Glycosyltransferase POMGNT1 deficiency strengthens N-cadherin-mediated cell–cell adhesion

Defects in protein O-mannosylation lead to severe congenital muscular dystrophies collectively known as ?-dystroglycanopathy. A hallmark of these diseases is the loss O-mannose-bound matriglycan on ?-dystroglycan, which reduces cell adhesion extracellular matrix. Mutations O-mannose ?1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), crucial for elongation O-mannosyl glycans, have mainly been associated with muscle–eye–brain (MEB) disease. In addition defects cell–extracellular matrix adhesion, aberrant cell–cell has occasionally observed response POMGNT1. However, specific molecular consequences POMGNT1 deficiency are largely unknown. We used knockout HEK293T cells and fibroblasts from an MEB patient gain deeper insight into changes deficiency. Biochemical biological techniques combined proteomics, glycoproteomics, glycomics revealed that a lack activity strengthens adhesion. demonstrate altered intrinsic properties due increased abundance N-cadherin (N-Cdh). addition, site-specific N-glycan structures domain N-Cdh were detected, positively impact homotypic interactions. Moreover, POMGNT1-deficient cells, ERK1/2 p38 signaling pathways activated transcriptional comparable epithelial–mesenchymal transition (EMT) triggered, defining possible mechanism underlying phenotype. Our study indicates cadherin-mediated other EMT-related processes may contribute complex clinical symptoms or ?-dystroglycanopathy general suggests N-glycosylation underestimated. The modification proteins by glycosylation ubiquitous feature all living organisms (1Corfield A.P. Berry M. Glycan variation evolution eukaryotes.Trends Biochem. Sci. 2015; 40: 351-359Abstract Full Text PDF PubMed Scopus (99) Google Scholar). Protein-linked glycans involved multitude cellular ranging monitoring folding state glycoproteins migration (2Moremen K.W. Tiemeyer Nairn A.V. Vertebrate glycosylation: Diversity, synthesis function.Nat. Rev. Mol. Cell Biol. 2012; 13: 448-462Crossref (1046) Among different types glycosylation, evolutionary conserved bacteria mammals. humans, those essential modifications inter alia can modulate immune responses, promote cancer metastasis, underlie pathophysiology disorders (3Pereira M.S. Alves I. Vicente Campar A. Silva M.C. Padrão N.A. Pinto V. Fernandes Â. Dias A.M. Pinho S.S. Glycans key checkpoints T function.Front. Immunol. 2018; 9: 2754Crossref (56) Scholar, 4Pinho Reis C.A. Glycosylation cancer: Mechanisms implications.Nat. Cancer. 15: 540-555Crossref (1432) 5Theodore Morava E. Congenital Sweet news.Curr. Opin. Pediatr. 2011; 23: 581-587Crossref (35) Both initiate at endoplasmic reticulum (ER), where target polypeptides donor saccharides synthesized eventually covalently linked Only properly glycosylated folded leave ER travel through Golgi apparatus reach their final destinations. On way, N-linked be further modified, leads diverse species- even cell-type-specific case N-glycosylation, dolichol-pyrophosphate-linked oligosaccharide Glc3Man9GlcNAc2 assembled membrane glycan moiety transferred en bloc Asn residues consensus sequon Asn-X-Ser/Thr/Cys (X: proline excluded). This vast majority enter secretory pathway N-glycosylated including many surface receptors molecules (6Zielinska D.F. Gnad F. Wisniewski J. Mann Precision mapping vivo N-glycoproteome reveals rigid topological sequence constraints.Cell. 2010; 141: 897-907Abstract (687) carbohydrate moieties then processed finally extended concerted action glycosyltransferases resulting three distinct N-glycans: high-mannose, complex-, hybrid-type, contain common core Man3GlcNAc2-Asn patterns highly dynamic differentially regulated both during development certain pathological conditions, often acquisition (4Pinho Classically, initiated PMT family O-mannosyltransferases (POMT1 POMT2 mammals), catalyze transfer mannose dolichol-phosphate-linked Ser Thr nascent (7Neubert P. Strahl S. Protein early pathway.Curr. 2016; 41: 100-108Crossref (32) Three built protein-linked (8Endo T. Mammalian glycans: Biochemistry glycopathology.Proc. Jpn. Acad. Ser. B Phys. 2019; 95: 39-51Crossref (24) Linear m1 branched m2 share inner GlcNAc-?1,2-Man-Ser/Thr, cis GlcNAc residue while surface. contrast, m3 already elongated (GalNAc-?1,3-GlcNAc-?1,4-(phosphate-6)-Man-Ser/Thr) modified sequential numerous ribulose-5-phosphate transferase fukutin (FKTN). polysaccharide structure, “matriglycan,” so far only found ?