N-Glycosylation Affects the Molecular Organization and Stability of E-cadherin Junctions
2006; Elsevier BV; Volume: 281; Issue: 32 Linguagem: Inglês
10.1074/jbc.m512621200
ISSN1083-351X
AutoresAneta Liwosz, Tianlei Lei, Maria A. Kukuruzinska,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoEpithelial cell-cell adhesion is mediated by E-cadherin, an intercellular N-glycoprotein adhesion receptor that functions in the assembly of multiprotein complexes anchored to the actin cytoskeleton named adherens junctions (AJs). E-cadherin ectodomains 4 and 5 contain three potential N-glycan addition sites, although their significance in AJ stability is unclear. Here we show that sparse cells lacking stable AJs produced E-cadherin that was extensively modified with complex N-glycans. In contrast, dense cultures with more stable AJs had scarcely N-glycosylated E-cadherin modified with high mannose/hybrid and limited complex N-glycans. This suggested that variations in AJ stability were accompanied by quantitative and qualitative changes in E-cadherin N-glycosylation. To further examine the role of N-glycans in AJ function, we generated E-cadherin N-glycosylation variants lacking selected N-glycan addition sites. Characterization of these variants in CHO cells, lacking endogenous E-cadherin, revealed that site 1 on ectodomain 4 was modified with a prominent complex N-glycan, site 2 on ectodomain 5 did not have a substantial oligosaccharide, and site 3 on ectodomain 5 was decorated with a high mannose/hybrid N-glycan. Removal of complex N-glycan from ectodomain 4 led to a dramatically increased interaction of E-cadherin-catenin complexes with vinculin and the actin cytoskeleton. The latter effect was further enhanced by the deletion of the high mannose/hybrid N-glycan from site 3. In MDCK cells, which produce E-cadherin, a variant lacking both complex and high mannose/hybrid N-glycans functioned like a dominant positive displaying increased interaction with γ-catenin and vinculin compared with the endogenous E-cadherin. Collectively, our studies show that N-glycans, and complex oligosaccharides in particular, destabilize AJs by affecting their molecular organization. Epithelial cell-cell adhesion is mediated by E-cadherin, an intercellular N-glycoprotein adhesion receptor that functions in the assembly of multiprotein complexes anchored to the actin cytoskeleton named adherens junctions (AJs). E-cadherin ectodomains 4 and 5 contain three potential N-glycan addition sites, although their significance in AJ stability is unclear. Here we show that sparse cells lacking stable AJs produced E-cadherin that was extensively modified with complex N-glycans. In contrast, dense cultures with more stable AJs had scarcely N-glycosylated E-cadherin modified with high mannose/hybrid and limited complex N-glycans. This suggested that variations in AJ stability were accompanied by quantitative and qualitative changes in E-cadherin N-glycosylation. To further examine the role of N-glycans in AJ function, we generated E-cadherin N-glycosylation variants lacking selected N-glycan addition sites. Characterization of these variants in CHO cells, lacking endogenous E-cadherin, revealed that site 1 on ectodomain 4 was modified with a prominent complex N-glycan, site 2 on ectodomain 5 did not have a substantial oligosaccharide, and site 3 on ectodomain 5 was decorated with a high mannose/hybrid N-glycan. Removal of complex N-glycan from ectodomain 4 led to a dramatically increased interaction of E-cadherin-catenin complexes with vinculin and the actin cytoskeleton. The latter effect was further enhanced by the deletion of the high mannose/hybrid N-glycan from site 3. In MDCK cells, which produce E-cadherin, a