EXPRESSION, PURIFICATION, AND LIGAND-BINDING ANALYSIS OF RECOMBINANT KERATINOCYTE LIPID-BINDING PROTEIN (MAL-1), AN INTRACELLULAR LIPID-BINDING PROTEIN FOUND OVEREXPRESSED IN NEOPLASTIC SKIN CELLS

The keratinocyte lipid-binding protein (KLBP) has been identified on t he basis of nucleotide sequence analysis of its cloned cDNA as a new m ember of the intracellular lipid-binding protein (iLBP) multigene fami ly. To characterize KLBP and determine its ligand-binding properties, its cDNA was subcloned into Escherichia coli, and the protein was over expressed and purified to homogeneity by a combination of acid extract ion, gel permeation, and ion-exchange chromatographies, Purified KT-BP exhibited high-affinity binding of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulfonate (1,8-ANS), displaying an apparent dis sociation constant of 390 +/- 90 nM (n = 0.74 +/- 0.2), Using an assay based upon displacement of the bound fluorophore, KLBP was found to b ind long chain fatty acids most avidly; oleic acid (18:1) bound with a n apparent K-d of 248 +/- 12 nM, and arachidonic acid (20:4) exhibited a dissociation constant of 318 +/- 14 nM. As the length of the fatty acid decreased, the binding affinity was reduced; myristic acid (14:0) bound with a K-d of 1409 +/- 423 nM, but medium-chain (decanoic acid, 10:0) and short-chain (octanoic acid, 8:0) lipids were not bound at a ll, The protein did not bind prostaglandin E(2) with any measurable af finity but did associate with eicosanoids such as 5-hydroperoxyeicosat etraenoic acid (5-HPETE; K-d of 848 +/- 211 nM) and 15-HPETE (K-d Of 4 63 +/- 243 nM) and to a lesser extent their hydroxy derivatives, 5-HET E and 15-HETE (K-d of 1560 +/- 115 nM and greater than 4 mu M, respect ively), all-trans-Retinoic acid was a weak ligand for KLBP, binding wi th a K-d of 3600 nM, and all-trans-retinol did not displace 1,8-ANS, M olecular modeling of the KLBP sequence upon the X-ray crystal structur es of several iLBP's suggested that thr side chains of one or more cys teine residues may reside within the putative ligand-binding cavity. C onsistent with this, sulfhydryl titration of purified KLBP with 5,5'-d ithiobis(2-nitrobenzoic acid) at pH 8.0 in the presence and absence of oleic acid revealed that at least one residue was protected from modi fication by the fatty acid. These results describe the first purificat ion and characterization of the ligand-binding properties of KLBP and indicate that the protein is a fatty acid binding protein with a terti ary structure likely to be similar to other members of the iLBP multig ene family.