New Insights into Glycosphingolipid Functions—Storage, Lipid Rafts, and Translocators

New Insights into Glycosphingolipid Functions—Storage, Lipid Rafts, and Translocators Dan J. Sillence Leicester School of Pharmacy, Hawthorne Building, De Montfort University, Leicester, LE1 9BH, United Kingdom

Glycosphingolipids are key components of eukaryotic cellular membranes. Through their propensity to form lipid rafts, they are important in membrane transport and signaling. At the cell surface, they are required for caveolar‐ mediated endocytosis, a process required for the action of many glycosphingolipid‐binding toxins. Glycosphingolipids also exist intracellularly, on both leaflets of organelle membranes. It is expected that dissecting the mechanisms of cell pathology seen in the glycosphingolipid storage diseases, where lysosomal glycosphingolipid degradation is defective, will reveal their functions. Disrupted cation gradients in Mucolipidosis type IV disease are interlinked with glycosphingolipid storage, defective rab 7 function, and the activation of autophagy. Relationships between drug translocators and glycosphingolipid synthesis are also discussed. Mass spectrometry of cell lines defective in drug transporters reveal clear differences in glycosphingolipid mass and fatty acid composition. The potential roles of glycosphingolipids in lipid raft formation, endocytosis, and cationic gradients are discussed. KEY WORDS: Glycosphingolipids lysosomal storage diseases, Endocytosis, Mass spectrometry, Niemann‐Pick type C, Autophagy, Membrane sorting, Multiple drug resistance, Mass spectrometry, Fatty acid composition, Translocators, Cation gradients. ß 2007 Elsevier Inc.

International Review of Cytology, Vol. 262 Copyright 2007, Elsevier Inc. All rights reserved.

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0074-7696/07 $35.00 DOI: 10.1016/S0074-7696(07)62003-8

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I. Introduction Eukaryotes and some bacteria contain a heterogeneous class of lipids called glycosphingolipids (GSLs). These lipids are composed of a ceramide backbone and a sugar headgroup (Fig. 1). The hydrophobic ceramide part, consisting of a sphingoid base and N‐linked fatty acid, inserts in a cellular membrane, whereas the hydrophilic sugar headgroup mostly faces the extracellular space. At the cellular level, GSLs organize protein function by forming glycosignaling domains, where clustering of specific proteins within bilayer‐spanning lipid rafts regulates their signal transduction (Hakomori and Igarashi, 1995). In addition, cells use the capacity of GSLs to form ordered domains to create selectivity in membrane sorting (Simons and van Meer, 1988).

A. Heterogeneity of Glycosphingolipids GSLs exhibit a huge heterogeneity of structure. The sphingoid bases can vary in length, saturation, hydroxylation, and branching. The main sphingoid base in mammals is sphingosine (Karlsson et al., 1973). Sphinganine, which corresponds to sphingosine without the trans double bond, and phytosphingosine (hydroxyl‐sphinganine) are also common sphingoid bases. The fatty acid is amide‐linked to the amino group of the sphingoid base. The fatty acid species are cell‐type–dependent, can vary in length, saturation, and hydroxylation, but are mostly long (
C16) and saturated. GSLs can be divided into two main classes based on the first sugar linked to the ceramide backbone, glucose or galactose.

O R

P O

O

O Glycerophospholipids

O

OH Gal

Glc O

n Glycosphingolipids (GSLs) FIG. 1

NH O

Structure of glycerphospholipids and glycosphingolipids.

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B. Synthesis of Glycosphingolipids 1. Recycling from Lysosomal Degradation Products The majority of glycosphingolipid synthesis in resting cells occurs from recycled breakdown products (e.g., sphingosine) derived from the lysosome (Gillard et al., 1998). It is unclear whether the lysosomal breakdown products, such as sphingosine, escape from lysosomes via a transporter or are able to passively slip across the lysosomal membrane. Transport to the endoplasmic reticulum (ER) may occur via discrete protein complexes in areas of close membrane contact between the late endosome and the ER (Holthuis and Levine, 2005; Ko et al., 2001). The accumulation of sphingosine in the lysosome of many GSL storage diseases (Rodriguez‐Lafrasse et al., 1994) suggests transport from the lysosome can be frustrated, but the consequences of this, if any, have yet to be determined. Once transported, sphingosine is phosphorylated at the ER, forming sphingosine‐1‐phosphate, an important intracellular messenger (Funato et al., 2003; Ghosh et al., 1994). Once dephosphorylated, sphingosine is then acylated to ceramide. In specialized cell types, luminal ceramide is then converted to GalCer. From the ER, ceramide can also follow the vesicular pathway to early Golgi compartments where it is converted to glucosylceramide (GlcCer). Alternatively, ceramide reaches the trans Golgi via CERT, a ceramide transfer protein that is able to extract ceramide from the ER and deliver it to the Golgi after docking to phosphatidylinositol‐4‐phosphate (Hanada et al., 2003). This pathway may also involve the membrane contacts between the ER and the trans Golgi (Ladinsky et al., 1994). Synthesis of the phosphosphingolipid sphingomyelin (SM) in the trans Golgi (Huitema et al., 2004) depends on this pathway for ceramide supply (Hanada et al., 2003). It has been reported that ER to Golgi transport of ceramide may be regulated via an ER sphingosine‐1‐phosphate phosphohydrolase. Anterograde transport of ceramide, probably via CERT, and at least some proteins are regulated by the degradation of ER sphingosine‐1‐phosphate (Giussani et al., 2006). Feedback regulation via the formation of sphingosine in the lysosome and lipid transport and synthesis is an interesting topic for future research. 2. De Novo Synthesis In actively dividing cells ‘‘de novo’’ synthesis is more active and starts with condensation of serine and palmitoyl‐CoA to 3‐ketosphinganine (Fig. 2). Formation of sphinganine and subsequent acylation and desaturation on the cytosolic side of the ER produces ceramide without production of sphingosine as an intermediate. A variety of ceramide synthases have been identified

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L-Serine, palmitoyl-coenzyme A

HO

O C

CH3

HN C C C H OH H

CH3

Ceramide

UDP-Glucose

UDP-Glucose: N-acylsphingosine glucosyltransferase

UDP O OH O HO HO

OH

C HN

H O

C

C H

CH3 CH3

C OH

Glucosylceramide FIG. 2

Synthesis of glucosylceramide de novo.

with both sphingoid base (Venkataraman et al., 2002) and fatty acid preferences (LASS1–LASS6). LASS1 and LASS4 expression preferentially increase C18 ceramide, LASS2 mainly increases C16 and C24:1 ceramide, and LASS5 and LASS6 produce C16 ceramide (Mizutani et al., 2005, 2006; Riebeling et al., 2003; Venkataraman et al., 2002). Ceramide is also the direct precursor for GlcCer. GlcCer is present in most eukaryotic cells and a few bacteria and serves as the major precursor for complex GSLs. It is synthesized by the UDP‐Glc:ceramide glucosyltransferase or GlcCer synthase CGlcT that occurs on the cytosolic side of Golgi membranes (Coste et al., 1986; Futerman and Pagano, 1991; Jeckel et al., 1992). Although the functions of GlcCer are unknown, in some yeast and bacteria, GlcCer may play an important role in the modulation of pH gradients; knocking out GlcCer synthase reduces growth in alkaline conditions (Rittershaus et al., 2006). Complex GSLs are made by the stepwise addition of individual sugars from their activated nucleotide precursors onto GlcCer. In mammals, the first reaction is the conversion of GlcCer to lactosylceramide by the LacCer synthase. This enzyme, like all further glycosyltransferases and sulfotransferases involved in the glycosylation of GSLs, acts in the Golgi lumen (Lannert et al., 1994). Sialoglycosphingolipids are also called ‘‘gangliosides,’’ and abbreviations have been assigned to them according to the number of sialic acids present and to their migration order

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in chromatography. Except for ganglioside GM4, which corresponds to NeuAca2‐3GalCer, all gangliosides have LacCer as a precursor. 3. Glycosphingolipid Degradation and Signaling In contrast to sphinganine1‐phosphate, the synthesis of sphingosine‐ 1‐phosphate is dependent on the degradation of GSLs and sphingomyelin in late endosomes and lysosomes (Berdyshev et al., 2006). Once formed, sphingosine‐1‐phosphate is involved in signaling events such as the regulation of calcium gradients (Bagshaw et al., 2005; Masgrau et al., 2003). Termination of the signal proceeds via a sphingosine‐1‐phosphate phosphohydrolase (Le StunV et al., 2002). The observation that exogenous addition of sphingosine‐1‐phosphate regulates cell proliferation (Zhang et al., 1991) is also consistent with the role of this second messenger in the control of proliferation. Overexpression of sphingosine kinase has been shown to activate autophagy, a process that occurs constitutively but is also activated in neurodegenerative diseases such as the secondary glycosphingolipid storage diseases (see Section IV.C). Whether there is any relationship between GSL storage and sphingosine‐1‐phosphate signaling has yet to be determined. Both sphingosine kinase 1 and sphingosine‐1‐phosphate phosphohydrolase modulate de novo sphingolipid synthesis (Berdyshev et al., 2006; Le StunV et al., 2002). The interrelationships between sphingosine‐1‐phosphate signaling and the regulation of new membrane sphingolipid has yet to be determined.

