Internalization and Sorting of Plasma Membrane Sphingolipid Analogues in Differentiating Oligodendrocytes

Watanabe, Rikio; Asakura, Koichi; Rodriguez, Moses; Pagano, Richard E.

doi:10.1046/j.1471-4159.1999.0731375.x

Cited by 29 publications

(20 citation statements)

References 37 publications

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“…As shown in Fig. 9, exogenous BODIPY-SGC by itself could also be taken up by H9C2 cells into an organelle that is distinct from mitochondria, presumably the Golgi complex or endoplasmic reticulum, as shown in other studies using oligodendrocytes and fibroblasts (50,51). However, when CTX A3 was included in the solution, significant amounts of exogenously introduced BODIPY-SGC co-localized with mitochondria ( Fig.…”

Section: Sgc-facilitated Ctx Internalization Occurs After Ctx Pore Fosupporting

confidence: 57%

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Glycosphingolipid-facilitated Membrane Insertion and Internalization of Cobra Cardiotoxin

Wang

Liu

Lee

et al. 2006

Journal of Biological Chemistry

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Cobra cardiotoxins, a family of basic polypeptides having lipidand heparin-binding capacities similar to the cell-penetrating peptides, induce severe tissue necrosis and systolic heart arrest in snakebite victims. Whereas cardiotoxins are specifically retained on the cell surface via heparan sulfate-mediated processes, their lipid binding ability appears to be responsible, at least in part, for cardiotoxin-induced membrane leakage and cell death. Although the exact role of lipids involved in toxin-mediated cytotoxicity remains largely unknown, monoclonal anti-sulfatide antibody O4 has recently been shown to inhibit the action of CTX A3, the major cardiotoxin from Taiwan cobra venom, on cardiomyocytes by preventing cardiotoxin-induced membrane leakage and CTX A3 internalization into mitochondria. Here, we show that anti-sulfatide acts by blocking the binding of CTX A3 to the sulfatides in the plasma membrane to prevent sulfatide-dependent CTX A3 membrane pore formation and internalization. We also describe the crystal structure of a CTX A3-sulfatide complex in a membrane-like environment at 2.3 Å resolution. The unexpected orientation of the sulfatide fatty chains in the structure allows prediction of the mode of toxin insertion into the plasma membrane. CTX A3 recognizes both the headgroup and the ceramide interfacial region of sulfatide to induce a lipid conformational change that may play a key role in CTX A3 oligomerization and cellular internalization. This proposed lipid-mediated toxin translocation mechanism may also shed light on the cellular uptake mechanism of the amphiphilic cell-penetrating peptides known to involve multiple internalization pathways.Glycosphingolipids in eukaryotic cells have important biological functions in membrane trafficking and cell signaling (1, 2). The number of disease-related proteins found to interact specifically with glycosphingolipids is also increasing (3-5). For instance, recent studies have shown that natural killer cells can be activated by glycosylceramides from the cell surface of Gram-negative bacteria that do not contain lipopolysaccharide (6, 7). There is also evidence indicating that, in response to tumor necrosis factor-␣, trafficking of ganglioside GD3 from the plasma membrane to mitochondria can trigger several cell death signaling responses (3,8,9). Although the molecular mechanisms accounting for the observed glycosphingolipid transport and glycolipidtriggered cell responses remain elusive, recent progress in determining the three-dimensional structure of protein⅐glycosphingolipid complexes has shed some light on the process (10 -13). These glycosphingolipid-binding proteins function by either extracting lipids from the membrane and forming a water-soluble protein⅐lipid complex, as in the cases of glycosphingolipid transfer protein, saposin B, and lipid-presenting CD1a (10 -12), or by localizing to a glycosphingolipid domain of the plasma membrane and eventually being internalized via the endocytic pathway, as seen for cholera and Shiga toxins (13,14). Aft...

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Section: Sgc-facilitated Ctx Internalization Occurs After Ctx Pore Fosupporting

confidence: 57%

“…To test whether CTX A3 internalization is indeed a membrane-delimited process, we examined the effects of CTX A3 on SGC internalization using fluorescently labeled SGC (BODIPY-SGC) (49,50). As shown in Fig.…”

Section: Sgc-facilitated Ctx Internalization Occurs After Ctx Pore Fomentioning

confidence: 99%

Glycosphingolipid-facilitated Membrane Insertion and Internalization of Cobra Cardiotoxin

Wang

Liu

Lee

et al. 2006

Journal of Biological Chemistry

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“…C 5 -BODIPY-fatty acid labeled analogs of galactosylceramide (GalCer), LacCer, globoside, GM 1 , and sulfatide, and 6-[(7-nitro-benz-2-oxa-1,3-diazol-4-yl)amino]caproic acid labeled-LacCer (NBD-LacCer) were synthesized and purified as described (Martin and Pagano, 1994;Watanabe et al, 1999;Puri et al, 2001). The d-isomer of NBD-phosphatidylcholine (NBD-d-PC) was prepared as described (Martin and Pagano, 1987).…”

