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.

Apolipophorin II/I, apolipoprotein B, vitellogenin, and microsomal triglyceride transfer protein genes are derived from a common ancestor.

Large lipid transfer proteins (LLTP) are nonexchangeable apolipoproteins and intracellular lipid-exchange proteins involved in the assembly, secretion, and metabolism of lipoproteins. We have identified contiguous conserved sequence motifs in alignments of insect apolipophorin II/I precursor (apoLp-II/I), human apolipoprotein B (apoB), invertebrate and vertebrate vitellogenins (VTG), and the large subunit of mammalian microsomal triglyceride transfer protein (MTP). Conserved motifs present in the N-terminal part of nonexchangeable apolipoproteins encompass almost completely the large subunit of MTP, suggesting a derivation from a common ancestral functional unit, termed large lipid transfer (LLT) module. Divergence of LLTP from a common ancestor is supported by (1) the statistical significance of the combined match scores obtained after motif-based database searches, (2) the presence of several identical amino acid residues in all LLTP sequences currently available, (3) the conservation of hydrophobic clusters in an alpha-helical domain, (4) the phylogenetic analysis of the conserved sequences related to the von Willebrand factor D (VWD) module identified in nonexchangeable apolipoproteins, and (5) the presence of four and one ancestral exon boundaries in the LLT and VWD modules, respectively. Our data indicate that the genes coding for apoLp-II/I, apoB, VTG, and the MTP large subunit are members of the same multigene superfamily. LLTP have emerged from an ancestral molecule designed to ensure a pivotal event in the intracellular and extracellular transfer of lipids and liposoluble substances.

An insect homolog of the vertebrate very low density lipoprotein receptor mediates endocytosis of lipophorins.

A novel member of the low density lipoprotein (LDL) receptor family was identified, which is expressed in locust oocytes, fat body, brain, and midgut. This receptor appeared to be a homolog of the mammalian very low density lipoprotein receptor as it contains eight cysteine-rich repeats in its putative ligand-binding domain. When transiently expressed in COS-7 or stably expressed in LDL receptor-deficient CHO cells, the receptor mediates endocytic uptake of high density lipophorin (HDLp), an abundant lipoprotein in the circulatory compartment of insects. Moreover, in the latter cell line, we demonstrated that an excess of unlabeled HDLp competed with fluorescent labeled HDLp for uptake whereas an excess of human LDL did not affect uptake. Expression of the receptor mRNA in fat body cells is down-regulated during adult development, which is consistent with the previously reported down-regulation of receptor-mediated endocytosis of lipophorins in fat body tissue (Dantuma, N. P., M.A.P. Pijnenburg, J. H. B. Diederen, and D. J. Van der Horst. 1997. J. Lipid Res. 38: 254-265). The expression of this receptor in various tissues that internalize circulating lipophorins and its capability to mediate endocytosis of HDLp indicate that this novel member of the LDL receptor family may function as an endocytic lipophorin receptor in vivo.

Preferential release of newly synthesized, exportable neuropeptides by insect neuroendocrine cells and the effect of ageing of secretory granules.

The release of newly synthesized neuropeptides was studied in an in vitro system using the adipokinetic hormone (AKH)-producing cells of an insect (Locusta migratoria) as a model system. Tritiated phenylalanine incorporated into three hormonal neuropeptides, AKH I, II and III, was used to distinguish newly synthesized hormones from older, preexisting ones. After pulse-chase labeling experiments of varying duration, the secretion of AKHs by the AKH cells was stimulated. Both hormones released into the incubation medium after stimulation and non-released hormones extracted from the tissue were separated by reversed-phase high performance liquid chromatography. Their radioactivity was measured by scintillation counting of the column eluate. The ratio between the specific radioactivities of the released and the non-released neuropeptides was always greater than 1.0, which indicates that the newly synthesized peptides are preferentially released. The percentages of newly synthesized (radioactive) AKHs which are released, increased until 8 1/4 h and decreased thereafter. The results indicate that after the packaging of the prohormones into secretory granules and their processing to bioactive AKHs, some further maturation of the secretory granules is required before they can release their content. After an 8 1/4 h incubation, secretory granules with radioactive AHKs enter a non-releasable pool consisting of older secretory granules.

Spectroscopic and lipid binding studies on the amino and carboxyl terminal fragments of Locusta migratoria apolipophorin III.