C. Physical Properties of GSLs Structural diVerences between GSLs and glycerolipids (Fig. 1) underlie the special behavior of GSLs in membranes. In GSLs, the region between the polar headgroup and the hydrophobic backbone contains chemical groups that can function both as hydrogen bond donor and hydrogen bond acceptor, in contrast to the glycerolipids, which only have hydrogen bond accepting properties in that part of the molecule (Pascher, 1976). Additional hydrogen bonding can occur between the sugar headgroups of GSLs. Another striking diVerence between GSLs and most glycerolipids is that the lipid chains are saturated over at least the first 15 carbons of both chains. In combination with the higher hydrogen bonding, the saturated nature results in denser packing. This is measured as an increased melting temperature, or Tm, which corresponds to the temperature above which a bilayer of a single lipid switches from a frozen state, the gel or solid‐ordered (so) phase, to a fluid state, the liquid‐ crystalline or liquid‐disordered (ld) phase. In addition, cholesterol, a rigid and flat cylindrical lipid, also interacts preferentially with sphingolipids via van der Waals interactions (Boggs, 1987). In model membranes containing mixtures of a high Tm lipid and cholesterol, a fluid‐fluid phase separation has been observed

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between the ld and a liquid‐ordered lo phase (Recktenwald and McConnell, 1981). Unfortunately, most evidence to date that domains enriched in GSLs exist in biological membranes is indirect (Harder, 2003). However, GSLs are clustered on erythrocytes (Thompson and Tillack, 1985), gangliosides GM1 and GM3 are concentrated in domains (Parton, 1994), and GM1 can be enriched in diVerent domains than GM3 on the same cell (Gomez‐ Mouton et al., 2001). Most indications for the lipid domain association have been obtained by using the detergent‐resistance criterion. Although this technique has had great prospective value for how membrane signaling may work (Harder, 2003), physical studies have suggested there is no straightforward physical basis for why extraction at 4  C would provide information concerning the situation at 37  C (Heerklotz, 2002). To avoid detergent extraction, pulse electron paramagnetic resonance (EPR) spin‐labeling methods and single‐molecule optical techniques have been applied (Dietrich et al., 2002; Kenworthy et al., 2000; Subczynski et al., 2003) to monitor the entry and exit of probe molecules in domains in model membranes or in living cells. Results obtained using single molecule microscopy of saturated fluorescent analogues of phosphatidylethanolamine (Schutz et al., 2000) suggest that lipid analogues can be used to detect small (50 nm), stable (0.5–2 min) cell surface microdomains that may be related to the well‐established annular lipids that surround membrane proteins. Overall results have provided evidence for small/unstable rafts in unstimulated cells and for larger stabilized rafts induced by oligomerization of GPI‐anchored proteins or ligand binding (Harder, 2003), in which some proteins preferentially partition. The size of the confining domain for a GPI‐anchored protein is reduced when cells are treated with inhibitors of GSL synthesis; other studies have shown increases in phospholipase C susceptibility as well as changes in expression, suggesting GSLs contribute to the physical properties of microdomains. Using a fluorescent analogue of lactosylceramide, which is taken up by caveolar‐dependent mechanism, has shown that this specific form of endocytosis is dependent on the level of endogenous GSL, suggesting that some level of cell surface GSL is necessary for this mechanism of endocytosis, which may be lipid raft‐mediated (Cheng et al., 2006c). These processes are discussed in Section II.B and C. In conclusion, many studies suggest GSL can be heterogeneously organized on living cells, and this has functional consequences, although there is no strong consensus yet on the size, shape, and dynamics of lipid rafts.

D. Methods Used in Glycosphingolipid Analysis One of the major setbacks in glycosphingolipid research has been a lack of tools with which to analyze them. Glycosphingolipids show increasing hydrophilicity with increasing size of the sugar headgroup, this leads to an

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increasing ability to form monomers in aqueous solution, decreasing solubility in organic solvents. Hence, diYculty arises with complete extraction from tissues and cells. Once extracted, utility of a phase split to purify the lipid components; higher GSLs escape into the aqueous phase which then leads to unacceptable losses. Higher GSLs are also more prone to adhere to the side of plastic tubes. The following techniques overcome many of these problems. 1. Extraction and Purification of GSLs For tissue homogenates, GSLs can be extracted by the addition of 3.2 vol of CHCl3/MeOH (1:2.2) for 10 min at room temperature, and the phases are split by the addition of 1 vol of CHCl3 and 1 vol of H2O (Bligh and Dyer, 1959). This quick extraction procedure gives similar recoveries of GSLs to other commonly used methods of lipid extraction (Miller Podraza et al., 1992; van Echten et al., 1990). Upper phase GSLs can then be recovered by SePak C18 columns (Sillence et al., 2000). Briefly, C18 SepPak columns (Waters, Milford, MA) are washed with 1 mL MeOH and 1 mL water. The CHCl3 phase was dried down and loaded in 50‐mL of CHCl3/MeOH/H2O (1:2.2:1). The corresponding aqueous phase was loaded onto the columns and washed with 5  1 mL water. GSLs are then eluted with 5  1 mL CHCl3:MeOH (1:3) and 1 mL of MeOH, and the samples are dried under nitrogen. At this point, the cholesterol and GSLs can be quantified as the following describes. Sometimes further purification is necessary (e.g., for mass spectrometry), in which case glycerophospholipids are removed from samples by saponification by the addition of 1 mL of chloroform and 1 mL of 0.2 M NaOH in methanol and incubated overnight at 37  C and further purified by silicic acid chromatography (Vance and Sweeley, 1967). Columns are washed with 5 mL of CHCl3 to remove fatty acids, nonalkali labile sterols, and alkylglycerols. Neutral GSLs are eluted with 6 mL acetone/ MeOH (9:1). Gangliosides are eluted with 6 mL CHCl3:MeOH (1:3) and the eluates dried under nitrogen. The samples are resuspended with 1  50 mL and then 1  100 mL of CHCl3:MeOH (1:2.2) and transferred to a 1.5‐mL tube. For mass spectrometry, the samples are dried down in a SpeedVac and resuspended in 20 mL MeOH. 2. Mass Spectrometry Matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry can be performed as described previously (Hunnam et al., 2001). Briefly, a mixture of the sample (1 mL, 100 pmol) and matrix (1 mL of a saturated solution of 2,5‐dihydroxybenzoic acid in acetonitrile) are crystallized on the MALDI target. Positive ion reflectron MALDI spectra can then be obtained using a time‐of‐flight (TOF) mass spectrometer calibrated externally with hydrolyzed dextran sugars.

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3. Higher Glycosphingolipid Mass Assay To dried lipid extracts, 10 mL incubation buVer (1 mg/mL sodium cholate in 50 mmol sodium acetate pH 5.0) was added. After vigorous vortexing and spinning in a benchtop picofuge, 10 mL of 50 mU/10 mL of ceramide glycanase (E.C. 3.2.1.123, Calbiochem, La Jolla, CA) in incubation buVer is added to cleave the glycans. Glucosylceramide is only partially digested due to the specificity of the glycanase (Wing et al., 2001). After this period, 10 mL of water is added to each enzyme digest followed by 80 mL of anthranilic acid and sodium cyanoborohydride to each digest and incubated in the 80  C oven for 1 h. Derivatized oligosaccharides can be purified on DPA‐6S (Supelco, Ballafonte, PA) columns preequilibrated with 2  1 mL CH3CN. One milliliter 97:3 CH3CN:H2O is added to each sample and vortexed prior to loading the samples onto the columns. Columns are washed with 4  1 mL 99:1 CH3CN:H2O and then with 0.5‐mL 97:3 CH3CN:H2O and the derivatized oligosaccharides eluted with 2  0.6 mL water into screw‐cap Eppendorfs and stored at 4  C in the dark until ready for NP‐HPLC. 4. Glucosylceramide Mass Assay Because of the specificity of the glycanase, GlcCer cannot be quantitated using the procedure applied for higher GSL assay. Fortunately GlcCer can be quantitated using glucocerebrosidase instead of the ceramide glycanase. Due to the preponderance of free glucose in most samples, GlcCer is first purified on silicic acid columns. After GSL extraction and sample loading (see earlier), columns are washed with 2 mL of CHCl3 and 2 mL of CHCl3: MeOH 98:2. GlcCer is then eluted with 2 mL of CHCl3:MeOH 97:3, 2 mL of CHCl3:MeOH 96:4, 2 mL of CHCl3:MeOH 95:5, and 2 mL of CHCl3:MeOH 94:6. Samples are dried down and resuspended in 15 mL of 50 mmol sodium acetate buVer (pH 5) with 0.1% triton and 0.25% taurocholate; 0.4 U of purified glucocerebrosidase (Cerezyme or Ceredase) is added and incubated at 37  C for 18 h. Samples of glucocerebrosidase can have significant oligosaccharide contamination; therefore, the enzyme has to be purified using a spin column (MW cut oV 10 kDa). Samples are then derivatized using the same method as for the higher GSLs.