variant lacking both complex and high mannose/hybrid N-glycans functioned like a dominant positive displaying increased interaction with γ-catenin and vinculin compared with the endogenous E-cadherin. Collectively, our studies show that N-glycans, and complex oligosaccharides in particular, destabilize AJs by affecting their molecular organization. E-cadherin is an N-glycoprotein cell-cell adhesion receptor that plays pivotal roles in epithelial tissue formation, cell polarity, and differentiation (1Gumbiner B.M. J. Cell Biol. 1996; 84: 345-357Scopus (2924) Google Scholar, 2Takeichi M. Curr. Opin. Cell Biol. 1995; 5: 806-811Crossref Scopus (829) Google Scholar, 3Gumbiner B.M. Nat Rev Mol. Cell. Biol. 2005; 6: 622-634Crossref PubMed Scopus (1210) Google Scholar). E-cadherin mediates cell-cell adhesion through the assembly of multiprotein complexes linked to the actin cytoskeleton referred to as adherens junctions (AJs) 2The abbreviations used are: AJ, adherens junction; EC, extracellular domain; ER, endoplasmic reticulum; GPT, dolichol phosphate-dependent N-acetylglucosamine 1-phosphotransferase; PNGaseF, Peptide:N-glycosidase F; EndoH, endoglycosidase H; CHO, Chinese hamster ovary; MDCK, Madin-Darby canine kidney; DSA, Datura stramonium agglutinin; GNA, Galanthus nivalis agglutinin; SNA, Sambucus nigra agglutinin.2The abbreviations used are: AJ, adherens junction; EC, extracellular domain; ER, endoplasmic reticulum; GPT, dolichol phosphate-dependent N-acetylglucosamine 1-phosphotransferase; PNGaseF, Peptide:N-glycosidase F; EndoH, endoglycosidase H; CHO, Chinese hamster ovary; MDCK, Madin-Darby canine kidney; DSA, Datura stramonium agglutinin; GNA, Galanthus nivalis agglutinin; SNA, Sambucus nigra agglutinin. (4Jamora C. Fuchs E. Nat Cell Biol. 2002; 4: E101-E108Crossref PubMed Scopus (490) Google Scholar, 5Brieher W.M. Yap A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (264) Google Scholar, 6Wheelock M.J. Johnson K.R. Ann. Rev. Cell Dev. Biol. 2003; 19: 207-235Crossref PubMed Scopus (530) Google Scholar, 7Wheelock M.J. Johnson K.R. Curr. Opin. Cell Biol. 2003; 15: 509-514Crossref PubMed Scopus (233) Google Scholar, 8Gumbiner B.M. J. Cell Biol. 2000; 148: 399-404Crossref PubMed Scopus (688) Google Scholar). The AJs are formed by the binding of E-cadherin extracellular domains (ECs) on adjacent cells in a calcium-dependent manner (5Brieher W.M. Yap A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (264) Google Scholar), whereas the intracellular domains anchor the cadherin to the actin cytoskeleton via catenins. β-Catenin and γ-catenin (plakoglobin) bind directly to the distal region of the cadherin cytoplasmic tail in a mutually exclusive manner. They also recruit α-catenin, which links the cadherin, either directly or indirectly, to the actin cytoskeleton (4Jamora C. Fuchs E. Nat Cell Biol. 2002; 4: E101-E108Crossref PubMed Scopus (490) Google Scholar, 5Brieher W.M. Yap A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (264) Google Scholar, 6Wheelock M.J. Johnson K.R. Ann. Rev. Cell Dev. Biol. 2003; 19: 207-235Crossref PubMed Scopus (530) Google Scholar, 7Wheelock M.J. Johnson K.R. Curr. Opin. Cell Biol. 2003; 15: 509-514Crossref PubMed Scopus (233) Google Scholar). The linkage of AJs to the actin cytoskeleton can be further stabilized through the binding of cytoskeletal proteins such as vinculin. Although the binding between E-cadherin ECs does not require association with the actin filaments, the interaction via catenins and cytoskeletal proteins provides strength to AJs by holding together the clustered receptors at cell-cell contacts (8Gumbiner B.M. J. Cell Biol. 2000; 148: 399-404Crossref PubMed Scopus (688) Google Scholar). E-cadherin can be