Glycolipid Transfer Protein Research Articles

Nonvesicular lipid transfer drives myelin growth in the central nervous system.

Oligodendrocytes extend numerous cellular processes that wrap multiple times around axons to generate lipid-rich myelin sheaths. Myelin biogenesis requires an enormously productive biosynthetic machinery for generating and delivering these large amounts of newly synthesized lipids. Yet, a complete understanding of this process remains elusive. Utilizing volume electron microscopy, we demonstrate that the oligodendroglial endoplasmic reticulum (ER) is enriched in developing myelin, extending into and making contact with the innermost myelin layer where growth occurs. We explore the possibility of transfer of lipids from the ER to myelin, and find that the glycolipid transfer protein (GLTP), implicated in nonvesicular lipid transport, is highly enriched in the growing myelin sheath. Mice with a specific knockout of Gltp in oligodendrocytes exhibit ER pathology, hypomyelination and a decrease in myelin glycolipid content. In summary, our results demonstrate a role for nonvesicular lipid transport in CNS myelin growth, revealing a cellular pathway in developmental myelination.

Superfamily of glycolipid transfer proteins (GLTPs): Accelerated cell death 11-like (ACD11) enhances salt tolerance in apple

For adaptation to challenging environments, plants have evolved various response mechanisms, such as inducing the expression of many stress genes, thereby increasing the resistance. Accelerated cell death (ACD) genes have been widely studied in plant senescence and defense responses. However, there are few reports on the role of ACD genes in abiotic stresses in plants. In this experiment, ACD11 was successfully isolated from apples (Malus baccata) and found to be mainly expressed in apple fruits and roots. The response elements of abiotic stresses were also found in the promoter of MbACD11. Furthermore, MbACD11 expression was induced by various abiotic stresses, especially salt treatment. After overexpression of MbACD11 in apple seedlings, callis and Arabidopsis thaliana, all showed enhanced tolerance to salt stress. Under salt conditions, MbACD11-OE showed higher fresh weight and chlorophyll content compared to the WT. It also exhibited lower relative electrical conductivity, malondialdehyde (MDA) content, and reactive oxygen species (ROS) accumulation than the WT. The expression of salt stress-related genes was higher in MbACD11-OE apple seedlings than in WT. In salt stress condition, the apple MbACD11 gene reduced ROS accumulation and affected the expression of salt stress-related genes, hence enhancing tolerance.

Exploration of the mechanism of reinfusion of fresh autologous blood in type 2 diabetes mice to induce macrophage polarization and inhibit erythrocyte damage.

Type 2 diabetes triggers an inflammatory response that can damage red blood cells. M2 macrophages have inhibitory effects on inflammation, and play an important role in tissue damage repair and fibrosis. Autologous blood transfusion has the potential to inhibit red blood cell damage by mediating macrophage polarization. Swiss mice were used to establish a suitable type 2 diabetes model, and autologous blood transfusion was carried out. The mice were killed, the blood of the mice was collected and CD14+ monocytes were sorted. The expression levels of phenotypic molecules CD16, CD32 and CD206 in CD14+ monocytes were analyzed by flow cytometry. The proportion of M1 and M2 macrophages were analyzed by flow cytometry. The Q value, P50 , 2,3-diphosphoglycerate and Na+ -K+ -ATPase of red blood cells were detected. The red blood cell osmotic fragility test analyzed the red blood cell osmotic fragility. Western blot analysis was used to analyze the expression changes of erythrocyte surface membrane proteins or transporters erythrocyte membrane protein band 4.1, sphingosine-1-phosphate, glycolipid transfer protein and signal peptide peptidase-like 2A. Autologous blood transfusion induced a significant increase in the number of macrophages. The state and capacity of blood cells improved with autologous blood transfusion. Reinfusion of fresh autologous blood in type 2 diabetes mice made erythrocytes shrink. The expression of erythrocyte-related proteins proved that the erythrocyte injury in the reinfusion of fresh autologous blood + type 2 diabetes group was significantly reduced. The reinfusion of fresh autologous blood into the body of patients with type 2 diabetes can induce macrophage polarization to M2, thereby inhibiting red blood cell damage.