II. Traffic of Glycosphingolipids A. Subcellular Distribution of Glycosphingolipids Consistent with the topology of their synthesis, many GSLs have been found at the cell surface where they constitute a small proportion of the total plasma membrane lipid. One exception is the apical plasma membrane of

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apical cells, where GSLs are found in relatively high concentrations (Brasitus and Schachter, 1980; Forstner et al., 1968). At the cellular level, the simplest and evolutionarily most ancient GSL, glucosylceramide GlcCer, has been found to be enriched within intracellular membranes, in contrast to more complex GSLs that are predominantly present on the cell surface (Warnock et al., 1993). Consistent with a more diverse distribution, GlcCer has been found to be important in endocytosis (Sillence et al., 2002; Singh et al., 2003; Smith et al., 2006) and melanosome formation (Sprong et al., 2001). Previous work has suggested that extensive (>50%) GSL depletion disrupts endocytic sorting of BODIPY lactosylceramide (Sillence et al., 2002). Although BODIPY lactosylceramide was sorted to the Golgi apparatus in control cells, in GSL‐depleted cells BODIPY lactosylceramide was sorted to punctate endocytic structures. As a first step in elucidating the mechanism underlying this phenomenon, it has been observed that GlcCer levels appear to correlate with changes in endocytosis (Sillence et al., 2002), suggesting a conserved and important role for this simple GSL. Although many GSLs are present at the cell surface, many studies have shown that specific GSLs are predominantly enriched at other locations (Chatterjee et al., 1983; Keenan et al., 1972; Matyas and Morre, 1987; Symington et al., 1987; van Genderen et al., 1991; Weinstein et al., 1970). Intracellular membranes show distinct GSL compositions (Muthing et al., 1994, 1998), and distinct localizations for complex and simple GSLs have been reported (Matyas and Morre, 1987; Warnock et al., 1993). However, these studies are not quantitative or complete. Because GlcCer depletion has eVects on the endocytic pathway (Sillence et al., 2002; Vruchte et al., 2004), do endosomes contain significant amounts of GlcCer? As depletion of GlcCer also aVects the formation of rafts at the ER (Smith et al., 2006), do significant amounts of GlcCer occur in the ER? Although complex globosides are not present in mitochondria or peroxisomes in normal cells (van Genderen et al., 1991), the mitochondria‐ associated membrane (MAM) is an ER subcompartment closely associated with mitochondria and has been reported to be enriched in enzymes of early GSL biosynthesis (Ardail et al., 2003). Hence, it will be important to clarify the GSL composition of mitochondria, especially given that mitochondrial GD3 has been reported to have roles in apoptosis (Malisan et al., 2002). Determining the subcellular distribution of GSLs within cells will be critical to address specific questions on the role of GSLs. Apart from GSLs, gradients of cholesterol in the Golgi apparatus (Orci et al., 1981) may dictate the localization of integral membrane proteins within the Golgi stack (Munro, 1998). It should be noted, however, that many of the studies that have addressed the subcellular distribution of cholesterol are open to interpretation due to the unreliability of fluorescent markers such as filipin (Orci et al., 1981) or failure to remove contaminating lipoproteins (Taylor et al., 1984).

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B. Glycosphingolipid Transport and Sorting Although the majority of GSL transport is likely to be solely via vesicular transport, the fact that GlcCer is synthesized on the cytosolic surface of the Golgi implies it can in principle follow three transport pathways: (1) If it moves into budding transport vesicles, it can reach the cytosolic surface of all membranes of the exocytic/endocytic membrane system. (2) It may be extracted from the membrane by a transfer protein and transferred to the cytosolic surface of other organelles (e.g., the mitochondria or peroxisomes). A glycolipid transfer protein has been identified (Bloj and Zilversmit, 1981), and cell fractionation has suggested vesicle independent transport of GlcCer between Golgi and plasma membrane can occur (Warnock et al., 1994). The function of this process is presently unclear; interestingly, FAPP2, a protein important in the transport of cargo from the trans‐Golgi network (TGN) to the plasma membrane contains a glycolipid‐transfer protein homology domain, suggesting a link to transport to the plasma membrane (Godi et al., 2004). (3) GlcCer may flip to the luminal leaflet of the Golgi membrane, where it becomes available for the LacCer synthase or where it can enter the luminal aspect of transport vesicles. Evidence for fast, energy‐independent GlcCer translocation across the Golgi membrane has been presented (Buton et al., 2002). What is the identity of such a transporter? Evidence has been presented of a relationship between GlcCer levels and the ABC‐transporters MDR1 P‐glycoprotein and MRP1 (Raggers et al., 1999). Whether GlcCer is directly transported by MDR1 or MRP1 or via an indirect eVect of the changes in their expression has yet to be determined. Preliminary experiments using a specific inhibitor of MDR1 do not show changes in GSL levels (see Section VI). One of the major mysteries in cell organization is how the distinct lipid compositions of organelles are maintained. Although the subcellular organization of sphingolipid synthesis is complex (Lala et al., 2000; Venkataraman et al., 2002), few compositional diVerences may be explained solely on the basis of local synthesis and degradation. Apart from the possibility of unidentified translocators that may actively flip GlcCer into specific organelles, a theory has been put forward to explain cosorting of GSLs by their selective partitioning and hence enrichment in microdomains, platforms composed of clustered sphingolipid, and cholesterol molecules in the plane of the bilayer (Simons and van Meer, 1988). However, evidence for selective enrichment has been lacking, due to the methodological diYculties in studying the transport of endogenous lipids within the cell and microdomain purification. Consequently, lipid sorting has been analyzed using lipid analogues that can be rapidly incorporated into membranes because they can form monomers in aqueous solution. To form extended bilayers, membrane lipids are composed of two lipid tails and a hydrophilic headgroup; substitution of part of one lipid tail with a

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hydrophobic fluorophore (such as BODIPY) leads to the generation of a lipid analogue whose traYcking can be followed in live cells. Uptake of many fluorescent analogues and toxins has been shown to be taken up in a temperature‐ and energy‐dependent fashion and to use specific endocytic pathways (Hoekstra and Kok, 1992; Koval and Pagano, 1989, 1990). BODIPY analogues show concentration‐dependent changes in the wavelength of light they emit due to resonance energy transfer (Pagano and Chen, 1998). Use of these analogues can lead to the assessment of whether membranes in living cells can have local increases in specific lipids in a similar fashion to what has been reported for proteins (Van Meer, 2002; Zacharias et al., 2002). Selective enrichment of BODIPY sphingomyelin or lactosylceramide has also been detected by changes in the emission from green to red in early recycling endosomes (Chen et al., 1997; Puri et al., 2001), probably by partitioning into distinct domains during endocytosis. It should be noted that BODIPY lipid analogues do not always behave like their natural counterparts with respect to detergent‐resistant membrane association (Wang and Silvius, 2000), hence the importance of verifying the results by studying endogenous sphingolipids. Such a comparison is partially made possible by labeled toxins that bind to natural sphingolipids and trace endogenous lipid transport pathways (Puri et al., 2001; Sandvig et al., 1989; Schapiro et al., 1998). It should be noted that such toxins are multivalent, and therefore, they report the behavior of glycosphingolipid oligomers rather than individual lipid molecules. Using lipid probes, it has been possible to discern further complexities in that lipid segregation can occur laterally, in the plane of the bilayer, between sphingomyelin and lactosylceramide, and even between acidic glycosphingolipids such as GM1 and GM3 or GD3 (Gomez‐Mouton et al., 2001; Vyas et al., 2001). In addition, lipid probes suggest lateral segregation in endosomes can also depend on the fluidity of the acyl chains; more fluid lipid probes are sorted into recycling endosomes, whereas more rigid structures are sorted toward the late endosome, (Mukherjee et al., 1999). The previous findings may indicate the subtle mechanisms underlying the inclusion of specific lipids in various endocytic microdomains.

