Glycosyltransferase POMGNT1 deficiency strengthens N-cadherin-mediated cell–cell adhesion
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100433
ISSN1083-351X
AutoresSina Ibne Noor, Marcus Hoffmann, Natalie Rinis, Markus F. Bartels, Patrick R. Winterhalter, Christina Hoelscher, René Hennig, Nastassja Himmelreich, Christian Thiel, Thomas Ruppert, Erdmann Rapp, Sabine Strahl,
Tópico(s)Galectins and Cancer Biology
ResumoDefects in protein O-mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on α-dystroglycan, which reduces cell adhesion to the extracellular matrix. Mutations in protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, have mainly been associated with muscle–eye–brain (MEB) disease. In addition to defects in cell–extracellular matrix adhesion, aberrant cell–cell adhesion has occasionally been observed in response to defects in POMGNT1. However, specific molecular consequences of POMGNT1 deficiency on cell–cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined with proteomics, glycoproteomics, and glycomics revealed that a lack of POMGNT1 activity strengthens cell–cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with the epithelial–mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell–cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O-mannosylation on N-glycosylation has been underestimated. Defects in protein O-mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on α-dystroglycan, which reduces cell adhesion to the extracellular matrix. Mutations in protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, have mainly been associated with muscle–eye–brain (MEB) disease. In addition to defects in cell–extracellular matrix adhesion, aberrant cell–cell adhesion has occasionally been observed in response to defects in POMGNT1. However, specific molecular consequences of POMGNT1 deficiency on cell–cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined with proteomics, glycoproteomics, and glycomics revealed that a lack of POMGNT1 activity strengthens cell–cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with the epithelial–mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell–cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O-mannosylation on N-glycosylation has been underestimated. The modification of proteins by glycosylation is a ubiquitous feature of all living organisms (1Corfield A.P. Berry M. Glycan variation and evolution in the eukaryotes.Trends Biochem. Sci. 2015; 40: 351-359Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Protein-linked glycans are involved in a multitude of cellular processes ranging from monitoring the folding state of glycoproteins to cell adhesion and migration (2Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: Diversity, synthesis and function.Nat. Rev. Mol. Cell Biol. 2012; 13: 448-462Crossref PubMed Scopus (1046) Google Scholar). Among the different types of glycosylation, N-glycosylation and O-mannosylation are evolutionary conserved from bacteria to mammals. In humans, changes in those essential protein modifications inter alia can modulate immune responses, promote cancer cell metastasis, and underlie the pathophysiology of severe congenital disorders (3Pereira M.S. Alves I. Vicente M. Campar A. Silva M.C. Padrão N.A. Pinto V. Fernandes Â. Dias A.M. Pinho S.S. Glycans as key checkpoints of T cell activity and function.Front. Immunol. 2018; 9: 2754Crossref PubMed Scopus (56) Google Scholar, 4Pinho S.S. Reis C.A. Glycosylation in cancer: Mechanisms and clinical implications.Nat. Rev. Cancer. 2015; 15: 540-555Crossref PubMed Scopus (1432) Google Scholar, 5Theodore M. Morava E. Congenital disorders of glycosylation: Sweet news.Curr. Opin. Pediatr. 