Stabilization of Plakoglobin and Enhanced Keratinocyte Cell-Cell Adhesion by Intracellular O-Glycosylation

O-Glycosylation modifies and regulates a variety of intracellular proteins. Plakoglobin, which functions in both cell-cell adhesion and signal transduction, is modified by O-glycosylation; however, the significance is unknown. To investigate the functional consequence of plakoglobin O-glycosylation, we cloned and overexpressed in keratinocytes murine O-GlcNAc transferase (mOGT). Over expression of mOGT in murine keratinocytes resulted in (i) glycosylation of plakoglobin and (ii) increased levels of plakoglobin due to post-translational stabilization of plakoglobin. Additionally, overexpression of mOGT in keratinocytes correlated with increased staining for cell-cell adhesion proteins and greater cell-cell adhesion. These observations suggest that O-glycosylation functions to regulate the post-translational stability of plakoglobin and keratinocyte cell-cell adhesion. O-Glycosylation modifies and regulates a variety of intracellular proteins. Plakoglobin, which functions in both cell-cell adhesion and signal transduction, is modified by O-glycosylation; however, the significance is unknown. To investigate the functional consequence of plakoglobin O-glycosylation, we cloned and overexpressed in keratinocytes murine O-GlcNAc transferase (mOGT). Over expression of mOGT in murine keratinocytes resulted in (i) glycosylation of plakoglobin and (ii) increased levels of plakoglobin due to post-translational stabilization of plakoglobin. Additionally, overexpression of mOGT in keratinocytes correlated with increased staining for cell-cell adhesion proteins and greater cell-cell adhesion. These observations suggest that O-glycosylation functions to regulate the post-translational stability of plakoglobin and keratinocyte cell-cell adhesion. Plakoglobin is a component of (i) adherens junctions, linking E-cadherin to the actin binding protein α-catenin, and (ii) desmosomes, linking desmogleins to desmoplakin, plakophilin, and in turn, keratin intermediate filaments. The suggestion that the association of plakoglobin with the adherens junction nucleates the formation of desmosomes may explain why plakoglobin is a component of both adherens junctions and desmosomes (1Lewis J.E. Jensen P.J. Wheelock M.J. Journal of Investigative Dermatology. 1994; 102: 870-877Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 2Lewis J.E. Wahl 3rd, J.K. Sass K.M. Jensen P.J. Johnson K.R. Wheelock M.J. Journal of Cell Biology. 1997; 136: 919-934Crossref PubMed Scopus (215) Google Scholar), whereas β-catenin is limited to adherens junctions. Phosphorylation regulates the adhesion (3Hu P. O'Keefe E.J. Rubenstein D.S. Journal of Investigative Dermatology. 2001; 117: 1059-1067Abstract Full Text Full Text PDF PubMed Google Scholar) and signaling (4Hu P. Berkowitz P. O'Keefe E.J. Rubenstein D.S. Journal of Investigative Dermatology. 2003; 121: 242-251Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar) functions of plakoglobin. Plakoglobin is also modified by intracellular O-glycosylation (5Hatsell S. Medina L. Merola J. Haltiwanger R. Cowin P. J. Biol. Chem. 2003; 278: 37745-37752Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar); however, the functional consequence of plakoglobin O-glycosylation is not known. Intracellular protein O-glycosylation (6Torres C.R. Hart G.W. Journal of Biological Chemistry. 1984; 259: 3308-3317Abstract Full Text PDF PubMed Google Scholar) modifies and regulates a variety of substrates including transcription factors, nuclear pore and cytoskeletal proteins, and enzymes (7Wells L. Vosseller K. Hart G.W. Science. 2001; 291: 2376-2378Crossref PubMed Scopus (816) Google Scholar). N-Acetylglucosamine (GlcNAc) 2The abbreviations used are: GlcNAc, N-acetylglucosamine; dsg1, desmoglein 1; OGT, O-GlcNAc transferase; mOGT, murine OGT; PBS, phosphate-buffered saline; CHAPS, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonic acid; PP2A, protein phosphatase 2A; MOPS, 4-morpholinepropanesulfonic acid. modification of serine and threonine is catalyzed by the enzyme UDP-N-acetylglucosamine-polypeptide β-N-acetylglucosaminyl transferase (O-GlcNAc transferase, OGT) (8Haltiwanger R.S. Blomberg M.A. Hart G.W. Journal of Biological Chemistry. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar), whereas, GlcNAc is removed by O-GlcNAc-selective N-acetyl-β-d-glucosaminidase (9Gao Y. Wells L. Comer F.I. Parker G.J. Hart G.W. J. Biol. Chem. 2001; 276: 9838-9845Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). OGT is highly conserved across species including humans, rodents, and nematodes (10Kreppel L.K. Blomberg M.A. Hart G.W. Journal of Biological Chemistry. 