Egghead and Brainiac Are Essential for Glycosphingolipid Biosynthesis in Vivo
2004; Elsevier BV; Volume: 280; Issue: 6 Linguagem: Inglês
10.1074/jbc.c400571200
ISSN1083-351X
AutoresHans H. Wandall, Sandrine Pizette, Johannes W. Pedersen, Heather Eichert, Steven B. Levery, Ulla Mandel, Stephen M. Cohen, Henrik Clausen,
Tópico(s)Sphingolipid Metabolism and Signaling
ResumoThe Drosophila genes, brainiac and egghead, encode glycosyltransferases predicted to act sequentially in early steps of glycosphingolipid biosynthesis, and both genes are required for development in Drosophila. egghead encodes a β4-mannosyltransferase, and brainiac encodes a β3-N-acetylglucosaminyltransferase predicted by in vitro analysis to control synthesis of the glycosphingolipid core structure, GlcNAcβ1–3Manβ1–4Glcβ1-Cer, found widely in invertebrates but not vertebrates. In this report we present direct in vivo evidence for this hypothesis. egghead and brainiac mutants lack elongated glycosphingolipids and exhibit accumulation of the truncated precursor glycosphingolipids. Furthermore, we demonstrate that despite fundamental differences in the core structure of mammalian and Drosophila glycosphingolipids, the Drosophila egghead mutant can be rescued by introduction of the mammalian lactosylceramide glycosphingolipid biosynthetic pathway (Galβ1–4Glcβ1-Cer) using a human β4-galactosyltransferase (β4Gal-T6) transgene. Conversely, introduction of egghead in vertebrate cells (Chinese hamster ovary) resulted in near complete blockage of biosynthesis of glycosphingolipids and accumulation of Manβ1–4Glcβ1-Cer. The study demonstrates that glycosphingolipids are essential for development of complex organisms and suggests that the function of the Drosophila glycosphingolipids in development does not depend on the core structure. The Drosophila genes, brainiac and egghead, encode glycosyltransferases predicted to act sequentially in early steps of glycosphingolipid biosynthesis, and both genes are required for development in Drosophila. egghead encodes a β4-mannosyltransferase, and brainiac encodes a β3-N-acetylglucosaminyltransferase predicted by in vitro analysis to control synthesis of the glycosphingolipid core structure, GlcNAcβ1–3Manβ1–4Glcβ1-Cer, found widely in invertebrates but not vertebrates. In this report we present direct in vivo evidence for this hypothesis. egghead and brainiac mutants lack elongated glycosphingolipids and exhibit accumulation of the truncated precursor glycosphingolipids. Furthermore, we demonstrate that despite fundamental differences in the core structure of mammalian and Drosophila glycosphingolipids, the Drosophila egghead mutant can be rescued by introduction of the mammalian lactosylceramide glycosphingolipid biosynthetic pathway (Galβ1–4Glcβ1-Cer) using a human β4-galactosyltransferase (β4Gal-T6) transgene. Conversely, introduction of egghead in vertebrate cells (Chinese hamster ovary) resulted in near complete blockage of biosynthesis of glycosphingolipids and accumulation of Manβ1–4Glcβ1-Cer. The study demonstrates that glycosphingolipids are essential for development of complex organisms and suggests that the function of the Drosophila glycosphingolipids in development does not depend on the core structure. Invertebrates, Caenorhabditis elegans and Drosophila melanogaster, have recently attracted considerable attention as model organisms for deciphering specific biological roles of complex carbohydrates. One elegant example of this was a number of studies leading to the identification of a series of glycosylation genes critical for vulval invagination in C. elegans, which were all shown to affect a common biosynthetic pathway for the assembly of the O-linked oligosaccharide linker region common for all proteoglycans (1Selleck S.B. Trends Genet. 2000; 16: 206-212Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Another example was the role of the O-linked fucose glycosylation pathway on the Notch receptor function (2Haltiwanger R.S. Lowe J.B. Annu. Rev. Biochem. 