The Drosophila Neurally Altered Carbohydrate Mutant Has a Defective Golgi GDP-Fucose Transporter

Journal of Biological Chemistry (Impact Factor: 4.6). 06/2012;

ABSTRACT Studying genetic disorders in model organisms can provide insights into heritable human diseases. The Drosophila neurally altered carbohydrate (nac) mutant is deficient for neural expression of the HRP epitope, which consists of N-glycans with core α1,3-linked fucose residues. Here, we show that a conserved serine residue in the Golgi GDP-fucose transporter (GFR) is substituted by leucine in nac1 flies, which abolishes GDP-fucose transport in vivo and in vitro. This loss of function is due to a biochemical defect, not to destabilization or mis-targeting of the mutant GFR protein. Mass spectrometry and HPLC analysis showed that nac1 mutants lack not only core α1,3-linked, but also core α1,6-linked fucose residues on their N-glycans. Thus, the nac1 Gfr mutation produces a previously unrecognized general defect in N-glycan core fucosylation. Transgenic expression of a wild-type Gfr gene restored the HRP epitope in neural tissues, directly demonstrating that the Gfr mutation is solely responsible for the neural HRP epitope deficiency in the nac1 mutant. These results validate the Drosophila nac1 mutant as a model for the human congenital disorder of glycosylation, CDG-IIc (also known as LAD-II), which is also the result of a GFR deficiency.

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    ABSTRACT: Antibodies specific for horseradish peroxidase (HRPeroxase) bind to neuronal membranes in Drosophila and serve as a specific neuronal marker. Immunocytochemical staining with these antibodies marks sensory neurons, peripheral nerves, and fiber tracks in the central nervous system of embryos, larvae, and adult flies. Similar patterns of staining also were seen in embryos of the grasshopper. It appears that an antigen associated with the nervous system and appearing early in differentiation is recognized by antibodies to HRPeroxase. Using this staining method, we followed embryogenesis of the central nervous system in Drosophila and found that the organization of central fiber tracks resembled that in the previously well-characterized grasshopper. We have used the anti-HRPeroxase antibodies to show that mutations affecting segmentation in Drosophila affect the organization of the embryonic nervous system.
    Proceedings of the National Academy of Sciences 05/1982; 79(8):2700-4. · 9.81 Impact Factor
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    ABSTRACT: Antiserum raised against horseradish peroxidase (HRP) recognizes a neural specific carbohydrate antigen in Drosophila and other insects. The epitopic activity of the carbohydrate moiety of HRP recognized by anti-HRP antiserum was measured by a newly developed enzyme-linked immunosorbent assay, in which HRP glycopeptides conjugated with bovine serum albumin were coated onto the wells and then reacted with goat anti-HRP antiserum. HRP sugar moieties released by almond glycopeptidase A digestion of HRP pepsin digests were subjected to pyridylamination. Pyridylamino oligosaccharides were separated into seven fractions by reverse-phase high performance liquid chromatography. The major fraction, which comprised about 80% of the total sugars, reacted strongly with anti-HRP antiserum. The carbohydrate structure of this fraction was determined by sugar composition analysis and 600-MHz 1H NMR spectroscopy as follows: Man alpha 1----6(Man alpha 1----3)(Xyl beta 1----2)Man beta 1----4GlcNAc beta 1----4(Fuc alpha 1----3)GlcNAc. Analyses of reactivity with anti-HRP antiserum of various oligosaccharide derivatives obtained from the major fraction by exoglycosidase digestion and partial acid hydrolysis indicated that alpha 1----6-linked mannose and alpha 1----3-linked fucose are predominantly involved in the epitopic structure.
    Journal of Biological Chemistry 04/1991; 266(7):4168-72. · 4.60 Impact Factor
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    ABSTRACT: Antiserum against horseradish peroxidase (anti-HRP Ab) labels the surfaces of neurons in both Drosophila and grasshopper (Jan and Jan, 1982). Here we show that the anti-HRP Ab (1) immunoprecipitates at least 17 different membrane glycoproteins from the Drosophila embryo CNS (and a similar array from grasshopper), and (2) recognizes a neural-specific carbohydrate moiety expressed by most if not all of these proteins. Although the anti-HRP Ab stains all axon pathways, 2 of the anti-HRP glycoproteins, fasciclin I and II, are expressed on specific subsets of axon pathways in the grasshopper embryo.
    Journal of Neuroscience 01/1988; 7(12):4137-44. · 6.75 Impact Factor

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