1997; 272: 9308-9315Abstract Full Text Full Text PDF PubMed Scopus (615) Google Scholar, 11Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). To investigate the functional consequence of plakoglobin O-glycosylation, we cloned and expressed in murine keratinocytes the murine OGT. We demonstrate that increased O-glycosylation is associated with increased plakoglobin protein levels, increased post-translational stability of plakoglobin, and greater keratinocyte cell-cell adhesion. Greater cell-cell adhesion in OGT overexpressing keratinocytes is likely because of both the increased amounts of and enhanced cell membrane localization of cell-cell adhesion proteins. Generation of Murine OGT Expression Vectors—Oligonucleotide primers flanking the published coding region of the murine OGT cDNA (12Hanover J.A. Yu S. Lubas W.B. Shin S.H. Ragano-Caracciola M. Kochran J. Love D.C. Arch. Biochem. Biophys. 2003; 409: 287-297Crossref PubMed Scopus (184) Google Scholar) were employed in a polymerase chain reaction-based strategy to clone the full-length nucleocytoplasmic OGT cDNA from murine keratinocytes. The pCS2myc-mOGT expression vector was constructed by cloning the full-length mOGT into the expression vector pCS2 (13Turner D.L. Weintraub H. Genes Dev. 1994; 8: 1434-1447Crossref PubMed Scopus (956) Google Scholar) with six tandem copies of the human myc epitope in-frame with the N terminus of mOGT (see Fig. 1A). The pTRE2myc-mOGT expression vector was constructed by subcloning myc-tagged mOGT from pCS2mycmOGT into the BamH1 and NotI sites of pTRE-2 (Clontech). Cell Culture and Transfection—The murine keratinocyte cell line PAM212 was cultured in RPMI 1640 (Invitrogen, Inc.) supplemented with 10% fetal bovine serum. The pCS2myc-mOGT expression vector was transiently transfected into subconfluent PAM212 cells using Effectene (Qiagen, Inc.). Stably transfected inducible cultures were obtained by cotransfection of pTRE2myc-mOGT and pTet-on (Clontech) into PAM212 cells as above and selected with G418 (Invitrogen, Inc.) and hygromycin B (Roche Diagnostics). mOGT expression was induced in stably transfected cells with doxycycline (2 mg/ml) for 24h. Experiments described utilized the permanently transfected cells (referred to as OGT cells); however, similar increases in plakoglobin protein levels were obtained with multiple independent transient transfections. Antibodies—Immunoblotting, immunoprecipitation, and immunostaining were performed with primary antibodies against plakoglobin, β-catenin, E-cadherin, desmoglein-1 (dsg1) (BD Biosciences, Inc.), α-catenin (Zymed Laboratories, Inc.), desmoplakin 1/2 (Abcam, Inc), plakophilin 1 (Santa Cruz Biotechnology, Inc.), O-GlcNAc (clone RL-2, Affinity Bioreagents, Inc.), O-GlcNAc (clone CTD110.6, Covance), lactate dehydrogenase V (Cortex Biochem, Inc.), actin (Calbiochem, Inc.), and myc (Clone 9B11, Cell Signaling Technology, Inc.). Immunofluorescence and Confocal Microscopy—Cells grown on glass coverslips were fixed in 4% paraformaldehyde, permeabilized in 0.25% Triton X-100, blocked in 2% bovine serum albumin, and incubated with primary antibodies in PBS, 2% bovine serum albumin for 1 h. After washing with PBS, cells were incubated for 1 h with Cy2- or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch laboratory, Inc.), washed, and mounted. Images were analyzed using a Leica SP2 AOBS confocal microscope as previously described (14Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. Rubenstein D.S. J. Biol. Chem. 2005; 280: 23778-23784Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Immunoblotting and Immunoprecipitation—Monolayer cells grown to confluence were extracted in cell lysis buffer (1% Nonidet P-40, 150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 1 mm EDTA, 10 μm E64, 100 μm leupeptin, 10 μm pepstatin, and 1 mm phenylmethylsulfonyl fluoride) at 4 °C for 1 h with rotating and then centrifuged at 13,700 × g for 15 min at 4 °C. The supernatants were collected as detergent-soluble fractions. The pellets were washed twice with PBS, resuspended by incubation in urea lysis buffer (8 m urea, 4% CHAPS, 10 mm Tris-HCl, pH 7.4) for 1 h at 4 °C, and then centrifuged as above; the supernatant was used as the detergent-insoluble fraction. Samples were equally loaded on and separated by SDS-PAGE. For immunoprecipitation, detergent-soluble