post-translationally modified by phosphorylation, O-glycosylation and N-glycosylation. Casein kinase II, a serine-threonine kinase, phosphorylates the cytosolic tail of E-cadherin and enhances binding to β-catenin (9Lickert H. Bauer A. Kemler R. Stappert J. J. Biol. Chem. 2000; 275: 5090-5095Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Cytoplasmic O-glycosylation of the E-cadherin cytosolic tail has been shown to occur in response to endoplasmic reticulum (ER) stress and inactivate E-cadherin-mediated intercellular adhesion by preventing its transport to the cell membrane (10Zhu W. Leber B. Andrews D.W. EMBO J. 2001; 20: 5999-6007Crossref PubMed Scopus (122) Google Scholar). In addition, E-cadherin can be N-glycosylated: the mouse E-cadherin has three N-glycan addition sites, one in EC4 and two in EC5, whereas human and canine E-cadherins have an additional site, each in different parts of the extracellular region. Even though N-glycans represent the most prominent modification, contributing up to 20% of the total mass of the E-cadherin molecule, virtually nothing is known about their role in the stability of AJs. N-Glycans modify proteins at asparagine residues within the consensus sequence NX(S/T), where X can be any amino acid with the exception of proline (11Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1978) Google Scholar, 12Rademacher T.W. Parekh R.B. Dwek R.A. Ann. Rev. Biochem. 1988; 57: 785-838Crossref PubMed Scopus (1194) Google Scholar, 13Kukuruzinska M.A. Lennon K. Crit. Rev. Oral Biol. Med. 1998; 9: 415-448Crossref PubMed Scopus (124) Google Scholar). However, not every potential N-glycan addition site on a given N-glycoprotein is modified, and frequently there are variations in the number of sites occupied by N-glycans, their overall sizes, and composition. This microheterogeneity reflects regulated changes in the activities of key enzymes that function in the synthesis and processing of N-glycans (11Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1978) Google Scholar, 12Rademacher T.W. Parekh R.B. Dwek R.A. Ann. Rev. Biochem. 1988; 57: 785-838Crossref PubMed Scopus (1194) Google Scholar, 14Mendelsohn R.D. Cippolo J.F. Helmerhorst E. Kukuruzinska M.A. Biochim. Biophys. Acta. 2005; 1723: 33-44Crossref PubMed Scopus (20) Google Scholar, 15Dennis J.W. Laferte S. Waghorne C. Breitman M.C. Kerbel R. Science. 1987; 236: 582-585Crossref PubMed Scopus (857) Google Scholar). For instance, N-glycan site occupancy has been shown to be regulated with growth and development by changes in the expression of GPT, the enzyme that initiates the synthesis of the lipid-linked oligosaccharide precursor in the ER (14Mendelsohn R.D. Cippolo J.F. Helmerhorst E. Kukuruzinska M.A. Biochim. Biophys. Acta. 2005; 1723: 33-44Crossref PubMed Scopus (20) Google Scholar). A high level of GPT expression results in abundant protein N-glycosylation during cell proliferation and tissue morphogenesis, and down-regulation of GPT leads to diminished N-glycosylation in growth-arrested cells and cytodifferentiated tissues (14Mendelsohn R.D. Cippolo J.F. Helmerhorst E. Kukuruzinska M.A. Biochim. Biophys. Acta. 2005; 1723: 33-44Crossref PubMed Scopus (20) Google Scholar, 16Fernandes R.P. Cotanche D.A. Lennon-Hopkins K. Erkan F. Menko A.S. Kukuruzinska M.A. Histochem. Cell Biol. 1999; 111: 153-162Crossref PubMed Scopus (17) Google Scholar). N-Glycans have a documented role in protein folding, and they have been shown to inhibit protein aggregation, increase protein solubility, and influence the binding of chaperones (11Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1978) Google Scholar, 12Rademacher T.W. Parekh R.B. Dwek R.A. Ann. Rev. Biochem. 