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Membrane penetration depth of intestinal fatty acid binding protein (IFABP)
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Abstracts / Chemistry and Physics of Lipids 163S (2010) S50–S56
Acknowledgment This work was supported by the Spanish “Ministerio de Ciencia e Innovación (Project MAT2008-04722)” and from “Xunta de Galicia (Project INCITE08PXIB206030PR)”. doi:10.1016/j.chemphyslip.2010.05.160 PO 99 Sphingolipid profiling in plasma and FPLC-separated lipoprotein fractions by LC–MS/MS Max Scherer ∗ , Gerd Schmitz, Gerhard Liebisch Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany Sphingolipids comprises a highly diverse and complex class of molecules that serve not only as structural components of membranes but also as signalling molecules. These metabolites are known to act as intracellular and extracellular messengers eliciting important cell functions including apoptosis, differentiation and chemotaxis. To understand the differential role of sphingolipids in a regulatory network it is important to use structure-specific and quantitative methods. We developed a new liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the rapid, simultaneous quantification of sphingolipids and metabolites such as sphingosine, sphinganine, di- and trimethyl-sphingosine, phyto-sphingosine, sphingosylphosphorylcholine, sphingosine-1-phosphate, hexosylceramide, lactosyl-ceramide and ceramide-1-phosphate in plasma and FPLC-separated lipoprotein fractions. Appropriate internal standards were added prior to lipid extraction and the mass spectrometer was operated in the multiple reaction monitoring. We used hydrophilic interaction liquid chromatography (HILIC) and achieved good peak shape, short analysis times and most important coelution of the analytes and their respective internal standards. Peak areas were corrected regarding isotope overlap. Quantification was achieved by standard addition. Data processing was highly automated by the use of Analyst® and self programmed Excel macros. Screening of human EDTA-plasma samples revealed a backbone specific glycol-sphingolipid lipid pattern. We determined beside the major sphingoid-backbone d18:1, significant amounts of d16:1 and d18:2 sphingoid bases. FPLC separation by size exclusion chromatography showed a sphingolipid class distribution according to the three main lipoprotein classes VLDL, LDL and HDL, respectively. Sphingolipid profiling in plasma and lipoprotein classes provide a powerful tool to search for novel diagnostic biomarkers in large clinical studies. doi:10.1016/j.chemphyslip.2010.05.161 PO 100 Membrane penetration depth of intestinal fatty acid binding protein (IFABP) Eduardo De Gerónimo 1 , Judith Storch 2 , Betina Córsico 1 1
Instituto de Investigaciones Bioquímicas de La Plata (CONICETUNLP), Facultad de de Ciencias Médicas, UNLP, Argentina 2 Department of Nutritional Sciences, Rutgers, The State University of New Jersey, 08901-8525 New Brunswickm NJ, USA Intestinal FABP (IFABP) belongs to a family of small cytosolic proteins involved in lipid transport and metabolism that share a common structure, consisting of ten antiparallel -strands that
form a -barrel, capped by two short ␣-helices. The helical domain is postulated to be part of a small portal at the top of the barrel, wherein a hydrophobic ligand enters and exits the cavity of the barrel. Fatty acid transfer from IFABP to phospholipid membranes is proposed to occur during protein-membrane collisional interactions. Employing a helix-less variant of IFABP, we have shown that the ␣-helical region of IFABP is involved in membrane interactions, and appears to play a primary role in the collisional mechanism of fatty acid transfer from IFABP to membranes. The objective of this study was to determine the membrane penetration depth of the IFABP portal region by fluorescence quenching techniques and analyze how it is affected by the charge of the membrane surface and by the ionic strength. We have engineered a series of IFABP singletryptophan mutants in the portal region. The Trp fluorescence of these mutants is quenched by brominated phosphatidylcholine localized in large unilamellar vesicles, with bromines at different positions along the fatty acid acyl chain. The results clearly show that the IFABP portal region is the membrane binding domain and is located within the lipid bilayer. Moreover, increasing the negative charge of the membrane could affect the depth and angle of insertion. doi:10.1016/j.chemphyslip.2010.05.162 PO 101 Fatty acid binding proteins from cestodes Gabriela Alvite, Alejandra Kun, Adriana Esteves ∗ Facultad de Ciencias, UdelaR, Montevideo, Uruguay; IIBCE, Montevideo, Uruguay Functional role of FABPs is one of the issues still unresolved. These proteins are key molecules in the biology of cestodes as these organisms are unable to synthesize de novo most of their own lipids. Our group has isolated two genes encoding binding proteins for fatty acids of Echinococcus granulosus, the parasite responsible of Hydatid disease (EgFABP1 and EgFABP2) and Mesocestoides vogue (MvFABPa and MvFABPb), another cestode used as a model organism. These proteins are highly similar at amino acid level and clusters with H-FABP subfamily. In order to find structural determinants of functional differences between them we have we set as our objectives to determine the intracellular location of EgFABPs and MvFABPs and putative ligands. Using subcellular fractionation techniques coupled with western blot of one and two dimensions electrophoresis coupled to mass spectroscopy identified the proteins under study in different subcellular compartments. In toto immunomicroscopy allowed us to verify the previous results using molecular markers. Molecular modelling was applied to build 3D structures using 3D solved structure of EgFABP1 as template. In silico docking analysis allowed us to identify and describe interaction energies and binding cavities. Unlike the expected, M. vogae FABPs seems to have an ubiquous localization, since both proteins are present in all the compartments studied. A small difference in the relative concentrations at the different compartments could indicate a differential behavior however. Concerning EgFABPs localization, our results are still umbiguous. Interaction energies analyzed so far not appear to explain the searched differences. doi:10.1016/j.chemphyslip.2010.05.163
