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.
Highlights
Glycolipid transfer proteins (GLTPs)2 are small (ϳ24 kDa), soluble, single-polypeptide proteins that selectively accelerate the intermembrane transfer of glycolipids [1]
Similar results were obtained with HEK 293T cells, indicating that human GLTP can be transcribed from more than one start sites but that TSS1 represents the major start site
The data show regulation of GLTP expression via Sp1/Sp3 by a complex mechanism that responds to elevated ceramide levels. This investigation represents the first characterization of any Gltp gene promoter and provides the first insights into the regulation of human GLTP gene expression
Summary
Glycolipid transfer proteins (GLTPs) are small (ϳ24 kDa), soluble, single-polypeptide proteins that selectively accelerate the intermembrane transfer of glycolipids [1]. The GLTP fold occurs widely among eukaryotes [12] and endows larger proteins, i.e. FAPP2 (phosphoinositol 4-phosphate adaptor protein-2) [13], with key functionality during synthesis of complex glycosphingolipids (GSLs), which serve as important signaling and structural components of raft microdomains in plasma membranes (14 –16). Our goal was to evaluate GLTP gene expression within the context of GSL metabolic homeostasis by determining if alterations in key sphingolipid metabolites trigger changes in GLTP transcription, as regulated by its previously uncharacterized GLTP gene promoter. The present study provides the first insights into human GLTP transcriptional regulation, including characterization of constitutive and basal GLTP promoter Regulation of Human GLTP Expression by Ceramide tion of Sp1/Sp3 transcription factors in a manner influenced by ceramide but not by related sphingolipid metabolites
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