C. Glycosphingolipids and Endocytosis 1. Regulation of Endocytic Transport by Luminal Glycosphingolipid Endocytic mechanisms can be broadly separated into clathrin‐dependent and clathrin‐independent pathways. Several clathrin‐independent pathways exist such as caveolar‐, RhoA‐, and Cdc42‐dependent pathways (Sabharanjak et al., 2002). It has been shown that one of the clathrin‐independent pathways is glycosphingolipid‐dependent (Cheng et al., 2006c). At least some

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glycosphingolipids have been shown to be taken up selectively by caveolar‐ dependent endocytosis (Singh et al., 2003). Exogenous addition of simple GSLs with short chains or complex GSLs with more natural fatty acid compositions (both of which are suYciently hydrophilic to exist as monomers in aqueous solution and incorporate onto the cell surface) specifically stimulates the caveolar‐dependent pathway (Sharma et al., 2004). Consistent with this, inhibition of GSL synthesis using pharmacological inhibitors suppresses the caveolar‐dependent uptake and reduces the appearance of caveolae and plasma membrane caveolin (Cheng et al., 2006c). GSL and/or cholesterol storage selectively disrupts caveolar‐dependent endocytosis, suggesting a balance is necessary for this specific form of endocytosis (Cheng et al., 2006b). It would appear caveolae‐mediated endocytosis requires cell surface GSL, probably by the formation of a specific lipid raft on the surface. 2. Regulation of Endocytic Transport by Cytosolic Glycosphingolipid As mentioned in Section II.A , glucosylceramide depletion in a macrophage cell line also induces changes in endocytosis (Sillence et al., 2002). Because glucosylceramide is the only GSL known to have both luminal and cytosolic pools (Burger et al., 1996; Futerman and Pagano, 1991; Jeckel et al., 1992), it is still an open question whether the eVects of glucosylceramide depletion are dependent on the cytosolic or luminal GlcCer populations. However, the eVects are reversed by the addition of exogenous glucosylsphingosine (which can be directly acylated to glucosylceramide in a variety of tissue culture cell lines [Farrer and Dawson, 1990]) but not glucosylceramide. Glucosylceramide would not be expected to access cytosolic GSL pools by virtue of its hydrophobicity. Hence, these changes in endocytosis may depend on changes in cytosolic GlcCer. Alternatively, it is also possible that glucosylsphingosine acts in a similar way to other short‐chain glycosphingolipids and stimulates caveolae and/or lipid raft‐dependent endocytosis and reverses the altered endocytosis by directly stimulation (Cheng et al., 2006b). Cytosolic glucosylceramide has also been suggested to be necessary for other vesicular transport processes, such as between the Golgi and the melanosome (Sprong et al., 2001). Whether the eVects of altered glucosylceramide expression are directly involved in membrane transport via uncharacterized lectins or via eVects on phosphoinositide metabolizing enzymes (Shayman et al., 1990) that have specific roles in endosomal transport (Huijbregts et al., 2000) remains to be established. In any case, glucosylceramide’s ubiquitous expression in the tissues of mammals, insects, and nematodes as well as severe phenotypes in some GSL knockout mice is consistent with a potentially important function at the cellular level

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(Yamashita et al., 1999). In yeast, GlcCer expression may be linked to pH regulation (Rittershaus et al., 2006).

D. Glycosphingolipids and Protein Sorting 1. Glycosphingolipids in Toxin Transport One subset of toxins bind to GSLs on the cell surface and appear to use GSLs to gain access to specific subcellular locations (Smith et al., 2004). At least in some cases, endogenous GSL is necessary for toxin transport to a particular ER microdomain (Smith et al., 2006). Toxin action involves transport from the cell surface to the ER, translocation to the cytosol, and inhibition of protein synthesis (Sandvig and van Deurs, 2002). Toxins bind to a variety of GSLs (Globotriaosylceramide Gb3, Globotetraosylceramide Gb4, GM1, GD1a, GD1b, and GT1b) with high specificity (Lencer et al., 1999). GSL structure is important for toxin action because transferring a GSL carbohydrate chain to phosphatidylethanolamine can drastically decrease toxin potency (Pacuszka et al., 1991). Transposition of dissimilar GSL‐binding motifs between toxins (so that cholera toxin binds GD1a instead of GM1, for instance) can also drastically decrease toxicity (Badizadegan et al., 2000a; Rodighiero et al., 2001; Wolf et al., 1998). Decreases in toxin potency correlate with detergent solubility, suggesting that microdomain association is important for toxin potency (Falguieres et al., 2001; Wolf et al., 1998). Specificity of toxin binding and action may be explained by the fact that GSLs are not equally prone to microdomain association and specific GSLs may occupy distinct domains in the plane of the membrane (Gomez‐Mouton et al., 2001; Vyas et al., 2001). The physical characteristics underlying diVerences in GSL behavior are not yet known. However, not only the carbohydrate portion of the GSL is important because toxin binding to GSLs with shorter fatty acids also correlates with increased potency and correct transport from the cell surface to the Golgi (Arab and Lingwood, 1998; Badizadegan et al., 2000b). Perhaps shorter acyl chains sort to thinner membranes that are sorted to specific ER subdomains in a similar fashion to Golgi proteins (Munro, 1995). Previously it has been proposed that ceramide synthesized de novo may occupy distinct ER subdomains to ceramide synthesized from recycling pathways via the degradation of sphingosine‐1‐ phosphate (Giussani et al., 2006). Possibly C16 and C18 sphingolipids are particularly important for retrograde traYcking, and recycling of ceramide is necessary to maintain this ER subdomain. In any case, GSL depletion can inhibit the ability of Shiga‐like toxin to associate with lipid rafts both at the plasma membrane and in the ER subdomain (Cheng et al., 2006b; Smith et al., 2006), inhibiting its activity. Despite multiple rounds of fusion events

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between intracellular organelles, microdomain association at the plasma membrane appears to have long‐lived consequences throughout the cell. 2. Glycosphingolipids in Tyrosinase Transport Using the glycolipid‐deficient melanoma cell line GM95, the importance of GSLs in the sorting of two melanosomal proteins, tyrosinase and tyrosinase‐ related protein 1 (TRP1), has been determined (Sprong et al., 2001). Tyrosinase was retained in the perinuclear region, whereas TRP1 could still reach the melanosomes but was misrouted via the cell surface. Sorting of melanomal enzymes and pigmentation could be restored when glycolipid‐deficient cells were transfected with the CGlcT or incubated with glucosylsphingosine (GlcSph). It is still not clear which GSL is involved in melanosomal protein sorting: it could be GlcSph itself, GlcCer synthesized by the cells when GlcSph is added, or a derivative or degradation product. Possibly, pumping GlcCer from the cytosolic side of membranes could be required for sorting. Alternatively, GlcCer could be part of domains necessary for the correct sorting of these enzymes.

III. Glycosphingolipid Storage Sphingolipids are degraded in late endosomes and lysosomes by the action of acid hydrolases and their cofactors. If a defect is inherited in a gene encoding a protein essential for lysosomal catabolism, storage of the substrate ensues. These diseases are termed the GSL storage diseases. To date, approximately 40 genetically distinct forms of GSL storage diseases have been described. They are inherited either as autosomal recessive or X‐linked traits and have a collective frequency of 1:8000 live births (Meikle et al., 1999). They are often associated with severe neurodegenerative pathology, and the majority of the GSL storage diseases involve the primary storage of one particular species of GSL and are termed the glycosphingolipidoses (Fig. 3). Despite a clear understanding of the biochemical and genetic defects that cause the majority of these disorders, we do not understand how storage leads to cellular dysfunction. How cell dysfunction in turn causes the pathological features of these diseases also remains unclear. For the most part, these diseases are characterized by specific storage of an endogenously synthesized sphingolipid. This explains why, for instance, in the gangliosidoses (gangliosides are GSLs with sialic acid residues) some neurons in the brain store, while others are spared. However, one exception, macrophages, actively phagocytose senescent/apoptotic cells and acquire sphingolipids from the cells they ingest. It is becoming clear that macrophage

165

GLYCOLIPID STORAGE, LIPID RAFTS, AND TRANSLOCATORS [GM1]

Galβ3GalNAcβ4 Galβ4GlcCer GM1 Gangliosidosis

NeuAcα3

(Globoside)

β-galactosidase

GalNAcβ3Galα4Galβ4GlcCer GalNAcβ4 Sandhoff β-hexosaminidase A & B Galβ4GlcCer [GM2] disease Tay-Sachs disease NeuAcα3 Galα4Galβ4GlcCer α-galactosidase Galβ4GlcCer β-hexosaminidase A Fabry disease NeuAcα3 [GM3] Galβ4GlcCer neuraminidase β-galactosidase GlcCer β-glucocerebrosidase Sulphatase A

SO3H-GalCer

β-galactosidase

GalCer

Metachromatic leukodystrophy

Gaucher disease Sphingomyelinase

Ceramide Krabbe disease

Sphingomyelin

Niemann-Pick A/B disease

FIG. 3 Sphingolipid storage diseases.

activation following storage is a characteristic of many of these diseases. The classic example is type 1 Gaucher disease, an inflammatory disease of the periphery in which only macrophages store (Beutler and Grabowski, 2001). However, even some storage diseases of the brain show an inflammatory component characterized by microglial/macrophage activation (Jeyakumar et al., 2003; Wada et al., 2000). Precisely how storage in macrophages leads to activation and whether once activated these cells play a significant role in disease progression is still unknown. All the disorders have unique although frequently overlapping and highly variable clinical phenotypes. They clearly pose a challenge to elucidating pathogenic mechanisms, and it would appear that to understand these diseases each disorder would have to be investigated individually. In Krabbe disease (Globoid cell leukodystrophy), in which galactocerebrosidase activity is diminished, the pathogenic mechanism may be partially understood. In this disease, lyso‐galactosylceramide (galactosylsphingosine or psychosine) accumulates and probably mediates neuropathology (Suzuki, 1998). However, a more general storage phenotype has been uncovered. Studies on GSL traYcking have uncovered new endocytic pathways targeted to the Golgi specifically disrupted in sphingolipid storage diseases (Puri et al., 1999, 2001). The precise mechanism of how GSL traYcking is disrupted has yet to be determined, but normalization of both cholesterol (Puri et al., 1999) and GSL levels (Vruchte et al., 2004) reverses the altered traYcking. This discovery oVers the prospect of understanding

