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The Drosophila melanogaster brainiac Protein Is a Glycolipid-specific β1,3N-Acetylglucosaminyltransferase

2002; Elsevier BV; Volume: 277; Issue: 36 Linguagem: Inglês

10.1074/jbc.c200381200

1083-351X

Reto Mu ̈ller, Friedrich Altmann, Dapeng Zhou, Thierry Hennet,

Lysosomal Storage Disorders Research

Mutations at the Drosophila melanogaster brainiac locus lead to defective formation of the follicular epithelium during oogenesis and to neural hyperplasia. Thebrainiac gene encodes a type II transmembrane protein structurally similar to mammalian β1,3-glycosyltransferases. We have cloned the brainiac gene from D. melanogastergenomic DNA and expressed it as a FLAG-tagged recombinant protein in Sf9 insect cells. Glycosyltransferase assays showed thatbrainiacis capable of transferringN-acetylglucosamine (GlcNAc) to β-linked mannose (Man), with a marked preference for the disaccharide Man(β1,4)Glc, the core of arthro-series glycolipids. The activity of brainiactoward arthro-series glycolipids was confirmed by showing that the enzyme efficiently utilized glycolipids from insects as acceptors whereas it did not with glycolipids from mammalian cells. Methylation analysis of the brainiac reaction product revealed a β1,3 linkage between GlcNAc and Man, proving thatbrainiac is a β1,3GlcNAc-transferase. Human β1,3GlcNAc-transferases structurally related tobrainiac were unable to transfer GlcNAc to Man(β1,4)Glc-based acceptor substrates and failed to rescue a homozygous lethal brainiac allele, indicating that these proteins are paralogous and not orthologous tobrainiac. Mutations at the Drosophila melanogaster brainiac locus lead to defective formation of the follicular epithelium during oogenesis and to neural hyperplasia. Thebrainiac gene encodes a type II transmembrane protein structurally similar to mammalian β1,3-glycosyltransferases. We have cloned the brainiac gene from D. melanogastergenomic DNA and expressed it as a FLAG-tagged recombinant protein in Sf9 insect cells. Glycosyltransferase assays showed thatbrainiacis capable of transferringN-acetylglucosamine (GlcNAc) to β-linked mannose (Man), with a marked preference for the disaccharide Man(β1,4)Glc, the core of arthro-series glycolipids. The activity of brainiactoward arthro-series glycolipids was confirmed by showing that the enzyme efficiently utilized glycolipids from insects as acceptors whereas it did not with glycolipids from mammalian cells. Methylation analysis of the brainiac reaction product revealed a β1,3 linkage between GlcNAc and Man, proving thatbrainiac is a β1,3GlcNAc-transferase. Human β1,3GlcNAc-transferases structurally related tobrainiac were unable to transfer GlcNAc to Man(β1,4)Glc-based acceptor substrates and failed to rescue a homozygous lethal brainiac allele, indicating that these proteins are paralogous and not orthologous tobrainiac. N-acetylglucosaminyltransferase brainiac thin-layer chromatography p-nitrophenyl ceramide Gal(β1,4)Glc-Cer GlcNAc(β1,3)Gal(β1,4)Glc-Cer high performance liquid chromatography The importance of glycoconjugates in regulating developmental processes is continually being supported by studies performed in various model organisms like Caenorhabditis elegans (1Herman T. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 974-979Crossref PubMed Scopus (120) Google Scholar),Drosophila melanogaster (2Perrimon N. Bernfield M. Nature. 2000; 404: 725-728Crossref PubMed Scopus (662) Google Scholar), and the mouse (3Hennet T. Ellies L.G. Biochim. Biophys. Acta. 1999; 1473: 123-136Crossref PubMed Scopus (28) Google Scholar). TheDrosophila genes sugarless, sulfateless, pipe, tout-velu, anddally participate in the formation of proteoglycans. Loss of function mutations in some of these genes produce polarity phenotypes mechanistically connected to incorrect diffusion of the signaling proteins wingless and hedgehog (4Sen J. Goltz J.S. Stevens L. Stein D. Cell. 1998; 95: 471-481Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 5Hacker U. Lin X. Perrimon N. Development (Camb.). 