,2, Lucia Sticco1 and Giovanni D’Angelo1,2,* ABSTRACT anchoring. Specifically at the PM, sphingolipids participate in Glycosphingolipids (GSLs) are ubiquitous components of eukaryotic signaling events by recruiting signaling molecules to, or plasma membranes that consist of a ceramide backbone linked to a sequestering them at, membrane microdomains for the modulation glycan moiety. Both the ceramide and the glycan parts of GSLs display of their activities and for their processing into the endocytic cycle structural variations that result in a remarkable repertoire of diverse (Holthuis and Menon, 2014; Holthuis et al., 2001; Simons and compounds. This diversity of GSLs is exploited during embryogenesis, Ikonen, 1997). Given these properties, sphingolipids are proposed to when different GSLs are produced at specific developmental stages and function as fundamental membrane organizers and to make up along several differentiation trajectories. Importantly, plasma membrane the fabric of eukaryotic PMs in order to influence the interaction receptors interact with GSLsto modify their activities. Consequently, two with the extracellular environment (Hannun and Obeid, 2018; otherwise identical cells can respond differently to the same stimulus Holthuis et al., 2001). owing to their different GSL composition. The metabolic reprograming of Interestingly, different cell types exhibit a specific sphingolipid GSLs is in fact a necessary part of developmental programs, as its array at their PMs (Hakomori, 2003; Ngamukote et al., 2007) impairment results in developmental failure or tissue-specific defects. (Table S1). Indeed, sphingolipids are subjected to remarkable Moreover, single-cell variability is emerging as a fundamental player in structural variations that lead to the production of hundreds of development: GSL composition displays cell-to-cell variability in different species (Hannun and Obeid, 2008, 2018). A substantial syngeneic cell populations owing to the regulatory gene expression part of this variability derives from the heterogeneous elongation circuits involved in microenvironment adaptation and in differentiation. of glycan chains that are covalently linked to the sphingolipid Here, we discuss how GSLs are synthesized and classified and review backbone in the synthesis of the class of compounds known as the role of GSLs in the establishment and maintenance of cell identity. glycosphingolipids (GSLs). GSL-associated glycans range have We further highlight the existence of the regulatory circuits that between one and more than 20 sugar residues, with 11 different ’ modify GSL pathways and speculate how GSL heterogeneity might monosaccharide types being used in vertebrates (D Angelo et al., contribute to developmental patterning. 2013a). Importantly, the elongation of glycans in GSLs is not driven by a template; instead, it entirely depends on the relative expression KEY WORDS: Differentiation, Glycosphingolipid, Golgi complex and organization of their specific synthetic enzymes (Bieberich et al., 2002; Giraudo and Maccioni, 2003). Still, GSL production Introduction is tightly controlled during differentiation programs; as a result, Cellular membranes serve as both barriers and interfaces between specific GSLs are used as differentiation stage or cell-type-specific topologically distinct biological spaces. The lipid composition of markers (D’Angelo et al., 2013a). In addition, GSL composition can these membranes varies at different cellular locations. For example, substantially vary among single cells in syngeneic cell populations the plasma membrane (PM) is rich in sphingolipids compared to (Majoul et al., 2002; Russo et al., 2018; Snijder et al., 2009). intracellular membranes, which results in the PM having distinct Furthermore, specific GSL glycans appear to organize interactions biophysical properties (Holthuis and Menon, 2014). Sphingolipids with receptors that are located at the PM in order to modulate their contain a hydrophobic ceramide (Cer) backbone that is composed of activity (Bremer and Hakomori, 1982; Bremer et al., 1984; Coskun a saturated fatty acid and sphingoid base. This allows sphingolipids et al., 2011; Farooqui et al., 1999; Liu et al., 2008; Mirkin et al., to establish lateral interactions (both homotypic and with sterols) 2002; Mutoh et al., 1995; Park et al., 2012; Toledo et al., 2004). to yield a tightly packed and thick membrane structure (Hannun This occurs, for instance, in the case of the GM3-dependent and Obeid, 2018; Holthuis et al., 2001). Owing to this lipid inhibition of epidermal growth factor receptor (EGFR) signaling, composition, the PM is less permeable