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the disease process in more detail, gaining insights into normal cell biology, and potentially identifying novel clinical intervention points. However, the function of these GSL traYcking processes in normal cells are not yet clear, and so it is as yet unknown which clinical phenotypes are due to altered traYcking. One further popular theory for how GSL storage mediates pathology is that the storage material escapes the lysosome, disrupting the function of the cell more widely than altered endocytosis and lysosome function. This leads to the question of whether the stored glycosphingolipid escapes the lysosome and disrupts the functions of other organelles. Accumulation of GSLs within the ER in GM1 gangliosidosis has been linked to the stimulation of the unfolded protein response (Tessitore et al., 2004). However, the utility of many anti‐GSL antibodies for subcellular localization is still controversial. The consequences of an increased nonlysosomal GSL are not yet clear. A knockout mouse for a bile acid b‐glucosidase has been generated. Surprisingly, knocking out this enzyme was found to lead to the accumulation of GlcCer in diverse tissues such as the testes, brain, and liver (Yildiz et al., 2006). Subsequently, this enzyme has been characterized as the nonlysosomal glucocerebrosidase (Boot et al., 2006). There is currently no agreement on the subcellular localization of this enzyme, as it has been determined as being ‘‘close to the plasma membrane’’ (Boot et al., 2006) or in the ER (Yildiz et al., 2006). Despite nonlysosomal GlcCer storage, the only phenotype uncovered in these mice has been disrupted sperm acrosome formation, leading to infertility with no phenotypes associated with Gaucher disease. The identification of a new mouse model for Gaucher’s disease generated by the disruption of the lysosomal glucocerebrosidase (Enquist et al., 2006) should help determine whether increases in GSLs such as GlcCer at nonlysosomal locations really do occur and lead to disruption of calcium homeostasis (Korkotian et al., 1999) and altered phospholipid synthesis (Bodennec et al., 2002).

IV. Secondary Storage Diseases A. Mucolipidosis Type IV In contrast to many lysosomal storage diseases, some do not show deficiencies in any enzyme activity or cofactor (Table I). In many of these diseases, the underlying reason for GSL storage is unclear. One example of secondary storage is a‐mannosidosis, where the lysosomal accumulation of glycoconjugates is thought to inhibit the activity of acid hydrolases and induce the accumulation of GSL. In other diseases with secondary GSL accumulation (e.g., Niemann‐Pick type C [NPC] and mucolipidosis type IV [MLIV] disease),

167

GLYCOLIPID STORAGE, LIPID RAFTS, AND TRANSLOCATORS TABLE I Lysosomal Diseases with Primary and Secondary GSL Accumulation

Primary GSL storage diseases Disease

Deficit/Storage

Lysosomal diseases with secondary storage of GSL in neurons Disease

Deficit/Storage

Gaucher (type 1, 2, and 3)

Glucocerebrosidase/ GlcCer

Niemann-Pick type A

Acid sphingomyelinase/SM

Fabry

a-galactosidase/Gb3

Niemann-Pick type C

NPC1 or NPC2/SM, sphingosine, GM2, GM3, LacCer, GlcCer

Tay-Sachs

b-hexosaminidase A/GM2

MPS I, IIIA, IIIB, VI, VIIa

Sugar sulphatases/GM2, GM3

SandhoV b

b-hexosaminidase B/GM2 GA2

a-mannosidosisc

a-mannosidase/GM2

GM1 gangliosidosis

b-galactosidase/GM1

a Mucopolysaccharidoses (MPS), primary storage of glycosaminoglycans such as dermatan sulphate and heparin sulphate which may inhibit GSL degradation. b SandhoV disease (b hexosaminidase) also leads to the accumulation of glycosaminoglycans. c Storage of mannose-containing protein polysaccharides Man(a1 ! 3)Man and Man(a1 ! 2) Man may then inhibit GSL degradation.

the reason for storage is not known. MLIV is a neurodegenerative disorder resulting from a defective multispanning membrane protein mucolipin‐1. Mucolipin‐1 is a member of transient receptor potential (TRP) cation channels. So far, studies have suggested mucolipin‐1 is a nonspecific lysosomal cation channel modulated by Ca2þ concentration and pH (LaPlante et al., 2002, 2004, 2006). Although the mucolipin‐1 has also been suggested to be specifically a proton channel (Soyombo et al., 2006), studies in MLIV fibroblasts report conflicting data on whether there is a decrease, increase, or no change in lysosomal pH (Bach et al., 1999; Pryor et al., 2006; Soyombo et al., 2006), suggesting a complicated relationship between lysosomal Ca2þ and Hþ concentrations (Lopez et al., 2005). Changes in lysosomal Ca2þ may clearly aVect sorting because it is necessary for late endosomal fusion events (Pryor et al., 2000). Hence, the changes in lysosomal calcium may well be linked to the changes in GSL traYcking observed in MLIV fibroblasts (Chen et al., 1998).

B. Niemann‐Pick Type C NPC is an autosomal recessive disorder, the hallmark of which is the storage of unesterified cholesterol in endosomes/lysosomes (Pentchev et al., 1994). Similar to MLIV disease, NPC disease shows a broader GSL accumulation

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than many of the primary storage diseases (Table I). The genetic lesion is in the NPC1 gene and less commonly the NPC2 gene. The two proteins are believed to play a role in a common pathway responsible for the correct late endosomal sorting of cholesterol, GSLs, and sphingosine related to the decreased motility of the late endosome (Higgins et al., 1999; Ko et al., 2001; Neufeld et al., 1999; Patel et al., 1999). NPC2 is a luminal protein suggested to have a lipid carrier function (Naureckiene et al., 2000). Why do NPC1‐deficient late endosomes show decreased motility? The specific intracellular steps slowed in NPC deficient cells have become clearer, including the sorting of multiple types of cargo from rab 7‐positive late endosomes to the TGN (Kobayashi et al., 1999; Neufeld et al., 1999). Overexpression of rab 7 can correct changes in lipid traYcking that occur in NPC fibroblasts (Choudhury et al., 2002). Moreover, cholesterol accumulation decreases the ability of rab‐GDI to extract rab 7 (Holtta‐Vuori et al., 2000; Lebrand et al., 2002). Rab 7 extraction from the bilayer by GDI is necessary for association of the late endosome with kinesin and the movement from pericentriolar to peripheral regions of the cell. Because cholesterol accumulates in many other lysosomal disorders (Puri et al., 1999), it would also be interesting to determine whether rab 7 function is widely aVected in the GSL storage diseases. Whether cholesterol accumulates due to GSL accumulation or the other way around is still to be resolved; however, in NPC mice which are also a knockout for GM2 synthesis, cholesterol accumulation in neurons was attenuated, suggesting that the accumulation of cholesterol may be secondary to the accumulation GSLs. Why only specific GSLs are aVected is not yet known, perhaps specific GSLs are localized to specific parts of the endosomal system in NPC cells (Zhang et al., 2001). Part of the answer may lie in the fact that specific GSLs have specific traYcking pathways which can vary between cell types (Gillard et al., 1998). However, the function of NPC1 and the mechanism by which a loss of function mutation in this protein leads to GSL, sphingosine, and cholesterol accumulation remains unclear. Because it appears the accumulation of GSL shows only a modest contribution toward overall NPC pathology (Liu et al., 2000; Zervas et al., 2001), other possibilities for the mediation of NPC disease are now discussed. NPC1 has been suggested to be a lipid permease that transports fatty acid‐like compounds (Davies et al., 2000). So far, many of the GSL storage diseases show alterations in the distribution of NPC1 (Puri et al., 1999). Altered function of NPC1 correlates with increased lysosomal sphingosine in spleen and liver samples in NPC and perhaps with other storage disease patients (Goldin et al., 1992; Rodriguez‐Lafrasse et al., 1994). It is also interesting to note that sphingoid bases and other agents that induce changes in lysosomal proton gradients induce an NPC phenotype (RoV et al., 1991). So how may the accumulation of sphingoid bases and altered cation

GLYCOLIPID STORAGE, LIPID RAFTS, AND TRANSLOCATORS

169

gradients be related to the altered endocytic traYcking seen in NPC cells? Along with cholesterol, sphingoid bases have also been reported to play an important role in yeast endocytosis (Friant et al., 2000, 2001; Zanolari et al., 2000). It is also interesting to note that sphingosine‐1‐phosphate releases intracellular calcium in yeast as well as mammalian cells (Birchwood et al., 2001; Ghosh et al., 1994). It is therefore possible that the increased lysosomal sphingosine found in NPC cells leads to decreased cytosolic sphingosine and sphingosine‐1‐phosphate (Ghosh et al., 1994). Decreased sphingosine or sphingosine‐1‐phosphate levels may then lead to altered lysosomal Ca2þ regulation (Itagaki and Hauser, 2003; Lee et al., 2006) similar to phagosomal calcium (Malik et al., 2003; Taha et al., 2006) and to defective late endocytic fusion. Alternatively, the accumulation of cholesterol in the lysosomes and late endosomes may eVectively starve the other organelles of cholesterol, and this may also have widespread eVects on cation gradients including the ER.

