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• W2024115143 abstract "Sialylation is an important carbohydrate modification of glycoconjugates in the deuterostome lineage of animals. By contrast, the evidence for sialylation in protostomes has been scarce and somewhat controversial. In the present study, we characterize a Drosophila sialyltransferase gene, thus providing experimental evidence for the presence of sialylation in protostomes. This gene encodes a functional α2-6-sialyltransferase (SiaT) that is closely related to the vertebrate ST6Gal sialyltransferase family, indicating an ancient evolutionary origin for this family. Characterization of recombinant, purified Drosophila SiaT revealed a novel acceptor specificity as it exhibits highest activity toward GalNAcβ1-4GlcNAc carbohydrate structures at the non-reducing termini of oligosaccharides and glycoprotein glycans. Oligosaccharides are preferred over glycoproteins as acceptors, and no activity toward glycolipid acceptors was detected. Recombinant Drosophila SiaT expressed in cultured insect cells possesses in vivo and in vitro autosialylation activity toward β-linked GalNAc termini of its own N-linked glycans, thus representing the first example of a sialylated insect glycoconjugate. In situ hybridization revealed that Drosophila SiaT is expressed during embryonic development in a tissue- and stage-specific fashion, with elevated expression in a subset of cells within the central nervous system. The identification of a SiaT in Drosophila provides a new evolutionary perspective for considering the diverse functions of sialylation and, through the powerful genetic tools available in this system, a means of elucidating functions for sialylation in protostomes. Sialylation is an important carbohydrate modification of glycoconjugates in the deuterostome lineage of animals. By contrast, the evidence for sialylation in protostomes has been scarce and somewhat controversial. In the present study, we characterize a Drosophila sialyltransferase gene, thus providing experimental evidence for the presence of sialylation in protostomes. This gene encodes a functional α2-6-sialyltransferase (SiaT) that is closely related to the vertebrate ST6Gal sialyltransferase family, indicating an ancient evolutionary origin for this family. Characterization of recombinant, purified Drosophila SiaT revealed a novel acceptor specificity as it exhibits highest activity toward GalNAcβ1-4GlcNAc carbohydrate structures at the non-reducing termini of oligosaccharides and glycoprotein glycans. Oligosaccharides are preferred over glycoproteins as acceptors, and no activity toward glycolipid acceptors was detected. Recombinant Drosophila SiaT expressed in cultured insect cells possesses in vivo and in vitro autosialylation activity toward β-linked GalNAc termini of its own N-linked glycans, thus representing the first example of a sialylated insect glycoconjugate. In situ hybridization revealed that Drosophila SiaT is expressed during embryonic development in a tissue- and stage-specific fashion, with elevated expression in a subset of cells within the central nervous system. The identification of a SiaT in Drosophila provides a new evolutionary perspective for considering the diverse functions of sialylation and, through the powerful genetic tools available in this system, a means of elucidating functions for sialylation in protostomes. Sialic acids compose a large family of negatively charged nine-carbon α-keto acids and are typically located at the non-reducing termini of glycans. In mammals, sialic acids have been implicated in a number of important biological processes, such as the regulation of turnover of circulating glycoproteins and erythrocytes, pathogen-host recognition, immune system functioning, and nervous