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• W2012244893 abstract "The neurogenic Drosophila genesbrainiac and egghead are essential for epithelial development in the embryo and in oogenesis. Analysis ofegghead and brainiac mutants has led to the suggestion that the two genes function in a common signaling pathway. Recently, brainiac was shown to encode a UDP-N-acetylglucosamine:βManβ1,3-N-acetylglucosaminyltransferase (β3GlcNAc-transferase) tentatively assigned a key role in biosynthesis of arthroseries glycosphingolipids and forming the trihexosylceramide, GlcNAcβ1–3Manβ1–4Glcβ1–1Cer. In the present study we demonstrate that egghead encodes a Golgi-located GDP-mannose:βGlcβ1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac, Manβ1–4Glcβ1–1Cer. Egghead is unique among eukaryotic gly- cosyltransferase genes in that homologous genes are limited to invertebrates, which correlates with the exclusive existence of arthroseries glycolipids in invertebrates. We propose that brainiac and egghead function in a common biosynthetic pathway and that inactivating mutations in either lead to sufficiently early termination of glycolipid biosynthesis to inactivate essential functions mediated by glycosphingolipids. The neurogenic Drosophila genesbrainiac and egghead are essential for epithelial development in the embryo and in oogenesis. Analysis ofegghead and brainiac mutants has led to the suggestion that the two genes function in a common signaling pathway. Recently, brainiac was shown to encode a UDP-N-acetylglucosamine:βManβ1,3-N-acetylglucosaminyltransferase (β3GlcNAc-transferase) tentatively assigned a key role in biosynthesis of arthroseries glycosphingolipids and forming the trihexosylceramide, GlcNAcβ1–3Manβ1–4Glcβ1–1Cer. In the present study we demonstrate that egghead encodes a Golgi-located GDP-mannose:βGlcβ1,4-mannosyltransferase tentatively assigned a biosynthetic role to form the precursor arthroseries glycosphingolipid substrate for Brainiac, Manβ1–4Glcβ1–1Cer. Egghead is unique among eukaryotic gly- cosyltransferase genes in that homologous genes are limited to invertebrates, which correlates with the exclusive existence of arthroseries glycolipids in invertebrates. We propose that brainiac and egghead function in a common biosynthetic pathway and that inactivating mutations in either lead to sufficiently early termination of glycolipid biosynthesis to inactivate essential functions mediated by glycosphingolipids. UDP-N-acetylglucosamine:acceptor β1,3-N-acetylglucosaminyltransferase ceramide lactosylceramide mactosylceramide total correlation spectroscopy gradient-enhanced correlation spectroscopy rotating frame Overhauser spectroscopy Chinese hamster ovary 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid The Drosophila genes brainiac andegghead play essential roles in epithelial development in the embryo and during oogenesis (1Goode S. Wright D. Mahowald A.P. Development (Camb.). 1992; 116: 177-192Crossref PubMed Google Scholar, 2Goode S. Melnick M. Chou T.B. Perrimon N. Development (Camb.). 1996; 122: 3863-3879Crossref PubMed Google Scholar). Brainiac andegghead encode proteins that are required in the germline to allow for normal interaction between germ line and somatic cells in the developing ovary (2Goode S. Melnick M. Chou T.B. Perrimon N. Development (Camb.). 1996; 122: 3863-3879Crossref PubMed Google Scholar). In the absence of brainiac oregghead in the germ line defects are observed in the overlying follicular epithelium, which is of somatic origin (1Goode S. Wright D. Mahowald A.P. Development (Camb.). 1992; 116: 177-192Crossref PubMed Google Scholar, 2Goode S. Melnick M. Chou T.B. Perrimon N. Development (Camb.). 1996; 122: 3863-3879Crossref PubMed Google Scholar). On one hand, these follicular epithelial defects resemble defects in epidermal growth factor receptor signaling between germ line and follicle cell layers. On the other hand, they resemble a subset of the follicular defects associated with Notch mutants (1Goode S. Wright D. Mahowald A.P. Development (Camb.). 