fractions were incubated with antibodies and recombinant protein G-Sepharose 4B conjugate beads (Zymed Laboratories, Inc.) with rotating for 16 h at 4 °C. Immunoprecipitates were washed three times with PBS, denatured with SDS sample buffer, and separated by SDS-PAGE. Immunoblotting was performed according to established protocols and developed by enhanced chemiluminescence (ECL) reaction (Amersham Biosciences). Signal intensity from the ECL reaction for each band was quantified with a GeneGnome scanner (Syngene Bio Imaging) using GeneSnap software. Two-dimensional Gel Electrophoresis—Cell extracts (100 μg) were prepared and separated in the first dimension using 13-cm pH 3-10, non-linear IPGphor strips (Amersham Biosciences, Inc) and in the second dimension by 10% SDS-PAGE followed by immunoblotting as described (14Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. Rubenstein D.S. J. Biol. Chem. 2005; 280: 23778-23784Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Protein Phosphatase 2A (PP2A) Treatment—Cell extracts were prepared in 100 mm NaCl, 20 mm MOPS, pH 7.5, 1 mg/ml bovine serum albumin, 60 mm β-mercaptoethanol, 10 μm E64, 100 μm leupeptin, 10 μm pepstatin, 1 mm phenylmethylsulfonyl fluoride, and 0.5 μm okadaic acid (okadaic acid was omitted from buffer for PP2A-treated extracts) by sonication for 5 s × 2 on ice, followed by centrifugation at 14,000 rpm in a microcentrifuge, and supernatants were collected in 500-μg protein aliquots. For samples treated with PP2A, 500-μg protein extracts were treated with 0.1 units PP2A (Upstate, Inc.) for 30 min at 30 °C. For two-dimensional gel electrophoresis, samples were desalted on Centricon 30 spin filters (Amicon Bioseparations, Inc.) and resuspended in 8 m urea, 4% CHAPS, 10 mm Tris-HCl, pH 7.4. Dithiothreitol was added to a final concentration of 2.5 mm and IPG buffer (pH 4-7, linear, Amersham Biosciences) was added to a final concentration of 0.5%, and the samples were separated in the first dimension using 7-cm, pH 4-7, linear IPGphor strips (Amersham Biosciences, Inc) and in the second dimension by 10% SDS-PAGE followed by immunoblotting as described (14Berkowitz P. Hu P. Liu Z. Diaz L.A. Enghild J.J. Chua M.P. Rubenstein D.S. J. Biol. Chem. 2005; 280: 23778-23784Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Dispase-based Dissociation Assay—A dispase-based dissociation assay was performed as described (15Huen A.C. Park J.K. Godsel L.M. Chen X. Bannon L.J. Amargo E.V. Hudson T.Y. Mongiu A.K. Leigh I.M. Kelsell D.P. Gumbiner B.M. Green K.J. J. Cell Biol. 2002; 159: 1005-1017Crossref PubMed Scopus (123) Google Scholar, 16Ishii K. Harada R. Matsuo I. Shirakata Y. Hashimoto K. Amagai M. J. Invest Dermatol. 2005; 124: 939-946Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Briefly, cells grown to confluence in triplicate on 100-mm dishes were washed twice with PBS, incubated in 5 ml of dispase II (2.4 units/ml, Roche Diagnostics) at 37 °C for 1 h, and rocked back and forth 10 times on a ClayAdams nutator, and the number of fragments was counted. 3HGlcNAc Radiolabeling—80% confluent cultures were incubated with d-6-3Hglucosamine hydrochloride (Amersham Biosciences) at 1 μCi/ml in glucose-free RPMI 1640 with 10% fetal bovine serum for 16 h at 37 °C, washed 3× in PBS, and extracted in cell lysis buffer as above. Soluble fractions containing equal amounts of protein were subjected to immunoprecipitation, separated by SDS-PAGE, and transferred to nitrocellulose, and the radioactive signal was visualized on a Molecular Dynamics Storm 840 phosphoimager. Galactosyltransferase Labeling—Galactosyltransferase labeling was as described (5Hatsell S. Medina L. Merola J. Haltiwanger R. Cowin P. J. Biol. Chem. 2003; 278: 37745-37752Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 17Haltiwanger R.S. Philipsberg G.A. Journal of Biological Chemistry. 