1988; 57: 785-838Crossref PubMed Scopus (1194) Google Scholar). In addition, N-glycans function in protein secretion, turnover, and bioactivity, and increasing evidence points to their role in intracellular signaling events (17Klebl B. Kozian D. Leberer E. Kukuruzinska M.A. Biochem. Biophys. Res. Commun. 2001; 286: 575-581Crossref Scopus (8) Google Scholar, 18Lee B.N. Elion E.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 98: 12679-12684Crossref Scopus (129) Google Scholar, 19Cullen P.J. Schultz J. Horecka J. Stevenson B.J. Jigami Y. Sprague Jr., G.F. Genetics. 2000; 155: 1005-1018Crossref PubMed Google Scholar, 20Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 21Guo H.B. Lee I. Kamar M. Akiyama S.K. Pierce M. Cancer Res. 2002; 62: 6837-6845PubMed Google Scholar). Recent studies have also revealed that N-glycans are important players in cell adhesion. Aberrant N-glycosylation of skeletal muscle dystrophin has been shown to cause dysfunction of neuromuscular junctions (22Yoshida 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. Toda T. Endo T. Dev. Cell. 2001; 1: 717-724Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar). Increased β(1Gumbiner B.M. J. Cell Biol. 1996; 84: 345-357Scopus (2924) Google Scholar,6Wheelock M.J. Johnson K.R. Ann. Rev. Cell Dev. Biol. 2003; 19: 207-235Crossref PubMed Scopus (530) Google Scholar)glycan branching on β1 integrin subunit leads to diminished α5β1 integrin clustering and increased cell migration (21Guo H.B. Lee I. Kamar M. Akiyama S.K. Pierce M. Cancer Res. 2002; 62: 6837-6845PubMed Google Scholar). A similar increase of β(1Gumbiner B.M. J. Cell Biol. 1996; 84: 345-357Scopus (2924) Google Scholar,6Wheelock M.J. Johnson K.R. Ann. Rev. Cell Dev. Biol. 2003; 19: 207-235Crossref PubMed Scopus (530) Google Scholar)glycan branching on N-cadherin has been shown to lead to a marked decrease of cell-cell adhesion in human fibrosarcoma HT1080 and mouse NIH3T3 cells (20Guo H.B. Lee I. Kamar M. Pierce M. J. Biol. Chem. 2003; 278: 52412-52424Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). In the yeast Saccharomyces cerevisiae, attenuation of protein N-glycosylation causes increased cell aggregation (23Kukuruzinska M.A. Lennon K. Biochim. Biophys. Acta. 1995; 1247: 51-59Crossref PubMed Scopus (30) Google Scholar). We have been interested in examining how the presence of, and changes in N-glycan structures on E-cadherin ECs affected its adhesive function. Previously, inhibition of protein N-glycosylation with tunicamycin had been shown not to interfere with the formation of E-cadherin-mediated AJs, indicating that N-glycans themselves were not the adhesive structures (24Shirayoshi Y. Nose A. Iwasaki K. Takeichi M. Cell Struct. Funct. 1986; 11: 245-252Crossref PubMed Scopus (143) Google Scholar). Our earlier studies with the postnatally developing hamster and mouse submandibular glands, however, provided evidence that, during tissue morphogenesis, when GPT expression was high, E-cadherin was primarily in unstable cell-cell contacts, whereas during cytodifferentiation, which was accompanied by down-regulation of GPT expression, there was increased association of E-cadherin with stable AJs (16Fernandes R.P. Cotanche D.A. Lennon-Hopkins K. Erkan F. Menko A.S. Kukuruzinska M.A. Histochem. Cell Biol. 1999; 111: 153-162Crossref PubMed Scopus (17) Google Scholar, 25Menko A.S. Zhang L. Schiano F. Kreidberg J.A. Kukuruzinska M.A. Dev. Dyn. 2002; 224: 321-333Crossref PubMed Scopus (29) Google Scholar). These data suggested that N-glycans might function in modulating E-cadherin adhesive stability. Here, we provide evidence that reduced N-glycosylation of E-cadherin with complex N-glycans leads to its preferential association with the actin cytoskeleton. We further