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• W2138947900 abstract "Cell-cell and cell-matrix adhesion are crucial during many stages of eukaryotic development. Here, we provide the first example that mucin-type O-linked glycosylation is involved in a developmentally regulated cell adhesion event in Drosophila melanogaster. Mutations in one member of the evolutionarily conserved family of enzymes that initiates O-linked glycosylation alter epithelial cell adhesion in the Drosophila wing blade. A transposon insertion mutation in pgant3 or RNA interference to pgant3 resulted in blistered wings, a phenotype characteristic of genes involved in integrin-mediated cell interactions. Expression of wild type pgant3 in the mutant background rescued the wing blistering phenotype, whereas expression of another family member (pgant35A) did not, revealing a unique requirement for pgant3. pgant3 mutants displayed reduced O-glycosylation along the basal surface of larval wing imaginal discs, which was restored with wild type pgant3 expression, suggesting that reduced glycosylation of basal proteins is responsible for disruption of adhesion in the adult wing blade. Glycosylation reactions demonstrated that PGANT3 glycosylates certain extracellular matrix (ECM) proteins. Immunoprecipitation experiments revealed that PGANT3 glycosylates tiggrin, an ECM protein known to bind integrin. We propose that this glycosyltransferase is uniquely responsible for glycosylating tiggrin in the wing disc, thus modulating proper cell adhesion through integrin-ECM interactions. This study provides the first evidence for the role of O-glycosylation in a developmentally regulated, integrin-mediated, cell adhesion event and reveals a novel player in wing blade formation during Drosophila development. Cell-cell and cell-matrix adhesion are crucial during many stages of eukaryotic development. Here, we provide the first example that mucin-type O-linked glycosylation is involved in a developmentally regulated cell adhesion event in Drosophila melanogaster. Mutations in one member of the evolutionarily conserved family of enzymes that initiates O-linked glycosylation alter epithelial cell adhesion in the Drosophila wing blade. A transposon insertion mutation in pgant3 or RNA interference to pgant3 resulted in blistered wings, a phenotype characteristic of genes involved in integrin-mediated cell interactions. Expression of wild type pgant3 in the mutant background rescued the wing blistering phenotype, whereas expression of another family member (pgant35A) did not, revealing a unique requirement for pgant3. pgant3 mutants displayed reduced O-glycosylation along the basal surface of larval wing imaginal discs, which was restored with wild type pgant3 expression, suggesting that reduced glycosylation of basal proteins is responsible for disruption of adhesion in the adult wing blade. Glycosylation reactions demonstrated that PGANT3 glycosylates certain extracellular matrix (ECM) proteins. Immunoprecipitation experiments revealed that PGANT3 glycosylates tiggrin, an ECM protein known to bind integrin. We propose that this glycosyltransferase is uniquely responsible for glycosylating tiggrin in the wing disc, thus modulating proper cell adhesion through integrin-ECM interactions. This study provides the first evidence for the role of O-glycosylation in a developmentally regulated, integrin-mediated, cell adhesion event and reveals a novel player in wing blade formation during Drosophila development. Cell interactions and adhesion are critical in many diverse processes, from events occurring during embryogenesis and organogenesis to wound healing and the alterations in cell adhesion seen upon tumor formation and metastasis (1Hynes R.O. Zhao Q. J. Cell Biol. 2000; 150: 89-96Crossref PubMed Google Scholar). Drosophila wing development has been used as a model system to identify factors responsible for regulating cell adhesion (2Brower D.L. Curr. Opin. Cell Biol. 2003; 15: 607-613Crossref PubMed Scopus (56) Google Scholar, 3Brower D.L. Bunch T.A. Mukai L. Adamson T.E. Wehrli M. Lam S. Friedlander E. Roote C.E. Zusman S. Development. 