2011; 23: 581-587Crossref PubMed Scopus (35) Google Scholar). Both modifications initiate at the endoplasmic reticulum (ER), where the target polypeptides and the donor saccharides are synthesized and eventually covalently linked (2Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: Diversity, synthesis and function.Nat. Rev. Mol. Cell Biol. 2012; 13: 448-462Crossref PubMed Scopus (1046) Google Scholar). Only properly glycosylated and folded proteins can leave the ER and travel through the Golgi apparatus to reach their final cellular destinations. On their way, N-linked and O-mannosyl glycans can be further modified, which leads to diverse species- or even cell-type-specific glycans (2Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: Diversity, synthesis and function.Nat. Rev. Mol. Cell Biol. 2012; 13: 448-462Crossref PubMed Scopus (1046) Google Scholar). In the case of N-glycosylation, the dolichol-pyrophosphate-linked oligosaccharide Glc3Man9GlcNAc2 is assembled at the ER membrane and the glycan moiety is transferred en bloc to Asn residues of the consensus sequon Asn-X-Ser/Thr/Cys (X: proline is excluded). This way, the vast majority of proteins that enter the secretory pathway are N-glycosylated including many cell surface receptors and cell adhesion molecules (6Zielinska D.F. Gnad F. Wisniewski J. Mann M. Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints.Cell. 2010; 141: 897-907Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar). Protein-linked carbohydrate moieties are then further processed and finally extended in the Golgi through the concerted action of diverse specific glycosyltransferases resulting in three distinct types of N-glycans: high-mannose, complex-, and hybrid-type, which contain the common core Man3GlcNAc2-Asn (2Moremen K.W. Tiemeyer M. Nairn A.V. Vertebrate protein glycosylation: Diversity, synthesis and function.Nat. Rev. Mol. Cell Biol. 2012; 13: 448-462Crossref PubMed Scopus (1046) Google Scholar). The diverse glycan structures and glycosylation patterns on cell surface molecules are highly dynamic and can be differentially regulated both during development and in certain pathological conditions, often associated with the acquisition of altered cellular properties (4Pinho S.S. Reis C.A. Glycosylation in cancer: Mechanisms and clinical implications.Nat. Rev. Cancer. 2015; 15: 540-555Crossref PubMed Scopus (1432) Google Scholar). Classically, O-mannosylation is initiated by the conserved PMT family of protein O-mannosyltransferases (POMT1 and POMT2 in mammals), which catalyze the transfer of mannose from dolichol-phosphate-linked mannose to Ser and Thr residues of nascent proteins (7Neubert P. Strahl S. Protein O-mannosylation in the early secretory pathway.Curr. Opin. Cell Biol. 2016; 41: 100-108Crossref PubMed Scopus (32) Google Scholar). Three different core structures can be built on the protein-linked mannose (8Endo T. Mammalian O-mannosyl glycans: Biochemistry and glycopathology.Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 2019; 95: 39-51Crossref PubMed Scopus (24) Google Scholar). Linear core m1 and branched core m2 glycans, which share the common inner core GlcNAc-β1,2-Man-Ser/Thr, are initiated in the cis Golgi by the addition of a GlcNAc residue by the protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), and are further extended while proteins travel through the Golgi to the cell surface. In contrast, core m3 glycans are already elongated in the ER (GalNAc-β1,3-GlcNAc-β1,4-(phosphate-6)-Man-Ser/Thr) and then further modified in the Golgi by the sequential action of numerous glycosyltransferases including the ribulose-5-phosphate transferase fukutin (FKTN). The resulting complex polysaccharide structure, known as “matriglycan,” is so far only found on α-dystroglycan (α-DG), a central member of the dystrophin glycoprotein complex family in peripheral membranes and enables its interaction with extracellular matrix (ECM) components such as laminin (9Yoshida-Moriguchi T. Campbell K.P. Matriglycan: A novel polysaccharide that links dystroglycan to the basement membrane.Glycobiology. 