1992; 116: 177-192Crossref PubMed Google Scholar, 2Goode S. Melnick M. Chou T.B. Perrimon N. Development (Camb.). 1996; 122: 3863-3879Crossref PubMed Google Scholar, 3Goode S. Morgan M. Liang Y.P. Mahowald A.P. Dev. Biol. 1996; 178: 35-50Crossref PubMed Scopus (64) Google Scholar). Defects in female fertility have also been described (4Rubsam R. Hollmann M. Simmerl E. Lammermann U. Schafer M.A. Buning J. Schafer U. Mech. Dev. 1998; 72: 131-140Crossref PubMed Scopus (20) Google Scholar). The diversity of defects caused by brainiac and egghead mutants suggests that they may be involved in communication between cells at a fundamental level and that they can affect multiple signaling pathways. Brainiac and egghead mutants exhibit similar and non-additive phenotypes, leading to the proposal that they function in a common signaling pathway. Based on sequence analysis, Yuan et al. (5Yuan Y.P. Schultz J. Mlodzik M. Bork P. Cell. 1997; 88: 9-11Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) originally proposed that brainiac together with the distant homologous gene fringe encoded glycosyltransferases. This hypothesis has subsequently proved correct and both represent glycosyltransferases with functionally conserved mammalian homologs (6Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (593) Google Scholar, 7Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (721) Google Scholar, 8Schwientek T. Keck B. Levery S.B. Jensen M.A. Pedersen J.W. Wandall H.H. Stroud M. Cohen S.M. Amado M. Clausen H. J. Biol. Chem. 2002; 277: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9Muller R. Altmann F. Zhou D. Hennett T. J. Biol. Chem. 2002; 277: 32417-32420Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Brainiac encodes a UDP-N-acetylglucosamine:βManβ1,3-N-acetylglucosaminyltransferase (β3GlcNAc-transferase)1with a predicted function in biosynthesis of arthroseries glycosphingolipids in the Drosophila (8Schwientek T. Keck B. Levery S.B. Jensen M.A. Pedersen J.W. Wandall H.H. Stroud M. Cohen S.M. Amado M. Clausen H. J. Biol. Chem. 2002; 277: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9Muller R. Altmann F. Zhou D. Hennett T. J. Biol. Chem. 2002; 277: 32417-32420Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Brainiac was shown to catalyze addition of the third monosaccharide residue to form the trihexosylceramide glycolipid, GlcNAcβ1–3Manβ1–4Glcβ1–1Cer. Arthroseries glycolipids have only been found in invertebrates and differ fundamentally from mammalian glycolipids by having a core disaccharide structure based on Manβ1–4Glcβ1-Cer (MacCer) rather than Galβ1–4Glcβ1-Cer (LacCer) (10Seppo A. Tiemeyer M. Glycobiology. 2000; 10: 751-760Crossref PubMed Scopus (62) Google Scholar). Interestingly, brainiac was found to transfer β1–3 linked GlcNAc to both MacCer and LacCer, while mammalian homologs only transfer to LacCer (8Schwientek T. Keck B. Levery S.B. Jensen M.A. Pedersen J.W. Wandall H.H. Stroud M. Cohen S.M. Amado M. Clausen H. J. Biol. Chem. 2002; 277: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 9Muller R. Altmann F. Zhou D. Hennett T. J. Biol. Chem. 2002; 277: 32417-32420Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 11Togayachi A. Akashima T. Ookubo R. Kudo T. Nishihara S. Iwasaki H. Natsume A. Mio H. Inokuchi J. Irimura T. Sasaki K. Narimatsu H. J. Biol. Chem. 2001; 276: 22032-22040Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Sequence analysis of eggheadindicates that it could encode a type II transmembrane glycosyltransferase. Homologous genes appear limited to invertebrates, and no similar genes are found in the mammalian databases. In the present study we tested the hypothesis that egghead encodes a unique invertebrate glycosyltransferase activity in the same biosynthetic pathway as brainiac and present evidence thategghead indeed encodes