1995; 121: 1311-1320Crossref PubMed Google Scholar, 4Prout M. Damania Z. Soong J. Fristrom D. Fristrom J.W. Genetics. 1997; 146: 275-285Crossref PubMed Google Scholar, 5Walsh E.P. Brown N.H. Genetics. 1998; 150: 791-805Crossref PubMed Google Scholar). Aberrant adhesion of the two epithelial cell layers that comprise the adult wing blade results in separation of the cell layers, creating a localized blister shortly after eclosion. Mutations in the integrin family of cell surface receptors (2Brower D.L. Curr. Opin. Cell Biol. 2003; 15: 607-613Crossref PubMed Scopus (56) Google Scholar, 3Brower D.L. Bunch T.A. Mukai L. Adamson T.E. Wehrli M. Lam S. Friedlander E. Roote C.E. Zusman S. Development. 1995; 121: 1311-1320Crossref PubMed Google Scholar, 5Walsh E.P. Brown N.H. Genetics. 1998; 150: 791-805Crossref PubMed Google Scholar, 6Araujo H. Negreiros E. Bier E. Development. 2003; 130: 3851-3864Crossref PubMed Scopus (29) Google Scholar, 7Bloor J.W. Brown N.H. Genetics. 1998; 148: 1127-1142Crossref PubMed Google Scholar) as well as proteins that interact with integrins (4Prout M. Damania Z. Soong J. Fristrom D. Fristrom J.W. Genetics. 1997; 146: 275-285Crossref PubMed Google Scholar, 5Walsh E.P. Brown N.H. Genetics. 1998; 150: 791-805Crossref PubMed Google Scholar, 8Brown N.H. Gregory S.L. Rickoll W.L. Fessler L.I. Prout M. White R.A. Fristrom J.W. Dev Cell. 2002; 3: 569-579Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 9Fogerty F.J. Fessler L.I. Bunch T.A. Yaron Y. Parker C.G. Nelson R.E. Brower D.L. Gullberg D. Fessler J.H. Development. 1994; 120: 1747-1758Crossref PubMed Google Scholar, 10Graner M.W. Bunch T.A. Baumgartner S. Kerschen A. Brower D.L. J. Biol. Chem. 1998; 273: 18235-18241Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 11Lee S.B. Cho K.S. Kim E. Chung J. Development. 2003; 130: 4001-4010Crossref PubMed Scopus (40) Google Scholar, 12Subramanian A. Wayburn B. Bunch T. Volk T. Development. 2007; 134: 1269-1278Crossref PubMed Scopus (79) Google Scholar, 13Torgler C.N. Narasimha M. Knox A.L. Zervas C.G. Vernon M.C. Brown N.H. Dev. Cell. 2004; 6: 357-369Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) have been shown to produce wing blisters, highlighting the central role of integrin-mediated cell adhesion events during wing blade formation. Interestingly, many cell surface and extracellular matrix (ECM) 2The abbreviations used are: ECM, extracellular matrix; ppGalNAcT or PGANT or pgant, UDP GalNAc:polypeptide N-acetylgalactosaminyltransferase; IR, inverted repeat; JNK, c-Jun NH2-terminal kinase; RNAi, RNA interference; Ab, antibody. 2The abbreviations used are: ECM, extracellular matrix; ppGalNAcT or PGANT or pgant, UDP GalNAc:polypeptide N-acetylgalactosaminyltransferase; IR, inverted repeat; JNK, c-Jun NH2-terminal kinase; RNAi, RNA interference; Ab, antibody. proteins that influence wing blistering also interact with integrins in other developmentally regulated cell adhesion events (2Brower D.L. Curr. Opin. Cell Biol. 2003; 15: 607-613Crossref PubMed Scopus (56) Google Scholar, 14Bunch T.A. Graner M.W. Fessler L.I. Fessler J.H. Schneider K.D. Kerschen A. Choy L.P. Burgess B.W. Brower D.L. Development. 