system development (reviewed in Refs. 1Schauer R. Glycoconj. J. 2000; 17: 485-499Crossref PubMed Scopus (508) Google Scholar and 2Angata T. Varki A. Chem. Rev. 2002; 102: 439-469Crossref PubMed Scopus (1048) Google Scholar). In addition, alterations in sialic acid biosynthesis often correlate with malignant transformation and tumor progression (3Fukuda M. Cancer Res. 1996; 56: 2237-2244PubMed Google Scholar, 4Kim Y.J. Varki A. Glycoconj. J. 1997; 14: 569-576Crossref PubMed Scopus (494) Google Scholar). In vertebrates, the biosynthesis of sialylated glycoconjugates includes a number of enzymatic reactions that convert the precursor sugar, uridine-diphosphate-N-acetylglucosamine, into the donor sugar, cytidine monophosphate N-acetylneuraminic acid (CMP-Neu5Ac) 1The abbreviations used are: CMP-Neu5Accytidine monophosphate N-acetylneuraminic acidNeu5AcN-acetylneuraminic acidLacNAcGalβ1-4GlcNAcLacdiNAcGalNAcβ1-4GlcNAcHPTLChigh performance thin layer chromatographyPNGaseFpeptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase FHAhemagglutininRCA-IR. communis agglutinin ISiaTα2-6-sialyltransferaseLNnTlacto-N-neotetraoseLNTlacto-N-tetraose. (2Angata T. Varki A. Chem. Rev. 2002; 102: 439-469Crossref PubMed Scopus (1048) Google Scholar). This donor molecule is used by sialyltransferases for the linkage-specific sialylation of glycoconjugates. Sialyltransferases usually exhibit strict acceptor specificity, and synthesize one type of linkage between sialic acid and acceptor substrates. Their expression is often regulated, and specific patterns of sialylation may vary at different developmental stages (5Harduin-Lepers A. Vallejo-Ruiz V. Krzewinski-Recchi M.A. Samyn-Petit B. Julien S. Delannoy P. Biochimie (Paris). 2001; 83: 727-737Crossref PubMed Scopus (438) Google Scholar). The combined characterization of the biochemical activity and the expression pattern of each sialyltransferase provide an indication of the structure and spatial-temporal distribution of sialylated glycoconjugates, and is thus essential for understanding the in vivo functions of sialic acid. cytidine monophosphate N-acetylneuraminic acid N-acetylneuraminic acid Galβ1-4GlcNAc GalNAcβ1-4GlcNAc high performance thin layer chromatography peptide-N4-(N-acetyl-β-glucosaminyl)asparagine amidase F hemagglutinin R. communis agglutinin I α2-6-sialyltransferase lacto-N-neotetraose lacto-N-tetraose. Twenty different sialyltransferases have been cloned in mammals. They synthesize different linkages (α2-6, α2-3, and α2-8) and differ in their acceptor specificities (5Harduin-Lepers A. Vallejo-Ruiz V. Krzewinski-Recchi M.A. Samyn-Petit B. Julien S. Delannoy P. Biochimie (Paris). 2001; 83: 727-737Crossref PubMed Scopus (438) Google Scholar, 6Takashima S. Ishida H.K. Inazu T. Ando T. Ishida H. Kiso M. Tsuji S. Tsujimoto M. J. Biol. Chem. 2002; 277: 24030-24038Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 7Takashima S. Tsuji S. Tsujimoto M. J. Biol. Chem. 2002; 277: 45719-45728Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 8Krzewinski-Recchi M.A. Julien S. Juliant S. Teintenier-Lelievre M. Samyn-Petit B. Montiel M.D. Mir A.M. Cerutti M. Harduin-Lepers A. Delannoy P. Eur. J. Biochem. 2003; 270: 950-961Crossref PubMed Scopus (62) Google Scholar). Structurally, all vertebrate sialyltransferases have a similar molecular architecture. (i) They are type II transmembrane glycoproteins that predominantly reside in the trans-Golgi compartment and have a short N-terminal cytoplasmic tail that is not important for their catalytic activity. (ii) They have a transmembrane anchor domain that contributes to Golgi retention. (iii) They include a stem region of highly variable length (from 20 to 200 amino acids) that is followed by a large C-terminal catalytic domain (5Harduin-Lepers A. Vallejo-Ruiz V. Krzewinski-Recchi M.A. Samyn-Petit B. Julien S. Delannoy P. Biochimie (Paris). 