a β1,4-mannosyltransferase predicted to form the MacCer precursor glycolipid substrate for brainiac. An expression construct of the full coding region of egghead was prepared by reverse transcriptase-PCR using Drosophila melanogastermRNA and the sense primer Egh001 (5′-AGCAGATCTCAAGATGAACTCCACCACAAAG-3′) with a BglII restriction site and the antisense primer Egh002 (5′-AATAGTCTAGACAGTCTCCAGTACGCG-3′) with a XbaI restriction site. The resulting 1.37-kb fragment was cloned into theBglII/XbaI sites of pVL1393 (PharMingen) and pVL1393-MYC. Baculovirus expression constructs, pVL-egghead-full and pVL-egghead-Myc-full, were co-transfected with Baculo-GoldTM DNA (PharMingen) in Sf9 cells as described (12Wandall H.H. Hassan H. Mirgorodskaya E. Kristensen A.K. Roepstorff P. Bennett E.P. Nielsen P.A. Hollingsworth M.A. Burchell J. Taylor-Papadimitriou J. Clausen H. J. Biol. Chem. 1997; 272: 23503-23514Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Control constructs included pVL-GalNAc-T4-full (13Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) and pVL-brainiac-full (8Schwientek T. Keck B. Levery S.B. Jensen M.A. Pedersen J.W. Wandall H.H. Stroud M. Cohen S.M. Amado M. Clausen H. J. Biol. Chem. 2002; 277: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Standard enzyme assays were performed in 50-μl reaction mixtures containing 25 mm HEPES-KOH (pH 7.4), 10 mmMgCl2, 0.1% n-octylgalactoside (Sigma), and 100 μm GDP-[14C]Man (2,000 cpm/nmol) (AmershamBiosciences), and varying concentration of acceptor substrates (purchased from Fluka, Merck, Sigma, and Toronto Research Chemicals Inc.; see Table I for structures). Assays with brainiac were carried out in the same reaction mixture except for addition of UDP-[14C]GlcNAc (3,000 cpm/nmol) (Amersham Biosciences) and MnCl2. Enzyme sources were microsomal fractions of baculovirus-infected Sf9 and High FiveTM cells prepared essentially as described (14Lee J. Sundaram S. Shaper N.L. Raju T.S. Stanley P. J. Biol. Chem. 2001; 276: 13924-13934Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Briefly, cells were lysed in lysis buffer (25 mm Tris-HCl (pH 7.4), 250 mmsucrose); after incubation 30 min on ice cells were homogenized and lysate centrifuged at 1,000 × g. Glycerol was added to 20%, and membrane pellets were obtained by 100,000 ×g. Pellets were used at 10 mg/ml (protein concentration determined by BCA, Pierce). Reaction products of soluble acceptors were quantified by chromatography on Dowex AG1-X8 (Sigma). Assays with glycosphingolipids included 5 mm2-acetamido-2-deoxy-d-glucono-1,5-lactone (inhibitor of hexosaminidase activity), and products were purified on octadecyl-silica cartridges (Supelco) and analyzed by high performance thin-layer chromatography followed by autoradiography.Table ISubstrate specificities of Egghead β1–4-mannosyltransferaseSubstrateEgghead1-aEnzyme sources were microsomal preparations of pVL-egghead infected High Five™ cells (see “Experimental Procedures”). Background values obtained with microsomes of cells infected with an irrelevant construct (GalNAc-T4) were subtracted.1 mm5 mmnmol/h/mgGlcβ1-MeUmb1-bBzl, benzyl; MeUmb, 4-methyl-umbelliferyl; Nph, nitrophenyl.330.0611.4Glcα1-MeUmb1.212.9Manβ1-MeUmb97171.4Galβ1-MeUmb0.0ND1-cND, not determined.GlcNAcβ1-MeUmb0.20.0Xylβ1-MeUmb0.0NDManβ1–4GlcNAc0.00.0Manα-1-Bzl0.0NDGlcβ1-pNph378.6561.4Manβ1-pNph98.6182.9GlcNAcβ1-pNph0.0NDGalβ1-pNph0.0NDGalNAcβ1-pNph0.0NDFucβ1-pNph0.00.0Galβ1-n-octyl0.00.0Glcα1-n-octyl3.66.5Glcβ1-n-octyl442.8732.6Manβ1–4Glcβ1-n-octyl0.00.01-a Enzyme sources were microsomal preparations of pVL-egghead infected High Five™ cells (see “Experimental Procedures”). Background values obtained with microsomes of cells infected with an irrelevant construct (GalNAc-T4) were subtracted.1-b Bzl, benzyl; MeUmb, 4-methyl-umbelliferyl; Nph, nitrophenyl.1-c ND, not determined. Open table in a new tab The 1.37-kb fragment used for baculo constructs was cloned into