2004; 73: 491-537Crossref PubMed Scopus (645) Google Scholar). The Drosophila neurogenic genes brainiac and egghead encode glycosyltransferases essential for epithelial development during oogenesis and in the embryo (3Goode S. Melnick M. Chou T.B. Perrimon N. Development (Camb.). 1996; 122: 3863-3879Crossref PubMed Google Scholar, 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). egghead and brainiac mutants display similar, non-additive defects, which has led to the proposal that they act in the same pathway (3Goode S. Melnick M. Chou T.B. Perrimon N. Development (Camb.). 1996; 122: 3863-3879Crossref PubMed Google Scholar). In previous reports we demonstrated that brainiac encodes a UDP-N-acetylglucosamine: βMan β1,3-N-acetylglucosaminyltransferase (β3GlcNAc-transferase), and egghead encodes a GDP-mannose:βGlc β1,4-mannosyltransferase, with putative functions in sequential steps in the biosynthesis of the core structure of arthro-series glycosphingolipids (GlcNAcβ1–3Manβ1–4Glcβ1-Cer) as predicted by in vitro analysis (Fig. 1) (5Schwientek 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, 6Wandall H.H. Pedersen J.W. Park C. Levery S.B. Pizette S. Cohen S.M. Schwientek T. Clausen H. J. Biol. Chem. 2003; 278: 1411-1414Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 7Muller R. Altmann F. Zhou D. Hennet T. J. Biol. Chem. 2002; 277: 32417-32420Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Loss of either gene is predicted to abrogate glycosphingolipid biosynthesis at the di- or monosaccharide-ceramide step.Fig. 1egghead mutants lack mannosyltransferase activity and elongated glycosphingolipids. A, glycosphingolipid core biosynthetic pathways of Drosophila and human. Glcβ1-Cer is a common precursor for most extended glycosphingolipids. Glcβ1-Cer is synthesized by a single homologous glucosyltransferase. Invertebrate elongation is initiated with a mannose residue catalyzed by Egghead followed by elongation with GlcNAc catalyzed by Brainiac and additional monosaccharide residues catalyzed by distinct glycosyltransferases. Phosphoethanolamine linked to GlcNAc residues are found only in invertebrates (data not shown). Vertebrate elongation is initiated with a galactose residue, catalyzed by two homologous β4-galactosyltransferases (β4Gal-T5 and β4Gal-T6) forming LacCer (Galβ1–4Glcβ1-Cer) (8Nomura T. Takizawa M. Aoki J. Arai H. Inoue K. Wakisaka E. Yoshizuka N. Imokawa G. Dohmae N. Takio K. Hattori M. Matsuo N. J. Biol. Chem. 1998; 273: 13570-13577Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). After this step, elongation branches into three pathways: ganglio-series catalyzed by the GM2 synthase, globo-series catalyzed by α4Gal-T1, and lacto-series catalyzed by multiple homologous β3GlcNAc-transferases. Highly conserved homologous enzymes carry out most biosynthetic steps in the glycosphingolipid pathways of vertebrates, insects, and nematodes. One exception is the addition of mannose in invertebrate glycosphingolipids catalyzed by Egghead. This saccharide linkage is not found in vertebrate glycosphingolipids, and no homologue of the egghead gene is found in vertebrates. B, mannosyltransferase activity (counts/min) measured in larvae extracts of wild-type, brainiac1.6P6, and the indicated egghead mutants using GDP-[C14]Man as donor substrate and Glcβ1-Octyl as acceptor substrate. C, analysis of glycosphingolipids from wild-type (control) and mutant larvae by high performance thin layer chromatography demonstrated accumulation of GlcCer in all four egh mutant larvae and of MacCer in brn mutant larvae. Dark bands appearing in mutant lanes near the bottom of the plate are not glycosphingolipids, indicated by failure to stain with primulin, a lipid indicator. D, immunostaining of follicle cells surrounding the Drosophila oocyte. GFP is shown in green, and anti-MacCer is shown in red. Nuclei labeled with DAPI are shown in blue. Upper panel, cells lacking Brainiac activity (brn1.6P6 clones marked by the absence of GFP) showed strong MacCer labeling (red). Lower panel, cells lacking Egghead activity (egh62d18 clones marked by the absence of GFP) did not show elevated levels of MacCer.