2001; 83: 727-737Crossref PubMed Scopus (438) Google Scholar, 9Paulson J.C. Colley K.J. J. Biol. Chem. 1989; 264: 17615-17618Abstract Full Text PDF PubMed Google Scholar). The catalytic region of all vertebrate sialyltransferases contains three conserved motifs: L (Large), S (Small), and VS (Very Small) (10Datta A.K. Paulson J.C. Indian J. Biochem. Biophys. 1997; 34: 157-165PubMed Google Scholar, 11Geremia R.A. Harduin-Lepers A. Delannoy P. Glycobiology. 1997; 7: v-viiCrossref PubMed Google Scholar). These motifs are involved in substrate binding, formation of essential disulfide bonds (L and S (12Datta A.K. Paulson J.C. J. Biol. Chem. 1995; 270: 1497-1500Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 13Datta A.K. Sinha A. Paulson J.C. J. Biol. Chem. 1998; 273: 9608-9614Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar)), and catalysis (VS (11Geremia R.A. Harduin-Lepers A. Delannoy P. Glycobiology. 1997; 7: v-viiCrossref PubMed Google Scholar, 14Kitazume-Kawaguchi S. Kabata S. Arita M. J. Biol. Chem. 2001; 276: 15696-15703Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar)). In contrast to deuterostomes (vertebrates, ascidians, and echinoderms), very little is known about the presence and function of sialic acids in protostomes (annelids, arthropods, and mollusks). Data on the occurrence of this carbohydrate in protostomes has been scarce and somewhat controversial. Several efforts to detect sialyltransferase activity (15Butters T.D. Hughes R.C. Vischer P. Biochim. Biophys. Acta. 1981; 640: 672-686Crossref PubMed Scopus (130) Google Scholar, 16Hooker A.D. Green N.H. Baines A.J. Bull A.T. Jenkins N. Strange P.G. James D.C. Biotechnol. Bioeng. 1999; 63: 559-572Crossref PubMed Scopus (95) Google Scholar, 17Lopez M. Tetaert D. Juliant S. Gazon M. Cerutti M. Verbert A. Delannoy P. Biochim. Biophys. Acta. 1999; 1427: 49-61Crossref PubMed Scopus (56) Google Scholar), sialylated glycans (17Lopez M. Tetaert D. Juliant S. Gazon M. Cerutti M. Verbert A. Delannoy P. Biochim. Biophys. Acta. 1999; 1427: 49-61Crossref PubMed Scopus (56) Google Scholar, 18Marz L. Altmann F. Staudacher E. Kubelka V. Montreuil J. Schachter H. Vliegenthart J.F. Glycoproteins. Elsevier Science Publishers B.V., Amsterdam1995: 543-563Google Scholar), or the CMP-Neu5Ac donor sugar (16Hooker A.D. Green N.H. Baines A.J. Bull A.T. Jenkins N. Strange P.G. James D.C. Biotechnol. Bioeng. 1999; 63: 559-572Crossref PubMed Scopus (95) Google Scholar, 19Tomiya N. Ailor E. Lawrence S.M. Betenbaugh M.J. Lee Y.C. Anal. Biochem. 2001; 293: 129-137Crossref PubMed Scopus (173) Google Scholar) in insect cells have failed, but other papers have reported the occurrence of sialic acids or sialyltransferase activity in insects (20Hiruma K. Riddiford L.M. Dev. Biol. 1988; 130: 87-97Crossref PubMed Scopus (92) Google Scholar, 21Roth J. Kempf A. Reuter G. Schauer R. Gehring W.J. Science. 1992; 256: 673-675Crossref PubMed Scopus (145) Google Scholar, 22Vadgama M.R. Kamat D.N. Histochemie. 1969; 19: 184-188Crossref PubMed Scopus (3) Google Scholar, 23Malykh Y.N. Krisch B. Gerardy-Schahn R. Lapina E.B. Shaw L. Schauer R. Glycoconj. J. 1999; 16: 731-739Crossref PubMed Scopus (51) Google Scholar). Experiments with insect Sf9 cells stably expressing two mammalian glycosyltransferases, β1-4-galactosyltransferase and α2-6-sialyltransferase, revealed the biochemical potential of these cells to provide a sugar donor for sialylation (24Hollister J.R. Jarvis D.L. Glycobiology. 2001; 11: 1-9Crossref PubMed Scopus (104) Google Scholar). However, endogenous sialyltransferase activity was not detected (25Hollister J.R. Shaper J.H. Jarvis D.L. Glycobiology. 1998; 8: 473-480Crossref PubMed Scopus (108) Google Scholar, 26Jarvis D.L. Finn E.E. Nat. Biotechnol. 