-dystroglycan (?-DG), central member dystrophin glycoprotein peripheral membranes enables its interaction (ECM) components such laminin (9Yoshida-Moriguchi Campbell K.P. Matriglycan: novel links dystroglycan basement membrane.Glycobiology. 25: 702-713Crossref (119) this biosynthetic ?-DG consequently impair interactions between and, e.g., laminin, interferes formation defect recognized major patho-mechanism neuronal defects, (OMIM 236670; 253280; 253800; 606612; 607155; 608840) (10Endo Glycobiology dystrophy.J. 2014; 157: 1-12Crossref (95) glycosyltransferase role (11Yoshida Kobayashi K. Manya H. Taniguchi Kano Mizuno Inazu Mitsuhashi Takahashi Takeuchi Herrmann R. Straub Talim B. Voit Topaloglu et al.Muscular dystrophy disorder caused mutations glycosyltransferase.Pomgnt1. Dev. Cell. 2001; 1: 717-724Abstract (632) absence, not missing, also fails, since recruits FKTN maturing (12Xiong Tachikawa Takeda Chiyonobu Fujikake N. Wang Nishimoto Morris G.E. Nagai Y. Kanagawa Endo Toda Molecular POMGnT1 ?-dystroglycan.Biochem. Biophys. Res. Commun. 2006; 350: 935-941Crossref (65) 13Kuwabara Yamada Tateno Akasaka-Manya Hirose Ikeguchi Hirabayashi Senda Kato Carbohydrate-binding stem region modulates sites ?-dystroglycan.Proc. Natl. U. 113: 9280-9285Crossref (45) great disease (MEB; OMIM 253280), characterized additional brain malformations structural anomalies eye murine model, viable multiple developmental similar picture human patients (14Liu Ball S.L. Yang Mei Zhang L. Shi Kaminski H.J. Lemmon V.P. Hu genetic model muscle-eye-brain mice lacking 1,2-N-acetylglucosaminyltransferase (POMGnT1).Mech. 123: 228-240Crossref (106) 15Hu Eade Xiong Qi Breaches pial disappearance glia limitans cortical lamination mouse disease.J. Comp. Neurol. 2007; 501: 168-183Crossref (63) pathology functional control migration. For example, transgenic POMGNT1-based impaired cell–ECM results disruption overmigration neurons cerebral cortex (15Hu clusters granule failed migrate, frequently important mammalian development, recently progression glioblastoma, fatal primary tumors survival time 12–15 months, well resistance glioblastoma chemotherapeutic agent temozolomide (16Lan Guo Lin Mao Q. Ge Li X. Jiang Qiu Role PomGnT1 progression.Neuro Oncol. 17: 211-222Crossref (13) 17Liu Xue Chen Han C. Que Lou Lan enhances activating epithelial-mesenchymal glioblastoma.Oncol. Rep. 2017; 38: 2911-2918Crossref (8) Strikingly, models, when missing reasons just emerging. Very recently, glyco-engineered embryonic kidney (HEK) 293 turned out especially useful characterization known, identification new (18Larsen I.S.B. Narimatsu Joshi Siukstaite Harrison O.J. Brasch Goodman K.M. Hansen Shapiro Honig Vakhrushev S.Y. Clausen Halim Discovery selectively serving cadherins protocadherins.Proc. 114: 11163-11168Crossref (52) 19Narimatsu Schjoldager K.T. Hintze Steentoft Nason Mandel Bennett E.P. Exploring regulation O-glycosylation isogenic HEK293 differential O-glycoproteomics.Mol. Proteomics. 18: 1396-1409Abstract (29) present work, we took advantage gene-targeted combination glyco(proteo)mics classic biochemistry, biology resulted discovery mediated cadherin (N-Cdh) affected defined Similar effects patient-derived confirmed validity deficiencies. To implications deficiency, generated (?POMGnT1) detailed Experimental procedures Figure S1. was status endogenous substrate using matriglycan-directed antibody IIH6. Whereas O-linked absent POMGNT1-depleted reintroduction rescued verifying specificity system (Fig. 1A). General morphology confocal microscopy appear more rounded stronger aggregated compared wild-type (WT) show extensive spreading distribution. phenotype reverted upon 1B). characterize events responsible morphological differences, analyzed cell–matrix As expected, adhere ECM component interactor matriglycan, significantly lower extent WT 2A). Intriguingly, confluent monolayers incubated respectively, ?POMGnT1 increased. same result monolayer 2B). Since opposing forces balancing migration, xCELLigence real-time analysis allows live proliferation proliferate slower than slopes 0.07 0.09, respectively S2, B). agreement rate reduced factor 3 (Figs. 2C S2D). Taken together, increases, whereas negatively elongated. order identify determinants performed label-free quantitative proteomics whole-cell lysates cells. Five