1997; 124: 3565-3573Crossref PubMed Google Scholar, 6Bellaiche Y. The I. Perrimon N. Nature. 1998; 394: 85-88Crossref PubMed Scopus (437) Google Scholar). Therotated abdomen locus, whose disruption is associated with a helical rotation of the body, has been found to encode a potentialO-mannosyltransferase (7Martin-Blanco E. Garcia-Bellido A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6048-6052Crossref PubMed Scopus (92) Google Scholar), and fringe, which modulates the interaction of the Notch receptor with its ligands (8Panin V.M. Papayannopoulos V. Wilson R. Irvine K.D. Nature. 1997; 387: 908-912Crossref PubMed Scopus (508) Google Scholar), has recently been demonstrated to be a β1,3N-acetylglucosaminyltransferase (GlcNAcT)1 (9Moloney D.J. Panin V.M. Johnston S.H. Chen J.H. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (723) Google Scholar, 10Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (597) Google Scholar). The Drosophila gene brainiac (brn) (11Goode S. Wright D. Mahowald A.P. Development (Camb.). 1992; 116: 177-192Crossref PubMed Google Scholar) encodes a protein that shares structural motifs with β1,3glycosyltransferases (12Yuan Y.P. Schultz J. Mlodzik M. Bork P. Cell. 1997; 88: 9-11Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 13Hennet T. Dinter A. Kuhnert P. Mattu T.S. Rudd P.M. Berger E.G. J. Biol. Chem. 1998; 273: 58-65Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The brn gene is localized on the X chromosome. brn was shown to cooperate with the epidermal growth factor receptor and one of its ligands, theDrosophila TGFα homologue gurken (11Goode S. Wright D. Mahowald A.P. Development (Camb.). 1992; 116: 177-192Crossref PubMed Google Scholar) during oogenesis. Mutant brn alleles exhibit altered morphology of the follicular epithelium (11Goode S. Wright D. Mahowald A.P. Development (Camb.). 1992; 116: 177-192Crossref PubMed Google Scholar), female sterility (14Gans M. Audit C. Masson M. Genetics. 1975; 81: 683-704PubMed Google Scholar), and germ line loss (15Swan A. Hijal S. Hilfiker A. Suter B. Genome Res. 2001; 11: 67-77Crossref PubMed Scopus (8) Google Scholar). Furthermore, brn embryos develop neural hyperplasia and epidermal hypoplasia (11Goode S. Wright D. Mahowald A.P. Development (Camb.). 1992; 116: 177-192Crossref PubMed Google Scholar) as encountered withNotch hypomorphic alleles and other neurogenic mutants, suggesting implications of brn in Notch signaling (16Goode S. Perrimon N. Cold Spring Harbor Symp. Quant. Biol. 1997; 62: 177-184Crossref PubMed Google Scholar, 17Goode S. Melnick M. Chou T.B. Perrimon N. Development (Camb.). 1996; 122: 3863-3879Crossref PubMed Google Scholar). While the relationships between brn and specific signaling pathways have been examined genetically, the nature of these interactions remained elusive as long as the biochemical function ofbrn was unclear. In the present study, we show thatbrn has a β1,3N-acetylglucosaminyltransferase (GlcNAcT) activity directed toward the Man(β1,4)Glc core structure of arthro-series glycolipids. The brn gene was amplified by PCR from D. melanogaster OregonR genomic DNA during 30 cycles at 95 °C for 45 s, 55 °C for 30 s, 72 °C for 60 s using the primers 5′-TTTGGATCCGTCGCCATGCAAAGT-3′ and 5′-CCTGTTCTAGATGCTACGCGTAAT-3′. The resulting 1.0-kb fragment was digested with BamHI and XbaI and subcloned into the pFastbac-FLAG(a) vector (Invitrogen) linearized at the BamHI andXbaI sites. The FLAG-tagged brn gene was expressed as a recombinant baculovirus in insect cells as described previously (13Hennet T. Dinter A. Kuhnert P. Mattu T.S. Rudd P.M. Berger E.G. J. Biol. Chem. 1998; 273: 58-65Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Infected cells (107) were lysed at 72 h post-infection in 600 μl of 50 mm