Modulation of ligand binding affinity of the adipocyte lipid-binding protein by selective mutation. Analysis in vitro and in situ.

The crystal structure of the adipocyte lipid-binding protein (ALBP) with coordinated fatty acid shows the hydrophobic ligand bound within a water-filled central cavity with its carboxyl group engaged in a hydrogen bonding network involving, at least in part, the functional groups of residues R126 and Y128. We produced mutant forms of ALBP which altered these amino acids, expressed these in Escherichia coli as glutathione S-transferase (GST) fusion proteins, and examined their ligand-binding properties using the fluorescent fatty acids cis-parinaric acid (c-PA) and 12-(9-anthroyloxy)-oleate (12-AO). The wild-type and all mutated forms of GST-ALBP displayed similar binding affinities for 12-AO, with Kd,app values ranging from 0.5 to 2.4 microM. The binding affinity of ALBP forms R126Q and Y128W for c-PA were reduced about 30-50-fold in comparison to GST-ALBP, while that for the double mutation R126L + Y128F was below the limits of detection. To determine if the hydrogen bonding system functioned in situ, Chinese hamster ovary (CHO) cell transfectants expressing wild-type ALBP demonstrated a moderate (1.5-2-fold) increase in the total rate of [3H]oleate uptake and trafficking into the esterified lipid pools over that of untransfected cells, while the rate of [3H]oleate uptake of the transfected CHOs expressing the R126L + Y128F mutation was identical to that of the control CHOs. In summary, these results suggest that the primary factor contributing to binding affinity of ALBP for fatty acids such as c-PA or oleic acid both in vitro and in situ is the hydrogen bonding network involving at least R126, Y128, and the lipid carboxyl group. However, a ligand with sufficiently large hydrophobic character such as 12-AO can bind in the absence of a functional carboxylate hydrogen bonding network, presumably due to stabilizing entropic interactions with other cavity atoms.

Human adipocyte lipid-binding protein: purification of the protein and cloning of its complementary DNA.

Human adipocyte lipid-binding protein (H-ALBP) was purified from normal subcutaneous adipose tissue to greater than 98% homogeneity, utilizing a combination of acid fractionation, gel filtration, covalent chromatography on activated thiol-Sepharose 4B, and anion-exchange chromatography. Human ALBP comprised about 1% of total cytosolic protein in human adipose tissue, had a relative molecular mass of about 15 kDa, and existed as a monomer in solution. The amino terminus of H-ALBP was blocked to sequencing. When a liposome ligand delivery assay was used, H-ALBP saturably bound oleic acid with about 1 mol of ligand bound per mole of protein. Additionally, H-ALBP saturably bound retinoic acid as determined by the quenching of intrinsic tryptophan fluorescence. A full-length H-ALBP cDNA has been cloned; the sequence predicts a 649-base mRNA comprised of a 62-base 5'-noncoding region containing an 18S ribosome-binding site, a single 396-base open-reading frame, and a 191-base 3'-noncoding region. Comparative sequence analysis indicated that the 132 amino acid H-ALBP is a member of a multigene family of intracellular lipid-binding proteins and contains the consensus substrate phosphorylation sequence for tyrosyl kinases.