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Point mutational analysis of the liganding site in human glycolipid transfer protein. Functionality of the complex.

ABSTRACT: Mammalian glycolipid transfer proteins (GLTPs) facilitate the selective transfer of glycolipids between lipid vesicles in vitro. Recent structural determinations of the apo- and glycolipid-liganded forms of human GLTP have provided the first insights into the molecular architecture of the protein and its glycolipid binding site (Malinina, L., Malakhova, M. L., Brown, R. E., and Patel, D. J. (2004) Nature 430, 1048-1053). In the present study, we have evaluated the functional consequences of point mutation of the glycolipid liganding site of human GLTP within the context of a carrier-based mechanism of glycolipid intermembrane transfer. Different approaches were developed to rapidly and efficiently assess the uptake and release of glycolipid by GLTP. They included the use of glass-immobilized, glycolipid films to load GLTP with glycolipid and separation of GLTP/glycolipid complexes from vesicles containing glycolipid (galactosylceramide or lactosylceramide) or from monosialoganglioside dispersions by employing nickel-nitrilotriacetic acid-based affinity or gel filtration strategies. Point mutants of the sugar headgroup recognition center (Trp-96, Asp-48, Asn-52) and of the ceramide-accommodating hydrophobic tunnel (Phe-148, Phe-183, Leu-136) were analyzed for their ability to acquire and release glycolipid ligand. Two manifestations of point mutation within the liganding site were apparent: (i) impaired formation of the GLTP/glycolipid complex; (ii) impaired acquisition and release of bound glycolipid by GLTP. The results are consistent with a carrier-based mode of GLTP action to accomplish the intermembrane transfer of glycolipid. Also noteworthy was the inefficient release of glycolipid by wtGLTP into phosphatidylcholine acceptor vesicles, raising the possibility of a function other than intermembrane glycolipid transfer in vivo.

SUBMITTER: Malakhova ML

PROVIDER: S-EPMC1393170 | biostudies-literature | 2005 Jul

REPOSITORIES: biostudies-literature

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Point mutational analysis of the liganding site in human glycolipid transfer protein. Functionality of the complex.

Malakhova Margarita L ML Malinina Lucy L Pike Helen M HM Kanack Alexander T AT Patel Dinshaw J DJ Brown Rhoderick E RE

The Journal of biological chemistry 20050518 28

Mammalian glycolipid transfer proteins (GLTPs) facilitate the selective transfer of glycolipids between lipid vesicles in vitro. Recent structural determinations of the apo- and glycolipid-liganded forms of human GLTP have provided the first insights into the molecular architecture of the protein and its glycolipid binding site (Malinina, L., Malakhova, M. L., Brown, R. E., and Patel, D. J. (2004) Nature 430, 1048-1053). In the present study, we have evaluated the functional consequences of poin ...[more]

PMID: 15901739

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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

Point mutational analysis of the liganding site in human glycolipid transfer protein. Functionality of the complex.

Publications

Point mutational analysis of the liganding site in human glycolipid transfer protein. Functionality of the complex.

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