to ions and peptides which maintains EGFR in an inactive state in the absence of its compared to intracellular membranes, which matches with its ligand (Coskun et al., 2011). By contrast, GD1a and GM1 enhance ‘barrier’ function towards the extracellular environment (Holthuis EGFR activation (Li et al., 2001, 2000; Liu et al., 2004). Thus, two and Menon, 2014). Sphingolipids also show incomplete miscibility otherwise identical cells can react differently to the same stimulus with phospholipids, which results in lateral phase partitioning of the owing to their different composition in GSLs. membrane and thus in the formation of membrane microdomains Whereas the role of cell-to-cell variability in GSL composition (Simons and Ikonen, 1997). Such microdomains have different in differentiated cells remains to be understood, non-genetic affinities for proteins depending on the length and composition of heterogeneity has been proposed to contribute to cell-type their transmembrane domains, or on their lipid-based membrane diversification in developmental processes (Huang, 2009). Specifically, non-genetic heterogeneity provides cells with transitory ‘states’ to potentially orient their fates towards diverging directions 1Institute of Protein Biochemistry, National Research Council, Via P. Castellino 111, Napoli, Italy. 2Institute of Bioengineering, Laboratory of Lipid Cell Biology, École (Huang, 2009). Given the role of GSLs in modulating cell responses to polytechnique fédérale de Lausanne (EPFL) CH-1015 Lausanne, Switzerland. environmental cues, along with their extensive structural variation, cell-to-cell heterogeneity in GSL composition might therefore help in *Authors for correspondence ([email protected]; [email protected]) generating identity patterns during tissue morphogenesis. In this D.R., 0000-0003-2171-657X; G.D., 0000-0002-0734-4127 Review, we discuss the role of GSLs as cell-fate determinants, focusing Journal of Cell Science 1 REVIEW Journal of Cell Science (2018) 131, jcs219204. doi:10.1242/jcs.219204 on (1) how GSL diversity is generated, (2) what GSL changes occur the PM. For its metabolic conversions, Cer can be galactosylated in when cells differentiate toward alternative fates, and (3) how the the ER to produce galactosylceramide (GalCer), extracted from ER GSL metabolism is controlled by differentiation programs. Finally, membranes by the lipid-transfer protein ceramide transfer protein we will speculate on how GSLs can contribute to tissue patterning (CERT) and delivered to the trans-Golgi where SM is synthesized and morphogenesis. (Hanada et al., 2003), or transported in vesicles to the cis-Golgi where it is glucosylated to produce glucosylceramide (GlcCer) GSL synthesis (Funakoshi et al., 2000) (Fig. 1). Whereas SM cannot be further GSL synthesis is initiated at the cytosolic membrane leaflet of the processed in an anabolic direction, GalCer is the precursor of endoplasmic reticulum (ER), where Cer is produced from its GSLs from the gala-series, also known as sulfatides, which include precursor sphinganine by the consecutive action of enzymes that sulfo-GalCer, (α2-3)-sialylated GalCer (GM4), di-GalCer (i.e. catalyze its acylation and desaturation (Mullen et al., 2012). Cer can Gal-GalCer) and di-sulfo-GalCer, which are produced at the then be converted into several compounds that include sphingosine, Golgi complex where the enzymes for GalCer processing reside Cer-1-phosphate, acyl-Cers, sphingomyelin (SM) and GSLs (Merrill, 2011) (Fig. 1). (Hannun and Obeid, 2018; Holthuis et al., 2001; Merrill et al., Apart from the gala-series GSLs, all other GSLs have GlcCer 2005). SM and GSLs are synthesized at the interface between the as a precursor (D’Angelo et al., 2013a; Merrill, 2011). GlcCer is ER and Golgi complex and constitute the major sphingolipids at converted into lactosylceramide (LacCer; Gal-GlcCer) (Kumagai SSEA-4 Globo ST3GAL2 Gb5 FUT1/2 Globo-H B3GALT5(SSEA-3) fucosyl-Gb5 A4GALT B3GALNT1 GBGT1 Forssman Gb3 Gb4 FAPP2 antigen − − Ganglio SO3 SO3 P SM TGN sulfo-GalCer ST3GAL5 B4GALNT1 B3GALT4 ST3GAL1 GM3 GM2 GM1a GD1a GT1a (sulfatide) di-sulfo-GalCer Cer-1-P CERT ST8SIA1 GAL3ST1 GAL3ST1 GM4 B4GALNT1 B3GALT4 ST3GAL1 Golgi GD3 GD2 GD1b GT1b di-GalCer B4GALT5 ST8SIA5 GlcCer LacCer GSL precursors GCS B4GALNT1 B3GALT4 GT3 GT2 GT1c GQ1c GalCerS CerS GalCer Cer Sph ER Asialo GD1c acyl-Cer ST8SIA5 B4GALNT1 B3GALT4 ST3GAL1 ST6GALNAC4 GA2 GA1 GM1b GD1a Key Sia Fuc Glu Gal Cer P Phosphate y Lacto H antigen Lewis − GlcNac GalNac SO3 Sulfate Sph Fatty acid x LT5 LC4 SSEA-1(Lewis) B3GA B3GNT5 B3GALT1 LC3 nLC4 nLC5 Fig. 1. GSL synthesis and classification and schematic representation