theBamHI/XbaI sites of pcDNA3(+). CHO-K1 cells were stably transfected with the pcDNA3-egghead-Myc-full as described previously and clones selected with anti-Myc antibodies (13Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Cells were grown to subconfluence and fixed with 3% paraformaldehyde and immunostained with anti-Myc monoclonal antibody (Invitrogen). Transferase assays were performed in standard reaction mixtures with cell lysates. The product formed with n-octyl glucoside (1 mg) was purified on octadecyl-silica cartridges (Bakerbond, J. T. Baker), followed by stepwise elution with increasing concentrations of methanol in water. The purified glycolipid was deuterium-exchanged by repeated addition of CDCl3-CD3OD 2:1, sonication, and evaporation under nitrogen, then dissolved in 0.5 ml of Me2SO-d 6, 2% D2O (0.03% tetramethylsilane) for NMR analysis. One-dimensional1H, two-dimensional 1H-1H gCOSY, TOCSY, and ROESY NMR spectra were acquired on a Varian Inova 500 MHz spectrometer at 35 °C. tBLASTn searches performed with D. melanogaster egghead coding region (GenBankTM accession number NM_080313) of the National Center for Biotechnology Information data base and the whole genome data base GadFly released by the Berkeley Drosophila Genome Project revealed genes with significant similarity in flies (diptera) and nematodes, includingCaenorhabditis elegans. Low sequence similarity was found to the putative cellulose synthetase CelA (GenBankTM accession number AAC41435 ) from Agrobacterium tumefaciens as well as other bacterial genes predicted to be glycosyltransferases (GenBankTM accession numbers NP_348317 (Clostridium acetobutylicum) and NP_531181 (A. tumefaciens str. C58)). No significant similarity was found with mammalian genes.egghead is predicted to encode a protein of 457 amino acids with a putative N-terminal signal sequence and a putative hydrophobic transmembrane retention signal (3Goode S. Morgan M. Liang Y.P. Mahowald A.P. Dev. Biol. 1996; 178: 35-50Crossref PubMed Scopus (64) Google Scholar), which is typical for Golgi located glycosyltransferases. SDS-PAGE Western blot analysis with anti-Myc antibodies of lysates of baculovirus-infected High FiveTMcells or a stable CHO egghead transfectant revealed a single protein migrating with an apparent molecular weight of 52 kDa (not shown). Subcellular localization of egghead was analyzed by immunofluorescense staining of a stable CHO eggheadtransfectant, where immunoreactivity was limited to a supranuclear pattern characteristic for Golgi localization (not shown). A similar staining pattern was found for a stable CHO transfectant with human β3GnT2 (not shown), as well as transfectants with other human glycosyltransferases (13Bennett E.P. Hassan H. Mandel U. Mirgorodskaya E. Roepstorff P. Burchell J. Taylor-Papadamitriou J. Hollingsworth M.A. Merkx G. Geurts van Kessel A. Eiberg H. Steffensen R. Clausen H. J. Biol. Chem. 1998; 273: 30472-30481Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). The GadFly data base predicts that egghead contains a sugar nucleotide donor substrate binding site with potential DXD/E binding motifs (15Busch C. Hofmann F. Selzer J. Munro S. Jeckel D. Aktories K. J. Biol. Chem. 1998; 273: 19566-19572Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Initial assays of activity included a screen with high concentrations of monosaccharide substrates and different donor substrates as described previously (6Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (593) Google Scholar,8Schwientek T. Keck B. Levery S.B. Jensen M.A. Pedersen J.W. Wandall H.H. Stroud M. Cohen S.M. Amado M. Clausen H. J. Biol. Chem. 2002; 277: 32421-32429Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Microsomal fractions of infected High FiveTM cells expressing the full coding region of egghead exhibited a marked increase in GDP-Man transferase activity withd-glucose (Fig. 1). Egghead exhibited strict donor substrate specificity