support this finding by showing that, in Chinese hamster ovary (CHO) cells, an E-cadherin N-glycosylation variant lacking the major complex N-glycan exhibited greater interaction with vinculin and increased association with the actin cytoskeleton. Likewise, in MDCK cells, a variant missing both complex and high mannose/hybrid N-glycans displayed an enhanced association with γ-catenin and vinculin compared with either the wild-type or endogenous E-cadherin. Collectively, our data show that N-glycans influence the stability of AJs by affecting their molecular organization. We propose that extensive modification of E-cadherin with complex N-glycans renders the formation of dynamic but weak AJs, whereas diminished N-glycosylation of E-cadherin promotes the establishment of stable AJs. Reagents—Monoclonal antibody to the cytoplasmic region of human E-cadherin, as well as monoclonal antibodies α-catenin, β-catenin, and γ-catenin, were obtained from BD Transduction Laboratories. Monoclonal antibody to the Myc tag (clone 9B11) was purchased from Cell Signaling, to vinculin (clone V284) from Upstate Biotechnology, to actin (pan Ab-5, clone ACTN05) from NeoMarkers, and to α-tubulin (clone B-5-1-2) from Sigma. Rhodamine-tagged phalloidin, used for visualization of F-actin, and 4′,6-diamidino-2-phenylindole, for visualization of nucleic acids, were obtained from Molecular Probes. Secondary antibodies for immunostaining included goat anti-mouse IgG(Fc), which was derivatized with fluorescein isothiocyanate (Molecular Probes). For Western blot analyses horseradish peroxidase-conjugated secondary antibodies were obtained from Amersham Biosciences. Lectin blot analyses were performed using the DIG glycan differentiation kit (Roche Applied Science). Vector Construction and in Vitro Mutagenesis—Human E-cadherin (GenBank™ accession no. Z13009) was cloned into pCMV5Bmyc vector (Stratagene). To produce the E-cadherin N-glycan site mutations singly and in different combinations, a PCR-based site-directed mutagenesis was carried out using a QuikChange XL site-directed mutagenesis kit (Stratagene). The site-directed mutagenesis primers for replacement the of asparagine for glutamine in the N-glycosylation sites (Asn-404 → Gln, Asn-468 → Gln, and Asn-483 → Gln) were created as follows: for E-cadherin Asn-404 → Gln variant: 5′-GAGCACGTGAAGCAGAGCACGTACACAGCCCT and 3′-TAGGGCTGTGTACGTGCTCTGCTTCACGTGCTC; for E-cadherin Asn-468 → Gln variant: 5′-GACCTTCCTCCCCAGACATCTCCCTTCACAGCA and 3′-TGCTGTGAAGGGAGATGTCTGGGGAGGAAGGTC; and for E-cadherin Asn-483 → Gln variant: 5′-GGGGCGAGTGCCCAGTGGACCATTCAGTACAC and 3′-GTGTACTGAATGGTCCACTGTTCACTCGCCCC. All mutations were verified by sequencing. Cell Culture, Transfection, and Preparation of Cell Lysates—CHO cells were grown in minimum essential medium α medium (Invitrogen) supplemented with 10% fetal calf serum, penicillin, and streptomycin. MDCK cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, penicillin, and streptomycin in 5% CO2 at 37 °C. CHO cells were transiently transfected with cDNA encoding E-cadherin and its N-glycosylation variants using PolyFect transfection reagent (Qiagen), whereas MDCK cells were transfected using Lipofectamine 2000 (Invitrogen). Transfectants were cultured for 48 h, after which they were collected and extracted with 600 μl of ice-cold Triton X-100/β-octylglucoside buffer (10 mm imidazole, 100 mm NaCl, 1 mm MgCl2, 5 mm Na2EDTA, 1% Triton X-100, 0.87 mg/ml β-octylglucoside) containing 50 μg/ml aprotinin, 25 μg/ml soybean trypsin inhibitor, 100 μm benzamidine, 5 μg/ml leupeptin, and 0.5 μm phenylmethylsulfonyl fluoride). For determination of cytoskeletal association of E-cadherin and its N-glycosylation variants, CHO cells were