1996; 14: 1288-1292Crossref PubMed Scopus (117) Google Scholar). Thus, it was suggested that in insect cells the sialylation pathway may be present as a highly specialized biochemical process that occurs in a tissue- and/or developmental stage-specific manner, or perhaps that sialic acids are present in only a small number of protostome species (2Angata T. Varki A. Chem. Rev. 2002; 102: 439-469Crossref PubMed Scopus (1048) Google Scholar, 27Marchal I. Jarvis D.L. Cacan R. Verbert A. Biol. Chem. 2001; 382: 151-159Crossref PubMed Scopus (153) Google Scholar). The availability of complete genomic information recently made Drosophila a powerful model system for elucidating the biochemical components of insect glycosylation pathways. Searches of the Drosophila genome data base revealed the presence of several genes that encode putative orthologues of vertebrate enzymes of the sialic acid biosynthetic pathway, including Neu5Ac phosphate synthase, CMP-Neu5Ac synthase, CMP-Neu5Ac/CMP antiporter, and sialyltransferase (GadFly data base (28FlyBase Nucleic Acids Res. 2003; 31: 172-175Crossref PubMed Scopus (382) Google Scholar); CAZY data base (29Coutinho P.M. Henrissat B. Gilbert H.J. Davies G. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. Royal Society of Chemistry, Cambridge, UK1999: 3-12Google Scholar); 30Angata T. Varki A. J. Biol. Chem. 2000; 275: 22127-22135Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 31Kim K. Lawrence S.M. Park J. Pitts L. Vann W.F. Betenbaugh M.J. Palter K.B. Glycobiology. 2002; 12: 73-83Crossref PubMed Scopus (53) Google Scholar). 2V. M. Panin, unpublished data. A recent study confirmed that Drosophila has a functional Neu5Ac phosphate synthase with significant homology to the human orthologue, which indicates the possibility of a vertebrate-type pathway for CMP-sialic acid biosynthesis in Drosophila (31Kim K. Lawrence S.M. Park J. Pitts L. Vann W.F. Betenbaugh M.J. Palter K.B. Glycobiology. 2002; 12: 73-83Crossref PubMed Scopus (53) Google Scholar). Evidence for the presence of sialylated structures in Drosophila has, however, only been reported in one study, and the nature of these sialylated glycoconjugates has not been analyzed further (21Roth J. Kempf A. Reuter G. Schauer R. Gehring W.J. Science. 1992; 256: 673-675Crossref PubMed Scopus (145) Google Scholar). In the present study, we identify and characterize the apparently single vertebrate-type sialyltransferase gene of Drosophila melanogaster, SiaT. We demonstrate that this gene encodes a functional α2-6-sialyltransferase, which is structurally and functionally related to the ST6Gal family of vertebrate sialyltransferases, yet has a distinct acceptor specificity. Analysis of the Drosophila SiaT expression pattern during embryonic development reveals a distinct pattern of expression in the central nervous system, which suggests possible functions of the SiaT gene in neural development. Materials—Bovine α1-acid glycoprotein, fetuin, asialofetuin, bovine α-lactalbumin, bovine lactoferrin, N-acetyllactosamine, Galβ1-3GlcNAc, Galβ1-3GalNAc, asialo-GM1, asialo-GM2, monosialo-GM3, type III gangliosides mixture, and lactoceramide were from Sigma. GalNAc-benzyl, lacto-N-tetraose, and lacto-N-neotetraose were from Calbiochem. Disaccharides with a hydrophobic aglycone, LacNAc-Rg and LacdiNAc-Rg (where Rg is O-(CH2)8CO2CH3 (32Hindsgaul O. Kaur K.J. Srivastava G. Blaszczyk-Thurin M. Crawley S.C. Heerze L.D. Palcic M.M. J. Biol. Chem. 1991; 266: 17858-17862Abstract Full Text PDF PubMed Google Scholar)), were the generous gifts from Dr. Monica Palcic (University of Alberta, Canada). Gal-NAcβ1-4GlcNAcβ1-2Manα1-O(CH2)7CH3 was from Dr. Johannis P. Kamerling (Utrecht University, The Netherlands). Drosophila mucin-D glycoprotein (33Kramerov A.A. Arbatsky N.P. Rozovsky Y.M. Mikhaleva E.A. Polesskaya O.O. Gvozdev V.A. Shibaev V.N. FEBS Lett. 1996; 378: 213-218Crossref PubMed Scopus (42) Google Scholar) and Drosophila glycolipid mixture (upper phase (34Seppo A. Moreland M. Schweingruber H. Tiemeyer M. Eur. J. Biochem. 