Abstracts / Chemistry and Physics of Lipids 163S (2010) S50–S56
Acknowledgment This work was supported by the Spanish “Ministerio de Ciencia e Innovación (Project MAT2008-04722)” and from “Xunta de Galicia (Project INCITE08PXIB206030PR)”. doi:10.1016/j.chemphyslip.2010.05.160 PO 99 Sphingolipid profiling in plasma and FPLC-separated lipoprotein fractions by LC–MS/MS Max Scherer ∗ , Gerd Schmitz, Gerhard Liebisch Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Germany Sphingolipids comprises a highly diverse and complex class of molecules that serve not only as structural components of membranes but also as signalling molecules. These metabolites are known to act as intracellular and extracellular messengers eliciting important cell functions including apoptosis, differentiation and chemotaxis. To understand the differential role of sphingolipids in a regulatory network it is important to use structure-specific and quantitative methods. We developed a new liquid chromatography tandem mass spectrometry (LC–MS/MS) method for the rapid, simultaneous quantification of sphingolipids and metabolites such as sphingosine, sphinganine, di- and trimethyl-sphingosine, phyto-sphingosine, sphingosylphosphorylcholine, sphingosine-1-phosphate, hexosylceramide, lactosyl-ceramide and ceramide-1-phosphate in plasma and FPLC-separated lipoprotein fractions. Appropriate internal standards were added prior to lipid extraction and the mass spectrometer was operated in the multiple reaction monitoring. We used hydrophilic interaction liquid chromatography (HILIC) and achieved good peak shape, short analysis times and most important coelution of the analytes and their respective internal standards. Peak areas were corrected regarding isotope overlap. Quantification was achieved by standard addition. Data processing was highly automated by the use of Analyst® and self programmed Excel macros. Screening of human EDTA-plasma samples revealed a backbone specific glycol-sphingolipid lipid pattern. We determined beside the major sphingoid-backbone d18:1, significant amounts of d16:1 and d18:2 sphingoid bases. FPLC separation by size exclusion chromatography showed a sphingolipid class distribution according to the three main lipoprotein classes VLDL, LDL and HDL, respectively. Sphingolipid profiling in plasma and lipoprotein classes provide a powerful tool to search for novel diagnostic biomarkers in large clinical studies. doi:10.1016/j.chemphyslip.2010.05.161 PO 100 Membrane penetration depth of intestinal fatty acid binding protein (IFABP) Eduardo De Gerónimo 1 , Judith Storch 2 , Betina Córsico 1 1
Instituto de Investigaciones Bioquímicas de La Plata (CONICETUNLP), Facultad de de Ciencias Médicas, UNLP, Argentina 2 Department of Nutritional Sciences, Rutgers, The State University of New Jersey, 08901-8525 New Brunswickm NJ, USA Intestinal FABP (IFABP) belongs to a family of small cytosolic proteins involved in lipid transport and metabolism that share a common structure, consisting of ten antiparallel -strands that
form a -barrel, capped by two short ␣-helices. The helical domain is postulated to be part of a small portal at the top of the barrel, wherein a hydrophobic ligand enters and exits the cavity of the barrel. Fatty acid transfer from IFABP to phospholipid membranes is proposed to occur during protein-membrane collisional interactions. Employing a helix-less variant of IFABP, we have shown that the ␣-helical region of IFABP is involved in membrane interactions, and appears to play a primary role in the collisional mechanism of fatty acid transfer from IFABP to membranes. The objective of this study was to determine the membrane penetration depth of the IFABP portal region by fluorescence quenching techniques and analyze how it is affected by the charge of the membrane surface and by the ionic strength. We have engineered a series of IFABP singletryptophan mutants in the portal region. The Trp fluorescence of these mutants is quenched by brominated phosphatidylcholine localized in large unilamellar vesicles, with bromines at different positions along the fatty acid acyl chain. The results clearly show that the IFABP portal region is the membrane binding domain and is located within the lipid bilayer. Moreover, increasing the negative charge of the membrane could affect the depth and angle of insertion. doi:10.1016/j.chemphyslip.2010.05.162 PO 101 Fatty acid binding proteins from cestodes Gabriela Alvite, Alejandra Kun, Adriana Esteves ∗ Facultad de Ciencias, UdelaR, Montevideo, Uruguay; IIBCE, Montevideo, Uruguay Functional role of FABPs is one of the issues still unresolved. These proteins are key molecules in the biology of cestodes as these organisms are unable to synthesize de novo most of their own lipids. Our group has isolated two genes encoding binding proteins for fatty acids of Echinococcus granulosus, the parasite responsible of Hydatid disease (EgFABP1 and EgFABP2) and Mesocestoides vogue (MvFABPa and MvFABPb), another cestode used as a model organism. These proteins are highly similar at amino acid level and clusters with H-FABP subfamily. In order to find structural determinants of functional differences between them we have we set as our objectives to determine the intracellular location of EgFABPs and MvFABPs and putative ligands. Using subcellular fractionation techniques coupled with western blot of one and two dimensions electrophoresis coupled to mass spectroscopy identified the proteins under study in different subcellular compartments. In toto immunomicroscopy allowed us to verify the previous results using molecular markers. Molecular modelling was applied to build 3D structures using 3D solved structure of EgFABP1 as template. In silico docking analysis allowed us to identify and describe interaction energies and binding cavities. Unlike the expected, M. vogae FABPs seems to have an ubiquous localization, since both proteins are present in all the compartments studied. A small difference in the relative concentrations at the different compartments could indicate a differential behavior however. Concerning EgFABPs localization, our results are still umbiguous. Interaction energies analyzed so far not appear to explain the searched differences. doi:10.1016/j.chemphyslip.2010.05.163