A comprehensive investigation of lipid-transfer proteins from Cicer arietinum disentangles their role in plant defense against Helicoverpa armigera-infestation.

Lipid Transfer Proteins (LTPs) play a crucial role in synthesizing lipid barrier polymers and are involved in defense signaling during pest and pathogen attacks. Although LTPs are conserved with multifaceted roles in plants, these are not yet identified and characterized in Cicer arietinum. In this study, a genome-wide analysis of LTPs was executed and their physiochemical properties, biochemical function, gene structure analysis, chromosomal localization, promoter analysis, gene duplication, and evolutionary analysis were performed using in silico tools. Furthermore, tissue-specific expression analysis and gene expression analysis during pest attack was also conducted for the LTPs. A total of 48 LTPs were identified and named as CaLTPs. They were predicted to be small unstable proteins with "Glycolipid transfer protein" and "Alpha-Amylase Inhibitors, Lipid Transfer and Seed Storage" domains, that are translocated to the extracellular region. CaLTPs were predicted to possess 3-4 introns and were located on all the eight chromosomes of chickpea with half of the CaLTPs being localized on chromosomes 4, 5, and 6, and found to be closely related to LTPs of Arabidopsis thaliana and Medicago trancatula. Gene duplication and synteny analysis revealed that most of the CaLTPs have evolved due to tandem or segmental gene duplication and were subjected to purifying selection during evolution. The promoters of CaLTPs had development-related, phytohormone-responsive, and abiotic and biotic stress-related cis-acting elements. A few CaLTP transcripts exhibited differential expression in diverse tissue types, while others showed no/very low expression. Out of 20 jasmonate-regulated CaLTPs, 14 exhibited differential expression patterns during Helicoverpa armigera-infestation, indicating their role in plant defense response. This study identified and characterized CaLTPs from an important legume, C. arietinum, and indicated their involvement in plant defense against H. armigera-infestation, which can be further utilized to explore lipid signaling during plant-pest interaction and pest management.

Role of Gltp in Maturation of Oligodendrocytes Under the Regulation of Nkx2.2.

Myelin, a lipid-enriched multi-layer membrane structure, allows for rapid long-distance saltatory conduction of neuronal impulses. Although glycolipids are the predominant types of lipids in the myelin bilayer, the role of glycolipid transfer protein (GLTP), which selectively mediates the transfer of various glycolipids between phospholipid bilayer, in myelin development and maintenance remains unknown at present. In this study, we identified Gltp as the key lipid metabolism gene in myelin-forming oligodendrocytes (OLs) through integrated omics analysis across independent transcriptomic and single-cell sequencing studies. Gene expression analysis revealed that Gltp is selectively expressed in the differentiated OLs. Functional study demonstrated that its expression is essential for the differentiation of OLs, and promotes the outgrowth of OL membrane. Moreover, we found that the expression of Gltp is regulated by OL-lineage transcriptional factors, such as NKX2.2, OLIG2, SOX10, and MYRF. These findings provide important insights into the unrecognized functions of Gltp in OL differentiation and maturation.

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 invitro 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.

Adaptive Binding of Alkyl Glycosides by Nonpeptidic Helix Bundles in Water: Toward Artificial Glycolipid Binding Proteins.

Amphipathic water-soluble helices formed from synthetic peptides or foldamers are promising building blocks for the creation of self-assembled architectures with non-natural shapes and functions. While rationally designed artificial quaternary structures such as helix bundles have been shown to contain preformed cavities suitable for guest binding, there are no examples of adaptive binding of guest molecules by such assemblies in aqueous conditions. We have previously reported a foldamer 6-helix bundle that contains an internal nonpolar cavity able to bind primary alcohols as guest molecules. Here, we show that this 6-helix bundle can also interact with larger, more complex guests such as n-alkyl glycosides. X-ray diffraction analysis of co-crystals using a diverse set of guests together with solution and gas-phase studies reveals an adaptive binding mode whereby the apo form of the 6-helix bundle undergoes substantial conformational change to accommodate the hydrocarbon chain in a manner reminiscent of glycolipid transfer proteins in which the cavity forms upon lipid uptake. The dynamic nature of the self-assembling and molecular recognition processes reported here marks a step forward in the design of functional proteomimetic molecular assemblies.