The structural basis for the lipid binding capability of Locusta migratoria apolipophorin III (apoLp-III) was assessed by characterizing the amino and carboxyl terminal halves of the protein. The native molecule (approximately 20 kDa) was deglycosylated with endoglycosidase F (molecular mass of deglycosylated species approximately 18 kDa) and cleaved with endoproteinase Arg-C to yield two fragments with molecular masses of approximately 9 kDa each. The two fragments were purified by reversed-phase HPLC and identified by mass spectrometry, amino acid analysis and N-terminal sequencing as the amino terminal (N9) and carboxyl terminal (C9) halves. Due to the apparent discrepancy of the protease digestion pattern obtained compared to that expected from the deduced amino sequence of apoLp-III cDNA, we carried out partial amino acid sequencing of the fragments and cDNA sequencing for the entire protein. Circular dichroism spectroscopy of the N9 and C9 peptides revealed that both exist in buffer in a random coil state. However, addition of trifluoroethanol, a helix-inducing agent, resulted in the formation of an alpha-helix, reflecting an innate propensity of the peptides to adopt a helical conformation. When cosonicated with dimyristoylphosphatidylcholine (DMPC) both peptides assumed an alpha-helical conformation, indicative of interaction with the phospholipid. In the presence of phospholipids, a 22 nm blue shift in Trp fluorescence emission was observed in the case of the C9 peptide, suggesting that the Trp residues are located in a more hydrophobic environment. Electron microscopy revealed that, compared to native apoLp-III, both peptides possessed a reduced ability to transform DMPC vesicles to disklike complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning of three distinct cDNAs, each encoding a different adipokinetic hormone precursor, of the migratory locust, Locusta migratoria. Differential expression of the distinct adipokinetic hormone precursor genes during flight activity.

Three distinct cDNAs encoding the preproadipokinetic hormones I, II, and III (prepro-AKH I, II, and III), respectively, of Locusta migratoria have been isolated and sequenced. The three L. migratoria AKH precursors have an overall architecture similar to that of other precursors of the AKH/red pigment-concentrating hormone (RPCH) family identified so far. The AKH I and II precursors of L. migratoria are highly homologous to the Schistocerca gregaria and Schistocerca nitans AKH precursors. Although the L. migratoria AKH III precursor appears to be the least homologous to the Manduca sexta, Drosophila melanogaster, and Carcinus maenas AKH/RPCH precursors, we favor the opinion that the L. migratoria AKH III precursor is evolutionary more related to the M. sexta, D. melanogaster, and C. maenas AKH/RPCH precursors than to the AKH I and II precursors of S. gregaria, S. nitans, or L. migratoria. In situ hybridization showed signals for the different AKH mRNAs to be co-localized in cell bodies of the glandular lobes of the corpora cardiaca. Northern blot analysis revealed the presence of single mRNA species encoding the AKH I precursor (approximately 570 bases), AKH II precursor (approximately 600 bases), and AKH III precursor (approximately 670 bases), respectively. Interestingly, flight activity increased steady-state levels of the AKH I and II mRNAs (approximately 2.0 times each) and the AKH III mRNA (approximately 4.2 times) in the corpora cardiaca.

Adipokinetic peptide hormone content and biosynthesis during locust development.

The content and biosynthesis of adipokinetic hormones (Lom-AKH-I, -II. and -III) were studied in larval stages and adults of Locusta migratoria. The amount of all three AKHs increases with age, although the patterns found for AKH-I and -II differ from that for AKH-III. Biosynthetic capacity of the corpus cardiacum for the three AKHs increases with age, particularly in larvae, whereas in adults this increase is only observed for AKH-III. The amounts of AKH-I and -II stored and their active biosynthesis greatly surpass the small quantities needed for mobilization of fuels during flight. The data for AKH-III suggest that this hormone may be important also during larval stages.

Isolation and structure elucidation of a novel adipokinetic hormone (Lom-AKH-III) from the glandular lobes of the corpus cardiacum of the migratory locust, Locusta migratoria.

A new adipokinetic hormone (named Lom-AKH-III) was isolated from the glandular lobes of the corpora cardiaca of Locusta migratoria. At the N-terminus it is blocked by a 5-oxoproline (pyroglutamic acid) residue (less than Glu). After enzymatic deblocking, the amino acid sequence of the N-terminus was partly established by automatic Edman degradation to be [less than Glu]-Leu-Asn-Phe-Thr-Pro-. Fast-atom-bombardment spectrometry (FAB-MS) revealed that the new hormone is an octapeptide, which is amidated at the C-terminus, and has a relative molecular mass of 1072. Based on the FAB-MS data the complete sequence is less than Glu-Leu-Asn-Phe-Thr-Pro-Trp-Trp-NH2, which was confirmed by chemical synthesis. All characteristics from HPLC, FAB-MS and biological activity of the natural hormone and the synthetic peptide appeared to be identical. Although the structure of this new hormone resembles that of Lom-AKH-I (less than Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2), its amino acid sequence points to a completely different route for its biosynthesis, involving a third prohormone. High-[K+]-containing media can cause release of all three adipokinetic hormones in vitro. Interestingly, the new hormone is absent in another locust species. Schistocerca gregaria. Based on in vitro biosynthesis experiments the turnover for this hormone is very high, suggesting an important physiological function. Locusta migratoria is the first insect species in which three different adipokinetic hormones have been demonstrated.