C. Autophagy in MLIV and NPC Disease What are the potential interrelationships between decreased lysosomal sphingoid base and cholesterol release, altered rab 7 function, and cation gradients? Increases in autophagy have been observed in an NPC1 mouse model (Ko et al., 2005). In this study, autophagy was not seen in NPC1 cell culture models, only in dying Purkinje cells, and the activation was proposed to occur in response to cholesterol depletion. However, in MLIV constitutively elevated autophagy was found in cultured fibroblasts (Jennings et al., 2006). Autophagy refers to a specialized form of lysosomal degradation in which cytosolic components are degraded. Although this process occurs constitutively, it is particularly active under conditions of cell stress to protect cells from apoptosis by the sequestration of malfunctioning organelles (Cuervo, 2004). Rab 7 has been identified as necessary for the formation of late autophagic vacuoles (Gutierrez et al., 2004; Jager et al., 2004), and upregulation of sphingosine kinase 1 increases the formation of late autophagic vacuoles (Gutierrez et al., 2004; Jager et al., 2004). Cholesterol depletion remains a possibility as a mechanism of cell death (Cheng et al., 2006a) in NPC. In other storage diseases where the late endosomal cholesterol transport is less aVected, sphingosine escape from lysosomes leading to a depletion in cytosolic sphingosine‐1‐phosphate (Lavieu et al., 2007) may play a more important role, limiting the ability of aVected neurons to escape apoptosis by the activation of autophagy (Hong et al., 2004). If this is the case, the mechanism of neuronal cell death in primary storage disease would be expected to be via apoptosis (programmed cell death type 1) rather than autophagy (programmed cell death type 2).

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V. Effect of MDR1a Deficiency on Glycosphingolipids in Both Normal and Niemann‐Pick Type C Mice It has been speculated that there may be a close relationship between multiple drug resistance (MDR) and NPC1 as lipid translocators/cation permeases (Ioannou, 2001). Evidence that supports such a relationship includes the observation that NPC1 can mediate drug eZux (Gong et al., 2006) and that a deficiency in mdr1a has been shown to be able to correct NPC1‐induced sterility in NPC1/ female mice (Erickson et al., 2002). One further relationship between MDR and NPC1 is that they have both been implicated in the regulation of GSL synthesis and transport (Lala et al., 2000; Van Helvoort et al., 1996). Their possible interrelationships will be considered in this chapter. MDR P‐glycoproteins were originally identified as one of the mechanisms whereby cells remove therapeutic agents by translocating them across membrane bilayers. However, studies have also suggested a link with GSL metabolism. Drug‐resistant tumor cells have been shown to increase GlcCer levels (Lavie et al., 1996). Transfection of canine kidney cells with the MDR1 gene results in a marked elevation of GlcCer, LacCer, and Gb3 levels with a concomitant increase in sensitivity to a Gb3‐binding toxin. Both eVects were reversed in the presence of MDR1 inhibitors (Lala et al., 2000). It has been proposed that MDR1 functions as a GlcCer translocase (Van Helvoort et al., 1996). However, others have argued the lack of an overt phenotype for MDR knockout mice is not consistent with a physiological function (Borst et al., 2000), although mild phenotypes in mdr1a knockout mice have been found such as in inflammatory bowel disease (Panwala et al., 1998). To investigate the possible role of P‐glycoproteins in GSL metabolism, the changes in GSL expression that occur in ear fibroblasts derived from triple knockout mice, defective in mrp1//mdr1 a//b/, are described. In mrp1//mdr1 a//b/ fibroblasts, increases in GSL fatty acid unsaturation are found (Table II). In addition, lower levels of GSLs (particularly neutral GSLs) are found in mrp1//mdr1 a//b/ fibroblasts (Table III). Although changes in some acidic GSLs were also detected in individual KOT cell lines, both KOT fibroblasts showed decreases in neutral GSLs (Table III). Since altered fatty acid saturation has been shown to have functional consequences, possibly due to eVects on raft formation (Stulnig et al., 1998), whether mdr1a deficiency can alter the fatty acid composition of the GSLs that accumulate in a mouse model of NPC disease has been investigated. Because NPC is a predominantly neurological disease, brain GSLs have been studied. However, in a mouse model of NPC disease that was also deficient in mdr1a, no alterations were found in cerebral GSL level or fatty acid composition. These results suggest the eVect of mdr1a/ on GSL metabolism and/or traYcking is either strain‐ or cell‐type dependent

171

GLYCOLIPID STORAGE, LIPID RAFTS, AND TRANSLOCATORS TABLE II Triple Knockout Fibroblasts GSLs Show Increases in C24:1 Fatty Acid Sample MF

KOT‐11

KOT‐51

GSL

C24:1

GlcCer

17  5

LacCer

18  1

GM3

13  2

GlcCer

23  5

LacCer

42  6a

GM3

35  6a

GlcCer

45  2a

LacCer

52  1a

GM3

47  1

C16:0

20  3

18  3

23  2

a

Significantly diVerent from control p < 0.001. C24:1 as a percentage of total 24‐fatty acid and C16:0 as a percentage of total fatty acids in GSLs extracted from mpr1/ mdr1a//b/ triple knockout mouse ear fibroblast cell lines (KOT 11 and KOT 51). GSLs were extracted as described, and MALDI mass spectrometry was performed (n ¼ 10–12). MF, normal mouse fibroblasts. a

TABLE III GSL Composition of mpr1/ mdr1a//b/ Triple Knockout Mouse Ear Fibroblast Cell Lines Cell Line MF

LacCer

GA2

400  200 40  2

CTH

GM3

GM2

Gb4

30  2

640  13

230  7

80  1

KOT‐11

60  17

10  5

11  10 870  200 252  82

KOT‐51

64  30

22  13 —

446  92

95

47  17 24  12

GA1

GM1a GM1b

GD1a

30  2 60  11 20  1 230  50 6  3 22  11 11  4 4  2 70  27

70  40

3  1 345  113

GSLs were extracted and purified before digestion with ceramide glycanase before derivatization and quantitation by HPLC. Data are from three experiments and are expressed as pmol/mg protein  SE. Dash (—), not detected; <0.01 pmol/mg protein.

and is consistent with similar neurological symptoms in both NPC1/ or NPC1/mdr1a/ mice (Erickson et al., 2002) and previous reports that mdr1 is not expressed in neurons (Matsuoka et al., 1999). A. Mass Spectrometry of Fibroblast GSLs from mpr1/ mdr1a//b/ Triple Knockout Mouse Ear Fibroblasts To investigate possible changes in GSL fatty acid composition in mpr1/ mdr1a//b/ fibroblasts, MALDI mass spectrometry was performed on a purified GSL fraction (Hunnam et al., 2001). Although this analysis was not

172

DAN J. SILLENCE

Glc-Cer 834.5 100 722.4 24:0

MF GM3

16:0 806.6

Relative abundance (%)

50

884.5

0 100

24:0 1287.8 Na salt 16:0 1309.7 1175.6 1241.8 1259.7

Lac-Cer

771.3 793.4

968.5

996.6 24:0

834.5

806.5 50 722.3 771.3

996.7

16:0 968.5 884.4 871.3 909.3 931.4

1309.8 1259.6 1175.5 1197.5 1241.7 1131.6

1024.2

0 771.3

100 722.4

727.2

50

793.3

909.3 931.4 884.4

806.5 834.5

700

24:1 994.6

KOT 51

968.6 1287.71301.8

865.3

0

1287.7

KOT 11

800

1050.6

900

1000 m/z

1175.7 1189.61241.7

1100

1200

1309.8

1300

FIG. 4 Changes in GSL fatty acid composition of GSLs extracted from wild‐type and mpr1/ mdr1a//b/ triple knockout mouse ear fibroblasts. Representative MALDI mass spectra showing clear increases in C24:1 fatty acid.

designed to be quantitative, it should give a representation of the fatty acid composition of the diVerent GSLs. Figure 4 shows a typical spectrum obtained from wild‐type mice. The spectrum was dominated by ions at m/z 722.4, 806.6, 834.5, 884.5, 968.5, 996.7, 1175.6, 1259.8, and 1287.8, corresponding to C16:0, C22:0, and C24:0 GlcCer, LacCer, and GM3, respectively. GSL fatty acid composition was investigated using two separate cell lines designated KOT 11 and KOT 51, derived from mpr1/ mdr1a//b/  triple knockout mice (Allen et al., 2000). Mass spectra from GSL in these cell lines were populated by similar ions to those found in the wild type. However, increases were also seen in m/z 832.5, 994.6, and 1285.8, corresponding to C24:1 GlcCer, LacCer, and GM3 (Figs. 4 and 5). These results suggest a higher degree of GSL unsaturation in mpr1/ mdr1a//b/ fibroblasts. A quantitative analysis (Table II) found that these changes were statistically significant and corresponded to 2‐ to 3‐fold increases in the levels of 24:1 species when compared to control fibroblasts. It has been shown that the 24:1 species of GM1 have lower levels of microdomain or lipid raft association (Panasiewicz et al., 2003). In this light, the level of enrichment of GSLs in lipid rafts in mpr1/mdr1a//b/ fibroblasts were quantitated. Figure 6 shows the ratio of GSL in lipid rafts versus that found in the homogenate for both normal and triple knockout fibroblasts. In normal fibroblasts the ratio between the concentration of both LacCer and GM3 in lipid rafts versus that found in the homogenate was 75, reflecting a large enrichment of these GSLs in lipid rafts (Fig. 6). In contrast, in both KOT 11 and KOT 51 cells a much lower ratio was

173

GLYCOLIPID STORAGE, LIPID RAFTS, AND TRANSLOCATORS

Relative abundance (%)

50 0 100

24:0 1287.8 Na salt 1309.7

MF

100 16:0 1175.6 1147.6 1166.7

Na salt 1197.7

1225.7

1241.8

18:0 1259.7 1281.7

1323.8

1232.7

1287.7 24:0

KOT 11

24:1 1259.6 1285.7 1281.6 1197.5 1257.6 1273.7 1215.6 1241.7

50 1175.5 1157.6

0 100

1309.7 1307.8

1325.7

1301.6

1301.8

1299.7 1287.7 1267.8 1285.7

KOT 51

50

1157.6 1175.7

1189.6 1197.6

1309.8

1241.7 1259.8

1199.71213.7

1317.7 1328.7

1143.6

0 1140

1160

1180

1200

1220

1240 m/z

1260

1280

1300

1320

1340

FIG. 5 Detailed mass spectrometry of GM3 extracted from wild type and mpr1/ mdr1a//b/ triple knockout mouse ear fibroblasts.