GLTP Is a Potential Prognostic Biomarker and Correlates with Immunotherapy Efficacy in Cervical Cancer

Cervical cancer (CC) is the fourth most commonly diagnosed cancer in women worldwide. The prognosis of CC patients remains poor. The objective of our study was to explore the potential of glycolipid transfer protein (GLTP) in predicting the prognosis of CC and patients' response to immunotherapy. The expression of GLTP was determined using TCGA and GEO datasets. The prognostic value of GLTP in CC patients was analyzed using Kaplan-Meier analysis and multivariate analysis. The relationships between BTBD10 and immunological checkpoints, immune checkpoint genes, and ferroptosis-related genes were analyzed to explore the impact of GLTP on CC immunotherapy. According to the dysregulated expressions of BTBD10, the IC50 distribution of various targeted medicines was studied. In this study, we found that GLTP expression was distinctly upregulated in CC specimens. However, Kaplan-Meier assays showed that CC patients with low GLTP expressions tended to exhibit a shorter overall survival. Importantly, multivariate assays revealed that GLTP expression was an independent prognostic factor for CC patients. Moreover, we observed that GLTP expression was related to CD4+ T cells, macrophages, and dendritic cells (DCs). Meanwhile, GLTP expressions were associated with those of immune checkpoints, ferroptosis-related genes, and m6A-related genes. The IC50 of Cisplatin, Docetaxel, and Paclitaxel was lower in the high-GLTP-expressing group. Taken together, GLTP was expected to be a prognostic and immunotherapeutic marker for CC.

MiR-196a Upregulation Contributes to Gefitinib Resistance through Inhibiting GLTP Expression.

Tyrosine kinase inhibitor (TKI) therapy has greatly improved lung cancer survival in patients with epidermal growth factor receptor (EGFR) mutations. However, the development of TKI-acquired resistance is the major problem to be overcome. In this study, we found that miR-196a expression was greatly induced in gefitinib-resistant lung cancer cells. To understand the role and mechanism of miR-196a in TKI resistance, we found that miR-196a-forced expression alone increased cell resistance to gefitinib treatment in vitro and in vivo by inducing cell proliferation and inhibiting cell apoptosis. We identified the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2) bound to the promoter region of miR-196a and induced miR-196a expression at the transcriptional level. NRF2-forced expression also significantly increased expression levels of miR-196a, and was an upstream inducer of miR-196a to mediate gefitinib resistance. We also found that glycolipid transfer protein (GLTP) was a functional direct target of miR-196a, and downregulation of GLTP by miR-196a was responsible for gefitinib resistance. GLTP overexpression alone was sufficient to increase the sensitivity of lung cancer cells to gefitinib treatment. Our studies identified a new role and mechanism of NRF2/miR-196a/GLTP pathway in TKI resistance and lung tumor development, which may be used as a new biomarker (s) for TKI resistance or as a new therapeutic target in the future.

Human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling in pancreatic cancer cells.

Pancreatic cancer (PC) is a devastating solid malignancy with a dismal prognosis. The treatment of metastatic PC is a current challenge for medical oncologists due to a lack of early detection, drug resistance, and relapse. Therefore, potential biomarkers and effective therapeutic targets for PC are urgently required. Ceramide-1-phosphate transfer protein (CPTP) is a member of the glycolipid transfer protein family, which is associated with autophagy and inflammation regulation. The roles and mechanisms of CPTP in PC have not been clarified. In this study, by RT-qPCR and immunohistochemistry analysis, we found that CPTP is highly expressed in PC and is associated with a poor prognosis in PC patients. By using cell counting kit-8, colony formation, transwell and matrigel assays in vitro, as well as xenograft model assays in vivo, we further proved that CPTP enhanced PC cells growth and metastasis. In PC cells, human CPTP promotes growth and metastasis via sphingolipid metabolite ceramide and PI4KA/AKT signaling. Sp (specific protein)-1 and Sp3 transcription factors also act as upstream positive regulators of CPTP expression in PC cells. Collectively, these findings suggested that CPTP may function as a pro-tumorigenic gene in PC cells and could be a promising therapeutic target in PC.

