THE DEVELOPMENTALLY-REGULATED AVIAN CH21 LIPOCALIN IS AN EXTRACELLULAR FATTY-ACID-BINDING PROTEIN

Ch21, a developmentally regulated extracellular protein expressed in c hick embryos and in cultured chondrocytes, was expressed in the baculo virus system, and the recombinant protein was purified to homogeneity by gel-filtration chromatography. Separation of two isoforms was achie ved on an ion-exchange column. Previous work had shown that Ch21 belon gs to the superfamily of lipocalins, which are transport proteins for small hydrophobic molecules. Studies were performed to identify the Ch 21 ligand. By analysis of recombinant Ch21 on native polyacrylamide ge l electrophoresis and by Lipidex assay, the binding of fatty acid to t he protein was shown and a preferential binding of long-chain unsatura ted fatty acids was observed. Both isoforms had the same behavior. The binding was saturable. Stoichiometry was about 0.7 mol of ligand/mol of protein. The protein binds the ligand in its monomeric form. Calcul ated dissociation constants were 2 x 10(-7) M for unsaturated fatty ac ids and 5 x 10(-7) M for stearic acid. The binding was specific; other hydrophobic molecules, as retinoic acid, progesterone, prostaglandins , and long-chain alcohols and aldehydes did not bind to the protein. S hort-chain fatty acids did not bind to the protein. Ch21, also present in chicken serum, represents the first extracellular protein able to selectively bind and transport fatty acid in extracellular fluids and serum. We propose to rename the Ch21 protein as (e) under bar xtracell ular (f) under bar atty (a) under bar cid-(b) under bar inding (p) und er bar rotein (Ex-FABP).