Tris/HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, and a protein inhibitor mixture (complete, EDTA free, Roche Diagnostics) on ice. Post-nuclear supernatants were incubated with 240 μl of anti-FLAG M2-agarose beads (Sigma) under rotation for 2.5 h at 4 °C. Beads were washed three times with Tris-buffered saline and used as enzyme source for assays. All donor and acceptor substrates were from Sigma except Man(β1,4)Glc(β1-OpNP) (pNP =p-nitrophenyl), which was purchased from Toronto Research (North York, Canada). Glycosyltransferase activity was assayed for 60 min at 25 °C with 15 μl of beads, 5% Me2SO, 20 mm MnCl2, 0.08 mm UDP-GlcNAc including 5 × 104 cpm of UDP-[14C]GlcNAc (Amersham Biosciences), and various acceptors (see Table I). Reaction products were purified over C18 Sep-Pak cartridges (Waters) (18Malissard M. Borsig L., Di Marco S. Grutter M.G. Kragl U. Wandrey C. Berger E.G. Eur. J. Biochem. 1996; 239: 340-348Crossref PubMed Scopus (46) Google Scholar) and quantified in a Tri-Carb 2900TR liquid scintillation counter (Packard) with luminescence correction.Table IAcceptor substrate specificity of brnAcceptor substrate (20 mm)Mock1-aAnti-FLAG bead bound lysate from Sf9 cells infected with mock baculovirus.brn1-bAnti-FLAG bead bound lysate from Sf9 cells infected with brn baculovirus.pmol/min/mlGlc(α1-OpNP)7.25.2Glc(β1-OpNP)5.45.7Gal(α1-OpNP)8.411.1Gal(β1-OpNP)6.212.6GalNAc(α1-OpNP)6.77.2GalNAc(β1-OpNP)5.38.9Fuc(α1-OpNP)7.64.3Fuc(β1-OpNP)6.65.5Man(α1-OpNP)4.616.1Man(β1-OpNP)5.6400.0Man(β1,4)Glc(β1-OpNP)3.0855.1Gal(β1,4)Glc(β1-OpNP)4.524.91-a Anti-FLAG bead bound lysate from Sf9 cells infected with mock baculovirus.1-b Anti-FLAG bead bound lysate from Sf9 cells infected with brn baculovirus. Open table in a new tab D. melanogaster Schneider 2 cells, Spodoptera frugiperda Sf9 cells, and human colon carcinoma Caco-2 cells were washed three times in phosphate-buffered saline and extracted in isopropanol:hexane:H2O (55:25:20). Extracts were spun twice at 500 x g, and supernatants were dried under N2. Phospholipids were removed by saponification in 0.2m NaOH in methanol for 24 h at 37 °C. After neutralization with HCl, the extracts were expanded to theoretical upper phase (methanol:water:chloroform, 47:48:3), applied on C18 SepPak cartridges, and eluted with 5 ml of methanol. Eluates were dried under N2 and resuspended in 500 μl of methanol. The procedure yielded about 120 μg of mannose equivalents for 108 S2 and Sf9 cells and 20 μg of mannose equivalents from 107 Caco-2 cells as determined by the phenol sulfuric acid assay (19Dubois M. Gilles K.A. Hamilton J.K. Rebers P.A. Smith F. Anal. Chem. 1956; 28: 350-356Crossref Scopus (41197) Google Scholar). Glycolipids (5 μg of mannose equivalents per assay) were dried under N2 and incubated together with 10 μl of beads-bound enzyme in 50 μl of 50 mm cacodylate buffer, pH 7.1, 20 mmMnCl2, 0.06% Triton X-100, 2.5 × 104 cpm of UDP-[14C]GlcNAc for 90 min at 25 °C. Reaction products were expanded to theoretical upper phase and purified over C18 Sep-Pak cartridges as described above. After drying over N2, the eluates were taken up in 100 μl of methanol:chloroform (1:1) and separated on aluminum high-performance thin-layer chromatography plates (Merck, Darmstadt, Germany) using a solvent system of chloroform:methanol:0.25% CaCl2 (5:4:1). Plates were stained with orcinol sulfuric acid (Sigma). The [14C]GlcNAc(β1,3)Gal(β1,4)Glc-ceramide (Lc3) standard was produced enzymatically with the Lc3 synthase β1,3 GlcNacT protein (20Henion T.R. Zhou D. Wolfer D.P. Jungalwala F.B. Hennet T. J. Biol. Chem. 2001; 276: 30261-30269Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) using Gal(β1,4)Glc-ceramide (Lc2) (Sigma) as acceptor substrate. Human β3GnT1 (21Zhou D. Dinter A. Gutierrez Gallego R. Kamerling J.P. Vliegenthart J.F.G. Berger E.G. Hennet T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96 (, correction (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11673–11675): 406-411Crossref PubMed Scopus (87) Google Scholar), β3GnT4 (22Shiraishi 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 (118) Google Scholar) and β3GnT5 (20Henion T.R. Zhou D. Wolfer D.P. Jungalwala F.B. Hennet T. J. Biol. Chem. 2001; 276: 30261-30269Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) cDNAs and the Drosophila brn gene were subcloned into the pUAST vector (23Brand A.H. Perrimon N. Development (Camb.). 