Ceramide-1-phosphate transfer protein promotes sphingolipid reorientation needed for binding during membrane interaction

Lipid transfer proteins acquire and release their lipid cargoes by interacting transiently with source and destination biomembranes. In the GlycoLipid Transfer Protein (GLTP) superfamily, the two-layer all-α-helical GLTP-fold defines proteins that specifically target sphingolipids (SLs) containing either sugar or phosphate headgroups via their conserved but evolutionarily-modified SL recognitions centers. Despite comprehensive structural insights provided by X-ray crystallography, the conformational dynamics associated with membrane interaction and SL uptake/release by GLTP superfamily members have remained unknown. Herein, we report insights gained from molecular dynamics (MD) simulations into the conformational dynamics that enable ceramide-1-phosphate transfer proteins (CPTPs) to acquire and deliver ceramide-1-phosphate (C1P) during interaction with 1-palmitoyl-2-oleoyl phosphatidylcholine bilayers. The focus on CPTP reflects this protein’s involvement in regulating pro-inflammatory eicosanoid production and autophagy-dependent inflammasome assembly that drives interleukin (IL-1β and IL-18) production and release by surveillance cells. We found that membrane penetration by CPTP involved α-6 helix and the α-2 helix N-terminal region, was confined to one bilayer leaflet, and was relatively shallow. Large-scale dynamic conformational changes were minimal for CPTP during membrane interaction or C1P uptake except for the α-3/α-4 helices connecting loop, which is located near the membrane interface and interacts with certain phosphoinositide headgroups. Apart from functioning as a shallow membrane-docking element, α-6 helix was found to adeptly reorient membrane lipids to help guide C1P hydrocarbon chain insertion into the interior hydrophobic pocket of the SL binding site.These findings support a proposed ‘hydrocarbon chain-first’ mechanism for C1P uptake, in contrast to the ‘lipid polar headgroup-first’ uptake used by most lipid-transfer proteins.

Ceramide-1-phosphate transfer protein (CPTP) regulation by phosphoinositides

Ceramide-1-phosphate transfer proteins (CPTPs) are members of the glycolipid transfer protein (GLTP) superfamily that shuttle ceramide-1-phosphate (C1P) between membranes. CPTPs regulate cellular sphingolipid homeostasis in ways that impact programmed cell death and inflammation. CPTP downregulation specifically alters C1P levels in the plasma and trans-Golgi membranes, stimulating proinflammatory eicosanoid production and autophagy-dependent inflammasome-mediated cytokine release. However, the mechanisms used by CPTP to target the trans-Golgi and plasma membrane are not well understood. Here, we monitored C1P intervesicular transfer using fluorescence energy transfer (FRET) and showed that certain phosphoinositides (phosphatidylinositol 4,5 bisphosphate (PI-(4,5)P2) and phosphatidylinositol 4-phosphate (PI-4P)) increased CPTP transfer activity, whereas others (phosphatidylinositol 3-phosphate (PI-3P) and PI) did not. PIPs that stimulated CPTP did not stimulate GLTP, another superfamily member. Short-chain PI-(4,5)P2, which is soluble and does not remain membrane-embedded, failed to activate CPTP. CPTP stimulation by physiologically relevant PI-(4,5)P2 levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P2 increasing membrane equilibrium binding affinity less effectively than PS. Functional mapping of mutations that led to altered FRET lipid transfer and assessment of CPTP membrane interaction by surface plasmon resonance indicated that di-arginine motifs located in the α-6 helix and the α3-α4 helix regulatory loop of the membrane-interaction region serve as PI-(4,5)P2 headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P2 headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P2 interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.

In Silico Docking of Vitamin E Isomers on Transport Proteins.

