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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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Expression of GLTP in GlcCerS knockdown HSF cells.The GLTP levels were analyzed with both qPCR and Western blot in HSF cells with 80% down-regulated expression of the GlcCerS gene. The GLTP gene level was normalized to the level in mock-transfected HSF cells, and β-actin was used as a loading control in the GLTP protein expression analysis.
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pone-0070283-g008: Expression of GLTP in GlcCerS knockdown HSF cells.The GLTP levels were analyzed with both qPCR and Western blot in HSF cells with 80% down-regulated expression of the GlcCerS gene. The GLTP gene level was normalized to the level in mock-transfected HSF cells, and β-actin was used as a loading control in the GLTP protein expression analysis.

Mentions: GlcCer is synthesized from ceramide by GlcCerS at the cytosolic leaflet of early Golgi membranes. GlcCerS (also called UGCG, NM_003358) was down-regulated using the manufacturers instructions. The expression of the GlcCerS gene was down-regulated by approximately 80% compared to the expression of GlcCerS in normal HSF cells (Fig. 7A). The GlcCerS expression levels were analyzed using reverse transcription qPCR. The synthesis of GlcCer, GalCer and LacCer was significantly decreased, and the Cer and SM levels increased (Fig. 7B). Inline with the GlcCer synthesis inhibitor experiments (Figure 6A), the expression of GLTP was significantly reduced by the down-regulation of GlcCerS, both on mRNA and protein levels (Figure 8).


Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

Kjellberg MA, Mattjus P - PLoS ONE (2013)

Expression of GLTP in GlcCerS knockdown HSF cells.The GLTP levels were analyzed with both qPCR and Western blot in HSF cells with 80% down-regulated expression of the GlcCerS gene. The GLTP gene level was normalized to the level in mock-transfected HSF cells, and β-actin was used as a loading control in the GLTP protein expression analysis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3722133&req=5

pone-0070283-g008: Expression of GLTP in GlcCerS knockdown HSF cells.The GLTP levels were analyzed with both qPCR and Western blot in HSF cells with 80% down-regulated expression of the GlcCerS gene. The GLTP gene level was normalized to the level in mock-transfected HSF cells, and β-actin was used as a loading control in the GLTP protein expression analysis.
Mentions: GlcCer is synthesized from ceramide by GlcCerS at the cytosolic leaflet of early Golgi membranes. GlcCerS (also called UGCG, NM_003358) was down-regulated using the manufacturers instructions. The expression of the GlcCerS gene was down-regulated by approximately 80% compared to the expression of GlcCerS in normal HSF cells (Fig. 7A). The GlcCerS expression levels were analyzed using reverse transcription qPCR. The synthesis of GlcCer, GalCer and LacCer was significantly decreased, and the Cer and SM levels increased (Fig. 7B). Inline with the GlcCer synthesis inhibitor experiments (Figure 6A), the expression of GLTP was significantly reduced by the down-regulation of GlcCerS, both on mRNA and protein levels (Figure 8).

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