1998; 125: 1679-1689Crossref PubMed Google Scholar, 15Prokop A. Martin-Bermudo M.D. Bate M. Brown N.H. Dev. Biol. 1998; 196: 58-76Crossref PubMed Scopus (102) Google Scholar). Cell surface, secreted, and ECM proteins undergo a number of post-translational modifications as they transit the secretory apparatus to their final destinations. Although the roles of classical N-linked glycans and proteoglycans are widely appreciated, recent studies have elucidated crucial roles for other types of glycans during development. The disaccharide GlcNAcβ1–3Fuc on the Notch receptor and its ligands has been shown to regulate receptor/ligand interactions and downstream signaling events (16Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (592) Google Scholar, 17Haines N. Irvine K.D. Nat. Rev. Mol. Cell Biol. 2003; 4: 786-797Crossref PubMed Scopus (336) Google Scholar, 18Moloney 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 (719) Google Scholar, 19Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Protein O-fucosylation and the fucosyltransferase catalyzing this modification have roles in protein folding, trafficking (20Sasaki N. Sasamura T. Ishikawa H.O. Kanai M. Ueda R. Saigo K. Matsuno K. Genes Cells. 2007; 12: 89-103Crossref PubMed Scopus (61) Google Scholar, 21Sasamura T. Ishikawa H.O. Sasaki N. Higashi S. Kanai M. Nakao S. Ayukawa T. Aigaki T. Noda K. Miyoshi E. Taniguchi N. Matsuno K. Development. 2007; 134: 1347-1356Crossref PubMed Scopus (69) Google Scholar), and secretion (22Ricketts L.M. Dlugosz M. Luther K.B. Haltiwanger R.S. Majerus E.M. J. Biol. Chem. 2007; 282: 17014-17023Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Most recently, O-linked glucose has been shown to have a modulatory role in Notch signaling events, influencing lateral inhibition and cell-fate specification (23Acar M. Jafar-Nejad H. Takeuchi H. Rajan A. Ibrani D. Rana N.A. Pan H. Haltiwanger R.S. Bellen H.J. Cell. 2008; 132: 247-258Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Taken together, these studies highlight the diversity of glycans found on proteins and the unique functional roles they play during development. Studies from our laboratory and others have demonstrated the abundant presence of another type of glycosylation, known as mucin-type O-linked glycosylation (referred to as O-linked glycosylation) throughout development in diverse species (24Kingsley P.D. Ten Hagen K.G. Maltby K.M. Zara J. Tabak L.A. Glycobiology. 2000; 10: 1317-1323Crossref PubMed Scopus (54) Google Scholar, 25Ten Hagen K.G. Tran D.T. Gerken T.A. Stein D.S. Zhang Z. J. Biol. Chem. 2003; 278: 35039-35048Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 26Tian E. Ten Hagen K.G. Glycobiology. 2007; 17: 820-827Crossref PubMed Scopus (48) Google Scholar, 27Tian E. Ten Hagen K.G. Glycobiology. 2006; 16: 83-95Crossref PubMed Scopus (51) Google Scholar). In contrast to the previously mentioned types of glycosylation, mucin-type O-glycosylation is initiated by a large family of enzymes known as the UDP-GalNAc: polypeptide N-acetylgalactosaminyltranferases (PGANTs in Drosophila or ppGalNAcTs in mammals) that transfer N-acetylgalactosamine (GalNAc) to serine or threonine residues of proteins destined to be membrane-bound or secreted (28Hang H.C. Bertozzi C.R. Bioorg. Med. Chem. 2005; 13: 5021-5034Crossref PubMed Scopus (229) Google Scholar, 29Ten Hagen K.G. Fritz T.A. Tabak L.A. Glycobiology. 2003; 13: 1-16Crossref PubMed Scopus (409) Google Scholar). Members of this family have unique but overlapping developmental expression patterns, and many show distinct substrate preferences in vitro, suggesting that each enzyme may be responsible for glycosylating a unique subset of proteins in vivo (25Ten Hagen K.G. Tran D.T. Gerken T.A. Stein D.S. Zhang Z. J. Biol. Chem. 2003; 278: 35039-35048Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 28Hang H.C. Bertozzi C.R. Bioorg. Med. Chem. 2005; 13: 5021-5034Crossref PubMed Scopus (229) Google Scholar, 29Ten Hagen K.G. Fritz T.A. Tabak L.A. Glycobiology. 