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Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

Kjellberg MA, Mattjus P - PLoS ONE (2013)

Bottom Line: Cells that does not synthesize glucosylceramide neither express GLTPs.We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide.We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry, Department of Biosciences, Åbo Akademi University, Turku, Finland.

ABSTRACT
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.

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GLTP expression, GlcCer, Galcer, LacCer, ceramide and sphingomyelin synthesis in HSF cells as a function of BFA or monensin treatment.A) 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) 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. C) 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.
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pone-0070283-g001: GLTP expression, GlcCer, Galcer, LacCer, ceramide and sphingomyelin synthesis in HSF cells as a function of BFA or monensin treatment.A) 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) 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. C) 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.

Mentions: In order to analyze how GLTP is affected by changes in GSL metabolism, we first examined if and how GLTP expression is affected in HSF cells as a result of treatment with BFA and monensin. We examined the expression of GLTP as a function of different concentrations of BFA and monensin after a 24-hour treatment (Figure 1A). GLTP expression levels were analyzed using reverse transcription qPCR, using 18S rRNA as an internal control. The results show that both compounds increase GLTP expression significantly in a concentration dependent manner. For BFA, GLTP expression reaches a plateau at concentrations as low as 0.01 µg/ml, whereas monensin induced GLTP expression appears to have a more linear increase, reaching a plateau at around 5 µg/ml.


Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

Kjellberg MA, Mattjus P - PLoS ONE (2013)

GLTP expression, GlcCer, Galcer, LacCer, ceramide and sphingomyelin synthesis in HSF cells as a function of BFA or monensin treatment.A) 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) 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. C) 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3722133&req=5

pone-0070283-g001: GLTP expression, GlcCer, Galcer, LacCer, ceramide and sphingomyelin synthesis in HSF cells as a function of BFA or monensin treatment.A) 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) 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. C) 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.
Mentions: In order to analyze how GLTP is affected by changes in GSL metabolism, we first examined if and how GLTP expression is affected in HSF cells as a result of treatment with BFA and monensin. We examined the expression of GLTP as a function of different concentrations of BFA and monensin after a 24-hour treatment (Figure 1A). GLTP expression levels were analyzed using reverse transcription qPCR, using 18S rRNA as an internal control. The results show that both compounds increase GLTP expression significantly in a concentration dependent manner. For BFA, GLTP expression reaches a plateau at concentrations as low as 0.01 µg/ml, whereas monensin induced GLTP expression appears to have a more linear increase, reaching a plateau at around 5 µg/ml.

Bottom Line: Cells that does not synthesize glucosylceramide neither express GLTPs.We found that GLTP expression, both at the mRNA and protein levels, is elevated in cells that accumulate glucosylceramide.We also found that an 80% loss of glucosylceramide due to glucosylceramide synthase knockdown resulted in a significant reduction in the expression of GLTP.

View Article: PubMed Central - PubMed

Affiliation: Biochemistry, Department of Biosciences, Åbo Akademi University, Turku, Finland.

ABSTRACT
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.

Show MeSH