2015; 25: 702-713Crossref PubMed Scopus (119) Google Scholar). Defects in this complex biosynthetic pathway lead to the loss of the matriglycan on α-DG and consequently impair interactions between α-DG and, e.g., laminin, which interferes with the formation of basement membranes (9Yoshida-Moriguchi T. Campbell K.P. Matriglycan: A novel polysaccharide that links dystroglycan to the basement membrane.Glycobiology. 2015; 25: 702-713Crossref PubMed Scopus (119) Google Scholar). This defect has been recognized as a major patho-mechanism of severe congenital muscular dystrophies with neuronal migration defects, known as α-dystroglycanopathy (OMIM 236670; 253280; 253800; 606612; 607155; 608840) (10Endo T. Glycobiology of α-dystroglycan and muscular dystrophy.J. Biochem. 2014; 157: 1-12Crossref PubMed Scopus (95) Google Scholar). The glycosyltransferase POMGNT1 has a key role in the elongation of O-mannosyl glycans (11Yoshida A. Kobayashi K. Manya H. Taniguchi K. Kano H. Mizuno M. Inazu T. Mitsuhashi H. Takahashi S. Takeuchi M. Herrmann R. Straub V. Talim B. Voit T. Topaloglu H. et al.Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase.Pomgnt1. Dev. Cell. 2001; 1: 717-724Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). In its absence, not only core m1 and m2 structures are missing, also formation of the matriglycan fails, since POMGNT1 recruits FKTN to maturing core m3 structures (12Xiong H. Kobayashi K. Tachikawa M. Manya H. Takeda S. Chiyonobu T. Fujikake N. Wang F. Nishimoto A. Morris G.E. Nagai Y. Kanagawa M. Endo T. Toda T. Molecular interaction between fukutin and POMGnT1 in the glycosylation pathway of α-dystroglycan.Biochem. Biophys. Res. Commun. 2006; 350: 935-941Crossref PubMed Scopus (65) Google Scholar, 13Kuwabara N. Manya H. Yamada T. Tateno H. Kanagawa M. Kobayashi K. Akasaka-Manya K. Hirose Y. Mizuno M. Ikeguchi M. Toda T. Hirabayashi J. Senda T. Endo T. Kato R. Carbohydrate-binding domain of the POMGnT1 stem region modulates O-mannosylation sites of α-dystroglycan.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 9280-9285Crossref PubMed Scopus (45) Google Scholar). The great majority of mutations in POMGNT1 have been linked to muscle–eye–brain disease (MEB; OMIM 253280), a congenital muscular dystrophy in humans, which is characterized by additional brain malformations and structural anomalies in the eye (11Yoshida A. Kobayashi K. Manya H. Taniguchi K. Kano H. Mizuno M. Inazu T. Mitsuhashi H. Takahashi S. Takeuchi M. Herrmann R. Straub V. Talim B. Voit T. Topaloglu H. et al.Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase.Pomgnt1. Dev. Cell. 2001; 1: 717-724Abstract Full Text Full Text PDF PubMed Scopus (632) Google Scholar). In the murine model, knockout of POMGNT1 is viable with multiple developmental defects, similar to the clinical picture of human MEB patients (14Liu J. Ball S.L. Yang Y. Mei P. Zhang L. Shi H. Kaminski H.J. Lemmon V.P. Hu H. A genetic model for muscle-eye-brain disease in mice lacking protein O-mannose 1,2-N-acetylglucosaminyltransferase (POMGnT1).Mech. Dev. 2006; 123: 228-240Crossref PubMed Scopus (106) Google Scholar, 15Hu H. Yang Y. Eade A. Xiong Y. Qi Y. Breaches of the pial basement membrane and disappearance of the glia limitans during development underlie the cortical lamination defect in the mouse model of muscle-eye-brain disease.J. Comp. Neurol. 2007; 501: 168-183Crossref PubMed Scopus (63) Google Scholar). The pathology of MEB suggests a functional role for POMGNT1 in control of cell adhesion and migration. For example, in the transgenic POMGNT1-based MEB mouse model, impaired cell–ECM adhesion results in disruption of basement membranes and overmigration of neurons during development of the cerebral cortex (15Hu H. Yang Y. Eade A. Xiong Y. Qi Y. Breaches of the pial basement membrane and disappearance of the glia limitans during development underlie the cortical lamination defect in the mouse model of muscle-eye-brain disease.J. Comp. Neurol. 2007; 501: 168-183Crossref PubMed Scopus (63) Google Scholar). However, also clusters of granule cells, which failed to migrate, have been frequently observed (15Hu H. Yang Y. Eade A. Xiong Y. Qi Y. Breaches of the pial basement membrane and disappearance of the glia limitans during development underlie the cortical lamination defect in the mouse model of muscle-eye-brain disease.J. Comp. Neurol. 