1993; 118: 401-415Crossref PubMed Google Scholar). The rescue constructs pUAST-β3GnT1, pUAST-β3GnT4, pUAST-β3GnT5, and pUAST-brn were injected together with the pUChspΔ2–3 P-element helper plasmid (Flybase accession FBmc0000938) intoyellow white Drosophila embryos using standard procedures. Then, white+ progeny was selected and X-chromosomal insertions of the transgene excluded. The GAL4 lines, driving ubiquitous expression of the UAS-transgenes in anarmadillo pattern (24Sanson B. White P. Vincent J.P. Nature. 1996; 383: 627-630Crossref PubMed Scopus (320) Google Scholar), carry Bloomington Stock numbers 1560 and 1561. Males of the genotype yellow white/Y; transgene/+ were mated to virgins forked brn1.6P6/FM6-w1; 1560 GAL4/+ and forked brn1.6P6/FM6-w1; 1561 GAL4/+ and the progeny examined for males carrying the forked mutation for 8 days after eclosion of the first flies. At least two independent lines of each transgene were used for the complementation assay, which were repeated four times. A mixture of substrate and of 10 nmol of product was separated by reversed phase HPLC on a 3 × 250 mm column filled with 5 μm ODS Hypersil (Shandon) at a flow rate of 0.6 ml/min. The column was eluted with a linear gradient from 6 to 24% of methanol during 18 min in 0.1 m ammonium acetate, pH 4.0. p-Nitrophenylglycosides were monitored at 245 nm. The mixture was also analyzed after incubation withN-acetyl-β-hexosaminidase from jack beans (Sigma) (25Wilson I.B. Zeleny R. Kolarich D. Staudacher E. Stroop C.J. Kamerling J.P. Altmann F. Glycobiology. 2001; 11: 261-274Crossref PubMed Scopus (210) Google Scholar). The fraction of interest was collected in a screw capped glass vial and dried in a speed-vac concentrator. A small aliquot was used for matrix assisted laser desorption mass spectrometry as described elsewhere (25Wilson I.B. Zeleny R. Kolarich D. Staudacher E. Stroop C.J. Kamerling J.P. Altmann F. Glycobiology. 2001; 11: 261-274Crossref PubMed Scopus (210) Google Scholar). The sample was dried over phosphorus pentoxide in vacuo and permethylated using NaOH (26Ciucanu I. Kerek F. Carbohydr. Res. 1984; 131: 209-217Crossref Scopus (3215) Google Scholar). Partially permethylated alditol acetates were prepared using NaBD4 as the reducing agent and analyzed by gas chromatography/mass spectrometry using a 60 m SP2330 (Restek) (27Doares S.H. Albersheim P. Darvill A.G. Carbohydr. Res. 1991; 210: 311-317Crossref Scopus (51) Google Scholar) and a Finnigan Ion Trap ITD800. Derivatives of terminal and 3-substituted galactose served to compare retention times with the data given by Doares et al.(27Doares S.H. Albersheim P. Darvill A.G. Carbohydr. Res. 1991; 210: 311-317Crossref Scopus (51) Google Scholar). We have cloned the D. melanogaster brn gene by PCR amplification and expressed it as an N-terminally FLAG-tagged full-length protein in Sf9 insect cells. The recombinantbrn protein was bound to anti-FLAG-agarose beads, and cellular contaminants such as possible endogenous acceptor substrates were washed out before assaying for enzymatic activity. A GlcNAcT activity was only detected toward the Man(β1-OpNP) acceptor when monosaccharide substrates were assayed (TableI). Highest activity was measured toward the disaccharide acceptor Man(β1,4)Glc(β1-OpNP), whereas a slight activity was also detected toward Gal(β1,4)Glc(β1-OpNP) (Table I). The Man(β1,4)Glc structure represents the core of arthro-series glycolipids found in nematodes (28Gerdt S. Lochnit G. Dennis R.D. Geyer R. Glycobiology. 