Pre- and post-Golgi translocation of glucosylceramide in glycosphingolipid synthesis

Glycosphingolipids are controlled by the spatial organization of their metabolism and by transport specificity. Using immunoelectron microscopy, we localize to the Golgi stack the glycosyltransferases that produce glucosylceramide (GlcCer), lactosylceramide (LacCer), and GM3. GlcCer is synthesized on the cytosolic side and must translocate across to the Golgi lumen for LacCer synthesis. However, only very little natural GlcCer translocates across the Golgi in vitro. As GlcCer reaches the cell surface when Golgi vesicular trafficking is inhibited, it must translocate across a post-Golgi membrane. Concanamycin, a vacuolar proton pump inhibitor, blocks translocation independently of multidrug transporters that are known to translocate short-chain GlcCer. Concanamycin did not reduce LacCer and GM3 synthesis. Thus, GlcCer destined for glycolipid synthesis follows a different pathway and transports back into the endoplasmic reticulum (ER) via the late Golgi protein FAPP2. FAPP2 knockdown strongly reduces GM3 synthesis. Overall, we show that newly synthesized GlcCer enters two pathways: one toward the noncytosolic surface of a post-Golgi membrane and one via the ER toward the Golgi lumen LacCer synthase

Interaction of 3â-amino-5-cholestene with phospholipids in binary and ternary bilayer membranes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Langmuir, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/la203589u.3β-Amino-5-cholestene (aminocholesterol) is a synthetic sterol whose properties in bilayer membranes have been examined. In fluid palmitoyl sphingomyelin (PSM) bilayers, aminocholesterol and cholesterol were equally effective in increasing acyl chain order, based on changes in diphenylhexatriene (DPH) anisotropy. In fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, aminocholesterol ordered acyl chains, but slightly less efficiently than cholesterol. Aminocholesterol eliminated the PSM and DPPC gel-to-liquid crystalline phase transition enthalpy linearly with concentration, and the enthalpy approached zero at 30 mol% sterol. Whereas cholesterol was able to increase the thermostability of ordered PSM domains in a fluid bilayer, aminocholesterol under equal conditions failed to do this, suggesting that its interaction with PSM was not as favorable as cholesterol’s. In ternary mixed bilayers, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), PSM or DPPC, and cholesterol at proportions to contain a liquid-ordered phase (60:40 by mol of POPC and PSM or DPPC, and 30 mol% cholesterol), the average life-time of trans parinaric acid (tPA) was close to 20 ns. When cholesterol was replaced with aminocholesterol in such mixed bilayers, the average life-time of tPA was only marginally shorter (about 18 ns). This observation, together with acyl chain ordering data, clearly shows that aminocholesterol was able to form a liquid-ordered phase with saturated PSM or DPPC. We conclude that aminocholesterol should be a good sterol replacement in model membrane systems for which a partial positive charge is deemed beneficial

Effects of bile salts on glucosylceramide containing membranes

AbstractThe glycolipid transfer protein (GLTP) is capable of transporting glycolipids from a donor membrane, through the aqueous environment, to an acceptor membrane. The GLTP mediated glycolipid transfer from sphingomyelin membranes is very slow. In contrast, the transfer is fast from membranes composed of phosphatidylcholine. The lateral glycolipid membrane organization is known to be driven by their tendency to mix non-randomly with different membrane lipids. Consequently, the properties of the membrane lipids surrounding the glycolipids play an important role in the ability of GLTP to bind and transfer its substrates. Since GLTP transfer of glycolipids is almost nonexistent from sphingomyelin membranes, we have used this exceptionality to investigate if membrane intercalators can alter the membrane packing and induce glycolipid transfer. We found that the bile salts cholate, deoxycholate, taurocholate and taurodeoxycholate, cause glucosylceramide to become transferrable by GLTP. Other compounds, such as single chain lipids, ceramide and nonionic surfactants, that have membrane-perturbing effects, did not affect the transfer capability of GLTP. We speculate that the strong hydrogen bonding network formed in the interfacial region of glycosphingolipid–sphingomyelin membranes is disrupted by the membrane partition of the bile salts causing the glycosphingolipid to become transferrable

Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

Members of the glycolipid transfer protein superfamily (GLTP) are found from animals and fungi to plants and red micro-alga. Eukaryotes that encode the glucosylceramide synthase responsible for the synthesis of glucosylceramide, the precursor for most glycosphingolipids, also produce GLTPs. Cells that does not synthesize glucosylceramide neither express GLTPs. Based on this genetic relationship there must be a strong correlation between the synthesis of glucosylceramide and GLTPs. To regulate the levels of glycolipids we have used inhibitors of intracellular trafficking, glycosphingolipid synthesis and degradation, and small interfering RNA to down-regulate the activity of glucosylceramide synthase activity. We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide. Monensin and brefeldin A block intracellular vesicular transport mechanisms. Brefeldin A treatment leads to accumulation of newly synthesized glucosylceramide, galactosylceramide and lactosylceramide in a fused endoplasmic reticulum-Golgi complex. On the other hand, inhibiting glycosphingolipid degradation with conduritol-B-epoxide, that generates glucosylceramide accumulation in the lysosomes, did not affect the levels of GLTP. However, glycosphingolipid synthesis inhibitors like PDMP, NB-DNJ and myriocin, all decreased glucosylceramide and GLTP below normal levels. We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP. We show here that interfering with membrane trafficking events and simple neutral glycosphingolipid synthesis will affect the expression of GLTP. We postulate that a change in the glucosylceramide balance causes a response in the GLTP expression, and put forward that GLTP might play a role in lipid directing and sensing of glucosylceramide at the ER-Golgi interface

Correction: Glycolipid Transfer Protein Expression Is Affected by Glycosphingolipid Synthesis.

[This corrects the article DOI: 10.1371/journal.pone.0070283.]

Expression of GLTP in GlcCerS knockdown cells with impaired intracellular membrane trafficking caused by BFA and monensin.

<p><b>A)</b>³H-sphinganine incorporation into GlcCer, GalCer and Cer as well as GLTP mRNA levels (filled circles) in GlcCerS KD HSF cells treated with BFA (0.01 µg/ml) and <b>B)</b> treated with monensin (5 µg/ml). Two asterisks (**), p<0.01 and three asterisks (***), p<0.005 indicate the statistical significance compared to the controls.</p

GlcCer, GalCer and Cer and GLTP mRNA levels in HSF cells co-treated with BFA/monensin and different GlcCer synthesis inhibitors.

<p><b>A)</b>³H-sphinganine incorporation and GLTP mRNA levels (filled circles) in HSF cells treated with either BFA (0.01 µg/ml) as well as HSF cells co-treated with BFA in addition to PDMP (50 µM), NB-DNJ (250 µM) and myriocin (25 µM). <b>B)</b>³H-sphinganine incorporation and GLTP mRNA levels (filled circles) in HSF cells treated with either monensin (5 µg/ml) as well as HSF cells co-treated with monensin in addition to PDMP (50 µM), NB-DNJ (250 µM) and myriocin (25 µM). HSF cells treated with myriocin were labeled with ³H-palmitic acid. The results are expressed as means +/− SD of three independent experiments. Two asterisks (**), p<0.01 and three asterisks (***), p<0.005 indicate the statistical significance compared to the controls.</p

Effect of CBE treatment on GLTP mRNA and protein levels.

<p>HSF cells were treated with CBE (250 µM) for 5 days. <b>A)</b> Simple sphingolipid levels were determined by ³H-sphinganine incorporation and TLC analysis. Incorporation of ³H-sphinganine into GlcCer, GalCer, LacCer, SM and ceramide in untreated controls compared to CBE treated cells. GlcCer (***) levels in CBE treated cells are significantly higher than their controls (p<0.005). <b>B)</b> The GLTP mRNA expression was determined by qPCR in CBE treated HSF cells. Results are expressed as means +/− SD of three independent qPCR experiments. <b>C)</b> The total lipid mass of GlcCer, GalCer and LacCer as visualized by orcinol-sulphuric acid on a HPTLC plate, as well as lipid band intensities semi-quantified using ImageJ software and normalized to the controls. GlcCer (**) levels in CBE treated cells are significantly higher than their controls (p<0.01). <b>D)</b> Western blot of cells treated as described above, C = untreated controls, CBE = 5 day CBE treatment (250 µM). β-Actin was used as a loading control.</p

GLTP expression, GlcCer, Galcer, LacCer, ceramide and sphingomyelin synthesis in HSF cells as a function of BFA or monensin treatment.

<p><b>A)</b> HFS cells were treated with BFA (left panel) or monensin (right panel) with increasing concentrations for 24 hours. The GLTP mRNA expression levels were analyzed using qPCR and corrected to an 18S rRNA internal control. <b>B)</b> qPCR analysis of GLTP expression (filled circles) and sphingolipid levels in HSF cells treated with BFA (0.01 µg/ml, left panel) or monensin (5 µg/ml, right panel) for 6, 12 and 24 hours, ³H-sphinganine incorporation into the sphingolipids was analyzed using TLC. qPCR results are expressed as means +/− SD of at least three independent experiments. The data for the incorporation of the radiolabeled ³H-sphinganine are from at least three different experiments, and the results are normalized to the controls. Two asterisks (**), p<0.01 and three asterisks (***), p<0.005 indicate the statistical significance compared to the controls. <b>C)</b> Western blot analysis of GLTP levels in HSF cells treated with BFA (0.01 µg/ml) or monensin (5 µg/ml) for 24 hours. C = untreated control, B = BFA treatment, M = monensin treatment. β-Actin was used as a loading control. The representative blot shown here was chosen from one of three independent experiments with similar results.</p