Vitamin E is comprised of α, β, γ and δ-tocopherols (Ts) and α, β, γ and δ- tocotrienols (T3s). Vitamin E has neuroprotective antioxidant, anti-cancer, and cholesterol-lowering effects. Intracellular trafficking of these isomers remains largely unknown, except for αT which is selectively transported by αT transfer protein (αTTP). This study aimed to determine the binding of vitamin E isomers on transport proteins using in silico docking. Transport proteins were selected using AmiGo Gene Ontology tool based on the same molecular function annotation as αTTP. Protein structures were obtained from the Protein Data Bank. Ligands structures were obtained from ZINC database. In silico docking was performed using SwissDock. A total of 6 transport proteins were found: SEC14-like protein 2, glycolipid transfer protein (GLTP), pleckstrin homology domain-containing family A member 8, collagen type IV alpha-3-binding protein, ceramide-1-phosphate transfer protein and afamin. Compared with other transport proteins, αTTP had the highest affinities for all isomers except βT3. Binding order of vitamin E isomers toward αTTP was γT > βT > αT > δT > αT3 > γT3 > δT3 > βT3. GLTP had a higher affinity for tocotrienols than tocopherols. βT3 bound stronger to GLTP than αTTP. αTTP remained as the most preferred transport protein for most of the isomers. The binding affinity of αT toward αTTP was not the highest than other isomers suggested that other intracellular trafficking mechanisms of these isomers may exist. GLTP may mediate the intracellular transport of tocotrienols, especially βT3. Improving the bioavailability of these isomers may enhance their beneficial effects to human.

Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes.

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long "shelf life" after production (≥6 days) and resilience to freeze-thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

P.310 Possible association of genetic variant rs3800373 in fkbp5 gene with schizophrenia and bipolar disorder

Aureobasidium pullulans is an increasingly attractive host for bio-production of pullulan, heavy oil, polymalic acid, and a large spectrum of extracellular enzymes. To date, genetic manipulation of A. pullulans mainly relies on time-consuming conventional restriction enzyme digestion and ligation methods. In this study, we present a one-step homologous recombination-based method for rapid genetic manipulation in A. pullulans. Overlaps measuring >40 bp length and 10 μg DNA segments for homologous recombination provided maximum benefits to transformation of A. pullulans. This optimized method was successfully applied to PKSIII gene (encodes polyketide synthase) knock-out and gltP gene (encodes glycolipid transfer protein) knock-in. After disruption of PKSIII gene, secretion of melanin decreased slightly. The melanin purified from disruptant showed lower reducing capacity compared with that of the parent strain, leading to a decrease in exopolysaccharide production. Knock-in of gltP gene resulted in at least 4.68-fold increase in heavy oil production depending on the carbon source used, indicating that gltP can regulate heavy oil synthesis in A. pullulans.

Proteomics of the corpus callosum to identify novel factors involved in hypomyelinated Niemann-Pick Type C disease mice

Hypomyelination in the central nerves system (CNS) is one of the most obviously pathological features in Niemann-Pick Type C disease (NPC), which is a rare neurodegenerative disorder caused by mutations in the NPC intracellular cholesterol transporter 1 or 2 (Npc1 or Npc2). Npc1 plays key roles in both neurons and oligodendrocytes during myelination, however, the linkage between the disturbed cholesterol transport and inhibited myelination is unrevealed. In this study, mass spectrometry (MS)-based differential quantitative proteomics was applied to compare protein composition in the corpus callosum between wild type (WT) and NPC mice. In total, 3009 proteins from both samples were identified, including myelin structural proteins, neuronal proteins, and astrocyte-specific proteins. In line to hypomyelination, our data revealed downregulation of myelin structural and indispensable proteins in Npc1 mutant mice. Notably, the reduced ceramide synthase 2 (Cers2), UDP glycosyltransferase 8 (Ugt8), and glycolipid transfer protein (Gltp) indicate the altered sphingolipid metabolism in the disease and the involvement of Gltp in myelination. The identification of most reported myelin structural proteins and proteins from other cell types advocates the use of the corpus callosum to investigate proteins in different cell types that regulate myelination.