1997; 7: 265-275Crossref PubMed Scopus (53) Google Scholar) and insects (29Wiegandt H. Biochim. Biophys. Acta. 1992; 1123: 117-126Crossref PubMed Scopus (51) Google Scholar) among others. In Drosophila, the arthro-series Man(β1,4)Glc core is elongated with a β1,3-linked GlcNAc (30Seppo A. Moreland M. Schweingruber H. Tiemeyer M. Eur. J. Biochem. 2000; 267: 3549-3558Crossref PubMed Scopus (80) Google Scholar), suggesting thatbrn may represent the enzyme catalyzing this step. To test this hypothesis, we have isolated neutral glycolipids fromDrosophila S2 and Spodoptera Sf9 cells and assayed these glycolipids as acceptors for the anti-FLAG beads-boundbrn enzyme. A significant GlcNAc-transferase activity was detected when incubating brn together with insect glycolipids, whereas only a low activity was measured with glycolipids extracted from mammalian Caco-2 cells, likely reflecting the low specificity of brn for lactosylceramide. The reaction products were separated by TLC and plates were autoradiographed, revealing a [14C]GlcNAc-labeled band at the size of a trihexoside ceramide in S2 and Sf9 cells (Fig.1). The nature of the linkage between GlcNAc and the underlying β-linked Man residue was investigated by methylation analysis of thebrn reaction product GlcNAc-Man(β1-OpNP). In reversed phase HPLC, the presumed disaccharide product eluted slightly ahead of the substrate Man(β1-OpNP). The disaccharide peak disappeared upon incubation with N-acetyl-β-hexosaminidase (Fig.2A). The purified fraction corresponding to the disaccharide peak exhibited a pseudomolecular ion of m/z 513.5. Linkage analysis of the GlcNAc-Man(β1-OpNP) disaccharide product gave a peak at the relative retention time of 0.597, which suggests a 2- or a 3-substituted mannosyl residue (27Doares S.H. Albersheim P. Darvill A.G. Carbohydr. Res. 1991; 210: 311-317Crossref Scopus (51) Google Scholar). The fragment spectrum clearly identified the derivative as substituted in the 3-position (Fig.2B), thus confirming the identity of brn as a β1,3 GlcNAcT. The brn protein is structurally related to human β1,3 glycosyltransferase enzymes (12Yuan Y.P. Schultz J. Mlodzik M. Bork P. Cell. 1997; 88: 9-11Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 13Hennet T. Dinter A. Kuhnert P. Mattu T.S. Rudd P.M. Berger E.G. J. Biol. Chem. 1998; 273: 58-65Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). The acceptor specificity ofbrn for the arthro-series glycolipid core suggested that it represents a paralogous enzyme to the mammalian β1,3 glycosyltransferases, including β1,3 galactosyltransferases (13Hennet T. Dinter A. Kuhnert P. Mattu T.S. Rudd P.M. Berger E.G. J. Biol. Chem. 1998; 273: 58-65Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 31Miyazaki H. Fukumoto S. Okada M. Hasegawa T. Furukawa K. J. Biol. Chem. 1997; 272: 24794-24799Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar,32Kolbinger F. Streiff M.B. Katopodis A.G. J. Biol. Chem. 1998; 273: 433-440Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), β1,3 GlcNAcT (20Henion T.R. Zhou D. Wolfer D.P. Jungalwala F.B. Hennet T. J. Biol. Chem. 2001; 276: 30261-30269Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 22Shiraishi 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 (118) Google Scholar), and a β1,3-N-acetylgalactosaminyltransferase (33Okajima T. Nakamura Y. Uchikawa M. Haslam D.B. Numata S.I. Furukawa K. Urano T. Furukawa K. J. Biol. Chem. 2000; 275: 40498-40503Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) acting on GlcNAcβ-, Galβ-, and GalNAcβ-based acceptors. Although no mammalian β1,3 GlcNAcT has been described to act on β-linked Man acceptors, we have investigated whether the three human β1,3 GlcNAcT structurally closest to brn can complement the lethal phenotype of brn deficient Drosophila flies. To this end, we have expressed the human β3GnT-I (21Zhou D. Dinter A. Gutierrez Gallego R. Kamerling J.P. Vliegenthart J.F.G. Berger E.G. Hennet T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96 (, correction (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11673–11675): 406-411Crossref PubMed Scopus (87) Google Scholar), -IV (22Shiraishi 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 (118) Google Scholar), and -V (20Henion T.R. Zhou D. Wolfer D.P. Jungalwala F.B. Hennet T. J. Biol. Chem. 2001; 276: 30261-30269Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) in brn1.6P6 mutant flies (34Perrimon N. Engstrom L. Mahowald A.P. Genetics. 