80 70 60 LacCer GM3

50 40 30 20 10 0 MF

KOT 11

KOT 51

FIG. 6 Changes in microdomain association of GSLs between wild‐type and mpr1/ mdr1a// b/ triple knockout mouse ear fibroblasts. Cell pellets were incubated with TX‐100 before separation of the floating detergent insoluble and soluble portions by ultracentrifugation. GSLs were extracted and purified before digestion with ceramide glycanase before derivatization and quantitation by HPLC. Data are expressed as % detergent resistant.

obtained to around 15 and 21, respectively. These results suggest that the level of both these GSLs is lower in the raft fraction, consistent with the increase in unsaturation that occurs in mpr1/ mdr1a//b/ fibroblasts.

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DAN J. SILLENCE

Because changes in the level, fatty acid composition as well as raft association were clearly seen in mdr1a//b/ mrp1/ fibroblasts, changes in the brain GSL levels in NPC1/ mice versus mice deficient in NPC1 and mdr1a (NPC1/mdr1a/) were investigated. This was particularly of interest given that the accumulation of GSLs in microdomains are thought to be important in the pathogenesis of this disease and the double knockout has previously been reported to reverse the sterility of NPC mice (Erickson et al., 2002).

B. Mass Spectrometry of Brain GSLs Isolated from NPC1/ Mice The total fatty acid composition of GSLs in NPC1þ/þmdr1aþ/þ, mdr1a/, and NPC1/mdr1a/ mice were analyzed by MALDI mass spectrometry (Hunnam et al., 2001). GSLs were extracted and purified and fractionated into neutral and acidic GSL fractions on silicic acid columns (Vance and Sweeley, 1967). Because NPC is a neurodegenerative disease, our analysis concentrated on total brain GSLs. They were found to contain mainly C18:0 fatty acids with minor amounts of C20:0, similar to findings in bovine brain and other mammalian species (Sonnino et al., 1984). 1. Neutral GSLs Figure 7A shows the spectrum obtained from the GSLs extracted from a cerebrum of a normal Balb/C NPC1þ/þ mdr1aþ/þ mouse. The spectra were dominated by ions at m/z 832.6 and 934.6, which correspond to the masses of the 24:1 isoforms of galactosylceramide [M]þ and sulfatide [MþNa]þ, respectively. In the spectrum obtained from GSLs isolated from NPC1/ and NPC1/mdr1a/ mice (Fig. 7C and D), similar peaks at m/z 832.6 and 934.6 are present. However, additional peaks at m/z 912.5 and 1115.7 are also seen, corresponding to C18:0 lactosylceramide [M þ Na]þ and C18:0 GA2 [M þ Na]þ, respectively. Other peaks were also observed at 1143.8 and 940.6, corresponding to C20:0 GA2 and C20:0 LacCer, respectively, as expected from the GSL storage that occurs in NPC mice (Vruchte et al., 2004). 2. Acidic GSLs Figure 8A and B illustrates the spectrum obtained from NPC1þ/þmdr1aþ/þ GSLs which shows peaks at m/z 1590.8, 1903.8, 2217.7, and 2530.7 corresponding to the [MþNa]þ for C18:0 GM1, C18:0 GD1, C18:0 GT1, and C18:0 GQ1. In each of these cases, higher m/z ratios are obtained from additional Naþ salts due to the presence of sialic acid moieties and minor

175

GLYCOLIPID STORAGE, LIPID RAFTS, AND TRANSLOCATORS 832.7

100 50

757.5 750.6 766.6 801.5 792.6

822.7 848.7 874.5

0 100 50

757.5 750.7

832.8 801.5 822.7 850.7

0

872.6

910.7

1110.5

822.6 850.6 750.5 766.5792.6806.6 868.6

*1115.8

934.7 940.8 950.7 968.7

C

952.6

924.6 908.6

1110.6

912.6 832.6 750.5

792.5 822.6 850.6 806.6 872.5 908.6 766.5

750

800

850

900

B

1143.9

934.6

832.6

0 100 50

950.6 968.7

908.6

*912.7

0 100 50

A

934.7

D

934.6 936.6 940.7 952.6

1115.7 1143.7

996.6

950 m/z

1000

1050

1100

1150

FIG. 7 Neutral GSL fatty acid composition in NPC1þ/þ, mdr1a/, NPC1/, and NPC1/ mdr1a/ mice. Representative MALDI mass spectra of GSLs from mouse cerebra. (A) Neutral GSLs from NPC1þ/þmdr1aþ/þ mice; (B) Neutral GSLs from NPC1/mdr1aþ/þ mice; (C) Neutral GSLs from NPC1þ/þmdrla/ mice; (D) Neutral GSLs from NPC1/mdrla/ mice. GSLs were extracted and saponified in methanolic NaOH before separation into neutral and ganglioside fractions using silicic acid chromatography. GSLs were dried down and resuspended in methanol before MALDI mass spectrometry. DiVerences between NPC1þ/þ and NPC1/ are indicated by *.

amounts of C20:0. The spectrum also shows metastable (wider peak) ions at m/z 1278.8 and 1319.8 due to in‐source fragmentation. In both the NPC1/ mdr1aþ/þ and NPC1/mdr1a/ mice, additional peaks were observed at m/z 1204.6, 1406.6 corresponding to the [M þ Na]þ ions for C18:0 GM3 and C18:0 GM2 (Fig. 8C, D). Additional metastable ions are seen for GA2 at m/z 1115.7 and lactosylceramide at m/z 913.5, consistent with loss of sialic acid from GM2 and GM3, respectively. Hence by mass spectrometry clear increases in LacCer, GA2, GM3, and GM2 occurs in agreement with studies employing thin layer chromatography (Liu et al., 2000; Pentchev et al., 1980). C. Analysis of Total GSLs by HPLC Further structural confirmation and quantitation of GSLs were performed by digestion with ceramide glycanase and separation of the resultant oligosaccharides using HPLC (Fig. 9) (Wing et al., 2001). In all cases, mouse brain GSLs were found to be similar to those reported previously, with GM1a, GD1a, GD1b, GT1b, and GQ1b predominating and lower levels of LacCer,

176

DAN J. SILLENCE 1903.9 1568.9 1590.9 1881.9 1859.8 1320.6 1680.0

100 50

A

1279.0 878.6

1115.8

0 100 50 912.8

2176.9 2219.0

2530.9

2558.9

1277.9

*1116.8

*

2217.9

B

1225.8

0 100

*

1406.7 1524.9

1591.9 1881.8

1903.8

2217.9 2194.7 2244.9

2529.8

1278.5

975.5

0 100

C

1591.0 1568.9 1859.9 1903.9 1919.9 2151.8 2218.0 1525.7 1630.01683.0

50 878.8 1278.9 1115.9

50

1203.7 1406.7

913.6

1568.8

D

1903.8

1590.9 1881.8

2195.8

2217.9 2530.8

0 1000

1200

1400

1600

1800 m/z

2000

2200

2400

2600

FIG. 8 Acidic GSL fatty acid composition in NPC1þ/þ, mdr1a/, NPC1/, and NPC1/ mdr1a/ mice. Representative MALDI mass spectra of GSLs from mouse cerebra. (A) Acidic GSLs from NPC1þ/þmdrlaþ/þ mice; (B) Acidic GSLs from NPC1/mdrlaþ/þ mice; (C) Acidic GSLs from NPC1þ/þmdrla/ mice; (D) Acidic GSLs from NPC1/mdrla/ mice. DiVerences between NPC1þ/þ and NPC1/ are indicated by *.

mv

400:00

NPC+/+

200:00

mv

0:00 200:00

NPC−/−

100:00

LacCer

mv

0:00 400:00

GM3 GM2 GA2

GM1a

GD1a GD1b

GT1b GQ1b

NPC+/+ MDR−/−

200:00

mv

0:00 200:00

NPC−/− MDR−/−

100:00 0:00 6:00 8:00 10:00 12:00 14:00 16:00 18:00 20:00 22:00 24:00 26:00 28:00 30:00 32:00 34:00 36:00 38:00 Minutes

FIG. 9 Separation of brain GSLs from NPC1þ/þ, mdr1a/, NPC1/, and NPC1/mdr1a/ mice by HPLC. Top traces, Cerebra GSLs from normal NPC1þ/þ mice; Bottom traces, Cerebra GSLs from NPC1/ mice. Representative traces of GSLs after digestion with ceramide glycanase before the resultant oligosaccharides were derivatized with anthranilic acid before separation by HPLC. Apart from mass spectrometry of intact GSLs, the identity of some GSLs were confirmed by (a) removal of sialic acid residues by trifluoroacetic acid and (b) mass spectrometry of the same samples both before and after derivatization (not shown).