Up-regulation of glycolipid transfer protein by bicyclol causes spontaneous restriction of hepatitis C virus replication

Bicyclol is a synthetic drug for hepatoprotection in clinic since 2004. Preliminary clinical observations suggest that bicyclol might be active against hepatitis C virus (HCV) with unknown mechanism. Here, we showed that bicyclol significantly inhibited HCV replication in vitro and in hepatitis C patients. Using bicyclol as a probe, we identified glycolipid transfer protein (GLTP) to be a novel restrictive factor for HCV replication. The GLTP preferentially bound host vesicle-associated membrane protein-associated protein-A (VAP-A) in competition with the HCV NS5A, causing an interruption of the complex formation between VAP-A and HCV NS5A. As the formation of VAP-A/NS5A complex is essential for viral RNA replication, up-regulation of GLTP by bicyclol reduced the level of VAP-A/NS5A complex and thus inhibited HCV replication. Bicyclol also exhibited an inhibition on HCV variants resistant to direct-acting antiviral agents (DAAs) with an efficacy identical to that on wild type HCV. In combination with bicyclol, DAAs inhibited HCV replication in a synergistic fashion. GLTP appears to be a newly discovered host restrictive factor for HCV replication, Up-regulation of GLTP causes spontaneous restriction of HCV replication.

In Vitro Measurement of Sphingolipid Intermembrane Transport Illustrated by GLTP Superfamily Members.

Herein, we describe methodological approaches for measuring in vitro transfer of sphingolipids (SLs) between membranes. The approaches rely on direct tracking of the lipid. Typically, direct tracking involves lipid labeling via attachment of fluorophores or introduction of radioactivity. Members of the GlycoLipid Transfer Protein (GLTP) superfamily are used to illustrate two broadly applicable methods for direct lipid tracking. One method relies on Förster resonance energy transfer (FRET) that enables continuous assessment of fluorophore-labeled SL transfer in real time between lipid donor and acceptor vesicles. The second method relies on tracking of radiolabeled SL transfer by separation of lipid donor and acceptor vesicles at discrete time points. The assays are readily adjustable for assessing lipid transfer (1) between various model membrane assemblies (vesicles, micelles, bicelles, nanodiscs), (2) involving other lipid types by other lipid transfer proteins, (3) with protein preparations that are either crudely or highly purified, and (4) that is spontaneous and occurs in the absence of protein.

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.

Upregulation of human glycolipid transfer protein (GLTP) induces necroptosis in colon carcinoma cells

Human GLTP on chromosome 12 (locus 12q24.11) encodes a 24 kD amphitropic lipid transfer protein (GLTP) that mediates glycosphingolipid (GSL) intermembrane trafficking and regulates GSL homeostatic levels within cells. Herein, we provide evidence that GLTP overexpression inhibits the growth of human colon carcinoma cells (HT-29; HCT-116), but spares normal colonic cells (CCD-18Co). Mechanistic studies reveal that GLTP overexpression arrested the cell cycle at the G1/S checkpoint via upregulation of cyclin-dependent kinase inhibitor-1B (Kip1/p27) and cyclin-dependent kinase inhibitor 1A (Cip1/p21) at the protein and mRNA levels, and downregulation of cyclin-dependent kinase-2 (CDK2), cyclin-dependent kinase-4 (CDK4), cyclin E and cyclin D1 protein levels. Assessment of the biological fate of HCT-116 cells overexpressing GLTP indicated no increase in cell death suggesting induction of quiescence. However, HT-29 cells overexpressing GLTP underwent cell death by necroptosis as revealed by phosphorylation of human mixed lineage kinase domain-like protein (pMLKL) via receptor-interacting protein kinase-3 (RIPK-3), elevated cytosolic calcium, and plasma membrane permeabilization by pMLKL oligomerization. Overexpression of W96A-GLTP, an ablated GSL binding site mutant, failed to arrest the cell cycle or induce necroptosis. Sphingolipid assessment (ceramide, monohexosylceramide, sphingomyelin, ceramide-1-phosphate, sphingosine, and sphingosine-1-phosphate) of HT-29 cells overexpressing GLTP revealed large decreases (>5-fold) in sphingosine-1-phosphate with minimal change in 16:0-ceramide, tipping the ‘sphingolipid rheostat’ (S1P/16:0-Cer ratio) towards cell death. Depletion of RIPK-3 or MLKL abrogated necroptosis induced by GLTP overexpression. Our findings establish GLTP upregulation as a previously unknown suppressor of human colon carcinoma HT-29 cells via interference with cell cycle progression and induction of necroptosis.