title: Dissecting the biological role of mucin-type O-glycosylation using RNA interference in Drosophila cell culture.
abstract: Mucin type O-glycosylation is a highly conserved form of post-translational modification initiated by the family of enzymes known as the polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs in mammals and PGANTs in Drosophila). To address the cellular functions of the many PGANT family members, RNA interference (RNAi) to each pgant gene was performed in two independent Drosophila cell culture lines. We demonstrate that RNAi to individual pgant genes results in specific reduction in gene expression without affecting the expression of other family members. Cells with reduced expression of individual pgant genes were then examined for changes in viability, morphology, adhesion, and secretion to assess the contribution of each family member to these cellular functions. Here we find that RNAi to pgant3, pgant6, or pgant7 resulted in reduced secretion, further supporting a role for O-glycosylation in proper secretion. Additionally, RNAi to pgant3 or pgant6 resulted in altered Golgi organization, suggesting a role for each in establishing or maintaining proper secretory apparatus structure. Other subcellular effects observed included multinucleated cells seen after RNAi to either pgant2 or pgant35A, suggesting a role for these genes in the completion of cytokinesis. These studies demonstrate the efficient and specific knockdown of pgant gene expression in two Drosophila cell culture systems, resulting in specific morphological and functional effects. Our work provides new information regarding the biological roles of O-glycosylation and illustrates a new platform for interrogating the cellular and subcellular effects of this form of post-translational modification.
authors.affiliation: Developmental Glycobiology Unit, NIDCR, National Institutes of Health, Bethesda, Maryland 20892-4370, USA.
authors.forename: Liping
authors.initials: L
authors.lastname: Zhang
authors.forename: Kelly G
authors.initials: KG
authors.lastname: Ten Hagen
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D048749:Cytokinesis / Q000502:physiology*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D055785:Gene Knockdown Techniques; D006031:Glycosylation; D006056:Golgi Apparatus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D009077:Mucins / Q000235:genetics / Q000378:metabolism*; D017350:N-Acetylgalactosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D034622:RNA Interference
chemical_list: D029721:Drosophila Proteins; D009077:Mucins; D017350:N-Acetylgalactosaminyltransferases
references.citation: Mol Biol Cell. 2003 Jan;14(1):190-200
references.pmid: 12529436
references.citation: Glycobiology. 2007 Aug;17(8):820-7
references.pmid: 17522109
references.citation: Semin Cell Dev Biol. 2009 Sep;20(7):780-3
references.pmid: 19508857
references.citation: Am J Physiol Cell Physiol. 2006 Jan;290(1):C1-C10
references.pmid: 16338974
references.citation: J Biol Chem. 2007 Jan 5;282(1):606-14
references.pmid: 17098739
references.citation: Science. 2004 Feb 6;303(5659):832-5
references.pmid: 14764878
references.citation: J Biol Chem. 2010 Jun 18;285(25):19491-501
references.pmid: 20371600
references.citation: Traffic. 2007 Jun;8(6):758-73
references.pmid: 17488291
references.citation: J Biol Chem. 2002 Jun 21;277(25):22623-38
references.pmid: 11925450
references.citation: Glycoconj J. 2009 Apr;26(3):325-34
references.pmid: 18695988
references.citation: J Biol Chem. 2003 Sep 12;278(37):35039-48
references.pmid: 12829714
references.citation: Glycobiology. 2006 Feb;16(2):83-95
references.pmid: 16251381
references.citation: Glycobiology. 2003 Jan;13(1):1R-16R
references.pmid: 12634319
references.citation: Mol Cell Biol. 2007 Dec;27(24):8783-96
references.pmid: 17923703
references.citation: J Cell Biol. 2007 Dec 31;179(7):1583-97
references.pmid: 18166658
references.citation: Endocrinology. 2009 Jun;150(6):2543-50
references.pmid: 19213845
references.citation: J Biol. 2003;2(4):27
references.pmid: 14527345
references.citation: J Biol Chem. 2002 Jun 21;277(25):22616-22
references.pmid: 11925446
references.citation: Curr Biol. 2004 Sep 21;14(18):1685-93
references.pmid: 15380073
references.citation: Glycobiology. 2003 Jul;13(7):549-57
references.pmid: 12651884
references.citation: Bioorg Med Chem. 2005 Sep 1;13(17):5021-34
references.pmid: 16005634
references.citation: J Cell Biol. 2000 Apr 17;149(2):341-56
references.pmid: 10769027
references.citation: J Cell Sci. 2001 Apr;114(Pt 8):1455-71
references.pmid: 11282022
references.citation: J Biol Chem. 1998 Nov 27;273(48):32353-9
references.pmid: 9822716
references.citation: J Biol Chem. 1998 Apr 3;273(14):8268-77
references.pmid: 9525933
references.citation: Glycoconj J. 1996 Oct;13(5):717-26
references.pmid: 8909998
references.citation: Dev Biol. 2002 Nov 15;251(2):294-306
references.pmid: 12435359
references.citation: Semin Cell Dev Biol. 2009 Sep;20(7):770-9
references.pmid: 19508854
references.citation: Curr Biol. 2001 May 15;11(10):735-46
references.pmid: 11378383
references.citation: Nat Cell Biol. 2007 Jul;9(7):847-53
references.pmid: 17558392
references.citation: Nature. 2006 Feb 2;439(7076):604-7
references.pmid: 16452979
references.citation: Science. 2003 Mar 28;299(5615):2039-45
references.pmid: 12663920
references.citation: Glycobiology. 2000 Dec;10(12):1317-23
references.pmid: 11159923
references.citation: J Cell Sci. 2007 Dec 15;120(Pt 24):4367-76
references.pmid: 18057029
references.citation: Glycobiology. 2009 Feb;19(2):102-11
references.pmid: 18824561
references.citation: J Cell Sci. 2002 Dec 1;115(Pt 23):4447-55
references.pmid: 12414991
references.citation: Cell Death Differ. 2008 Aug;15(8):1279-90
references.pmid: 18369370
references.citation: J Biol Chem. 2008 Dec 5;283(49):34076-86
references.pmid: 18835818
references.citation: J Cell Sci. 2009 Dec 15;122(Pt 24):4526-34
references.pmid: 19934220
references.citation: Nature. 2006 Nov 9;444(7116):230-4
references.pmid: 17086199
references.citation: J Cell Biol. 2009 Dec 14;187(6):847-58
references.pmid: 19995935
references.citation: J Cell Biol. 1998 Jun 15;141(6):1311-22
references.pmid: 9628888
references.citation: Eukaryot Cell. 2004 Oct;3(5):1164-8
references.pmid: 15470244
references.citation: Semin Cell Dev Biol. 2009 Sep;20(7):810-6
references.pmid: 19508856