2003; 13: 1-16Crossref PubMed Scopus (409) Google Scholar). In vitro data further indicate that there exists a hierarchy of activity within the family, with some members initiating the glycosylation of unmodified substrates and others acting only on previously glycosylated substrates, adding GalNAc at positions vicinal to sites previously modified by other family members (30Pratt M.R. Hang H.C. Ten Hagen K.G. Rarick J. Gerken T.A. Tabak L.A. Bertozzi C.R. Chem. Biol. 2004; 11: 1009-1016Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 31Ten Hagen K.G. Bedi G.S. Tetaert D. Kingsley P.D. Hagen F.K. Balys M.M. Beres T.M. Degand P. Tabak L.A. J. Biol. Chem. 2001; 276: 17395-17404Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 32Ten Hagen K.G. Tetaert D. Hagen F.K. Richet C. Beres T.M. Gagnon J. Balys M.M. VanWuyckhuyse B. Bedi G.S. Degand P. Tabak L.A. J. Biol. Chem. 1999; 274: 27867-27874Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The unique spatial and temporal expression patterns, substrate preferences, and hierarchical action of members of this family suggest a highly regulated process governing the acquisition of this type of glycan. Mucin-type O-glycans on secreted and membrane-bound proteins are uniquely positioned to mediate many events regulating homeostasis and development. Indeed, mutations in ppGalNAc-T3 are thought to be responsible for familial tumoral calcinosis, a rare human disease characterized by hyperphosphatemia and the development of calcified “tumors” in cutaneous and subcutaneous tissues (33Topaz O. Shurman D.L. Bergman R. Indelman M. Ratajczak P. Mizrachi M. Khamaysi Z. Behar D. Petronius D. Friedman V. Zelikovic I. Raimer S. Metzker A. Richard G. Sprecher E. Nat. Genet. 2004; 36: 579-581Crossref PubMed Scopus (463) Google Scholar). Mice deficient in ppGalNAc-T1, although viable and fertile, display reduced lymphocyte homing and bleeding disorders (34Tenno M. Ohtsubo K. Hagen F.K. Ditto D. Zarbock A. Schaerli P. von Andrian U.H. Ley K. Le D. Tabak L.A. Marth J.D. Mol. Cell. Biol. 2007; 27: 8783-8796Crossref PubMed Scopus (90) Google Scholar). Studies from our laboratory have demonstrated a role for another member of this family (pgant35A) in the proper formation of the embryonic tracheal system in Drosophila (35Ten Hagen K.G. Tran D.T. J. Biol. Chem. 2002; 277: 22616-22622Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 36Tian E. Ten Hagen K.G. J. Biol. Chem. 2007; 282: 606-614Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Finally, mice deficient in the core 1 β3-galactosyltransferase, which adds a galactose to the O-GalNAc of mucin-type O-glycans, die embryonically from defective angiogenesis resulting in fatal brain hemorrhages (37Xia L. Ju T. Westmuckett A. An G. Ivanciu L. McDaniel J.M. Lupu F. Cummings R.D. McEver R.P. J. Cell Biol. 2004; 164: 451-459Crossref PubMed Scopus (143) Google Scholar); hypomorphic mutations result in thrombocytopenia and kidney defects later in development (38Alexander W.S. Viney E.M. Zhang J.G. Metcalf D. Kauppi M. Hyland C.D. Carpinelli M.R. Stevenson W. Croker B.A. Hilton A.A. Ellis S. Selan C. Nandurkar H.H. Goodnow C.C. Kile B.T. Nicola N.A. Roberts A.W. Hilton D.