Modification of the adipocyte lipid binding protein by sulfhydryl reagents and analysis of the fatty acid binding domain

Human epidermal-type fatty acid-binding protein (E-FABP) belongs to a family of intracellular 14–15kDa lipid-binding proteins, whose functions have been associated with fatty acid signalling, cell growth, regulation and differentiation. As a contribution to understanding the structure—function relationship, we report in the present study features of its solution structure and backbone dynamics determined by NMR spectroscopy. Applying multi-dimensional high-resolution NMR techniques on unlabelled and 15N-enriched recombinant human E-FABP, the 1H and 15N resonance assignments were completed. On the basis of 2008 distance restraints, the three-dimensional solution structure of human E-FABP was subsequently obtained (backbone atom root-mean-square deviation of 0.92±0.11Å; where 1Å = 0.1nm), consisting mainly of 10 anti-parallel β-strands that form a β-barrel structure. 15N relaxation experiments (T1, T2 and heteronuclear nuclear Overhauser effects) at 500, 600 and 800MHz provided information on the internal dynamics of the protein backbone. Nearly all non-terminal backbone amide groups showed order parameters S2>0.8, with an average value of 0.88±0.04, suggesting a uniformly low backbone mobility in the nanosecond-to-picosecond time range. Moreover, hydrogen/deuterium exchange experiments indicated a direct correlation between the stability of the hydrogen-bonding network in the β-sheet structure and the conformational exchange in the millisecond-to-microsecond time range. The features of E-FABP backbone dynamics elaborated in the present study differ markedly from those of the phylogenetically closely related heart-type FABP and the more distantly related ileal lipid-binding protein, implying a strong interdependence with the overall protein stability and possibly also with the ligand-binding affinity for members of the lipid-binding protein family.

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A novel lipid-binding protein from the cestode Moniezia expansa

Biochemical Journal ◽

10.1042/bj3110049 ◽

1995 ◽

Vol 311 (1) ◽

pp. 49-57 ◽

Cited By ~ 25

Author(s):

D Janssen ◽

J Barrett

Keyword(s):

Fatty Acid ◽

Amino Acid ◽

Binding Proteins ◽

Binding Protein ◽

Alpha Helix ◽

Lipid Binding ◽

Fatty Acid Binding ◽

Lipid Binding Protein ◽

Kd Values ◽

Moniezia Expansa

A lipid-binding protein (LBP) has been purified from the cytosol of the cestode Moniezia expansa. The native LBP was found to be an oligomer of approx. 250 kDa, consisting of 11 kDa monomers. The LBP bound saturated and unsaturated fatty acids, but not their CoA derivatives, with KD values in the range 0.68-7.8 microM. Cholesterol, dihydroergosterol, bilirubin and retinoids were also bound, but alpha-tocopherol, bile acids, alk-2-enals and alka-2,4-dienals were not. Evidence suggests that there are two binding sites per subunit, each with different specificities. The fluorescent fatty acid 11-[(5-dimethylaminonaphthalene-1-sulphonyl)amino]undecanoic acid (DAUDA) and retinol both showed an additional high-affinity binding site with a density of approximately 0.1 per subunit, suggesting specific binding to the oligomer. The amino acid composition of Moniezia LBP was distinct from that of previously characterized fatty acid-binding proteins (FABPs). The protein was not N-terminally blocked and yielded a unique amino acid sequence, unrelated to that of any known FABP; there was also evidence of microheterogeneity. Polyclonal antibodies raised to the Moniezia protein did not cross-react with mammalian, nematode or digenean FABP. The Gibbs free energy for protein folding (13.02 kJ/mol; 3.1 kcal/mol), determined by urea denaturation, was identical for both the native and ligand-bound Moniezia LBP. CD spectra showed that the Moniezia protein contained 36% alpha-helix and that the secondary structure underwent only minor changes on ligand binding. Moniezia LBP binds a range of anthelmintics, with KD values again in the range 0.66-7.3 microM. It is possible that, in helminths, binding proteins may play a role in determining the specificity and site of action of anthelmintics.