1989; 121: 333-352Crossref PubMed Google Scholar) using the UAS-GAL4 transgenesis system (23Brand A.H. Perrimon N. Development (Camb.). 1993; 118: 401-415Crossref PubMed Google Scholar). The human β3GnT transgenes and a brn transgene were expressed in flies carrying the allele brn1.6P6, which causes lethality at the late pupal stage. The transgenes were expressed ubiquitously using armadillo GAL4 transactivator lines. The brn transgene did rescuebrn1.6P6 mutant males from their hemizygous late pupal lethality, whereas the human β3GnT transgenes did not (TableII). The rescue ofbrn1.6P6 males was confirmed by detection of theforked marker, whose gene is located besides thebrn1.6P6 allele on the X chromosome. Control crosses of females carrying brn1.6P6 with yellow white males did not yield any living brn1.6P6forked/Y males either. The inability of human β3GnT enzymes to compensate for the loss of brn activity in mutant flies suggested that the former enzymes cannot elongate the arthro-series glycolipid core in vivo. This was confirmedin vitro by showing that the human β3GnT enzymes did not exhibit significant activity toward the Man(β1,4)Glc(β1-OpNP) acceptor (Table II).Table IIComplementation of Drosophila brn1.6P6β1,3GlcNAcT gene2-aβ3GnT-I (21), β3GnT-IV (22), and β3GnT-V (20).Lines2-bNumber of independent lines per transgene tested.brn1.6P6 rescue2-cNumber of independent lines per transgene rescueing brn1.6P6 (34). Rescue was scored by counting males alive with a forkedphenotype within 8 days after eclosion of control animals.GlcNAc->Man(β1,4)Glc activity2-dIn vitro GlcNAc-transferase activity towards Man(β1,4)Glc(β1-OpNP) given in percentage of the activity measured with brn.%Drosophila brn22100Human β3GnT-I206.3Human β3GnT-IV201.6Human β3GnT-V608.6Rescue of the brn1.6P6 late pupal lethal phenotype by ubiquitous expression of Drosophila brn and human β1,3GlcNAcT transgenes.2-a β3GnT-I (21Zhou D. Dinter A. Gutierrez Gallego R. Kamerling J.P. Vliegenthart J.F.G. Berger E.G. Hennet T. Proc. Natl. Acad. Sci. U. S. A. 1999; 96 (, correction (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11673–11675): 406-411Crossref PubMed Scopus (87) Google Scholar), β3GnT-IV (22Shiraishi 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 (118) Google Scholar), and β3GnT-V (20Henion T.R. Zhou D. Wolfer D.P. Jungalwala F.B. Hennet T. J. Biol. Chem. 2001; 276: 30261-30269Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).2-b Number of independent lines per transgene tested.2-c Number of independent lines per transgene rescueing brn1.6P6 (34Perrimon N. Engstrom L. Mahowald A.P. Genetics. 1989; 121: 333-352Crossref PubMed Google Scholar). Rescue was scored by counting males alive with a forkedphenotype within 8 days after eclosion of control animals.2-d In vitro GlcNAc-transferase activity towards Man(β1,4)Glc(β1-OpNP) given in percentage of the activity measured with brn. Open table in a new tab Rescue of the brn1.6P6 late pupal lethal phenotype by ubiquitous expression of Drosophila brn and human β1,3GlcNAcT transgenes. We have shown that Drosophila brn, a member of the β1,3 glycosyltransferase family, encodes a β1,3 GlcNAcT enzyme with a specificity for the Man(β1,4)Glc disaccharide found in arthro-series glycolipids (29Wiegandt H. Biochim. Biophys. Acta. 1992; 1123: 117-126Crossref PubMed Scopus (51) Google Scholar). Several mammalian enzymes structurally related to brn have been suggested to represent homologues (35Egan S. Cohen B. Sarkar M. Ying Y. Cohen S. Singh N. Wang W. Flock G. Goh T. Schachter H. Glycoconj. J. 2000; 17: 867-875Crossref PubMed Scopus (5) Google Scholar, 36Cole S.E. Mao M.S. Johnston S.H. Vogt T.F. Mamm. Genome. 