Abstract
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 (~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.
Highlights
Glycolipid transfer protein is the prototypical and founding member of the new GLTP superfamily distinguished by a novel conformational fold and glycolipid binding motif
Organization of Human GLTP Genes To identify the human gene(s) encoding GLTP, the sequences of cDNAs that we previously cloned from GLTP mRNA of human skin fibroblasts and glioma cells (GenBank AF209074, AY372530, AY372531, AY372532) were used in BLAST searches of the NCBI Human Genome
When adjoined in 5'-to-3' fashion, the five exons encoded the same 209 amino acid sequence that we previously determined for human GLTP by reverse transcription polymerase chain reaction (RT-PCR) using purified mRNA [16,27]
Summary
Glycolipid transfer protein is the prototypical and founding member of the new GLTP superfamily distinguished by a novel conformational fold and glycolipid binding motif. Glycolipid transfer protein (GLTP) is a soluble protein (209 amino acids; ~24 kDa) that can selectively transfer GSLs between membranes [7,8,9,10,11,12,13]. Acquisition of glycolipid occurs via an adaptive recognition process involving a sugar headgroup recognition center that forms multiple hydrogen bonds and van der Waals contacts to selectively anchor the sugar-amide moieties to amino acid side chains of the protein surface as well as a 'molded-to-fit', hydrophobic tunnel to accommodate the hydrocarbon chains of the ceramide moiety [13,16,17,18]
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