2000; 267: 3549-3558Crossref PubMed Scopus (80) Google Scholar)) were kindly provided, respectively, by Dr. Andrei Kramerov (UCLA) and Dr. Michael Tiemeyer (Complex Carbohydrate Research Center, University of Georgia). Biotinylated Sambucus nigra (SNA), Ricinus communis agglutinin I (RCA-I), and Wistaria floribunda (WFA) lectins were from Vector Laboratories (Burlingame, CA). Merck HPTLC Silica Gel 60 F254 plates were from EM Science (Gibbstown, NJ). IgG-Sepharose 6 Fast Flow beads were from Amersham Biosciences. CMP-[14C]Neu5Ac (12.0 GBq/mmol, 740 KBq/ml) was from PerkinElmer Life Sciences. PNGaseF and β-N-acetylhexosaminidasef were from New England Biolabs (Beverly, MA). Purified rabbit IgG was from Jackson ImmunoResearch (West Grove, PA). Asialo-α1-acid glycoprotein was prepared by acid hydrolysis in 0.02 n HCl for 1 h at 80 °C. Expression Constructs—Construct for the expression of HA-tagged full-length Drosophila SiaT (designated D. SiaT) protein was prepared as follows: (i) by introducing an XbaI site after the last codon of D. SiaT open reading frame in cDNA clone GH27778 by PCR; (ii) in-frame ligation of double-stranded oligonucleotide encoding HA tag (35Niman H.L. Houghten R.A. Walker L.E. Reisfeld R.A. Wilson I.A. Hogle J.M. Lerner R.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4949-4953Crossref PubMed Scopus (319) Google Scholar) followed by 6 histidines and a stop codon into this XbaI site; and (iii) by subcloning the resulting DNA construct into pRmHA-3 expression vector (36Bunch T.A. Grinblat Y. Goldstein L.S. Nucleic Acids Res. 1988; 16: 1043-1061Crossref PubMed Scopus (369) Google Scholar). In order to make a construct expressing D. SiaT with N-terminal protein A tag, we PCR-amplified a DNA fragment encoding the IgG-binding domain of Staphylococcus aureus protein A from plasmid pRL715 (a gift from Dr. K. Severinov (37Opalka N. Mooney R.A. Richter C. Severinov K. Landick R. Darst S.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 617-622Crossref PubMed Scopus (26) Google Scholar)), ligated this DNA in-frame with PCR-amplified DNA fragment of GH27778 encoding 86-451 amino acids of D. SiaT protein, and then inserted the resulting DNA into pMT/BiP/V5-HisA vector (Invitrogen) in-frame with the BiP signal sequence (38Kirkpatrick R.B. Ganguly S. Angelichio M. Griego S. Shatzman A. Silverman C. Rosenberg M. J. Biol. Chem. 1995; 270: 19800-19805Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The final construct expressed a D. SiaT fusion protein in which the first 85 amino acids, including the endogenous signal peptide and a part of the stem region, were replaced with BiP signal peptide followed by the IgG-binding domain of S. aureus protein A. Cell Culture—Drosophila S2 cells were maintained and transfected as described (39Panin V.M. Shao L. Lei L. Moloney D.J. Irvine K.D. Haltiwanger R.S. J. Biol. Chem. 2002; 277: 29945-29952Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Spodoptera frugiperda Sf9 cells were grown as a suspension culture in Erlenmeyer flasks with orbital shaking at 110 rpm using the media described (26Jarvis D.L. Finn E.E. Nat. Biotechnol. 