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 16442-16447Crossref PubMed Scopus (66) Google Scholar). Altogether, these studies highlight the diverse consequences of alterations in O-glycosylation that have only recently been discovered due to the inherent complexity of the ppGalNAcT family. Here, we examine the developmental role of another member of this family, pgant3. Previous work from our laboratory demonstrated that PGANT3 is one of the initiating glycosyltransferases, transferring GalNAc to previously unmodified substrates (25Ten Hagen K.G. Tran D.T. Gerken T.A. Stein D.S. Zhang Z. J. Biol. Chem. 2003; 278: 35039-35048Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Additionally, pgant3 gene expression is highly regulated during development (27Tian E. Ten Hagen K.G. Glycobiology. 2006; 16: 83-95Crossref PubMed Scopus (51) Google Scholar). In this study we find that a transposon mutation in pgant3 or RNA interference (RNAi) to pgant3 results in wing blistering, implicating O-glycosylation in integrin-mediated cell adhesion occurring during wing blade formation. A reduction in O-glycoproteins was seen along the basal surface of the pgant3 mutant wing imaginal discs along with altered disc morphology. Based on our studies, we propose that O-glycosylation of specific ECM proteins known to bind integrin is required for proper epithelial cell adhesion in the wing disc. This finding provides the first direct evidence for the role of mucin type O-glycans in a developmentally regulated cell adhesion event and identifies a novel protein modification required for proper wing blade formation in the fly. Fly Strains Used—The stocks used in this study are as follows: Bloomington stocks #5138 (y1, w*; P{w+mC = tubP-GAL4}LL7/TM3, Sb1) (the tubulin-Gal4 driver line); #8860 (w1118, P{w+mW.hs = GawB}BxMS1096) (the wing-specific Gal4 driver line); #1561 (w*; P{w+mW.hs = Gal4-arm}4a, P{w+mW.hs = Gal4-arm}4b/TM3, Sb1, Ser1) (the armadillo-Gal4 driver line); #7748 (w1118; Df(2R)Exel 6283, PXP-UExel6263); #8283 (w1118; CyO, P{w+mc = FRT (w+)Tub-PBac™}2/wgsp-1); #8795 (w*; TigA1/CyO,P{lacZ-un3}276); #8796 (w*; TigX/CyO,P{lacZ-un3}276). Additionally, the following stocks from other sources were used: PBac{PB}pgant3c01318 from the Exelixis Drosophila Stock Collection (39Thibault S.T. Singer M.A. Miyazaki W.Y. Milash B. Dompe N.A. Singh C.M. Buchholz R. Demsky M. Fawcett R. Francis-Lang H.L. Ryner L. Cheung L.M. Chong A. Erickson C. Fisher W.W. Greer K. Hartouni S.R. Howie E. Jakkula L. Joo D. Killpack K. Laufer A. Mazzotta J. Smith R.D. Stevens L.M. Stuber C. Tan L.R. Ventura R. Woo A. Zakrajsek I. Zhao L. Chen F. Swimmer C. Kopczynski C. Duyk G. Winberg M.L. Margolis J. Nat. Genet. 2004; 36: 283-287Crossref PubMed Scopus (649) Google Scholar); w; Dr/TM3, Sb1, twi-2XGFP stock (the kind gift of D. Andrew); w; TM6C, cu, Sb, e, ca/Su(Tpl)s1, red, e stock (the kind gift of J. Kennison). Construction of Gal4-inducible pgant3 and pgant3IR Vectors and Transgenic Lines—The pUAST plasmid (40Brand A.H. Perrimon N. Development. 1993; 118: 401-415Crossref PubMed Google Scholar) was used to generate a Gal4-inducible construct expressing wild type pgant3 cDNA that was then used to create transgenic flies. The complete coding region from the wild type pgant3 gene was excised from the GH09147 cDNA clone (Invitrogen) using EcoRI and XbaI and cloned into the same sites of pUAST to generate the plasmid pUAS-pgant3. To generate the Gal4-inducible pgant3IR construct, sense (taatacctaggAAGGTGAATGTTACGGAGCGTGTGG) and antisense (taatacctaggCTGCGCCAGCATTACATTCGAAGTG) primers were used to amplify a 500-bp fragment from the catalytic region of pgant3. The PCR product was then cloned stepwise into the AvrII and NheI sites on either side of the white intron in the vector pWIZ (41Lee Y.S. Carthew R.W. Methods. 