Abstract
Ch21, a developmentally regulated extracellular protein expressed in chick embryos and in cultured chondrocytes, was expressed in the baculovirus system, and the recombinant protein was purified to homogeneity by gel-filtration chromatography. Separation of two isoforms was achieved on an ion-exchange column. Previous work had shown that Ch21 belongs to the superfamily of lipocalins, which are transport proteins for small hydrophobic molecules. Studies were performed to identify the Ch21 ligand. By analysis of recombinant Ch21 on native polyacrylamide gel electrophoresis and by Lipidex assay, the binding of fatty acid to the protein was shown and a preferential binding of long-chain unsaturated 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. Calculated dissociation constants were 2 X 10(-7) M for unsaturated fatty acids 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. Short-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 extracellular fatty acid-binding protein (Ex-FABP).
Highlights
By analysis of recombinant Ch21 on native polyacrylamide gel electrophoresis and by Lipidex assay, the binding of fatty acid to the protein was shown and a preferential binding of long-chain unsaturated fatty acids was observed
Hypertrophic chondrocytes synthesize and secrete large amounts of Ch21 protein, a low molecular weight protein belonging to the lipocalin superfamily (19 –22)
We present evidence that the recombinant Ch21 expressed in baculovirus-infected cells selectively binds long chain fatty acids
Summary
Materials [1-14C]Linoleic acid, [1-14C]oleic acid, [1-14C]arachidonic acid, [1-14C]stearic acid (50 – 60 mCi/mmol) were from Amersham International Srl., Buckinghamshire, United Kingdom; [U-14C]linoleic acid (1 Ci/mmol) and [1-14C]myristic acid (50 – 60 mCi/mmol) were purchased from NEN Du Pont De Nemours, Dreiech, Germany. Polyacrylamide gel electrophoresis reagents were purchased from Bio-Rad. Ethyl acetate was HPLC grade from BDH, Poole, United Kingdom. SF900 medium was from purchased Life Technologies, Inc., S. Recombinant baculoviruses were obtained by cotransfection of Sf9 cells with wild type AcMNPV and pACyMI/pDr5 and purified by limiting dilution and dot hybridization. For production of Ch21 protein, Sf9 cells were grown in SF900 medium without fetal calf serum and infected with purified recombinant virus at 5 plaque-forming units/cell. At 3 days post-infection, the culture medium was recovered. After centrifugation, it was analyzed by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gel was stained with silver. 250 ml of medium were collected from 5– 6 ϫ 108 cells
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