1996; 14: 1288-1292Crossref PubMed Scopus (117) Google Scholar). Generation of recombinant baculovirus and production of recombinant protein in baculovirus-infected cells was performed using the Bac-to-Bac expression system (Invitrogen) according to the manufacturer's protocol. Protein Purification—For purification of protein A-tagged D. SiaT protein, we collected medium from Sf9 cells 72 h after infection with recombinant baculovirus. This medium was incubated with IgG-Sepharose beads on a rotator at 4 °C overnight. Afterward, the beads were extensively washed with phosphate-buffered saline and assay buffer and used in sialyltransferase assays. The amount of purified protein was quantified by Coomassie staining of SDS gels loaded with D. SiaT and a protein standard (bovine serum albumin). Sialyltransferase Assays—Unless otherwise stated, D. SiaT activity was measured in 50 mm cacodylate buffer, pH 6.5, 1% Triton CF-54 (v/v), 3 mm oligosaccharide acceptor, 50 μg of glycoprotein or 12.5 μg of glycolipids, and 108 μm CMP-[14C]Neu5Ac (22.7 mCi/mmol) in a total volume of 20 μlat37 °C for 2 h. Before the assays, IgG-SiaT beads were first autosialylated in the presence of 100 μm CMP-Neu5Ac for 1.5 h at 37 °C in order to eliminate competition between autosialylation and the tested acceptors. During assays, IgG-SiaT beads were kept in suspension using a shaking water bath. The reactions were within linearity range for at least 3 h, and no more than 10% of CMP-Neu5Ac and substrates were consumed in any assay. Oligosaccharide samples were spotted directly on HPTLC plates and developed in 1-propyl alcohol/aqueous ammonia/water (6:1:2.5) solvent system. Glycoproteins were analyzed by SDS-PAGE followed by autoradiography and quantification using the Storm imaging system (Amersham Biosciences). Gangliosides were desalted as described (40Williams M.A. McCluer R.H. J. Neurochem. 1980; 35: 266-269Crossref PubMed Scopus (405) Google Scholar) and analyzed by HPTLC in a solvent system of chloroform, methanol, and 0.02% CaCl2 (55:45:10). An aliquot of Drosophila glycolipids was also treated with hydrofluoric acid to remove phosphoethanolamine residues as described (34Seppo A. Moreland M. Schweingruber H. Tiemeyer M. Eur. J. Biochem. 2000; 267: 3549-3558Crossref PubMed Scopus (80) Google Scholar). After the sialyltransferase assays, Drosophila glycolipids were desalted on Sephadex G-25 columns (41Dahms N.M. Schnaar R.L. J. Neurosci. 1983; 3: 806-817Crossref PubMed Google Scholar) and analyzed by HPTLC in a buffer system of chloroform, methanol, and 0.25% aqueous potassium chloride (10:10:3). LacNAc-Rg and LacdiNAc-Rg acceptors were also analyzed by HPTLC in the same buffer system without prior desalting. When GalNAc-benzyl was used as an acceptor, the unincorporated donor was removed by Sep-Pak C18 (Waters) column, and then incorporated radioactivity was measured by scintillation counting (32Hindsgaul O. Kaur K.J. Srivastava G. Blaszczyk-Thurin M. Crawley S.C. Heerze L.D. Palcic M.M. J. Biol. Chem. 1991; 266: 17858-17862Abstract Full Text PDF PubMed Google Scholar). Km and Vmax values for asialofetuin and lactoferrin were estimated based on the calculated amount of terminal LacNAc or LacdiNAc structures, i.e. 8.8 LacNAc termini per asialofetuin molecule (based on Ref. 42Green E.D. Adelt G. Baenziger J.U. Wilson S. Van Halbeek H. J. Biol. Chem. 1988; 263: 18253-18268Abstract Full Text PDF PubMed Google Scholar). Terminal LacdiNAc concentration of lactoferrin was estimated at 2.6 LacdiNAc termini per lactoferrin based on Coddeville et al. (43Coddeville B. Strecker G. Wieruszeski J.M. Vliegenthart J.F. van Halbeek H. Peter-Katalinic J. Egge H. Spik G. Carbohydr. Res. 1992; 236: 145-164Crossref PubMed Scopus (99) Google Scholar). Because the degree of sialylation can vary between different batches of lactoferrin, we estimated the ratio of sialylated LacdiNAc termini in our lactoferrin sample by comparing D. SiaT-mediated incorporation of [14C]Neu5Ac into Vibrio cholerae sialidase-treated and non-treated lactoferrin. We did not detect any difference in the amount of Neu5Ac incorporation between these two samples; hence we assumed that the ratio of sialylated to non-sialylated LacdiNAc in our commercial lactoferrin