for GDP-mannose and did not utilize other donor sugar nucleotides tested (UDP-Gal, UDP-GalNAc, UDP-GlcNAc). Analysis of a panel of mono- and disaccharide derivatives showed that egghead exhibits strong preference for substrates containing terminal β-linked glucose (β-Glc) (TableI). Interestingly, some βMan monosaccharide derivatives also served as efficient substrates; however, no activity was found with the disaccharides Manβ1–4GlcNAc and Manβ1–4Glcβ1-n-Oct. Analysis of apparentK m for the most active substrates identified showed that n-octyl-β-Glc was the preferred acceptor substrate (apparent K m 0.67 ± 0.08 mm) with Glcβ1-pNph (apparent K m 1.10 ± 0.3 mm) being comparable and Manβ1-pNph (apparent K m 2.30 ± 0.5 mm) less preferred. The apparent K m for GDP-Man withn-octyl-β-Glc acceptor substrate was 58.0 ± 6.2 μm. Optimization of the enzyme assay using microsomal membranes demonstrated that Triton X-100, Triton CF-54, and Nonidet P-40 inhibited egghead activity at 0.1%, whilen-octylgalactoside at 3.4 mm (0.1%) and to a lesser extent CHAPS activated the enzyme. The pH optimum of egghead activity was pH 7–8. Addition of 5–10 mmMgCl2 and MnCl2 activated enzyme activity (Mg2+ being better than Mn2+), and CaCl2 had no effect, while addition of 10 mmEDTA destroyed the activity. Analysis of egghead activity in the established CHO transfectant cells showed the same properties as when egghead is expressed in insect cells (not shown). Attempts to visualize in vivo formed products by lectin staining with Vicia Faba (Sigma) was unsuccessful, and further characterization of the products formed await large scale production of cells for chemical analysis of glycolipids. Glycosphingolipids of the fruit fly are based on the arthroseries GlcNAcβ1–3Manβ1–4Glcβ1–1Cer core (10Seppo A. Tiemeyer M. Glycobiology. 2000; 10: 751-760Crossref PubMed Scopus (62) Google Scholar). The finding that egghead exhibits β-mannosyltransferase activity with βGlc acceptor substrates strongly suggested that egghead transfers Man to Glcβ1–1Cer to form MacCer. As shown in Fig. 2 egghead utilizes Glcβ1–1Cer as an acceptor substrate, whereas LacCer does not serve as substrate. In addition, Galβ1–1Cer was found not to serve as a substrate (not shown). Based on this result it was predicted that egghead functions as the MacCer synthase. Evidence in support hereof was provided by showing that brainiac utilizes the product formed by egghead (Fig. 3). This assay was carried out withn-octyl-β-Glc as initial acceptor substrate because it served as a better substrate than GlcCer under the assay conditions used.Figure 3The product formed by egghead withn-octyl-βGlc serves as a substrate for brainiac. High performance thin-layer chromatography analysis of product developments (2 h) with combinations of microsomal fractions of egghead(Egh), polypeptide GalNAc-T4 (GT4), andbrainiac (Brn) expressing High FiveTMcells and combinations of sugar nucleotides GDP-Man and UDP-GlcNAc are shown. The upper panel is stained with orcinol, and thelower panel represents an autoradiography. Plates were run in chloroform-methanol-water (60/30/8, v/v/v), and the migration ofn-octyl-βGlc (NOG) and the disaccharide and trisaccharide products hereof are indicated in the margins. Man-Glc-Oct is formed only in the presence of egghead and GDP-Man, and GlcNAc-Man-Glc-Oct is formed only in the presence both of egghead and brainiac as well as GDP-Man and UDP-GlcNAc. In lane 7, theasterisks indicate that the autoradiography assay was carried out with non-labeled GDP-Man to confirm that the initial added sugar was Man.