2007; 501: 168-183Crossref PubMed Scopus (63) Google Scholar). In addition to its important role during mammalian development, POMGNT1 has recently been linked to the progression of glioblastoma, fatal primary brain tumors with survival time of 12–15 months, as well as the resistance of glioblastoma cells to the chemotherapeutic agent temozolomide (16Lan J. Guo P. Lin Y. Mao Q. Guo L. Ge J. Li X. Jiang J. Lin X. Qiu Y. Role of glycosyltransferase PomGnT1 in glioblastoma progression.Neuro Oncol. 2015; 17: 211-222Crossref PubMed Scopus (13) Google Scholar, 17Liu Q. Xue Y. Chen Q. Chen H. Zhang X. Wang L. Han C. Que S. Lou M. Lan J. PomGnT1 enhances temozolomide resistance by activating epithelial-mesenchymal transition signaling in glioblastoma.Oncol. Rep. 2017; 38: 2911-2918Crossref PubMed Scopus (8) Google Scholar). Strikingly, in glioblastoma models, increased cell–cell adhesion has been observed when POMGNT1 is missing (16Lan J. Guo P. Lin Y. Mao Q. Guo L. Ge J. Li X. Jiang J. Lin X. Qiu Y. Role of glycosyltransferase PomGnT1 in glioblastoma progression.Neuro Oncol. 2015; 17: 211-222Crossref PubMed Scopus (13) Google Scholar). However, molecular reasons for the different consequences of POMGNT1 deficiency are just emerging. Very recently, glyco-engineered human embryonic kidney (HEK) 293 cells turned out to be especially useful for the characterization of known, as well as the identification of new glycosylation pathways (18Larsen I.S.B. Narimatsu Y. Joshi H.J. Siukstaite L. Harrison O.J. Brasch J. Goodman K.M. Hansen L. Shapiro L. Honig B. Vakhrushev S.Y. Clausen H. Halim A. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 11163-11168Crossref PubMed Scopus (52) Google Scholar, 19Narimatsu Y. Joshi H.J. Schjoldager K.T. Hintze J. Halim A. Steentoft C. Nason R. Mandel U. Bennett E.P. Clausen H. Vakhrushev S.Y. Exploring regulation of protein O-glycosylation in isogenic human HEK293 cells by differential O-glycoproteomics.Mol. Cell Proteomics. 2019; 18: 1396-1409Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In the present work, we took advantage of a gene-targeted POMGNT1 knockout in HEK293T cells to study the consequences of POMGNT1 deficiency. The combination of glyco(proteo)mics with classic biochemistry, molecular and cell biology resulted in the discovery that cell–cell adhesion mediated by neuronal cadherin (N-Cdh) is affected and defined a possible molecular mechanism underlying the observed phenotype. Similar effects in MEB patient-derived fibroblasts confirmed the validity of the HEK293T model to study molecular effects of O-mannosylation deficiencies. To gain insight into functional implications of POMGNT1 deficiency, we generated a gene-targeted knockout in HEK293T cells (ΔPOMGnT1) as detailed in Experimental procedures and Figure S1. The loss of POMGNT1 activity was confirmed by the O-mannosylation status of the endogenous substrate α-DG using the matriglycan-directed antibody IIH6. Whereas the O-linked matriglycan is absent in POMGNT1-depleted cells, reintroduction of human POMGNT1 rescued O-mannosylation of α-DG verifying the specificity of the system (Fig. 1A). General characterization of the morphology of POMGNT1 knockout cells by confocal microscopy revealed that POMGNT1-deficient cells appear more rounded and stronger aggregated compared with wild-type (WT) cells, which show extensive spreading and even distribution. This phenotype is also reverted upon reintroduction of POMGNT1 (Fig. 1B). To further characterize molecular events responsible for the morphological differences, we analyzed cell–matrix and cell–cell adhesion. As expected, POMGNT1-deficient cells adhere to laminin, a major ECM component and interactor of the α-DG matriglycan, to a significantly lower extent when compared with WT cells (Fig. 2A). Intriguingly, when confluent monolayers of WT cells were