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Fat depot origin affects fatty acid handling in cultured rat and human preadipocytes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.2001.280.2.e238 ◽

2001 ◽

Vol 280 (2) ◽

pp. E238-E247 ◽

Cited By ~ 53

Author(s):

Frank Caserta ◽

Tamara Tchkonia ◽

Vildan N. Civelek ◽

Marc Prentki ◽

Nicholas F. Brown ◽

...

Keyword(s):

Fatty Acid ◽

Binding Protein ◽

Lipid Binding ◽

Fatty Acid Binding Proteins ◽

Fatty Acid Binding ◽

Chain Acyl ◽

Human Preadipocytes ◽

Fat Depot ◽

Carnitine Palmitoyltransferase 1 ◽

Adipose Cells

Regional differences in free fatty acid (FFA) handling contribute to diseases associated with particular fat distributions. As cultured rat preadipocytes became differentiated, FFA transfer into preadipocytes increased and was more rapid in single perirenal than in epididymal cells matched for lipid content. Uptake by human omental preadipocytes was greater than uptake by abdominal subcutaneous preadipocytes. Adipose-specific fatty acid binding protein (aP2) and keratinocyte lipid binding protein abundance was higher in differentiated rat perirenal than in epididymal preadipocytes. This interdepot difference in preadipocyte aP2 expression was reflected in fat tissue in older animals. Carnitine palmitoyltransferase 1 activity increased during differentiation and was higher in perirenal than in epididymal preadipocytes, particularly the muscle isoform. Long-chain acyl-CoA levels were higher in perirenal than in epididymal preadipocytes and isolated fat cells. These data are consistent with interdepot differences in fatty acid flux ensuing from differences in fatty acid binding proteins and enzymes of fat metabolism. Heterogeneity among depots results, in part, from distinct intrinsic characteristics of adipose cells. Different depots are effectively separate miniorgans.

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Structural properties of the adipocyte lipid binding protein

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids ◽

10.1016/s1388-1981(99)00154-7 ◽

1999 ◽

Vol 1441 (2-3) ◽

pp. 106-116 ◽

Cited By ~ 39

Author(s):

Amy Reese-Wagoner ◽

James Thompson ◽

Leonard Banaszak

Keyword(s):

Structural Properties ◽

Binding Protein ◽

Lipid Binding ◽

Lipid Binding Protein ◽

Adipocyte Lipid Binding Protein

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The binding of cholesterol and bile salts to recombinant rat liver fatty acid-binding protein

Biochemical Journal ◽

10.1042/bj3200729 ◽

1996 ◽

Vol 320 (3) ◽

pp. 729-733 ◽

Cited By ~ 47

Author(s):

Alfred E. A. THUMSER ◽

David C. WILTON

Keyword(s):

Fatty Acid ◽

Bile Salts ◽

Fatty Acid Binding Protein ◽

Binding Protein ◽

Physiological Role ◽

Lipid Binding ◽

Liver Fatty Acid ◽

Fatty Acid Binding ◽

Acid Binding Protein ◽

Wide Range

The physiological role of liver fatty acid-binding protein (L-FABP) has yet to be clarified. An important feature of this member of the family of intracellular lipid-binding proteins is the wide range of compounds that have been identified as potential physiological ligands. By using recombinant L-FABP, the binding of cholesterol, bile salts and their derivatives has been investigated under conditions that allow a direct comparison of the binding affinities of these ligands for fatty acids. The results demonstrate an inability of L-FABP to bind cholesterol, although the anionic derivative, cholesteryl sulphate, will bind under similar assay conditions. Of the bile salts examined, lithocholate and taurolithocholate sulphate showed the greatest binding to L-FABP. It is proposed that an important function of L-FABP is to bind certain physiological amphipathic anions, thus preventing the ‘free’ concentrations of these compounds from exceeding their critical micelle concentration, which could result in cell damage.

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