Molecular characterization and gene expression in the eye of the apolipophorin II/I precursor from Locusta migratoria.

The transport of lipids via the circulatory system of animals constitutes a vital function that uses highly specialized lipoprotein complexes. In insects, a single lipoprotein, lipophorin, serves as a reusable shuttle for the transport of lipids between tissues. We have found that the two nonexchangeable apolipoproteins of lipophorin arise from a common precursor protein, apolipophorin II/I (apoLp-II/I). To examine the mechanisms of transport of lipids and liposoluble substances inside the central nervous system, this report provides the molecular cloning of a cDNA encoding the locust apoLp-II/I. We have recently shown that this precursor protein belongs to a superfamily of large lipid transfer proteins (Babin et al. [1999] J. Mol. Evol. 49:150-160). We determined that, in addition to its expression in the fat body, the locust apoLp-II/I is also expressed in the brain. Part of the signal resulted from fat body tissue associated with the brain; however, apoLp-II/I was strongly expressed and the corresponding protein detected, in pigmented glial cells of the lamina underlying the locust retina and in cells or cellular processes interspersed in the basement membrane. The latter finding strongly suggests an implication of apolipophorins in the transport of retinoids and/or fatty acids to the insect retina.

Primary structure and binding characteristics of locust and human muscle fatty-acid-binding proteins.

The conservation between muscle fatty-acid-binding proteins (M-FABP) of Locusta migratoria flight muscle and human skeletal muscle was investigated. The locust M-FABP cDNA (632 bp) was isolated by 5' and 3' rapid amplification of cDNA ends. The identities of the locust and human M-FABP on the cDNA and protein levels were 54% and 42%, respectively. The predicted amino acid sequence of locust M-FABP indicated a molecular mass of 14935 Da and isoelectric point 6.1. The locust M-FABP was expressed in Escherichia coli, purified by (NH4)2SO4 precipitation, anion-exchange and gel-filtration chromatographies and compared with the recombinant human M-FABP with respect to immunological and binding properties. In spite of the high sequence similarity, the proteins did not show immunological cross-reactivity. The binding parameters of locust M-FABP were analyzed with radiolabeled oleic acid by the Lipidex assay and titration microcalorimetry. Both methods revealed a Kd for oleic acid of 0.5 microM and a binding stoichiometry of 1 mol fatty acid/mol FABP. The delta H, delta G and delta S for oleic acid binding were -146 kJ.mol-1 and -36 J.mol-1 and -369 J.mol-1.K-1 respectively. All the information obtained from binding, fluorescence and displacement studies indicated that locust M-FABP has binding characteristics similar to human M-FABP. Finally the recombinant locust M-FABP was crystallized with and without oleic acid. All crystals were trigonal in the P3(1)21 space group. The unit cell dimensions were a = b = 5.89 nm and c = 14.42 nm.