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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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Related in: MedlinePlus

GLTP response to tunicamycin and heat shock.A) GLTP mRNA expression determined by qPCR in cells undergoing heat shock and ER-stress. HSF cells were either heat shocked at 42°C for one hour, following different recovery periods (0–24 hours) at 37°C, or treated with tunicamycin (10 µg/ml) for 24 hours. Results are expressed as means +/− SD of three independent experiments. B) GLTP protein levels in HSF cells analyzed with Western blot after one hour heat shock followed by recovery, or tunicamycin at 10 µg/ml for 24 hours. C = control and T = tunicamycin, β-actin was used as a loading control.
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pone-0070283-g010: GLTP response to tunicamycin and heat shock.A) GLTP mRNA expression determined by qPCR in cells undergoing heat shock and ER-stress. HSF cells were either heat shocked at 42°C for one hour, following different recovery periods (0–24 hours) at 37°C, or treated with tunicamycin (10 µg/ml) for 24 hours. Results are expressed as means +/− SD of three independent experiments. B) GLTP protein levels in HSF cells analyzed with Western blot after one hour heat shock followed by recovery, or tunicamycin at 10 µg/ml for 24 hours. C = control and T = tunicamycin, β-actin was used as a loading control.

Mentions: Since both monensin and BFA cause structural changes in the ER and Golgi compartments and subsequently place the cell under great stress, we examined how two other types of stresses affect GLTP. HSF cells were either heat shocked or treated with tunicamycin, a nucleoside antibiotic that inhibits protein glycosylation and induces ER-stress [40]. In Figure 10 it can be seen that neither heat shocking nor tunicamycin cause any significant changes in GLTP expression levels. This further strengthens the argument that the accumulation of simple glycosphingolipids indeed could be the cause of the increased GLTP amounts and not the ER-stress per se.


Glycolipid transfer protein expression is affected by glycosphingolipid synthesis.

Kjellberg MA, Mattjus P - PLoS ONE (2013)

GLTP response to tunicamycin and heat shock.A) GLTP mRNA expression determined by qPCR in cells undergoing heat shock and ER-stress. HSF cells were either heat shocked at 42°C for one hour, following different recovery periods (0–24 hours) at 37°C, or treated with tunicamycin (10 µg/ml) for 24 hours. Results are expressed as means +/− SD of three independent experiments. B) GLTP protein levels in HSF cells analyzed with Western blot after one hour heat shock followed by recovery, or tunicamycin at 10 µg/ml for 24 hours. C = control and T = tunicamycin, β-actin was used as a loading control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0070283-g010: GLTP response to tunicamycin and heat shock.A) GLTP mRNA expression determined by qPCR in cells undergoing heat shock and ER-stress. HSF cells were either heat shocked at 42°C for one hour, following different recovery periods (0–24 hours) at 37°C, or treated with tunicamycin (10 µg/ml) for 24 hours. Results are expressed as means +/− SD of three independent experiments. B) GLTP protein levels in HSF cells analyzed with Western blot after one hour heat shock followed by recovery, or tunicamycin at 10 µg/ml for 24 hours. C = control and T = tunicamycin, β-actin was used as a loading control.
Mentions: Since both monensin and BFA cause structural changes in the ER and Golgi compartments and subsequently place the cell under great stress, we examined how two other types of stresses affect GLTP. HSF cells were either heat shocked or treated with tunicamycin, a nucleoside antibiotic that inhibits protein glycosylation and induces ER-stress [40]. In Figure 10 it can be seen that neither heat shocking nor tunicamycin cause any significant changes in GLTP expression levels. This further strengthens the argument that the accumulation of simple glycosphingolipids indeed could be the cause of the increased GLTP amounts and not the ER-stress per se.

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
Related in: MedlinePlus