Glycolipid transfer protein knockout disrupts vesicle trafficking to the plasma membrane.

The glycolipid transfer protein (GLTP) has been linked to many cellular processes aside from its best-known in vitro function as a lipid transport protein. It has been proposed to act as a sensor and regulator of glycosphingolipid homeostasis in cells. Furthermore, through its previously determined interaction with the endoplasmic reticulum membrane protein VAP-A (vesicle-associated membrane protein-associated protein A), GLTP may also be involved in facilitating vesicular transport in cells. In this study, we characterized the phenotype of CRISPR/Cas9-mediated GLTP KO HeLa cells. We showed that motility, three-dimensional growth, and cellular metabolism were all altered by GLTP knockout. Expression of a GLTP mutant incapable of binding VAP disrupted cell spheroid formation, indicating that the GLTP-VAP interaction is linked to cellular adhesion, cohesion, and three-dimensional growth. Most notably, we found evidence that GLTP, through its interaction with VAP-A, affects vesicular trafficking, marking the first cellular process discovered to be directly impacted by a change in GLTP expression.

Who moves the sphinx? An overview of intracellular sphingolipid transport.

Lipid bilayers function as boundaries that enclose their content from the surrounding media, and the composition of different membrane types is accurately and dynamically tailored so that they can perform their function. To achieve this balance, lipid biosynthetic machinery and lipid trafficking events are intertwined into an elegant network. In this review, we focus on the intracellular movement of sphingolipids mediated by sphingolipid transfer proteins. Additionally, we will focus on the best characterized and understood mammalian sphingolipid transfer proteins and provide an overview of how they are hypothesized to function. Some are already well understood, while others remain enigmatic. A few are actual lipid transfer proteins, moving lipids from membrane to membrane, while others may have more of a sensor role, possibly reacting to changes in the concentrations of their ligands. Considering the substrates available for cytosolic sphingolipid transfer proteins, one open question that is discussed is whether galactosylceramide is a target. Another question is the exact mechanics by which sphingolipid transfer proteins are targeted to different organelles, such as how four phosphate adapter protein-2, FAPP2 is targeted to the endoplasmic reticulum. The aim of this review is to discuss what is known within the field today and to provide a basic understanding of how these proteins may work.

Indirect Lipid Transfer Protein Activity Measurements Using Quantification of Glycosphingolipid Production.

Here we summarize how glycosphingolipid production can be followed using metabolic labeling with radiolabeled lipid precursors. No assays are available yet that directly would address the lipid transfer protein activity in vivo. Therefore, these approaches can serve as tools to indirectly study the lipid transfer protein activity in cells, by monitoring their impact on the glycosphingolipid homeostasis.

Glucosylceramide acyl chain length is sensed by the glycolipid transfer protein.

The glycolipid transfer protein, GLTP, can be found in the cytoplasm, and it has a FFAT-like motif (two phenylalanines in an acidic tract) that targets it to the endoplasmic reticulum (ER). We have previously shown that GLTP can bind to a transmembrane ER protein, vesicle-associated membrane protein-associated protein A (VAP-A), which is involved in a wide range of ER functions. We have addressed the mechanisms that might regulate the association between GLTP and the VAP proteins by studying the capacity of GLTP to recognize different N-linked acyl chain species of glucosylceramide. We used surface plasmon resonance and a lipid transfer competition assay to show that GLTP prefers shorter N-linked fully saturated acyl chain glucosylceramides, such as C8, C12, and C16, whereas long C18, C20, and C24-glucosylceramides are all bound more weakly and transported more slowly than their shorter counterparts. Changes in the intrinsic GLTP tryptophan fluorescence blueshifts, also indicate a break-point between C16- and C18-glucosylceramide in the GLTP sensing ability. It has long been postulated that GLTP would be a sensor in the sphingolipid synthesis machinery, but how this mechanistically occurs has not been addressed before. It is unclear what proteins the GLTP VAP association would influence. Here we found that if GLTP has a bound GlcCer the association with VAP-A is weaker. We have also used a formula for identifying putative FFAT-domains, and we identified several potential VAP-interactors within the ceramide and sphingolipid synthesis pathways that could be candidates for regulation by GLTP.

Purification and Validation of Lipid Transfer Proteins.

Understanding the holistic picture of lipid homeostasis not only involves the analysis of synthesis and breakdown of lipids but also requires a thorough understanding of their transport. The transport of lipid monomers in an aqueous environment is facilitated by different lipid transfer proteins. Their universal feature is the shielding or encapsulation of the hydrophobic part of the lipid, consequently overcoming the poor solubility of lipids in water. Here we describe a method to purify lipid transfer proteins using bacterial expression. We also present three methods to validate their transfer activity.

Alternation in the glycolipid transfer protein expression causes changes in the cellular lipidome.

The glycolipid transfer protein (GLTP) catalyzes the binding and transport of glycolipids, but not phospholipids or neutral lipids. With its all-alpha helical fold, it is the founding member for a new superfamily, however its biological role still remains unclear. We have analyzed changes in the HeLa cell lipidome in response to down- and up-regulation of GLTP expression. We used metabolic labeling and thin layer chromatography analysis, complemented with a lipidomics mass spectroscopic approach. HeLa cells were treated with GLTP siRNA or were transiently overexpressing the GLTP gene. We identified eight different lipid classes that changed as a result of the GLTP down- or up-regulation treatments; glucosylceramide, lactosylceramide, globotriaosylceramide, ceramide, sphingomyelin, cholesterol-esters, diacylglycerol and phosphatidylserine. We discovered that the amount of globotriaosylceramide (Gb3) was extensively lowered after down-regulation of GLTP. Further, an up-regulation of GLTP caused a substantial increase in both the Gb3 and glucosylceramide levels compared to the controls. Total galactosylceramide levels remained unchanged. Both lactosylceramide and ceramide showed small changes, an increase with increasing GLTP and a decrease in the HeLa cell GLTP knockdowns. The cholesterol-esters and diacylglycerol masses increased in cells that had upregulated GLTP protein levels, wheras down-regulation did not affect their amounts. For the glycerophospholipids, phosphatidylserine was the only species that was lower in GLTP overexpressing cells. Phosphatidylethanolamine, phosphatidylglyerol and phosphatidylinositol remained unaltered. A total of 142 lipid species were profiled and quantified using shotgun lipidomics analyses. This work provides for the first time insights into how alternations in the levels of a protein that binds and transfers glycolipids affects the cellular lipid metabolism. We discuss the observed changes in the lipidome and how these relate to GLTP. We suggest, that GLTP not only could be a significant player in cellular sphingolipid metabolism, but also could have a much broader role in the overall lipid metabolism.