The Developmentally Regulated Avian Ch21 Lipocalin Is an Extracellular Fatty Acid-binding Protein

doi:10.1074/jbc.271.33.20163

The Developmentally Regulated Avian Ch21 Lipocalin Is an Extracellular Fatty Acid-binding Protein*

From the ^‡ Centro di Biotecnologie Avanzate, Istituto Nazionale per la Ricerca sul Cancro and
^∥ Istituto di Oncologia Clinica e Sperimentale, Universitá di Genova, 16132 Genova, Italy, and the
^§ Istituto Internazionale di Genetica e Biofisica and
^¶ Istituto per la Chimica di Molecole di Interesse Biologico, Consiglio Nazionale delle Ricerche, 80100 Napoli, Italy

^∥To whom correspondence should addressed:
Centro di Biotecnologie Avanzate, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi, 10, 16132 Genova, Italy.
Tel.: 39-10-5737398; Fax: 39-10-5737405.

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 × 10⁻⁷ M for unsaturated fatty acids and 5 × 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 rotein (Ex-FABP).

Footnotes

↵* This work was supported by grants from Progetti Finalizzati: “Ingegneria Genetica” and “Applicazioni Cliniche della Ricerca Oncologica” of the CNR, and by funds from the Associazione Italiana per la Ricerca sul Cancro and from Telethon, Italy. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ M. Riminucci, C. Gentili, J. N. Bradbeer, F. Descalzi, R. Cancedda, and P. Bianco, submitted for publication.
↵² The abbreviations used are:

PBS

phosphate-buffered saline

HPLC

high pressure liquid chromatography.
- Received October 25, 1995.
- Revision received May 20, 1996.