extracted with ice-cold Triton extraction buffer (10 mm imidazole, 100 mm NaCl, 1 mm MgCl2, 5 mm Na2EDTA, 1% Triton X-100, pH 7.4, containing 50 μg/ml aprotinin, 25 μg/ml soybean trypsin inhibitor, 100 μm benzamidine, 5 μg/ml leupeptin, and 0.5 μm phenylmethylsulfonyl fluoride). The Triton-soluble and -insoluble fractions were separated by centrifugation at 12,000 × g for 10 min. The resulting Tritoninsoluble pellet was re-extracted with β-octylglucosidase (0.87 mg/ml) in the above buffer in the absence of Triton. Protein concentrations were determined by using a BCA protein assay (Pierce). PNGaseF and EndoH Digestions—Total cell lysates were digested with 1 unit of PNGaseF and/or EndoH (New England Biolabs) for 1 h at 37°C, loaded onto a 7.5% SDS-PAGE, and examined by Western blotting. For controls, samples were incubated without the enzymes. Western Blotting—Total cell lysates were fractionated on either 4-12% or 7.5% SDS-PAGE and blotted onto nitrocellulose membranes (Invitrogen). For comparison of expression levels of the wild type E-cadherin and its N-glycosylation variants in CHO and MDCK cells, typically 10 μg of total cellular protein was used. For immunoblot analyses of Triton-soluble fractions, samples were loaded at equal amounts of protein (10 μg). Their respective insoluble fractions were loaded at volumes equal to the soluble fractions so that direct comparisons could be made. The samples were blocked in PBS-Tween (PBST) (20 mm Tris/137 mm NaCl/0.1% Tween 20, pH 7.6) with 10% milk, and membranes were incubated with primary antibodies at appropriate dilutions in PBST with 5% milk for 2 h at room temperature. Next, membranes were washed four times with PBST solution, followed by incubation with horseradish peroxidase-linked secondary antibody (1:3000) in PBST with 5% milk. The results were visualized with ECL Plus Detection Reagents (Amersham Biosciences). Immunoprecipitation—Equal amounts of total protein (200 μg) were precleared with 30 μl of protein G (Sigma) and incubated 2 h at 4°C. After centrifugation at 1200 rpm for 1 min, supernatant was incubated for 2 h at 4°C with 2.5 μg of antibodies against either E-cadherin, or β-catenin, or the myc tag and 30 μl of protein G. Next, the beads were washed three times with the lysis buffer (10 mm Tris HCl, pH 7.5, 1 mm EDTA, 1 mm EGTA, and 0.5% Triton X-100). Samples were resuspended in 100 μl of 2× SDS sample buffer and boiled 5 min at 95 °C prior to analyses by Western and lectin blotting. Lectin Blotting—Polyvinylidene difluoride membranes were incubated overnight in blocking reagents (Roche Applied Science) at 4 °C. Next the blots were washed twice with TBS (50 mm Tris, 150 mm NaCl, pH 7.5) and once with lectin buffer 1 (TBS, 1 mm MgCl2, 1 mm MnCl2, pH 7.5). Subsequently, the blots were incubated for 1 h with digoxigenin-labeled lectins Galanthus nivalis agglutinin (GNA), Sambucas nigra agglutinin (SNA), and Datura stramonium agglutinin (DSA) (1 μg/ml). Membranes were washed three times with TBS and incubated with anti-digoxigenin-AP conjugate (diluted 1:1,000 in TBS). Reactivities to specific lectins were detected using NBT/X phosphate in 0.1 m Tris/HCl, 0.05 m MgCl2, 0.1 m NaCl at pH 9.5. Immunofluorescence—For immunofluorescence analyses, CHO transfectants were grown in the presence of 0.8 μg/μl G418 (Invitrogen) for 2 weeks. MDCK transfectants were grown to 80% confluence. CHO and MDCK cells were fixed in 3.7% paraformaldehyde, permeabilized with 0.1% Triton X-100, and washed with PBS. The samples were blocked with goat serum, rinsed with PBS, and incubated with primary antibodies to either E-cadherin (for CHO cells) or the Myc tag (for MDCK cells) overnight at 4 °C. Cells were then incubated with fluorescein isothiocyanate-tagged secondary antibodies, rhodamine-tagged phalloidin, and 4′,6-diamidino-2-phenylindole. Next, cells were rinsed four times with PBS and mounted in Vectashield (Vector Laboratories, Inc.). The immunostained samples were analyzed with a Nikon Eclipse TE300 epifluorescence microscope. N-Glycosylation of E-cadherin Is Cell Density-dependent—Previous studies have shown that protein N-glycosylation is proliferation-dependent, being abundant in dividing cells and diminished in dense cultures (26Kukuruzinska M.A. Lennon K. Glycobiology. 