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Insect, nematode, and vertebrate glycosphingolipids share a common element consisting of Glcβ1-ceramide, after which they differ markedly in structure and complexity (Fig. 1A). Insect and nematode glycosphingolipids are built on Manβ1–4Glcβ1-ceramide (MacCer) 1The abbreviations used are: MacCer, Manβ1–4Glcβ1-ceramide; LacCer, Galβ1–4Glcβ1-ceramide; CHO, Chinese hamster ovary; CHO, Chinese hamster ovary; HPTLC, high performance thin layer chromatography; SPE, solid-phase extraction; MBL, mannan binding lection; DAPI, 4′,6-diamidino-2-phenylindole; GFP, green fluorescent protein; UAS, upstream activating sequence; CDH, ceramide dihexoside; GM2, GalNAcβ1–4(NeuAcα2–3)Galβ1–4Glcβ1-Cer; GM3, N-acetylneuraminylgalactosylceramide. predicted to be catalyzed by Egghead, while vertebrαte glycosphingolipids are built on Galβ1–4Glcβ1-ceramide (LacCer) catalyzed by the β4-galactosyltransferases, β4Gal-T5 and -T6 (8Nomura T. Takizawa M. Aoki J. Arai H. Inoue K. Wakisaka E. Yoshizuka N. Imokawa G. Dohmae N. Takio K. Hattori M. Matsuo N. J. Biol. Chem. 1998; 273: 13570-13577Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 9Amado M. Almeida R. Schwientek T. Clausen H. Biochim. Biophys. Acta. 1999; 1473: 35-53Crossref PubMed Scopus (263) Google Scholar). Despite considerable differences in overall structures of glycosphingolipids among insects and vertebrates, it is clear that homologous glycosyltransferase genes conserved throughout evolution catalyze most biosynthetic steps. Egghead is perhaps the only exception suggesting that MacCer-based glycosphingolipids represent a specific functional basis for the diversification of the underlying biosynthetic pathways. Importantly, vertebrate glycosphingolipids based on the LacCer core diverge at the third biosynthetic step to form different classes of structures (Fig. 1A), which are differentially expressed in cells and are differentially expressed during development and differentiation (10Fenderson B.A. Andrews P.W. Nudelman E. Clausen H. Hakomori S. J. Cell Biol. 1986; 103: A225Google Scholar). The vertebrate glycosphingolipid lacto-series is initiated by addition of β1,3GlcNAc to LacCer by brainiac orthologs designated β3GnTs (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 (106) Google Scholar, 12Shiraishi N. Natsume A. Togayachi A. Endo T. Akashima T. Yamada Y. Imai N. Nakagawa S. Koizumi S. Sekine S. Narimatsu H. Sasaki K. J. Biol. Chem. 2001; 276: 3498-3507Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 13Kudo T. Iwai T. Kubota T. Iwasaki H. Takayma Y. Hiruma T. Inaba N. Zhang Y. Gotoh M. Togayachi A. Narimatsu H. J. Biol. Chem. 2002; 277: 47724-47731Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 14Iwai T. Inaba N. Naundorf A. Zhang Y. Gotoh M. Iwasaki H. Kudo T. Togayachi A. Ishizuka Y. Nakanishi H. Narimatsu H. J. Biol. Chem. 2002; 277: 12802-12809Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 15Yeh J.C. Hiraoka N. Petryniak B. Nakayama J. Ellies L.G. Rabuka D. Hindsgaul O. Marth J.D. Lowe J.B. Fukuda M. Cell. 2001; 105: 957-969Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar). Interestingly, Drosophila brainiac functions both on the invertebrate and vertebrate precursor substrate MacCer and LacCer, while the vertebrate orthologs appear to only act on LacCer (5Schwientek 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). In this report we present direct evidence that Egghead and Brainiac do function in vivo in the glycosphingolipid pathway and are essential for glycosphingolipid biosynthesis in vivo. Furthermore, we demonstrate that despite the fundamental difference in the structure of core glycosphingolipid, the Drosophila egghead mutant can be rescued by introduction of the corresponding enzyme from the human glycosphingolipid biosynthetic pathway. In contrast the fly glycosphingolipid biosynthetic pathway is not elongated in vertebrate cells. The results show that glycosphingolipids are essential for development of complex organisms and suggest that the function of Drosophila glycosphingolipids in development does not depend on the core structure. Sequencing