1997; 272: 8752-8758Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Briefly, plakoglobin immunoprecipitation reactions were washed 6 times in buffer A (15 mm Tris, pH 7.4, 5 mm EDTA, 1 m NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100) and resuspended in 500 μl of buffer B (50 mm Hepes, 5 mm MnCl2, 10 mm galactose, 2% Triton X-100). The labeling reaction was initiated by adding 2.5 μCi of UDP-6-3Hgalactose (American Radiolabeled Chemicals) and 50 milliunits of galactosyltransferase (Sigma) and incubating at 37 °C for 1 h. The beads were washed four times with buffer A, resuspended in 20 μl of SDS-PAGE sample buffer, incubated at 37 °C for 10 min, and subjected to SDS-PAGE followed by autoradiography. Plakoglobin Half-life—The plakoglobin half-life was measured by treating confluent cells with the 20 mg/ml protein synthesis inhibitor cycloheximide (Sigma Aldrich) for 0, 2.5, 5.0, 7.5, and 10 h. Cells were harvested, and extracts were prepared as above. Soluble fractions containing equal amounts of protein (20 μg) were subjected to SDS-PAGE and immunoblot with antibodies to plakoglobin and actin. Blots were developed by ECL and quantified as above using a GeneGnome scanner (Syngene Bio Imaging) and GeneSnap software. The Cloned cDNA Encodes mOGT and Is Catalytically Active—Myc tagged OGT (Fig. 1A) localized to both the cytoplasm and nucleus (Fig. 1B) as expected for the described distribution of this intracellular enzyme (11Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). The expressed protein migrated on SDS-PAGE as a single band of 120,000 consistent with the predicted molecular weight for the full-length expressed OGT polypeptide fused to the 6-copy myc epitope tag (Fig. 1C). Increased reactivity with the GlcNAc-specific RL2 monoclonal antibody (18Snow C.M. Senior A. Gerace L. Journal of Cell Biology. 1987; 104: 1143-1156Crossref PubMed Scopus (401) Google Scholar) was detected by immunoblot of OGT cell extracts compared with control cells demonstrating that the expressed myctagged protein was catalytically active (Fig. 1, C and D). Plakoglobin Is a Substrate for mOGT—To demonstrate that the cloned cDNA encoded the enzyme that was in fact responsible for catalyzing the addition of GlcNAc to plakoglobin, plakoglobin was immunoprecipitated from control and OGT cell extracts, and the immunoprecipitation reactions were probed for plakoglobin and GlcNAc modification using anti-plakoglobin and anti-GlcNAc monoclonal antibodies, respectively (Fig. 2, A and B). Greater GlcNAc reactivity was detected in plakoglobin from the OGT cells using both the RL2 (Fig. 2A) and CTD110.6 (Fig. 2B) (19Comer F.I. Vosseller K. Wells L. Accavitti M.A. Hart G.W. Anal. Biochem. 2001; 293: 169-177Crossref PubMed Scopus (240) Google Scholar) GlcNAc-specific monoclonal antibodies. Quantitation of the signal intensity from replicates (n = 3) of the experiment shown in Fig. 2B demonstrated a 1.95 ± 0.03-fold increase in GlcNAc and a ± increase in plakoglobin in the plakoglobin from OGT control keratinocytes. the in OGT and control cells was The galactosyltransferase reaction the of from to plakoglobin from both control and OGT cells was with however, the signal was greater in OGT cells (Fig. compared with a both control and OGT keratinocytes were incubated in the of was detected in plakoglobin from OGT cells to (Fig. analyzed by two-dimensional gel electrophoresis, OGT cell extracts compared with (Fig. of by treating control and OGT cell extracts with PP2A the of plakoglobin the that the in OGT cells from the of likely because of the of the Increased Protein of Plakoglobin in OGT plakoglobin was immunoprecipitated from OGT control increased levels of plakoglobin in the OGT keratinocytes. was by immunoblot of plakoglobin in cell plakoglobin was detected in OGT keratinocytes (Fig. Expression of OGT the of modification of plakoglobin by O-glycosylation and the increased levels of plakoglobin in OGT cells that O-glycosylation of plakoglobin to increase the post-translational stability of plakoglobin. To investigate the of O-glycosylation on the half-life of plakoglobin, control and OGT cells were treated with the protein synthesis inhibitor cycloheximide and the levels of plakoglobin as a of by immunoblot of cell extracts (Fig. 10 h, plakoglobin levels to 30 of levels in control OGT cells that O-glycosylation of plakoglobin half-life was not a on protein as the half-life of actin in control and OGT cells not Is Increased in OGT plakoglobin functions in cell-cell adhesion as a component of both the keratinocyte adherens junction and we OGT cells demonstrated greater cell-cell adhesion using the dispase assay (15Huen A.C. Park J.K. Godsel L.M. Chen X. Bannon L.J. Amargo E.V. Hudson T.Y. Mongiu A.K. Leigh I.M. Kelsell D.P. Gumbiner B.M. Green K.J. J. Cell Biol. 2002; 159: 1005-1017Crossref PubMed Scopus (123) Google Scholar). this cells grown to confluence and the as using The cell then rocked back and forth a number of times on a nutator, which the to a Greater cell-cell adhesion to the and in of the cell The number of fragments a of cell-cell adhesion. subjected to this OGT cells and fragments of = ± control cells of = ± consistent with