sample was negligible. Thus, 2.6 mol of non-sialylated LacdiNAc termini were assumed to be present on each mole of lactoferrin. Linkage Analysis—The linkage and terminal position of incorporated sialic acids were examined using a battery of exoglycosidases. After modification by D. SiaT, glycoprotein and oligosaccharide acceptors were treated with α2-3-sialidase (Streptococcus pneumoniae), α2-3,6-sialidase (Clostridium perfringens), α2-3,6,8-specific (V. cholerae) or α2-3,6,8,9-sialidase (Arthrobacter ureafaciens) (all from Sigma). The position of sialic acids was also examined by β1-3,4,6-galactosidase (bovine testes, Sigma) treatment. The acceptor sugar residue on D. SiaT was determined by the combination of β1-2,3,4,6-N-acetylglucosaminidase from S. pneumoniae (Calbiochem) and β1-2,3,4,6-N-acetylhexosaminidasef (New England Biolabs) digestions. All experiments with glycosidase treatments included a control reaction (mock treatment) with identical conditions except for the absence of the corresponding glycosidase. The digestion products of oligosaccharides were analyzed by HPTLC, and those of glycoproteins were subjected to PAGE, followed by autoradiography. Lectin Blotting—SDS-PAGE-separated glycoproteins were transferred to nitrocellulose membrane, blocked in PBTS (phosphate-buffered saline, 0.05% Tween, 0.05% saponin) overnight, and incubated with the corresponding biotinylated lectins in PBTS at 5 μg/ml (SNA) or 10 μg/ml (WFA) concentrations for 1 h at room temperature. After extensive washes, lectin binding was visualized using horseradish peroxidase-conjugated streptavidin (ABC Vectastain Elite kit, Vector Laboratories) followed by chemiluminescence detection (Pierce). Large Scale Sialyltransferase Assay and 1H NMR—For the preparative scale sialyltransferase assay, 1 mg of D. SiaT (bound to IgG beads), 100 nmol of GalNAcβ1-4GlcNAcβ1-2Manα1-O(CH2)7CH3, and 150 nmol of CMP-NeuAc were incubated in 50 mm cacodylate buffer, pH 6.5, at 25 °C for 72 h in 1.5-ml final volume. Every 12 h an aliquot of CMP-NeuAc (70 nmol) was added to the reaction mixture. The sialylated product was desalted on Sep-Pak C18 cartridges as described (32Hindsgaul O. Kaur K.J. Srivastava G. Blaszczyk-Thurin M. Crawley S.C. Heerze L.D. Palcic M.M. J. Biol. Chem. 1991; 266: 17858-17862Abstract Full Text PDF PubMed Google Scholar), and the sample was lyophilized twice from 99.8% D2O (Cambridge Isotopes Labs) and finally dissolved in 99.96% D2O. 1H NMR spectra were collected at 14.1 tesla (600-MHz proton frequency) and 300 K on a Varian Inova NMR spectrometer. Chemical shifts are referenced relative to internal acetone (δ 2.225 ppm). NMR data were processed using a software package developed by Dr. J. A. van Kuik (Bijvoet Center, Department of Bio-organic Chemistry, Utrecht University). Immunostaining and in Situ Hybridization—Immunostaining of D. SiaT-expressing S2 cells was performed as described (44Panin V.M. Papayannopoulos V. Wilson R. Irvine K.D. Nature. 1997; 387: 908-912Crossref PubMed Scopus (508) Google Scholar). We used primary monoclonal mouse anti-HA antibody at 1:1000 (Babco), polyclonal rabbit anti-Lva antibody at 1:5000 (a gift from Dr. John Sisson, The University of Texas, Austin), Cy3-conjugated donkey anti-mouse (1:500), and fluorescein isothiocyanate-conjugated donkey anti-rabbit (1:140) antibodies (both from Jackson ImmunoResearch). D. melanogaster Canton-S embryos were collected on apple juice plates at 25 °Cas described (45Rothwell W.F. Sullivan W. Sullivan W. Ashburner M. Hawley R.S. Drosophila Protocols. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY2000: 141-157Google Scholar). In situ hybridization of Drosophila embryos was performed using a published protocol (46Lehmann R. Tautz D. Methods Cell Biol. 1994; 44: 575-598Crossref PubMed Scopus (252) Google Scholar) with digoxigenin-labeled RNA probe (produced with DIG RNA labeling mix from Roche Applied Science) and anti-DIG antibody conjugated with alkaline phosphatase (Roche Applied Science). Images were obtained with Axioplan 2 fluorescent microscope (Zeiss). Identification of a Putative Drosophila Sialyltransferase Gene—By searching the complete Drosophila genome data base with the L-sialyl motif consensus sequence (10Datta A.K. Paulson J.C. Indian J. Biochem. Biophys. 1997; 34: 157-165PubMed Google Scholar), we identified a gene, located at 60D14 on 2R chromosome (GadFly CG4871), with homology to known sialyltransferases. Searches of the Drosophila EST data base (47Rubin G.M. Hong L. Brokstein P. Evans-Holm M. Frise E. Stapleton M. Harvey D.A. Science. 2000; 287: 2222-2224Crossref PubMed Scopus (305) Google Scholar) identified a cDNA clone, GH27778, with homology in the 5′-prime sequence to the predicted mRNA of CG4871. The sequence of this clone revealed that it included an open reading frame corresponding to the predicted open reading frame of CG4871, confirming that hypothetical gene CG4871 indeed represents an active transcription unit. This gene has been referred to as ST6Gal in the Drosophila genome data base based on its sequence similarity to vertebrate sialyltransferases, but as this name does not accurately reflect its actual enzymatic activity (see below), we instead designate it as SiaT. Similar to vertebrate sialyltransferases, this gene encodes a putative type II transmembrane protein with an N-terminally located signal peptide/anchoring domain, a stem region, and a C-terminal presumptive catalytic domain with high homology to the corresponding domain of vertebrate sialyltransferases (Fig. 1, A and B). The closest vertebrate homologues of Drosophila SiaT (designated D. SiaT) are β-galactoside α2-6-sialyltransferases, including human ST6Gal II (7Takashima S. Tsuji S. Tsujimoto M. J. Biol. Chem. 2002; 277: 45719-45728Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 8Krzewinski-Recchi M.A. Julien S. Juliant S. Teintenier-Lelievre M. Samyn-Petit B. Montiel M.D. Mir A.M. Cerutti M. Harduin-Lepers A. Delannoy P. Eur. J. Biochem. 2003; 270: 950-961Crossref PubMed Scopus (62) Google Scholar) and chick and bovine ST6Gal I proteins (48Kurosawa N. Kawasaki M. Hamamoto T. Nakaoka T. Lee Y.C. Arita M. Tsuji S. Eur. J. Biochem. 1994; 219: 375-381Crossref PubMed Scopus (36) Google Scholar, 49Mercier D. Wierinckx A. Oulmouden A. Gallet P.F. Palcic M.M. Harduin-Lepers A. Delannoy P. Petit J.M. Leveziel H. Julien R. Glycobiology. 1999; 9: 851-863Crossref PubMed Scopus (20) Google Scholar) (BLAST E-values: 3e-50, 2e-47, and 2e-45, respectively). BLAST searches also revealed an Anopheles gambiae (mosquito) gene, agCG56989 (GenBank™ accession number EAA04038), encoding a putative sialyltransferase with high sequence homology to D. SiaT (E-value: 3e-89). Multiple sequence alignment with the catalytic domains of known vertebrate sialyltransferases confirmed a closer relationship of D. SiaT to ST6Gal sialyltransferases, as compared with ST3Gal, ST6GalNAc, or ST8Sia sialyltransferas" @default.
• W2024115143 created "2016-06-24" @default.
• W2024115143 creator A5071303454 @default.
• W2024115143 creator A5071660392 @default.
• W2024115143 creator A5077397465 @default.
• W2024115143 date "2004-02-01" @default.
• W2024115143 modified "2023-09-29" @default.
• W2024115143 title "Functional Characterization of Drosophila Sialyltransferase" @default.
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• W2024115143 doi "https://doi.org/10.1074/jbc.m309912200" @default.
• W2024115143 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14612445" @default.
• W2024115143 hasPublicationYear "2004" @default.
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