incubated with WT and knockout cells, respectively, cell–cell adhesion of ΔPOMGnT1 cells turned out to be significantly increased. The same result is observed using a monolayer of ΔPOMGnT1 cells (Fig. 2B). Since cell–matrix and cell–cell interactions are major opposing forces balancing cellular migration, we further took advantage of xCELLigence real-time cell analysis that allows live monitoring of cell proliferation and cell migration. ΔPOMGnT1 cells proliferate slower than WT cells with slopes of 0.07 and 0.09, respectively (Fig. S2, A and B). In agreement with increased cell–cell adhesion, the migration rate of ΔPOMGnT1 cells is reduced by a factor of 3 (Figs. 2C and S2D). Taken together, the POMGNT1 knockout HEK293T cell model revealed that cell–cell adhesion increases, whereas cell–matrix interactions and cell migration are negatively affected when O-mannosyl glycans are not further elongated. In order to identify determinants that underlie the observed phenotype in ΔPOMGnT1 cells, we performed label-free quantitative proteomics of whole-cell lysates from WT and ΔPOMGnT1 HEK293T cells. Five independent replicates were analyzed, and homoscedasticity and normal distribution were confirmed (Fig. S3, A and B, Tables S1–S3). Altogether, 86 out of 437 proteins with differential abundance in POMGNT1-deficient cells could be identified (Fig. 3A and S3D, Table S4). Interestingly, gene ontology term functional annotation of proteins with a significant regulation revealed enrichment for proteins under the molecular function term of "cadherin binding involved in cell-cell adhesion" (Figs. 3A and S3D, Table S4), pointing to an impact of POMGNT1 deficiency on cadherin-mediated cell–cell adhesion. In addition, the protein N-Cdh was found to be more abundant by a factor of ∼2.7 in ΔPOMGnT1 cells (Fig. 3A). This result was also confirmed by western blot (Fig. 3, B and C) and correlated well with increased mRNA levels of N-Cdh (Fig. 3D). The observed difference in the increase of N-Cdh abundance in proteomics (∼2.7-fold) and western blot analysis (∼1.4-fold) is most likely due to the different detection principles that could be influenced by changes in N-Cdh glycosylation (see below). To investigate the general validity of our findings, we took advantage of skin fibroblasts derived from an MEB patient who presented characteristic symptoms such as mental retardation and blindness due to variant c.535_751del (p.Asp179Argfs∗11) in the POMGNT1 gene (NM_017739.4). In accordance with our HEK293T model, protein and mRNA abundance of N-Cdh in MEB patient-derived fibroblasts showed increased values compared with fibroblasts from two healthy donors (Fig. 3, E–G). Cadherins are major players in the formation of cellular junctions (20Wheelock M.J. Johnson K.R. Cadherins as modulators of cellular phenotype.Annu. Rev. Cell Dev. Biol. 2003; 19: 207-235Crossref PubMed Scopus (523) Google Scholar). These membrane-anchored cell surface glycoproteins mediate cell–cell adhesion through homotypic interactions of their conserved extracellular domains. Thus, to determine whether elevated cell–cell adhesion observed in POMGNT1-deficient cells is directly linked to the increased abundance of N-Cdh, we performed cell–cell adhesion assays in presence of either N-Cdh blocking or IgG-directed antibodies. As shown in Figure 4, ΔPOMGnT1 cells show increased adhesion to WT and other ΔPOMGnT1 cells in the IgG-treated controls. This increase, however, is diminished upon incubation with an N-Cdh targeting antibody, demonstrating N-Cdh as a key molecular driver for increased cell–cell adhesion in the established ΔPOMGnT1 HEK293T cell model. N-Cdh is highly N-glycosylated and N-linked glycans affect its adhesive properties by modulating its homomeric interactions in cis and in trans (21Guo H.B. Johnson H. Randolph M. Pierce M. Regulation of homotypic cell-cell adhesion by branched N-glycosylation of N-cadherin extracellular EC2 and EC3 domains.J. Biol. Chem. 2009; 284: 