2001; 12: 177-179Crossref PubMed Scopus (9) Google Scholar, 37Vollrath B. Fitzgerald K.J. Leder P. Mol. Cell. Biol. 2001; 21: 5688-5697Crossref PubMed Scopus (18) Google Scholar). However, the specificity of brn for Man(β1,4)Glc, a disaccharide that has never been described in vertebrates, rather indicates that brn and mammalian β1,3 glycosyltransferases are paralogous proteins derived from a common ancestor gene. The functional disparity between the β1,3 GlcNAcT brn and mammalian β1,3 GlcNAcT enzymes is further supported by the inability of the latter to complement the lethal phenotype of the mutant allelebrn1.6P6 in Drosophila. The specificity of brn toward Manβ1,4Glc-Cer suggests the presence of functional homologues only in organisms harboring arthro-series glycolipids, whose core structure is GlcNAc(β1,3)Man(β1,4)Glc-Cer. A protein structurally related to brn has recently been described in C. elegans (38Griffitts J.S. Whitacre J.L. Stevens D.E. Aroian R.V. Science. 2001; 293: 860-864Crossref PubMed Scopus (181) Google Scholar), which express arthro-series glycolipids (28Gerdt S. Lochnit G. Dennis R.D. Geyer R. Glycobiology. 1997; 7: 265-275Crossref PubMed Scopus (53) Google Scholar). The loss of that gene, named bre-5 (39Marroquin L.D. Elyassnia D. Griffitts J.S. Feitelson J.S. Aroian R.V. Genetics. 2000; 155: 1693-1699Crossref PubMed Google Scholar), renders the animal resistant to high doses of Bacillus thuringiensis Bt toxin. Since Bt toxin binds to arthro-series glycolipids (40Dennis R.D. Wiegandt H. Haustein D. Knowles B.H. Ellar D.J. Biomed. Chromatogr. 1986; 1: 31-37Crossref PubMed Scopus (12) Google Scholar), it is possible that bre-5 participates in the formation of this class of glycolipids in C. elegans and thereby represents a true orthologue of brn. brn mutations affect follicle cell-germ line interactions and lead to neurogenic phenotypes in Drosophila embryos. Considering the involvement of brn in glycolipid biosynthesis, one can envision that arthro-series glycolipids may regulate cell adhesion, proliferation, and differentiation via carbohydrate-lectin interactions. On the other hand, arthro-series glycolipids may modulate specific signaling proteins in a way similar to gangliosides affecting the epidermal growth factor receptor (41Bremer E.G. Schlessinger J. Hakomori S. J. Biol. Chem. 1986; 261: 2434-2440Abstract Full Text PDF PubMed Google Scholar,42Miljan 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 (153) Google Scholar), insulin receptor (43Nojiri H. Stroud M. Hakomori S. J. Biol. Chem. 1991; 266: 4531-4537Abstract Full Text PDF PubMed Google Scholar), and platelet-derived growth factor receptor (44Van Brocklyn J. Bremer E.G. Yates A.J. J. Neurochem. 1993; 61: 371-374Crossref PubMed Scopus (59) Google Scholar) signaling cascades. The notion that brn glycolipid products interact with adhesion or signaling proteins implies that other mutant genes with phenotypes similar to those encountered inbrn mutant flies may encode partner lectin/signaling proteins. Along this line, Drosophila egghead mutants have similar and non-additive phenotypes to brn (17Goode S. Melnick M. Chou T.B. Perrimon N. Development (Camb.). 1996; 122: 3863-3879Crossref PubMed Google Scholar). Experiments aimed at characterizing the biochemical and functional relation betweenbrn products and the egghead protein should reveal the mechanisms how arthro-series glycolipids regulate morphogenic events during Drosophila development. We thank Erich Frei, Michael Daube, and Markus Noll from the Institute of Molecular Biology at the University of Zu¨rich for their assistance with the transgenic expression inDrosophila.