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The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.

ABSTRACT: Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. Here, the biophysical properties and glycolipid transfer specificity of FAPP2-C-terminal domain have been characterized and compared with other established GLTP-folds. Experimental evidence for a GLTP-fold includes: i) far-UV circular dichroism (CD) showing secondary structure with high alpha-helix content and a low thermally-induced unfolding transition (~41°C); ii) near-UV-CD indicating only subtle tertiary conformational change before/after interaction with membranes containing/lacking glycolipid; iii) Red-shifted tryptophan (Trp) emission wavelength maximum (?(max)~352nm) for apo-FAPP2-C-terminal domain consistent with surface exposed intrinsic Trp residues; iv) 'signature' GLTP-fold Trp fluorescence response, i.e., intensity decrease (~30%) accompanied by strongly blue-shifted ?(max) (~14nm) upon interaction with membranes containing glycolipid, supporting direct involvement of Trp in glycolipid binding and enabling estimation of partitioning affinities. A structurally-based preference for other simple uncharged GSLs, in addition to glucosylceramide, makes human FAPP2-GLTP more similar to fungal HET-C2 than to plant AtGLTP1 (glucosylceramide-specific) or to broadly GSL-selective human GLTP. These findings along with the distinct mRNA exon/intron organizations originating from single-copy genes on separate human chromosomes suggest adaptive evolutionary divergence by these two GLTP-folds.

SUBMITTER: Kamlekar RK

PROVIDER: S-EPMC3654534 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.

Kamlekar Ravi Kanth RK Simanshu Dhirendra K DK Gao Yong-guang YG Kenoth Roopa R Pike Helen M HM Prendergast Franklyn G FG Malinina Lucy L Molotkovsky Julian G JG Venyaminov Sergei Yu SY Patel Dinshaw J DJ Brown Rhoderick E RE

Biochimica et biophysica acta 20121116 2

Phosphoinositol 4-phosphate adaptor protein-2 (FAPP2) plays a key role in glycosphingolipid (GSL) production using its C-terminal domain to transport newly synthesized glucosylceramide away from the cytosol-facing glucosylceramide synthase in the cis-Golgi for further anabolic processing. Structural homology modeling against human glycolipid transfer protein (GLTP) predicts a GLTP-fold for FAPP2 C-terminal domain, but no experimental support exists to warrant inclusion in the GLTP superfamily. H ...[more]

PMID: 23159414

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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.

Dataset Information

The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.

Publications

The glycolipid transfer protein (GLTP) domain of phosphoinositol 4-phosphate adaptor protein-2 (FAPP2): structure drives preference for simple neutral glycosphingolipids.

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