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.

A new adipokinetic hormone (named Lom-AKH-111) 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 (< Glu). After enzymatic deblocking, the amino acid sequence of the N-terminus was partly established by automatic Edman degradation to be 1 < Glul-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 < Glu-Leu-Asn-Phe-ThrPro-Trp-Trp-NH,, 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 ( < Glu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH,), 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.The first physiological evidence for the presence of compound(s) with adipokinetic activity in the corpora cardiaca (CC) of the locusts Locusta migratoria and Schistocerca gregaria was reported independently by Beenakkers [l] and by Mayer and Candy 121, respectively. From both species an identical adipokinetic hormone (AKH) was isolated (Lom-AKH-I; for nomenclature see [3]) and sequenced ( < Glu-LeuAsn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH,) 141. Both species also contain a second adipokinetic hormone [5] and sequence analysis revealed that this hormone in Locusta (Lom-AKH-11) ( < Glu-Leu-Asn-Phe-Ser-Ala-Gly-Trp-NH,) differs in only one amino acid from that in Schistocerca (Scg-AKH-11) ( < Glu-Leu-Asn-Phe-Ser-Thr-Gly-Trp-NH2) [6].AKHs are synthesized and stored in the glandular lobe of the CC [7 -91 and are colocalized in the same secretory granCorrespondence to R.

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 (ϳ570 bases), AKH II precursor (ϳ600 bases), and AKH III precursor (ϳ670 bases), respectively. Interestingly, flight activity increased steady-state levels of the AKH I and II mRNAs (ϳ2.0 times each) and the AKH III mRNA (ϳ4.2 times) in the corpora cardiaca.Three peptide hormones with hyperlipemic activity, the adipokinetic hormones I, II and III (AKH 1 I, II and III; see Table I) (1-3), are synthesized by the glandular neurosecretory cells of the corpora cardiaca (CC) of the migratory locust, Locusta migratoria. These peptides are members of a large family of structurally related but functionally diverse peptides (the AKH/RPCH family) (4). In the adult locust, the AKHs I and II are released into the hemolymph during flight and are involved in the mobilization of lipid and carbohydrate from the fat body to serve as energy substrates for the flight muscles (4 -7). Data on the release and functioning of AKH III are lacking so far. Isolation and characterization of CC peptides revealed that two other locust species, Schistocerca gregaria and Schistocerca nitans, each contain two AKHs that are mutually identical (1, 8), whereas Manduca sexta and Drosophila melanogaster each contain only one AKH (9, 10) (see Table I).Molecular biological studies have resulted in the characterization of the structure of the AKH/RPCH precursors (a signal peptide, AKH/RPCH, a Gly-(Lys/Arg)-Arg sequence, and an AKH/RPCH-associated peptide (AAP/RAP), in this order) of S. gregaria, S. nitans, M. sexta, D. melanogaster,.The biosynthesis of the AKHs in S. gregaria has been elucidated in detail by O'Shea and co-workers (13, 18 -21). The signal peptide is co-translationally removed from prepro-AKH, generating pro-AKH. Next, proteolytic processing, which is preceded by dimerization of two pro-AKHs (I/I, I/II, or II/II) via their single COOH-terminal Cys residues, gives rise to two AKHs (I and/or II) and one homo-or hete...

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)

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