of egghead Mutants—Genomic DNA was purified from egh7, egh62d18, and egh9PP4 homozygous mutant larvae. A PCR product was generated by standard polymerase chain reaction using primers (5′-AAGCTCTGGAGGACCAAAGCC-3′) and (5′-TCCTCCCTCATCCAGTTCCAC-3′) (25 cycles of 95 °C for 45 s, 55 °C for 30 s, 72 °C for 2 min). The generated PCR product was purified and sequenced with primers: Eghs01 (5′-AAGATGAACTCCACCACA-3′), Eghs02 (5′-GTCAATCATAATACCGCC-3′), Eghs03 (5′-TCATCGAAGTGGTCACGG-3′), Eghs04 (5′-CGCAAGCAGCCGTTCCG-3′), Eghs05 (5′-TCAACTTCARCGAGGGCG-3′), and Eghs07 (5′-GAACATCATCTTTGCGGC-3′) using ABI sequencing. Mutations/deletions were verified by generation of a second independent PCR product. Enzymatic Activity in Extracts from Mutant Larvae—Mutant larvae were homogenized in extraction buffer: 100 mm Hepes, 1% n-octyl β1-glucoside, 10 mm NaCl, EDTA-free protease inhibitor mixture (Roche Applied Science), spun 1,000 × g for 10 min, and supernatant used for enzymatic assay. Extraction of Glycosphingolipids from Mature Flies, Larvae, and CHO Cells—glycosphingolipids were extracted and fractionated by methods similar to those described previously (16Bennion B. Park C. Fuller M. Lindsey R. Momany M. Jennemann R. Levery S.B. J. Lipid Res. 2003; 44: 2073-2088Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Freeze-dried flies (∼5–10 g) were homogenized (Polytron, Kinematica AG, Luzern, Switzerland) in ∼5 volumes of solvent A (2-isopropanol/hexane/water, 55:25:20, v/v/v, upper phase discarded), centrifuged, and the supernatant removed; this step was repeated with ∼5 volumes of solvent B (chloroform/methanol, 1:1, v/v) and then sequentially again with ∼5 volumes each of solvent A and solvent B. The four solvent extracts were combined, dried in a rotary evaporator, and treated with 10 ml of methanol/water/1-butanol (4:3:1, v/v/v) containing 25–30% methylamine at 55 °C for 4 h (flask tightly stoppered), with occasional agitation and sonication. The reagents were again reduced to dryness by rotary evaporation, resuspended in a minimal volume of solvent C (chloroform/methanol/water, 30:60:8, v/v/v), and applied to a column of DEAE-Sephadex A-25 (Ac-form). Neutral glycosphingolipids were eluted with 5 volumes of solvent C. Acidic glycosphingolipids were eluted with 5 volumes of 0.8 m sodium acetate in methanol, which was reduced to dryness, subjected to exhaustive dialysis (3,500 Da cutoff) against deionized water to remove salts, and recovered by lyophilization prior to HPTLC analysis. CHO cells (∼1–2 ml packed volume) were subjected to a similar protocol, except that extensive sonication (20–30 min per step) was used instead of Polytron homogenization to break up cells during extraction, and the order was solvent B (1×), solvent A (2×), and solvent B (1×). Frozen larvae (50–150 mg) were subjected to a similar but truncated micro-scale extraction procedure with the following differences: (i) solvent volumes were 3 ml each, and the order was solvent B (1×), solvent A (2×), and solvent B (1×); (ii) larvae were macerated with a glass rod and sonicated for 20 min at each step; (iii) 20–25% methylamine reagent volume was 1 ml; (iv) drying steps were carried under N2 stream at 35–40 °C; (v) the DEAE-Sephadex anion exchange fractionation was omitted, but instead total lipids were subjected to a solid-phase extraction (SPE) cleanup step to remove as much non-lipid carbohydrate material as possible. SPE was carried out on 0.5-g octadecyl-silica cartridges (Honeywell/Burdick & Jackson, Muskegon, MI), applying lipids sonicated thoroughly in 0.5 n NaCl (1 ml). The pass-through was collected and re-applied two times and the SPE cartridge then washed sequentially with 0.5 n NaCl (2 ml) and deionized water (4 ml). Lipids were eluted with methanol (2 ml), followed by solvent B (2 ml), combining both and drying under N2 stream prior to HPTLC analysis. High Performance Thin Layer Chromatography—Analytical HPTLC was performed on silica gel 60 plates (E. Merck, Darmstadt, Germany) using chloroform/methanol/water (60:35:8, v/v/v; solvent D) as mobile phase for neutral or total