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A one-dimensional 1H NMR spectrum of the diglycosyl product formed with n-octyl-β-glucoside exhibited resonances consistent with ∼55% conversion to Manβ1–4Glcβ1–1-n-octyl, i.e. anomeric signals at 4.477 and 4.143 ppm (3 J 1,2 = ∼1 and 7.9 Hz, respectively), corresponding to H-1 of Manβ1–4 and Glcβ1–1 residues of this glycolipid. H-1 of unreacted Glcβ1–1 is observed at 4.080 ppm (3 J 1,2 = 7.6 Hz) (Fig. 4). Following complete assignment of1H resonances from all three monosaccharide spin systems present (see Table II) by two-dimensional1H-1H gCOSY and TOCSY experiments (not shown), the connectivity between the β-Man and the more abundant β-Glc (spin system originating from the H-1 at 4.143 ppm) was established as a 1→4 linkage by a two-dimensional ROESY experiment, which showed a dipolar cross-relaxation correlation between β-Man H-1 and β-Glc H-4. This is consistent with the substantial downfield shift of H-4 compared with that observed for unreacted n-octyl β-glucoside (3.350 versus 3.016). Although other β-Glc resonances are affected by the glycosylation, H-4 is shifted downfield by the largest increment (ΔδH-4 = 0.334 ppm; ΔδH-3 = 0.244 ppm; ΔδH-5 = 0.151 ppm).Table II1 H chemical shifts (ppm) and 3 J1,2 coupling constants (Hz, in parentheses) for Glcβ1-n-octyl substrate and biosynthetic Manβ4-Glcβ1-n-octyl product.Manβ4-Glcβ1-n-octylGlcβ1-n-octylManβ4Glcβ1n-OctGlcβ1n-OctH-14.4774.1433.40, 3.73 (2)4.0803.40, 3.71 (2)3 J 1,2(∼1)(7.9)(7.6)H-23.6772.9741.494 (2)2.9121.494 (2)H-33.2393.316∼1.24 (10)3.111∼1.24 (10)H-43.2753.3443.016H-53.0943.2153.059H-6a3.3953.5003.410H-6b3.6953.5973.636CH30.842 (3)0.842 (3)Data were obtained in Me2SO-d 6, 2% D2O at 35 °C. Chemical shifts are referenced to internal tetramethylsilane (set to 0.000 ppm). Open table in a new tab Data were obtained in Me2SO-d 6, 2% D2O at 35 °C. Chemical shifts are referenced to internal tetramethylsilane (set to 0.000 ppm). The original prediction that the neurogenic genesbrainiac and egghead encoded proteins serving functions in a common pathway has been verified by demonstrating that both genes encode glycosyltransferases and that egghead can synthesize the immediate precursor glycolipid substrate for brainiac. The two enzymes function very early in glycosphingolipid biosynthesis at the second and third steps in build-up of the glycan chain, and it is likely that this reflects the severe phenotypes associated with inactivation of these genes. Glycosphingolipids ofDrosophila have been reported to be based on the arthroseries and exist as extended oligosaccharide structures such as Galβ1–3GalNAcα1–4GalNAcβ1–4GlcNAcβ1–3Manβ1–4Glcβ1–1Cer, which can be terminated by glucuronic acids and modified with phosphoethanolamine to give charged and zwitterionic glycolipids (10Seppo A. Tiemeyer M. Glycobiology. 2000; 10: 751-760Crossref PubMed Scopus (62) Google Scholar, 16Seppo A. Moreland M. Schweingruber H. Tiemeyer M. Eur. J. Biochem. 2000; 267: 3549-3558Crossref PubMed Scopus (80) Google Scholar). Specific biological functions of distinct glycolipid structures have not been elucidated in Drosophila, but it is conceivable that termination of glycolipid biosynthesis at GlcCer and at MacCer could block biological activity of glycolipids to similar effect. Genetic approaches to studying glycosphingolipid functions in mammals have so far provided some insight into defined biological activities. In contrast to invertebrate glycolipids that appear to be based on one class, mammalian glycolipids are based on multiple classes. Mice deficient in ganglioseries glycolipids built on GalNAcβ1–4Galβ1–4Glcβ1-Cer have yielded significant information (17Inoue M. Fujii Y. Furukawa K. Okada M. Okumura K. Hayakawa T. Furukawa K. Sugiura Y. J. Biol. Chem. 2002; 277: 29881-29888Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 18Chiavegatto S. Sun J. Nelson R.J. Schnaar R.L. Exp. Neurol. 