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177

GA2, GM3, and GM2 (Fig. 9) (Waki et al., 1994). In contrast to the changes in peripheral tissues, no large changes in GSL levels were seen in cerebra from mdr1a/ mice (Fig. 9). However, in brains from NPC1/mdr1aþ/þ and NPC1/mdr1a/ mice, additional GSLs were present, including GM2, GA2, GM3, and LacCer in agreement with changes seen by mass spectrometry. To analyze these changes further, membrane microdomains were purified and GSL levels were determined. 1. GSL and Cholesterol Accumulate in NPC1/mdr1aþ/þ and NPC1/mdr1a/ Mice to Similar Degrees Membrane microdomains were purified by the detergent procedure using sucrose gradients. All GSLs were found to be highly enriched in membrane microdomains. Large increases in total GSL levels were seen in microdomains isolated from cerebra from NPC1/ mice when compared to NPC1þ/þ mice (from 3 to 11 pmol/mg). In contrast to the results obtained in the triple knockout fibroblasts, no large changes in microdomain GSL levels were seen between mdr1aþ/þ and mdr1a/ mice (Table IV). Moreover similar GSL levels were seen in both NPC1/ and double knockout NPC1/mdr1a/ mice (Table IV). NPC has historically been viewed as a cholesterol storage disease, and cholesterol is an important structural component of membrane microdomains. Hence, it was also determined that the elevated cholesterol that occurs in NPC is enriched in membrane microdomains. Table IV shows that in membrane microdomains isolated from cerebra cholesterol levels were 0.9 mmol/mg protein. No significant change was detectable between mdr1aþ/þ and mdr1a/ mice. Additionally, in membrane microdomains isolated from NPC1/ cerebra, levels increased by about 50% in both NPC/ and NPC/mdr1a/ mice, suggesting that deficiency in mdr1a does not aVect the levels of total GSL or cholesterol. It still remained possible that the eVects of mdr1a were confined to a subset of GSL, so the individual GSLs were quantitated. 2. Glycosphingolipids in Both NPC1/ and NPC1/mdr1a/ Mice Accumulate in Membrane Microdomains to Equal Extents In the cerebra of both NPC1/mdr1aþ/þ and NPC1/mdr1a/ mice, clear increases could be detected in neutral GSLs and monosialylated GSLs such as LacCer, GA2, GM3, and GM2, in agreement with the mass spectrometric and HPLC analysis of the total levels (Figs. 7 and 9). The magnitude of the increases in the microdomain fraction were broadly similar to that found to total GSL levels in human NPC patients (Vanier, 1999), although the increase in GM2 level in both NPC1‐deficient mice was greater than that found in human patients (40‐fold vs. 5‐fold). Similar levels were seen in both NPC1/

178

DAN J. SILLENCE TABLE IV Total Microdomain GSL and Cholesterol Levels in MDR/NPC1 Double Knockout Mice Mouse strains

GSL (pmol/mg)

Cholesterol (mmol/mg)

NPC1þ/þmdr1aþ/þ

3  0.7

0.9  0.03

NPC1/mdr1aþ/þ

11  0.4

1.4  0.05

NPC1þ/þmdr1a/

31

0.9  0.07

NPC1/mdr1a/

11  0.6

1.6  0.1

Cerebra were dissected from normal and NPC mice and homogenized. The equivalent of 5 mg of tissue was incubated with TX‐100 before separation of the floating detergent insoluble and soluble portions by ultracentrifugation. GSLs from both portions were extracted and purified before digestion with ceramide glycanase before derivatization and quantitation by HPLC. Cholesterol was purified and quantitated by a cholesterol microassay. Sphingosine from both portions was extracted with acidified butanol before saponification and derivatization and separation and quantitation by HPLC. Data are from three to four experiments and are expressed as pmol or mmol/mg protein SE. TABLE V Microdomain GSLs in MDR/NPC1 Double Knockout Mice Mouse strains NPC1

þ/þ

mdr1a

þ/þ

LacCer

GA2

0.03  0.01 —

GM3

GM1a

GD1a

GD1b

GT1b

0.05  0.01 0.03  0.001 0.9  0.1 1.5  0.5 0.3  0.06 0.3  0.1

NPC1/mdr1aþ/þ 0.8  0.3 0.3  0.1 1.4  0.2 NPC1þ/þmdr1a/ 0.04  0.02 —

GM2

1.8  0.1

2.2  0.05 3.3  0.3 0.8  0.05 0.7  0.1

0.04  0.01 0.04  0.01 1.4  0.3 1.9  0.3 0.5  0.1 0.6  0.2

NPC1/mdr1a/ 0.9  0.6 0.4  0.2 0.9  0.1

1.5  0.2

2.9  0.3 2.6  0.5

1  0.07 0.8  0.3

Cerebra were dissected from mice and homogenized. The equivalent of 5 mg of tissue was incubated with TX‐100 before separation of the floating detergent insoluble and soluble portions by ultracentrifugation. GSLs were extracted and purified before digestion with ceramide glycanase before derivatization and quantitation by HPLC. Data are from four experiments and are expressed as pmol/mg protein SE. Dash (—), not detected; <0.01 pmol/mg protein.

and NPC1/mdr1a/, suggesting mdr1a does not significantly aVect GSL synthesis or degradation in mouse brain (Table V).

VI. Summary In this review, the significance of some of the subtleties of GSL transport and synthesis have been discussed. Multiple pathways for synthesis via de novo and recycling routes may lead to changes in the levels of signaling sphingosine‐1‐ phosphate. This may in turn aVect both anterograde (Giussani et al, 2006) and

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retrograde endocytic traYcking. GSLs can be internalized via a specific retrograde endocytic pathway utilized by toxins to gain access to ER subdomains. Moreover, this endocytic pathway is dependent on endogenous GSL (Smith et al., 2006), possibly on the cell surface (Cheng et al., 2006c). GlcCer is synthesized on the cytosolic leaflet of the Golgi and can be transported via exchange proteins to the cytosolic leaflet of other organelles, leading to potential functions on the cytosolic leaflet. Its topology of synthesis means it has to be translocated for higher GSL synthesis, and this translocation may itself have implications for cation gradients within the Golgi apparatus and other organelles. The unusual structure of the GSLs leads to self‐organizing properties and a propensity for the formation of lipid rafts involved in membrane sorting and traYcking. The GSL storage diseases shed light on the normal functions of GSLs because in these diseases their normal functions are disrupted via their accumulation within late endosomes and lysosomes. Hence, specific changes in endocytosis are found. These are either due to changes in raft formation/localization or altered cholesterol and sphingosine transport. Apart from the relocation of lipid rafts from the cell surface to the lysosome, it is possible decreases in GSL breakdown generally lead to alterations in the sphingosine/sphinganine ratio (Gillard et al., 1998), as is observed in SandhoV disease liver and spleen (Rodriguez‐Lafrasse et al., 1994). If so, subtle alterations in sphingoid base formation may play a general role in altered late endosomal calcium gradients (Pryor et al., 2000), leading to altered apoptotic signaling and autophagosome formation. Consistent with previous reports, decreased expression of mdr1 and mrp1 are associated with decreases in neutral GSL expression (De Rosa et al., 2004). Previously, it has been determined that increased expression of mdr1 and mrp1 are associated with increased expression of GlcCer and decreased expression of LacCer and gangliosides (Veldman et al., 2002). Increases in GSL 24:1 fatty acid occurs on decreased mdr1 and mrp1 expression in triple knockout fibroblasts. This change in fatty acid composition occurs in both neutral and acidic GSLs. In combination with previous results, it would appear that both decreases and increases in mdr1 expression lead to an increase in 24:1 fatty acid (Hinrichs et al., 2005). Clearly, there are potential interaction(s) between mdr1/mrp1 and GSL synthesis and/or breakdown. Whether there is a direct interaction (e.g., GlcCer flipping) or some indirect interaction (e.g., via changes in cation gradients) remains to be established. However, when normal mouse ear fibroblasts were treated with XR 9576 (an inhibitor of mdr1), for up to 1 week, no significant changes in GSL mass could be detected (Sillence, unpublished observations). This observation would suggest an indirect eVect. Further experiments with the triple knockout fibroblasts in which the mdr1 or mrp1 gene has been retransfected back in should help resolve this issue.

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