1994; 4: 437-443Crossref PubMed Scopus (36) Google Scholar). Similarly, the stability of AJs in cultured endothelial cells was shown to depend on cell density: sparse endothelial cells formed unstable AJs, whereas contact-inhibited cells produced AJs stably associated with the actin cytoskeleton (27Lampugnani M.G. Corada M. Andriopoulou P. Esser S. Risau W. Dejana E. J. Cell Sci. 1997; 110: 2065-2077Crossref PubMed Google Scholar). To determine whether the formation of E-cadherin-containing AJs in epithelial cells was also dependent on cell density, we first examined the association of E-cadherin with the actin cytoskeletal membrane fraction in sparse and dense MDCK cultures using Triton solubility criteria (25Menko A.S. Zhang L. Schiano F. Kreidberg J.A. Kukuruzinska M.A. Dev. Dyn. 2002; 224: 321-333Crossref PubMed Scopus (29) Google Scholar, 27Lampugnani M.G. Corada M. Andriopoulou P. Esser S. Risau W. Dejana E. J. Cell Sci. 1997; 110: 2065-2077Crossref PubMed Google Scholar, 28Walker J.L. Menko A.S. Dev. Biol. 1999; 210: 497-511Crossref PubMed Scopus (76) Google Scholar). We used this approach because Triton insolubility of cadherins has been correlated with linkage to the actin cytoskeleton and association with lipid rafts (29Leong L. Menko A.S. Grunwald G.B. Invest. Ophthalmol. Vis. Sci. 2000; 41: 3503-3510PubMed Google Scholar, 30Causeret M. Taulet N. Comunale F. Favard C. Gauthier-Rouviere C. Mol. Biol. Cell. 2005; 16: 2168-2180Crossref PubMed Scopus (78) Google Scholar). As shown in Fig. 1A, the majority of E-cadherin from sparse cultures was found in the actinun-associated (Triton-soluble) membrane fraction. Also, in sparse cells, β- and α-catenins distributed primarily into this actin-unassociated fraction (Fig. 1A). In contrast, in dense cultures, the abundance of E-cadherin in the actin-unassociated fraction was decreased while the level of E-cadherin in the cytoskeletal membrane fraction became augmented. Similarly, levels of β- and α-catenins in the cytoskeletal pools increased in dense cultures (Fig. 1A). Moreover, E-cadherin in the actin-unassociated fraction from sparse cultures migrated as a large and diffuse band and with an overall higher molecular size compared with E-cadherin in the cytoskeletal fraction (Fig. 1A). This was most probably due to the presence of extensively N-glycosylated E-cadherin glycoforms in the actin-unassociated fraction. In contrast, in dense cultures, E-cadherin in the actin-unassociated fraction was less diffuse and of a smaller size compared with E-cadherin from sparse cultures. These data suggested that scarcely N-glycosylated E-cadherin was preferentially associated with the actin-cytoskeletal membrane fraction. We next evaluated E-cadherin N-glycosylation from sparse and dense cultures using sensitivity to PNGaseF and EndoH. Both enzymes remove N-glycans from proteins, although they differ in glycan specificities: EndoH is an endoglycosidase that hydrolyzes only high mannose and some hybrid N-glycans at the chitobiose core, whereas PNGaseF is an amidase that cleaves most N-glycans, including high mannose, hybrid, and complex structures, at the asparagines residues. A noted