2003; 30: 322-329Crossref PubMed Scopus (307) Google Scholar) to generate a vector containing two inversely oriented pgant3 fragments flanking the white intron. Transformants were produced by Genetic Services Inc. (Cambridge, MA) using methodology based on the procedure described previously (42Rubin G.M. Spradling A.C. Science. 1982; 218: 348-353Crossref PubMed Scopus (2335) Google Scholar, 43Spradling A.C. Rubin G.M. Science. 1982; 218: 341-347Crossref PubMed Scopus (1169) Google Scholar). Fly Crosses—Rescue and overexpression experiments were performed using flies from a UAS-pgant35A transgenic line (36Tian E. Ten Hagen K.G. J. Biol. Chem. 2007; 282: 606-614Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) or three independent UAS-pgant3 transgenic lines (w; P{UAS-pgant3#3}, on the third chromosome) (w; P{UAS-pgant3#7}, on the X chromosome) (w; P{UAS-pgant3#10}, on the third chromosome) and the Gal4-driver stocks described herein. All stocks used in the rescue experiments were first crossed into the pgant3c01318 background to generate both Gal4 driver lines and P{UAS-pgant} transgenic lines heterozygous for pgant3c01318. These heterozygous lines were then crossed as follows, and rescue of the wing blister phenotype was assessed by scoring straight winged progeny with or without Sb1: pgant3c01318/CyO; P{UAS-pgant}/P{UAS-pgant} females X pgant3c01318/CyO;P{tubP-GAL4}LL7/TM3, Sb1 males. Crosses to assess the effect of pgant3 overexpression were performed as shown in Table 2.TABLE 2Over-expression of pgant3 results in lethality UAS-pgant3#3 and UAS-pgant3#10 are independent transgenic lines generated as described under “Experimental Procedures.”CrossesProgeny overexpressing pgant3aAssessed by the absence of Sb1Progeny without pgant3 overexpressionSurvival of pgant3 overexpressing flies(%)UAS-pgant3#3/UAS-pgant3#3 × Tub-Gal4/TM3, Sb103120UAS-pgant3#10/UAS-pgant3#10 × Tub-Gal4/TM3, Sb161714UAS-pgant3#3/UAS-pgant3#3 × arm-Gal4/TM3, Sb112621350UAS-pgant3#3/TM6C, Sb1 × MS1096-Gal4/MS1096-Gal4214211100a Assessed by the absence of Sb1 Open table in a new tab Crosses to generate RNAi to pgant3 were performed by crossing homozygous inverted repeat (IR) transgenic lines (w1118; P{UAS-pgant3IR2#2} or w1118; P{UAS-pgant3IR2#9}) to a tubulin-Gal4-driver line (P{tubP-GAL4}LL7/TM3, Sb1, twi-2XGFP) and comparing progeny with and without Sb1 and GFP. Crosses to the wing-specific driver (MS1096-Gal4) were performed using homozygous w1118, P{w+mW.hs = GawB}BxMS1096 females crossed to homozygous transgene-containing males. All Drosophila crosses were kept on MM media (KD Medical, Inc.) at 25 °C unless specified otherwise. Mutant Sequencing and Quantitative Reverse Transcription-PCR—The genomic region flanking each transposon insertion was amplified and sequenced according to the previously described procedures (39Thibault S.T. Singer M.A. Miyazaki W.Y. Milash B. Dompe N.A. Singh C.M. Buchholz R. Demsky M. Fawcett R. Francis-Lang H.L. Ryner L. Cheung L.M. Chong A. Erickson C. Fisher W.W. Greer K. Hartouni S.R. Howie E. Jakkula L. Joo D. Killpack K. Laufer A. Mazzotta J. Smith R.D. Stevens L.M. Stuber C. Tan L.R. Ventura R. Woo A. Zakrajsek I. Zhao L. Chen F. Swimmer C. Kopczynski C. Duyk G. Winberg M.L. Margolis J. Nat. Genet. 2004; 36: 283-287Crossref PubMed Scopus (649) Google Scholar). To examine the effect of the transposon on pgant3 gene expression levels, pgant3c01318/pgant3c01318 homozygotes, wild type, and transposon excision lines were used to isolate RNA and perform real-time PCR. Briefly, RNA was isolated using the FastRNA Pro Green kit (Q-BIOgene). cDNA synthesis was performed using the iScript cDNA synthesis kit (Bio-Rad). PCR primers were designed using Beacon Designer software (Bio-Rad). Quantitative reverse transcription-PCR was performed on a MyiQ real time PCR thermocycler (Bio-Rad) using the SYBR-Green PCR Master Mix (Bio-Rad). Quantitative reverse transcription-PCR to determine expression levels of all pgant family members was performed using the PCR primers listed in supplemental Table 1 with cDNA prepared from wild type, Tub>pgant3IR2#2, and Tub>pgant3IR2#9 larval wing discs. Wing Disc Fixation and Staining—Imaginal wing discs were stained according to standard procedures and analyzed by confocal microscopy. Mouse monoclonal anti-Tn antibody (Ca3638) (44Avichezer D. Springer G.F. Schechter B. Arnon R. Int. J. Cancer. 