A group of Calliphoru vicinu pupal glycolipids could be segregated from the neutral glycosphingolipids, according to their two-dimensional TLC migration properties and positive reactions toward ninhydrin and fluorescamine spray reagents. These classified zwitterionic glycolipids were isolated by silica-gel column chromatography and characterized by the presence of a N-acetyl-glucosamine-bound phosphoethanolamine residue. The structural elucidation of the oligosaccharide moieties was performed by the determination of constituent carbohydrates as alditol acetates, linkage analysis by permethylation, exoglycosidase cleavage, fast-atombombardment mass spectrometry and NMR spectroscopy. The dominant fatty acid and sphingoid base species of the ceramide moieties were C2,: , (arachidic acid) and C14: (tetradecasphing-4-enine), respectively. The chemical structures of the zwitterionic, biogenetic glycosphingolipid series were determined as :
The two major components of the acidic glycolipid fraction from the pupae of Calliphora vicina were isolated using high-performance liquid chromatography. The acidic moiety was identified as glucuronic acid by jglucuronidase cleavage and gas chromatographic analysis as the pentafluoropropionyl derivative. The structures of the carbohydrate moiety were elucidated by peracetylation, methylation, exoglycosidase cleavage, fast-atombombardment mass spectrometric and 'H-nuclear magnetic resonance spectroscopic analysis. The only difference between the two hexasaccharide variants was the presence, in one of them, of a phosphoethanolamine (AeP) sidechain on the third sugar of the sequence, i. e. N-acetylglucosamine. The composition of the ceramide moiety was dominated by a C2,, : fatty acid (arachidic acid) and a CI4 : sphingoid base (tetradecasphing-4-enine). The chemical structures of the two insect acidic glycosphingolipids were determined to be:Such glucuronic-acid-containing insect glycosphingolipids have been given the generic name arthrosides, with the implied synonymity to the gangliosides.Systematic structural elucidation of glycosphingolipids from members of the phyla Mollusca and Athropoda (class: Insecta) of the Invertebrata (Protostomia) have revealed major divergences in the carbohydrate moiety compared to those of the Vertebrata (Deuterostomia). The mollu series (Ml) of the Mollusca and arthro series (Ap) of the Arthropoda are characterised by a mactose-containing ceramide disaccharide in which the galactose residue of vertebrate lactose is replaced by mannose. Abbreviations. Gangliosides of the sialoganglio series are designated as was suggested previously [I] : GI,,I = I13NeuAc-LacCer, G,,,l = I13NeuAc-Gg,Cer. The shorthand abbreviations of glycosphingolipids (GSL) of the arthro series (Ap), characterized by the monosaccharide sequence of the arthroheptaose (Ap7) GlcNAcPl3GalP1 -3GalNAcal -4GalNAcP1 -4GlcNAcPI -3ManDl -4Glc, are N, Nz, A, and Az, where N stands for GSL with a neutral oligosaccharide chain, A for those with an additional acidic substituent (glucuronic acid); the lower case letter z is added for members that carry the zwitterionic substituent 2-aminoethylphosphate; arabic numerals 1-7 indicate the number of constituent neutral monosaccharides. In the case of two isomeric ceramide pentahexosides, a lower-case letter b or c distinguishes IV3Gala-Ap,Cer (N5b) and IV3GalP-Ap,Cer ( N~c ) , respectively. Mab, monoclonal antibody; Mac, mactose, i. e. Manp(1-4)Glc; FAB-MS, fast atom-bombardment mass spectrometry.Enzymes. P-Glucuronidase (EC 3.2.1.31), P-galactosidase (EC 3.2.1.23).
As a first approach to testing the working hypothesis that glycosphingolipids are functionally involved in the ontogeny of insects, their chemical distribution in larval organs was determined and any stadium-correlated differences documented. Selected organs, i.e., the fatbody, striated muscle, intestinal tract, salivary glands, imaginal discs, and central nervous system, were dissected from seven-day-old larvae of the blowfly, Calliphora vicina, and their glycolipids isolated. Two-dimensional, high-performance thin-layer chromatography was used to separate the neutral and acidic glycolipids of each organ. Significantly different total glycolipid component-patterns were obtained for the individual organs, whereby, except for a number of additional uncharacterized components in the intestinal tract, the neutral glycolipids of all organs were found to be qualitatively similar. However, major quantitative differences between the selected organs were found in their total glycolipid-carbohydrate contents, as well as the respective quantitative neutral glycosphingolipid-component distributions. The acidic glycolipids showed pronounced qualitative as well as quantitative organ-dependent variations. Whereas the highest proportion of uncharged glycolipids was characteristic of the fatbody, a high proportion of zwitterionic glycolipid-components was observed to be typical of the central nervous system and imaginal discs, i.e., of organs persisting during larval life and throughout metamorphosis. Imaginal disc glycolipids were distinguished by their high content of acidic glycolipids, a putative reflection of the functional role of these glycoconjugates in regulated cell reorganization during metamorphosis.
A cloned, hybridoma cell-line was established that secreted the monoclonal antibody CAF-I following stimulation of the donor Balb/c mouse spleen cells by the total acidic fraction glycolipids of the third-instar larvae of Calliphora vicina (Insecta:Diptera). The monoclonal antibody isotype was IgG3 By qualitative (TLC-immunostaining) and semi-quantitative (enzyme-linked immunosorbent assay) methods, and comparison with the cross-reactivity of known monoclonal antibodies, the epitope was specifically located on the terminal, non-reducing end of the oligosaccharide chain of most of the insect acidic glycolipids. Following isolation of the two main acidic glycolipids of C. vicina larvae (A5c and Az5c), exoglycosidase treatment characterized the terminal disaccharide CAF-I epitope as glucuronic acid bound to subterminal galactose, both in the beta-anomeric configuration: G1cA beta-Ga1 beta-. The immunohistological distribution of this epitope in the dipteran, Drosophila melanogaster, showed its main expression to be in the imaginal discs and brain of the third-instar larva, and the retinula cells of the ommatidial elements of the compound eye retina of the adult female.
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