lipids and chloroform/methanol/water (50:47: 14, v/v/v, containing 0.038% (w/v) CaCl2; solvent E) as mobile phase for acidic lipids. Detection was made by Bial's orcinol reagent (0.55% orcinol (w/v) and 5.5% H2SO4 (v/v) in ethanol/water 9:1 (v/v); the plate is sprayed and heated briefly to ∼200–250 °C). Preparative HPTLC was carried out on neutral lipids using solvent D as mobile phase, streaking crude fraction lengthwise on 10 × 20-cm plates; separated glycosphingolipid bands were visualized under UV after spraying with primulin (Aldrich; 0.01% in 80% aqueous acetone). Bands were marked by pencil and individually scraped from the plate. Glycosphingolipids were then isolated from the silica gel by repeated sonication in solvent B followed by centrifugation. Following concentration of the extract, primuline was removed by passage through a short column of DEAE-Sephadex A-25 in solvent C, which was removed under N2 stream prior to analysis by NMR spectroscopy. 1H NMR Spectroscopic Analysis of Glycosphingolipids—Individual glycosphingolipids isolated by preparative HPTLC were deuterium-exchanged by repeated addition of CD3OD, sonication, and evaporation under nitrogen, then dissolved in 0.5 ml of Me2SO-d6, 2% D2O (0.03% tetramethylsilane as internal chemical shift reference) for NMR analysis (17Dabrowski J. Hanfland P. Egge H. Biochemistry. 1980; 19: 5652-5658Crossref PubMed Scopus (130) Google Scholar). One-dimensional 1H NMR spectra were acquired on a Varian Inova 500 MHz spectrometer at 35 °C. Spectra were interpreted by comparison to those of authentic standards and published data (5Schwientek 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, 17Dabrowski J. Hanfland P. Egge H. Biochemistry. 1980; 19: 5652-5658Crossref PubMed Scopus (130) Google Scholar, 18Steffensen R. Carlier K. Wiels J. Levery S.B. Stroud M. Cedergren B. Sojka B.N. Bennett E.P. Jersild G. Clausen H. J. Biol. Chem. 2000; 275: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In Vitro Glycosylation Assays—Expression constructs of the full coding region of Drosophila egghead and brainiac were performed as described previously (6Wandall H.H. Pedersen J.W. Park C. Levery S.B. Pizette S. Cohen S.M. Schwientek T. Clausen H. J. Biol. Chem. 2003; 278: 1411-1414Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 18Steffensen R. Carlier K. Wiels J. Levery S.B. Stroud M. Cedergren B. Sojka B.N. Bennett E.P. Jersild G. Clausen H. J. Biol. Chem. 2000; 275: 16723-16729Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Expression constructs for human β4Gal-T6, β3GnT2, and β3GnT5 were prepared by reverse transcriptase-PCR using human brain and colon mRNA. The following sense primers with a BamHI restriction site and the antisense primers with a NotI restriction site were used: β4Gal-T6:B4GT601 (5′-AGCGGATCCAAGATGTCTGTGCTCAGGCGG-3′), B4GT602 (5′-CGCGGCCGCTTAATAGTCTTCGATTGGAGC-3′), β3GnT201 (5′-AGCGGATCCGAAATGAGTGTTGGACGTCG-3′), sol β3GnT201(5′-AGCGGATCCATGGAAGTCTCCAAAAGCAG-3′), β3GnT202 (5′-GCGCGGCCGCTTAGCATTTTAAATGAGC-3′), β3GnT501 (5′-AGCGGATCCGATATGAGAATGTTGGTTAGT-3′), and β3GnT502 (5′-GCGCGGCCGCATTCAAGTACTATTAGATAAACGC-3′). Fragments were cloned into the BamHI/NotI sites of pVL1393 (Pharmingen). Baculovirus expression constructs, pVL-egghead-full, pVL-brainiac full, pVL-β4GalT6-full, pVL-β3GnT2-full, and β3GnT5-full were co-transfected with Baculo-Gold™ DNA (Pharmingen) in Sf9 cells as described (19Wandall 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 (268) Google Scholar). Egghead enzyme assays were performed as described previously (6Wandall H.H. Pedersen J.W. Park C. Levery S.B. Pizette S. Cohen S.M. Schwientek T. Clausen H. J. Biol. Chem. 2003; 278: 1411-1414Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) in reaction mixtures containing 25 mm Hepes-KOH (pH 7.4), 10 mm MgCl2, 1% n-octyl glucoside (Sigma), and 100 μm GDP-[14C]Man (2,000–4,000 cpm/nmol) (Amersham Biosciences). Assays with brainiac, β3GnT5, and β3GnT2 were carried out in the same reaction mixture except for addition of UDP-[14C]GlcNAc (3,000 cpm/nmol)/UDP-GlcNAc (Amersham Biosciences) and MnCl2. Assays with