Section: Fluorescent Lipids Toxins and Other Reagentsmentioning

confidence: 99%

Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

Singh

Puri

Valiyaveettil

et al. 2003

MBoC

234

249

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We studied the endocytosis of fluorescent glycosphingolipid (GSL) analogs in various cell types using pathway-specific inhibitors and colocalization studies with endocytic markers and DsRed caveolin-1 (cav-1). Based on inhibitor studies, all GSLs tested were internalized predominantly (Ͼ80%) by a clathrin-independent, caveolar-related mechanism, regardless of cell type. In addition, fluorescent lactosylceramide (LacCer) colocalized with DsRed-cav-1 in vesicular structures upon endocytosis in rat fibroblasts. The internalization mechanism for GSLs was unaffected by varying the carbohydrate headgroup or sphingosine backbone chain length; however, a fluorescent phosphatidylcholine analog was not internalized via caveolae, suggesting that the GSL ceramide core may be important for caveolar uptake. Internalization of fluorescent LacCer was reduced 80 -90% in cell types with low cav-1, but was dramatically stimulated by cav-1 overexpression. However, even in cells with low levels of cav-1, residual LacCer internalization was clathrin independent. In contrast, cholera toxin B subunit (CtxB), which binds endogenous GM 1 , was internalized via clathrin-independent endocytosis in cells with high cav-1 expression, whereas significant clathrin-dependent uptake occurred in cells with low cav-1. Fluorescent GM 1 , normally internalized by clathrin-independent endocytosis in HeLa cells with low cav-1, was induced to partially internalize via the clathrin pathway in the presence of CtxB. These results suggest that GSL analogs are selectively internalized via a caveolar-related mechanism in most cell types, whereas CtxB may undergo "pathway switching" when cav-1 levels are low.

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“…Typically, such analogues contain one or two truncated chains, consisting of only 2 (acetyl; C 2 ), 6 (hexanoyl; C 6 ) or 8 (octanoyl; C 8 ) carbon atoms, and easily insert as monomers in a lipid bilayer. Being structurally closely related to their natural counterparts, these semisynthetic analogues appear to be transported and metabolised normally (Watanabe et al, 1999). Some lipid analogues elicit strong cellular responses.…”

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confidence: 99%

N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx

et al. 2004

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Anticancer drugs generally have intracellular targets, implicating transport over the plasma membrane. For amphiphilic agents, such as the anthracycline doxorubicin, this occurs by passive diffusion. We investigated whether exogenous membrane-permeable lipid analogues improve this drug influx. Combinations of drugs and lipid analogues were coadministered to cultured endothelial cells and various tumour cell lines, and subsequent drug accumulation in cells was quantified. We identified N-hexanoyl-sphingomyelin (SM) as a potent enhancer of drug uptake. Low micromolar amounts of this short-chain sphingolipid, being not toxic itself, enhanced the uptake of doxorubicin up to 300% and decreased its EC 50 toxicity values seven-to 14-fold. N-hexanoyl SM acts at the level of the plasma membrane, but was found not incorporated in (isolated) lipid rafts, and artificial disruption or elimination of raft constituents did not affect its drug uptake-enhancing effect. Further, any mechanistic role of the endocytic machinery, membrane leakage or ABCtransporter-mediated efflux could be excluded. Finally, a correlation was established between the degree of drug lipophilicity, as defined by partitioning in a two-phase octanol -water system, and the susceptibility of the drug towards the uptake-enhancing effect of the sphingolipid. A clear optimum was found for amphiphilic drugs, such as doxorubicin, epirubicin and topotecan, indicating that N-hexanoyl-SM might act by modulating the average degree of plasma membrane lipophilicity, in turn facilitating transbilayer drug diffusion. The concept of short-chain sphingolipids as amphiphilic drug potentiators provides novel opportunities for improving drug delivery technologies.

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Internalization and Sorting of Plasma Membrane Sphingolipid Analogues in Differentiating Oligodendrocytes

Cited by 29 publications

References 37 publications

Glycosphingolipid-facilitated Membrane Insertion and Internalization of Cobra Cardiotoxin

Glycosphingolipid-facilitated Membrane Insertion and Internalization of Cobra Cardiotoxin

Selective Caveolin-1–dependent Endocytosis of Glycosphingolipids

N-hexanoyl-sphingomyelin potentiates in vitro doxorubicin cytotoxicity by enhancing its cellular influx

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