34986-34997Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Langer M.D. Guo H. Shashikanth N. Pierce J.M. Leckband D.E. N-glycosylation alters cadherin-mediated intercellular binding kinetics.J. Cell Sci. 2012; 125: 2478-2485Crossref PubMed Scopus (51) Google Scholar). We therefore asked whether an altered N-glycosylation, as an indirect effect of POMGNT1 deficiency, could contribute to increased N-Cdh-mediated cell–cell adhesion. We performed comprehensive N-glycomics and N-glycoproteomics on N-Cdh. For that purpose, the extracellular domain (EC) of N-Cdh was recombinantly expressed and purified from WT and ΔPOMGnT1 cells (detailed in Experimental procedures; Fig. S4). N-glycomics analysis by multiplexed capillary gel electrophoresis with laser-induced fluorescence detection (xCGE-LIF) revealed that total abundance of fully galactosylated and sialylated (α2,6- and α2,3-NeuNAc) N-glycan structures is remarkably decreased on N-Cdh derived from ΔPOMGnT1 cells when compared with WT (Fig. 5A, normalized intensity of peaks 2, 4, 9, and 10; Fig. S5 and Table S5). In accordance, the abundance of nongalactosylated N-glycan structures is increased (Fig. 5A, normalized intensity of peaks 6 and 7). Moreover, fully galactosylated multiantennary N-glycans (Fig. 5A, normalized intensity of peak A3+F) are reduced on ΔPOMGnT1-derived N-Cdh, and a nongalactosylated complex-type N-glycan with a bisecting GlcNAc (GlcNAc(5)Man(3)+Fuc(1)) represents the dominating N-glycan structure. In order to gain even deeper insights, an exploratory site-specific approach was taken to comprehensively map glycosylation sites and the corresponding N-glycoforms of N-Cdh. Hydrophilic interaction liquid chromatography (HILIC)-enriched tryptic and proteinase K-generated N-Cdh N-glycopeptides were analyzed by nano-reverse-phase liquid chromatography coupled online to an electrospray ionization orbitrap tandem mass spectrometer (nano-RP-LC-ESI-OT MS/MS) (for details see Experimental procedures). All eight potential N-glycosylation sites (Asn190, 273, 325, 402, 572, 622, 651, 692) of N-Cdh were identified (Fig. 5B, indicated as red ribbons, and Table S6). With the exception of Asn651, which predominantly carries a high-mannose-type N-glycan, all sites feature complex-type N-glycans. Characterization of the N-glycan microheterogeneity based on intact N-glycopeptides revealed differences in relative abundance of the major N-glycoform at each site on N-Cdh derived from ΔPOMGnT1 compared with WT cells (Fig. 5C). All N-glycosylation sites (except for Asn651 and Asn692, which are both located at the stem region of the molecule) feature nongalactosylated complex-type N-glycans with a bisecting GlcNAc (GlcNAc(5)Man(3)±Fuc(1)) as the dominating composition on N-Cdh from ΔPOMGnT1 cells. Comparative and site-specific N-glycoproteomics of quantitative changes in the N-glycan microheterogeneity revealed a significant decrease in galactosylation (−27% on average) and sialylation (−16% on average) throughout the vast majority of N-glycosylation sites (only exception are Asn190 and Asn651) (Fig. 5C). This decrease in galactosylation and sialylation is associated with a significant increase of nongalactosylated N-glycan compositions that feature a bisecting GlcNAc (Fig. 5, B and C). The site-specific relative changes of N-glycan traits on N-Cdh are shown in detail in Supporting information SI1–SI3 (Figs S6–S13 and Tables S7–S17). Overall, N-glycosylation of N-Cdh in ΔPOMGnT1 cells exhibits a reduction in the degree of galactosylation and sialylation with nongalactosylated complex-type N-glycans with a bisecting GlcNAc dominating. To identify glycosyltransferases that could be responsible for the observed changes in the N-glycan profile of N-Cdh, we next measured the transcript levels of genes encoding for major glycosyltransferases of N-glycan processing using the nCounter technology (23Geiss G.K. Bumgarner R.E. Birditt B. Dahl T. Dowidar N. Dunaway D.L. Fell H.P. Ferree S. George R.D. Grogan T. James J.J. Maysuria M. Mitton J.D. Oliveri P. Osborn J.L. et al.Direct multiplexed measurement of gene expression with color-coded probe pairs.Nat. Biotechnol. 