2000; 166: 227-234Crossref PubMed Scopus (141) Google Scholar, 19Okada M. Itoh M.M. Haraguchi M. Okajima T. Inoue M. Oishi H. Matsuda Y. Iwamoto T. Kawano T. Fukumoto S. Miyazaki H. Furukawa K. Aizawa S. Furukawa K. J. Biol. Chem. 2002; 277: 1633-1636Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 20Takamiya K. Yamamoto A. Furukawa K. Yamashiro S. Shin M. Okada M. Fukumoto S. Haraguchi M. Takeda N. Fujimura K. Sakae M. Kishikawa M. Shiku H. Furukawa K. Aizawa S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10662-10667Crossref PubMed Scopus (340) Google Scholar, 21Kawai H. Allende M.L. Wada R. Kono M. Sango K. Deng C. Miyakawa T. Crawley J.N. Werth N. Bierfreund U. Sandhoff K. Proia R.L. J. Biol. Chem. 2001; 276: 6885-6888Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). Globoseries glycolipids built on Galα1–4Galβ1–4Glcβ1-Cer are dispensable in man as evidenced from the rare Pk and p blood groups (22Steffensen R. Carlier K. Wiels J. Levery S.B. Stroud M. Cedergren B. Nilsson S.B. Bennett E.P. Jersild C. Clausen H. J. Biol. Chem. 2000; 275: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 23Hellberg A. Poole J. Olsson M.L. J. Biol. Chem. 2002; 277: 29455-29459Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). While the biosynthesis of ganglioseries and globoseries glycolipids are carried out by unique single copy genes, each step in the biosynthesis of lacto- and neolactoseries glycolipids based on the fact that GlcNAcβ1–3Galβ1–4Glcβ1-Cer is carried out by multiple isoenzymes, many of which serve functions in the synthesis of glycoproteins as well (24Amado M. Almeida R. Schwientek T. Clausen H. Biochim. Biophys. Acta. 1999; 1473: 35-53Crossref PubMed Scopus (263) Google Scholar). Drosophila and C. elegans may in this respect constitute simpler systems for studies of functions of glycolipids. Recently, the β4GalNAc-transferase acting in sequence after brainiac to form GalNAcβ1–4GlcNAcβ1–3Manβ1–4Glcβ1–1Cer was characterized (25Kawar Z.S. Van D.I. Cummings R.D. J. Biol. Chem. 2002; 277: 34924-34932Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). An increasing number of genes involved in biosynthesis of glycoconjugates have been identified as essential or important for normal development of flies and nematodes. A number of genes involved in biosynthesis of the proteoglycan core region were identified through an elegant screen for defects in vulval invagination of C. elegans (26Herman T. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 974-979Crossref PubMed Scopus (119) Google Scholar), and these include glycosyltransferases functioning in precursor-product relationships and relevant sugar nucleotide transporters (27Bulik D.A. Robbins P.W. Biochim. Biophys. Acta. 2002; 1573: 247-257Crossref PubMed Scopus (26) Google Scholar), fringe, a distant homolog ofbrainiac, was found to encode a key enzyme controlling elongation of O-linked fucose directly on Notch (6Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (593) Google Scholar,7Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (721) Google Scholar), and precursor-product relationships with glycosyltransferases functioning after fringe have also been implicated (28Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar). To our knowledge, egghead and brainiac are currently the only available examples of essential genes in Drosophilawith functions in the biosynthesis of glycolipids. Glycolipids are known to serve important biological functions in mammals including modulation of receptor functions (29Hakomori S. Glycoconj. J. 2000; 17: 627-647Crossref PubMed Scopus (168) Google Scholar). Modulation may be mediated through direct lectin-carbohydrate interactions between the receptor and glycolipids (30Miljan E.A. Meuillet E.J. Mania-Farnell B. George D. Yamamoto H. Simon H.G. Bremer E.G. J. Biol. Chem. 2002; 277: 10108-10113Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) or through organization of lipid rafts, which are known to be enriched in MacCer in Drosophila (31Rietveld A. Neutz S. Simons K. Eaton S. J. Biol. Chem. 1999; 274: 12049-12054Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar).Egghead and brainiac offers new tools to decipher mechanisms of receptor modulation through glycolipids." @default.
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