Background: Glycolipid 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. Results: In 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. Conclusion: The 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.
Bibliographical noteFunding Information:
We thank Prof. Julian G. Molotkovsky (Russian Academy of Sciences) for synthesizing the fluorescent glycolipids used by Dr. Xiuhong Zhai to confirm the glycolipid intermembrane transfer activities of GLTP homologs obtained with radiolabeled glycolipids. We are grateful to Prof. Hervé Philippe (Université de Montréal) for recommendations regarding phyloge-netics and evolutionary bioinformatics analyses, to Dr. Anthony J. Winde-bank (Mayo Clinic) for enabling access to the Mayo Molecular Biology Core Facility, and to Drs. Joshua Liao, Peter Ruvolo, and Vivian Ruvolo (UMN-Hormel Institute) for helpful suggestions. The research was supported by NIH/NIGMS GM45928, NIH/NCI CA121493, and The Hormel Foundation. The authors have declared no conflict of interests regarding the findings, financing, and data reported herein.