β4Gal-T6 were performed with UDP-[14C]Gal/UDP-Gal. Enzyme sources were microsomal fractions of baculovirus-infected High Five™ cells prepared essentially as described (6Wandall H.H. Pedersen J.W. Park C. Levery S.B. Pizette S. Cohen S.M. Schwientek T. Clausen H. J. Biol. Chem. 2003; 278: 1411-1414Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Reaction products were purified on octadecyl-silica cartridges (Supelco) and analyzed either by scintillation counting and/or by high performance thin layer chromatography followed by detection with orcinol. Generation of Monoclonal Antibody Recognizing Manβ1–4Glcβ1-Cer—For production of the anti-MacCer monoclonal antibody BALB/c mice were immunized three times with 10 μg of purified MacCer isolated from High Five™ cells as described (5Schwientek 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). Hybridomas were selected by immunocytology on air-dried, acetone-fixed CHO cells stably transfected with full-length egghead as well as by ability to differentially recognize MacCer in enzyme-linked immunosorbent assay (20Mandel U. Hassan H. Therkildsen M.H. Rygaard J. Jakobsen M.H. Juhl B.R. Dabelsteen E. Clausen H. Glycobiology. 1999; 9: 43-52Crossref PubMed Scopus (106) Google Scholar). Stable Expression of Egghead in Chinese Hamster Ovary Cells—The 1.37-kb egghead-Myc-full fragment used for baculo constructs was cloned into the BamHI/XbaI sites of pcDNA3(+)Zeocin. Chinese hamster ovary (CHO-K1) cells were stably transfected with the pcDNA3-egghead-Myc-full as described previously and clones selected with anti-Myc antibodies (Invitrogen) (6Wandall H.H. Pedersen J.W. Park C. Levery S.B. Pizette S. Cohen S.M. Schwientek T. Clausen H. J. Biol. Chem. 2003; 278: 1411-1414Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Two rounds of screening and cloning were performed by limiting dilution cloning using immunoreactivity with anti-Myc monoclonal antibody. Immunolabeling—CHO cells were grown to subconfluence in the appropriate media as recommended by American Type Culture Collection and fixed in 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with undiluted anti-MacCer hybridoma supernatants for 18 h at 4 °C and detected with fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin (F261, Dako). Immunostaining with soluble mannan binding lectin (MBL) was performed on non-fixed cells using purified MBL from human serum detected with an anti-MBL monoclonal antibody (generous gift from P. Garred P, Copenhagen University Hospital, Copenhagen, Denmark) and fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin. Drosophila ovaries were dissected, fixed in 4% formaldehyde in phosphate-buffered saline, blocked in 0.1% bovine serum albumin, 0.05% Tween 20 in phosphate-buffered saline, and incubated with undiluted anti-MacCer antibody and detected with Cy5 anti mouse antibodies from Jackson ImmunoResearch Laboratories. Ovaries were mounted in 80% glycerol. DAPI was included in the washes to reveal nuclei. Fly Strains—Armadillo-Gal4 (II), actin-Gal4, and tubulin-Gal4 are described in flybase (fly.bio.Indiana.edu/gal4.htm). Brn1.6P6 is described in Goode et al. (21Goode S. Morgan M. Liang Y.P. Mahowald A.P. Dev. Biol. 1996; 178: 35-50Crossref PubMed Scopus (64) Google Scholar) and egh mutations in Goode et al. (3Goode S. Melnick M. Chou T.B. Perrimon N. Development (Camb.). 1996; 122: 3863-3879Crossref PubMed Google Scholar). We further characterized three egh mutant alleles at the molecular level. For isolation of genomic DNA and characterization of the enzymatic activity of egh and brn mutants, animals of the correct genotype were identified as follows: egh and brn alleles were balanced over a GFP-expressing FM7 balancer chromosome. Larvae were sexed, and mutant males were picked on the basis of their lack of GFP expression. In the case of the egh9PP4 and brn1.6P6 alleles, the cuticular marker yellow (y) present on these chromosomes was also used to identify mutants by the color of the head skeleton. Genetic Mosaic Analysis—brn and egh mutant alleles were recombined onto FRT18 and mitotic recombination clones