1997; 72: 119-127Crossref PubMed Scopus (44) Google Scholar) (dilution, 1:50) was the kind gift of Dr. Richard Cummings who had acquired the stocks of antibodies and hybridomas from the late Dr. Georg F. Springer. Immunopositive signals were developed using Cy3-conjugated donkey anti-mouse IgM antibody (dilution, 1:100) (Jackson Immuno-Research Laboratories). Stained wing discs were analyzed using the Zeiss LSM 510 confocal laser scanning microscope. Images were processed using the LSM Imager Browser and Photoshop. Measurements of wing disc thickness and O-glycan staining in x-z cross sections were performed in the center of each disc. Values were averaged, and S.D. were calculated. Statistical significance was determined using a Student's t test. Glycosylation Assays in Vitro—Assays for glycosyltransferase activity were performed as described previously (25Ten Hagen K.G. Tran D.T. Gerken T.A. Stein D.S. Zhang Z. J. Biol. Chem. 2003; 278: 35039-35048Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Briefly, media from COS7 cells expressing recombinant pgant3 or pgant35A (25Ten Hagen K.G. Tran D.T. Gerken T.A. Stein D.S. Zhang Z. J. Biol. Chem. 2003; 278: 35039-35048Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) was harvested and used in the in vitro reactions with [14C]UDP-GalNAc, and the acceptor substrates denoted in Table 3. Reaction products were purified by anion exchange chromatography, and [14C]GalNAc incorporation was measured. Reactions using media from cells expressing empty vector alone yielded background values that were subtracted from each experimental value. Adjusted experimental values were then averaged, and S.D. were calculated. All assays were performed in duplicate. Glycosyltransferase activity is expressed as dpm/h.TABLE 3Peptide substrates derived from proteins expressed in wing discs The bold residues are sites of potential O-glycosylation based on predictions performed by the NetOGlyc server. NA, no evidence from references.PeptideGeneProteinPeptide sequenceWing blisters in mutantsififαPS2 integrinEPQVNQTSFTTYSTSSSSSGyeslanA1LanALaminin ATLPPTTPTTTTTTTTTyesmewmewαPS1 integrinVGFFKRIRPTDPTLSGNLEyesscabscbαPS3 integrinVDPVEVTTTLSGGLERTVyestigAtigTiggrinLEGETARPRPPNPAPIVSTPKPyestigBtigTiggrinQQATKVEVEATSEPSFWEKLKyestenM1ten-mTenascin-majorFLLEGVTPTAPPDVPPRNPTNAtenM2ten-mTenascin-majorTSNSGTAQGLQSTSASAEATSSNAtsptspThrombospondinIQIKLVNSTEGPGPMMRNSNA Open table in a new tab Western Blotting—Protein extracts were prepared from 3rd instar larval wing discs of wild type, pgant3c01318 homozygotes, and transposon excision lines (pgant3c01318revertant#7 homozygotes) as described (45Pickup A.T. Banerjee U. Dev. Biol. 1999; 205: 254-259Crossref PubMed Scopus (37) Google Scholar). Samples were electrophoresed under reducing conditions in a 4–12% SDS-PAGE gradient gel. Gels were transferred to nitrocellulose; membranes were blocked with 1× blocking buffer (Sigma), incubated with Tn antibody (dilution, 1:500) or the tiggrin antibody (the kind gift of Drs. L. and J. Fessler) (9Fogerty F.J. Fessler L.I. Bunch T.A. Yaron Y. Parker C.G. Nelson R.E. Brower D.L. Gullberg D. Fessler J.H. Development. 