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Human glycolipid transfer protein gene (GLTP) expression is regulated by Sp1 and Sp3: involvement of the bioactive sphingolipid ceramide.

ABSTRACT: Glycolipid transfer protein (GLTP) accelerates glycolipid intermembrane transfer via a unique lipid transfer/binding fold (GLTP fold) that defines the GLTP superfamily and is the prototype for functional GLTP-like domains in larger proteins, i.e. FAPP2. Human GLTP is encoded by the single-copy GLTP gene on chromosome 12 (12q24.11 locus), but regulation of GLTP gene expression remains completely unexplored. Herein, the ability of glycosphingolipids (and their sphingolipid metabolites) to regulate the transcriptional expression of GLTP via its promoter has been evaluated. Using luciferase and GFP reporters in concert with deletion mutants, the constitutive and basal (225 bp; ?78% G+C) human GLTP promoters have been defined along with adjacent regulatory elements. Despite high G+C content, translational regulation was not evident by the mammalian target of rapamycin pathway. Four GC-boxes were shown to be functional Sp1/Sp3 transcription factor binding sites. Mutation of one GC-box was particularly detrimental to GLTP transcriptional activity. Sp1/Sp3 RNA silencing and mithramycin A treatment significantly inhibited GLTP promoter activity. Among tested sphingolipid analogs of glucosylceramide, sulfatide, ganglioside GM1, ceramide 1-phosphate, sphingosine 1-phosphate, dihydroceramide, sphingosine, only ceramide, a nonglycosylated precursor metabolite unable to bind to GLTP protein, induced GLTP promoter activity and raised transcript levels in vivo. Ceramide treatment partially blocked promoter activity decreases induced by Sp1/Sp3 knockdown. Ceramide treatment also altered the in vivo binding affinity of Sp1 and Sp3 for the GLTP promoter and decreased Sp3 acetylation. This study represents the first characterization of any Gltp gene promoter and links human GLTP expression to sphingolipid homeostasis through ceramide.

SUBMITTER: Zou X

PROVIDER: S-EPMC3020738 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Human glycolipid transfer protein gene (GLTP) expression is regulated by Sp1 and Sp3: involvement of the bioactive sphingolipid ceramide.

Zou Xianqiong X Gao Yongguang Y Ruvolo Vivian R VR Gardner Tawnya L TL Ruvolo Peter P PP Brown Rhoderick E RE

The Journal of biological chemistry 20101025 2

Glycolipid transfer protein (GLTP) accelerates glycolipid intermembrane transfer via a unique lipid transfer/binding fold (GLTP fold) that defines the GLTP superfamily and is the prototype for functional GLTP-like domains in larger proteins, i.e. FAPP2. Human GLTP is encoded by the single-copy GLTP gene on chromosome 12 (12q24.11 locus), but regulation of GLTP gene expression remains completely unexplored. Herein, the ability of glycosphingolipids (and their sphingolipid metabolites) to regulate ...[more]

PMID: 20974858

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Project description:BackgroundGlycolipid transfer protein is the prototypical and founding member of the new GLTP superfamily distinguished by a novel conformational fold and glycolipid binding motif. The present investigation provides the first insights into the organization, transcriptional status, phylogenetic/evolutionary relationships of GLTP genes.ResultsIn human cells, single-copy GLTP genes were found in chromosomes 11 and 12. The gene at locus 11p15.1 exhibited several features of a potentially active retrogene, including a highly homologous (approximately 94%), full-length coding sequence containing all key amino acid residues involved in glycolipid liganding. To establish the transcriptional activity of each human GLTP gene, in silico EST evaluations, RT-PCR amplifications of GLTP transcript(s), and methylation analyses of regulator CpG islands were performed using various human cells. Active transcription was found for 12q24.11 GLTP but 11p15.1 GLTP was transcriptionally silent. Heterologous expression and purification of the GLTP paralogs showed glycolipid intermembrane transfer activity only for 12q24.11 GLTP. Phylogenetic/evolutionary analyses indicated that the 5-exon/4-intron organizational pattern and encoded sequence of 12q24.11 GLTP were highly conserved in therian mammals and other vertebrates. Orthologs of the intronless GLTP gene were observed in primates but not in rodentiates, carnivorates, cetartiodactylates, or didelphimorphiates, consistent with recent evolutionary development.ConclusionThe results identify and characterize the gene responsible for GLTP expression in humans and provide the first evidence for the existence of a GLTP pseudogene, while demonstrating the rigorous approach needed to unequivocally distinguish transcriptionally-active retrogenes from silent pseudogenes. The results also rectify errors in the Ensembl database regarding the organizational structure of the actively transcribed GLTP gene in Pan troglodytes and establish the intronless GLTP as a primate-specific, processed pseudogene marker. A solid foundation has been established for future identification of hereditary defects in human GLTP genes.

Project description: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.

Dataset Information

Human glycolipid transfer protein gene (GLTP) expression is regulated by Sp1 and Sp3: involvement of the bioactive sphingolipid ceramide.

Publications

Human glycolipid transfer protein gene (GLTP) expression is regulated by Sp1 and Sp3: involvement of the bioactive sphingolipid ceramide.

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