were induced in adult females by heat shock for 60 min at 38 °C. The genotypes used are as follows: y w brn1.6P6 f FRT18/y UbiGFP FRT18; hs-FLP/+ (II), y egh9PP4 f FRT18/y UbiGFP FRT18, hs-FLP/+ (II), egh62d18 f FRT18/y UbiGFP FRT18, hs-FLP/+ (II), egh7 f FRT18/y UbiGFP FRT18, hs-FLP/+ (II). Clones were marked by the loss of GFP expression in follicular epithelial cells of the Drosophila ovary. Rescue of the egghead Mutant Flies—pUAS-β4GalT6 was constructed by cloning full-length cDNA into pUAST. The same construct was used for the in vitro glycosylation assay described above verifying the activity of β4GalT6. pUAST-egh was constructed by cloning the full-length coding sequence of egh into the BglII/XbaI sites of pUAST. Both constructs were used to generate transgenic flies. Stocks carrying a ubiquitous driver (armadillo-Gal4 or actin-Gal4) and a UAS line were established (four independent UAS insertions were tested). Their ability to rescue the lethality of egh9PP4 mutant was assayed by scoring the male progeny when these stocks were mated to heterozygous y egh9PP4 f females. egghead and brainiac Mutants Produce Truncated Glycosphingolipids—In vitro studies predicted that the enzymes encoded by the egghead and brainiac genes would be required for glycosphingolipid biosynthesis in vivo. To confirm this, we tested four different egghead (egh) mutants. As a first step, we sequenced three of the egh alleles to determine the nature of their molecular lesions. egh62d18 resulted from an 11 nucleotide deletion which caused a frameshift at amino acid 97 and deletion of most of the coding sequence. This allele is expected to cause a complete loss of enzymatic activity as the active site has been deleted. egh9PP4 resulted from a 15-base pair deletion that removed amino acids 113–117, of which two are conserved. egh7 resulted from a single nucleotide change that changes the conserved methionine at position 308 to lysine (M308K). Extracts were prepared from larvae mutant for these alleles as well as egh64h6 and tested for mannosyltransferase activity with n-octyl glucoside (Fig. 1B). All four mutants were devoid of significant detectable mannosyltransferase activity. We next asked whether brainiac (brn) and egghead mutants were blocked in glycosphingolipid biosynthesis in vivo, as would be predicted on the basis of their in vitro enzymatic functions, if no redundancy in these enzyme functions or alternate biosynthetic pathways exist. Characterization of glycosphingolipids from mutant larvae by thin layer chromatography showed accumulation of the truncated product Glcβ1-Cer in all four egh mutants, whereas MacCer accumulated in the brn mutant (Fig. 1C). We produced a monoclonal antibody that specifically recognizes MacCer but not further elongated glycosphingolipids, to provide a tool to visualize this biosynthetic intermediate in vivo. The specificity of the antibody was tested by immunostaining of glycosphingolipids separated by thin layer chromatography. The antibody detected MacCer but not LacCer or GlcNAcβ1–3Manβ1–4Glcβ1Cer (data not shown). The antibody was then used to test for the presence of MacCer in clones of cells lacking Egghead or Brainiac activity in the Drosophila ovary. Clones of cells lacking Brainiac activity, which accumulate MacCer, showed strong labeling (Fig. 1D). In contrast, cells lacking Egghead activity, which we expect to be blocked at the Glcβ1-Cer step, showed no labeling above background with this antibody (Fig. 1D). This indicates that Egghead and Brainiac are present and active in the follicular epithelial cells of egg chambers. The anti-MacCer antibody produced only background levels of labeling in the wild-type cells adjacent to the clones presumably reflecting low level of expression of the immediate precursor substrate for Brainiac (and subsequent enzymes), as co-expression of multiple intermediate species is a common feature found for glycosphingolipids. Taken together, these observations confirm the predicted functions of Egghead and Brainiac as enzymes required for sequen
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