independent replicates analyzed, homoscedasticity normal distribution S3, B, Tables S1–S3). Altogether, 86 437 could identified 3A S3D, Table S4). Interestingly, gene ontology term annotation significant enrichment under function "cadherin binding cell-cell adhesion" S4), pointing abundant ?2.7 3A). western blot 3, C) correlated mRNA levels 3D). difference increase (?2.7-fold) (?1.4-fold) most likely detection principles influenced (see below). investigate our findings, skin derived who presented characteristic mental retardation blindness variant c.535_751del (p.Asp179Argfs?11) (NM_017739.4). accordance showed values two healthy donors E–G). Cadherins players junctions (20Wheelock M.J. Johnson K.R. modulators phenotype.Annu. 2003; 19: 207-235Crossref (523) These membrane-anchored mediate domains. Thus, determine whether elevated directly N-Cdh, assays presence either blocking IgG-directed antibodies. shown 4, IgG-treated controls. increase, however, diminished incubation targeting antibody, demonstrating driver established model. affect adhesive modulating homomeric trans (21Guo H.B. Randolph Pierce Regulation EC2 EC3 domains.J. Chem. 2009; 284: 34986-34997Abstract (66) 22Langer M.D. Shashikanth J.M. Leckband D.E. alters intercellular kinetics.J. 125: 2478-2485Crossref (51) therefore asked indirect effect N-Cdh-mediated comprehensive N-glycomics N-glycoproteomics N-Cdh. purpose, (EC) recombinantly expressed purified (detailed procedures; Fig. multiplexed capillary gel electrophoresis laser-induced fluorescence (xCGE-LIF) total fully galactosylated sialylated (?2,6- ?2,3-NeuNAc) remarkably decreased 5A, normalized intensity peaks 2, 9, 10; S5 S5). accordance, nongalactosylated 6 7). multiantennary N-glycans peak A3+F) ?POMGnT1-derived complex-type bisecting (GlcNAc(5)Man(3)+Fuc(1)) represents dominating structure. insights, exploratory approach taken comprehensively map corresponding N-glycoforms Hydrophilic liquid chromatography (HILIC)-enriched tryptic proteinase K-generated N-glycopeptides nano-reverse-phase coupled online electrospray ionization orbitrap tandem mass spectrometer (nano-RP-LC-ESI-OT MS/MS) (for details see procedures). All eight potential (Asn190, 273, 325, 402, 572, 622, 651, 692) 5B, indicated red ribbons, S6). With exception Asn651, predominantly carries high-mannose-type N-glycan, N-glycans. Characterization microheterogeneity based intact differences relative N-glycoform each site 5C). (except Asn651 Asn692, located molecule) (GlcNAc(5)Man(3)±Fuc(1)) composition Comparative decrease galactosylation (?27% average) sialylation (?16% throughout (only Asn190 Asn651) compositions 5, C). traits detail Supporting information SI1–SI3 (Figs S6–S13 S7–S17). Overall, exhibits reduction degree dominating. profile next measured transcript genes encoding processing nCounter technology (23Geiss G.K. Bumgarner R.E. Birditt Dahl Dowidar Dunaway D.L. Fell H.P. Ferree George R.D. Grogan James J.J. Maysuria Mitton J.D. Oliveri Osborn J.L. al.Direct measurement expression color-coded probe pairs.Nat. Biotechnol. 2008; 26: 317-325Crossref (1488) Mild GlcNAc-transferases MGAT1 (by ?21%), MGAT2 ?13%), MGAT5 ?21%) Furthermore, ?1,4-galactosyltransferase B4GALT1 ?22%) sialyltransferases ST6GAL1 ?64%) ST3GAL3 ?69%) detected (Table S18). Basal transcription very low close limit applied Therefore, aimed validate qRT-PCR ?2,3-sialyltransferases (24Qi Isaji Duan Fukuda Gu ST3GAL3, ST3GAL4, ST3GAL6 differ functions via specificities ?2,3-sialylation proteins.FASEB 2020; 34: 881-897Crossref (22) 5D, ?31%) ?33%) explaining limited occurrence respective less consistent, possibly limiting amount transcripts. levels, no change ST3GAL4 transcripts ?31%). ?50%). view partial ?2,3-sialyltransferases, latter might indicate compensatory reactions summary, data changed alterations correlate modifying enzymes. N-glycans, nonelongated (25Winterhalter P.R. Lommel Ruppert non-canonical T-cadherin rabbit skeletal muscle single residues.FEBS Lett. 2013; 587: 3715-3721Crossref (20) 26Vester-Christensen M.B. Levery S.B. Mining glycoproteome O-mannosylated glycoproteins.Proc. 110: 21018-21023Crossref (116) Scholar), depend class (tetratricopeptide repeats (TPR)-containing proteins; TMTC 1–3), rather 15 sites—four reported before S6 SI4: Figs S14 S15). pattern excluding TMTCs. Hypo-N-glycosylation increases prevalence dimers membrane, thereby stabilization contacts