2008; 26: 317-325Crossref PubMed Scopus (1488) Google Scholar). Mild reduction in the mRNA levels of the Golgi GlcNAc-transferases MGAT1 (by ∼21%), MGAT2 (by ∼13%), and MGAT5 (by ∼21%) was observed in ΔPOMGnT1 cells. Furthermore, a decrease in transcript levels of β1,4-galactosyltransferase 1 B4GALT1 (by ∼22%) as well as sialyltransferases ST6GAL1 (by ∼64%) and ST3GAL3 (by ∼69%) was detected (Table S18). Basal transcription levels of sialyltransferases are very low and close to the detection limit of the applied nCounter technology (Table S18). Therefore, we aimed to validate our results by qRT-PCR analysis including other α2,3-sialyltransferases that could contribute to the modification of N-Cdh (24Qi F. Isaji T. Duan C. Yang J. Wang Y. Fukuda T. Gu J. ST3GAL3, ST3GAL4, and ST3GAL6 differ in their regulation of biological functions via the specificities for the α2,3-sialylation of target proteins.FASEB J. 2020; 34: 881-897Crossref PubMed Scopus (22) Google Scholar). As shown in Figure 5D, a significant reduction in B4GALT1 (by ∼31%) and ST6GAL1 (by ∼33%) mRNA was confirmed in ΔPOMGnT1 cells, explaining the limited occurrence of respective N-glycans on N-Cdh in ΔPOMGnT1 cells. The changes in the mRNA levels of α2,3-sialyltransferases were found to be less consistent, possibly due to the limiting amount of transcripts. For ST3GAL3 transcript levels, no significantly change could be detected, whereas ST3GAL4 transcripts were reduced (by ∼31%). The mRNA amount of ST3GAL6 was increased (by ∼50%). In view of the partial substrate specificities of α2,3-sialyltransferases, the latter might indicate even more complex compensatory reactions (24Qi F. Isaji T. Duan C. Yang J. Wang Y. Fukuda T. Gu J. ST3GAL3, ST3GAL4, and ST3GAL6 differ in their regulation of biological functions via the specificities for the α2,3-sialylation of target proteins.FASEB J. 2020; 34: 881-897Crossref PubMed Scopus (22) Google Scholar). In summary, our data demonstrate that in POMGNT1-deficient cells, the N-glycan profile of N-Cdh is changed and that the observed alterations correlate to a great extent with transcriptional changes of respective N-glycan modifying enzymes. In addition to N-glycans, N-Cdh carries nonelongated O-linked mannose residues (25Winterhalter P.R. Lommel M. Ruppert T. Strahl S. O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues.FEBS Lett. 2013; 587: 3715-3721Crossref PubMed Scopus (20) Google Scholar, 26Vester-Christensen M.B. Halim A. Joshi H.J. Steentoft C. Bennett E.P. Levery S.B. Vakhrushev S.Y. Clausen H. Mining the O-mannose glycoproteome reveals cadherins as major O-mannosylated glycoproteins.Proc. Natl. Acad. Sci. U. S. A. 2013; 110: 21018-21023Crossref PubMed Scopus (116) Google Scholar), which depend on a recently identified class of O-mannosyltransferases (tetratricopeptide repeats (TPR)-containing proteins; TMTC 1–3), rather than classic O-mannosylation (18Larsen I.S.B. Narimatsu Y. Joshi H.J. Siukstaite L. Harrison O.J. Brasch J. Goodman K.M. Hansen L. Shapiro L. Honig B. Vakhrushev S.Y. Clausen H. Halim A. Discovery of an O-mannosylation pathway selectively serving cadherins and protocadherins.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 11163-11168Crossref PubMed Scopus (52) Google Scholar). The O-mannose glycoproteome of N-Cdh revealed 15 O-mannose glycosylation sites—four of which have not been reported before (Table S6 and Supporting information SI4: Figs S14 and S15). However, no difference in the O-mannosylation pattern of N-Cdh between ΔPOMGnT1 and WT HEK293T cells was detected, excluding indirect effects on the activity of TMTCs. Hypo-N-glycosylation of N-Cdh increases the prevalence of cis N-Cdh dimers on the cell membrane, thereby stabilization of cell–cell contacts (21Guo H.B. Johnson H. Randolph M. Pierce M. Regulation of homotypic cell
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