exception to PNGaseF sensitivity includes a class of complex N-glycans modified by fucose at the chitobiose core. Based on the mobility shifts before and after EndoH and PNGaseF treatments, E-cadherin from sparse cultures was primarily PNGaseF-sensitive, with little EndoH sensitivity, indicating that it was modified with complex N-glycans and lacked high mannose structures (Fig. 1B, sparse). On the other hand, E-cadherin from dense cultures displayed detectable EndoH and some PNGase F sensitivity providing evidence that it was decorated with high mannose/hybrid and some complex N-glycans. The finding that E-cadherin from sparse cultures was more extensively modified with complex N-glycans than in dense cultures was confirmed by the reactivity of immunoprecipitated E-cadherins with SNA and DSA lectins (Fig. 1C). Both lectins recognize complex N-glycans, with SNA being specific for terminal sialic acid linked α(2Takeichi M. Curr. Opin. Cell Biol. 1995; 5: 806-811Crossref Scopus (829) Google Scholar, 3Gumbiner B.M. Nat Rev Mol. Cell. Biol. 2005; 6: 622-634Crossref PubMed Scopus (1210) Google Scholar, 4Jamora C. Fuchs E. Nat Cell Biol. 2002; 4: E101-E108Crossref PubMed Scopus (490) Google Scholar, 5Brieher W.M. Yap A.S. Gumbiner B.M. J. Cell Biol. 1996; 135: 487-496Crossref PubMed Scopus (264) Google Scholar, 6Wheelock M.J. Johnson K.R. Ann. Rev. Cell Dev. Biol. 2003; 19: 207-235Crossref PubMed Scopus (530) Google Scholar) to galactose, and DSA interacting with terminal galactose linked β(1Gumbiner B.M. J. Cell Biol. 1996; 84: 345-357Scopus (2924) Google Scholar, 2Takeichi M. Curr. Opin. Cell Biol. 1995; 5: 806-811Crossref Scopus (829) Google Scholar, 3Gumbiner B.M. Nat Rev Mol. Cell. Biol. 2005; 6: 622-634Crossref PubMed Scopus (1210) Google Scholar, 4Jamora C. Fuchs E. Nat Cell Biol. 2002; 4: E101-E108Crossref PubMed Scopus (490) Google Scholar) to GlcNAc residues in complex and hybrid structures. There was no significant difference in the reactivity of E-cadherins from sparse and dense cultures with GNA, a lectin specific for high mannose/hybrid N-glycans (Fig. 1C). This may reflect the presence of core mannose residues on complex N-glycans in sparse cells and high mannose/hybrid N-glycans as well as a few core mannose residues in complex type structures in dense cultures. Collectively, these data show that there were differences between the sizes and types of N-glycans that modified E-cadherin in sparse and dense cultures (Fig. 1, B and C). Notably, E-cadherin from sparse cultures that formed unstable AJs was modified with substantial complex N-glycans, whose abundance diminished in dense cultures. Also, there was a notable decrease in the overall levels of E-cadherin in dense compared with sparse cultures (Fig 1, A and B), suggesting that changes in N-glycosylation affected its stability. We concluded that E-cadherin N-glycosylation status is cell density-dependent. Generation of E-cadherin N-Glycosylation Variants—To examine the role of N-glycans and their site occupancy in the stability of AJs, we generated seven N-glycosylation variants lacking selected potential N-glycan addition sites singly and in different combinations (Fig. 2A; “Experimental Procedures”). Specifically, we focused on the three N-glycosylation sites located in EC4 and EC5 that are shared by the human, canine, and mouse E-cadherins. To avoid interfering with E-cadherin structure and/or function, individual potential N-glycan sites at Asn-404, EC4, and at Asn-468 and Asn-483, EC5, were deleted and substituted with Gln, where least perturbation was expected (Figs. 2A). The cytomegalovirus (CMV) promoter was used to drive transient expression of these myc-tagged E-cadherins (Fig. 2B). Firs
Referência(s)