1994; 120: 1747-1758Crossref PubMed Google Scholar) (dilution, 1:500), and developed with horse-radish peroxidase-conjugated secondary antibody (dilution, 1:10,000). Immunoprecipitation—Equivalent amounts of protein extracts were prepared from 3rd instar larval wing discs from wild type and pgant3c01318 homozygotes. 50 μl of immobilized protein L suspension (Thermo Scientific) was added to 500 μl of protein extract and incubated at 4 °C for 1 h to preclear. The mixtures were centrifuged, and the precleared supernatant was collected. 10 μl of the tiggrin antibody were added to the precleared supernatant and incubated at 4 °C overnight. The following day 50 μl of washed immobilized protein L suspension was added, and incubation was performed at 4 °C overnight. Immunocomplexed proteins were collected by pulse centrifugation. Pellets were washed three times with lysis buffer. The final pellet was resuspended in sample loading buffer, heated to 95 °C for 5 min, and analyzed by reducing SDS-PAGE followed by immunoblotting with the Tn Ab as described. Transposon Insertion Decreases pgant3 Gene Expression and Causes Wing Blistering—pgant3 is one of 12 members of the Drosophila gene family encoding the polypeptide GalNAc glycosyltransferases that are responsible for initiating mucin-type O-linked glycosylation of secreted and membrane-bound proteins (25Ten Hagen K.G. Tran D.T. Gerken T.A. Stein D.S. Zhang Z. J. Biol. Chem. 2003; 278: 35039-35048Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 27Tian E. Ten Hagen K.G. Glycobiology. 2006; 16: 83-95Crossref PubMed Scopus (51) Google Scholar). pgant3 gene expression is highly regulated both spatially and temporally during development (27Tian E. Ten Hagen K.G. Glycobiology. 2006; 16: 83-95Crossref PubMed Scopus (51) Google Scholar). We set out to examine the biological roles of O-glycosylation mediated by this glycosyltransferase using three putative transposon insertions in the pgant3 gene from the Exelixis Drosophila stock collection (39Thibault S.T. Singer M.A. Miyazaki W.Y. Milash B. Dompe N.A. Singh C.M. Buchholz R. Demsky M. Fawcett R. Francis-Lang H.L. Ryner L. Cheung L.M. Chong A. Erickson C. Fisher W.W. Greer K. Hartouni S.R. Howie E. Jakkula L. Joo D. Killpack K. Laufer A. Mazzotta J. Smith R.D. Stevens L.M. Stuber C. Tan L.R. Ventura R. Woo A. Zakrajsek I. Zhao L. Chen F. Swimmer C. Kopczynski C. Duyk G. Winberg M.L. Margolis J. Nat. Genet. 2004; 36: 283-287Crossref PubMed Scopus (649) Google Scholar). Genomic sequencing of the insertion sites revealed that only one transposon resided within the pgant3 gene (PBac{PB}pgant3c01318); the line containing this transposon is hereafter designated pgant3c01318. pgant3c01318 contains a piggyBac transposable element in the fourth intron of pgant3, separating exons that encode the conserved catalytic region of the enzyme (Fig. 1A). Quantitative PCR was performed on wild type and homozygous transposon insertion mutants to assess the effect of the transposon on the expression levels of pgant3 as well as the flanking tetraspanin genes (Tsp42Ep andTsp42Eq) (Fig. 1B). As shown in Fig. 1C, pgant3 gene expression 3′ to the transposon insertion site was significantly reduced in the transposon insertion line relative to wild type. Expression of the flanking tetraspanin genes was not affected. This result demonstrates that the transposon insertion specifically affects pgant3 gene expression, thus supporting the use of this line to invest" @default.
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• W2138947900 title "A Mucin-type O-Glycosyltransferase Modulates Cell Adhesion during Drosophila Development" @default.
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