greater cell-cell adhesion in OGT cells (Fig. Plakoglobin in OGT Cells Is with in and Protein the distribution of E-cadherin and desmoglein-1 (dsg1) by confocal (Fig. control both and membrane staining was for staining in control cells was a of membrane staining also E-cadherin and staining in OGT cells was localized to the cell membrane (Fig. plakoglobin staining was both and membrane in control whereas greater plakoglobin membrane staining was in OGT membrane staining for both plakoglobin and was in the OGT consistent with the staining expected for desmosomes (Fig. OGT cells also demonstrated staining for desmoplakin 1 and plakophilin 1, staining was and in control membrane staining was membrane staining for desmoplakin was in OGT staining was also (Fig. plakophilin staining in control cells was and whereas plakophilin membrane staining that with was in OGT cells (Fig. suggest increased membrane localization of both adherens junction and proteins in OGT overexpressing keratinocytes. Increased of Plakoglobin with and Is in OGT the protein levels of adherens junction and junctions in as P-40, whereas desmosomes in to the detergent-insoluble as a for and OGT cells were separated into detergent-soluble and and the fractions subjected to SDS-PAGE and immunoblotting using antibodies to adherens junction and proteins (Fig. levels of the adherens junction proteins E-cadherin, β-catenin, and α-catenin were detected in the detergent-soluble of both control and OGT cells (Fig. plakoglobin was in the detergent-soluble of OGT cells compared with from the detergent-soluble were used to amounts of E-cadherin were immunoprecipitated from the detergent-soluble of control and OGT cells (Fig. in the association of β-catenin with E-cadherin was as similar amounts of β-catenin with E-cadherin from control and OGT plakoglobin and α-catenin with E-cadherin from OGT detergent-soluble extracts were subjected to immunoprecipitation with monoclonal antibodies to plakoglobin. plakoglobin was immunoprecipitated from the detergent-soluble of OGT cells (Fig. E-cadherin as as the with plakoglobin in OGT cells that O-glycosylation was associated with increased formation of plakoglobin detergent-soluble and fractions were probed by immunoblotting for proteins. Increased amounts of plakoglobin and were in both the detergent-soluble and of OGT cells (Fig. The increased of the plakoglobin and in the detergent-insoluble increased similar increases in desmoplakin and plakophilin were not in the detergent-soluble and fractions of OGT the in the distribution of plakophilin and desmoplakin from cytoplasm to membrane by confocal These observations to suggest that desmoplakin and plakophilin the to into the above observations demonstrate that increased post-translational stability of plakoglobin is a functional consequence of plakoglobin and plakoglobin for the increase in plakoglobin stability because O-glycosylation of the protein in cells K. X. J.E. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). β-catenin is also to and by the greater amounts of β-catenin expected the were because of of in the levels of β-catenin were OGT and control cells that the of O-glycosylation on the post-translational stability of plakoglobin was the of in modification of plakoglobin by O-glycosylation is likely responsible for increased stability to and greater protein levels of plakoglobin in OGT is by plakoglobin O-glycosylation and plakoglobin levels were increased in OGT however, the of plakoglobin O-glycosylation to plakoglobin was similar in OGT and control each a plakoglobin polypeptide is the of plakoglobin in the cell is increased by the O-glycosylation of plakoglobin to threonine (5Hatsell S. Medina L. Merola J. Haltiwanger R. Cowin P. J. Biol. Chem. 2003; 278: 37745-37752Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) in the may by as of the for plakoglobin to the for or in increased O-Glycosylation and to the in proteins T.Y. Hart G.W. Journal of Biological Chemistry. Full Text Full Text PDF PubMed Scopus Google Scholar). overexpression of OGT in the modification of proteins as by the enhanced GlcNAc on extracts separated by two-dimensional may to suggest that modification of plakoglobin is the for the increase in cell-cell because O-glycosylation may a variety of proteins in adhesion. For shown to modified by O-glycosylation Journal of Biological Chemistry. 1992; 267: Full Text PDF PubMed Google Scholar), and enhanced GlcNAc was in the region of the two-dimensional to which (Fig. increased cell-cell adhesion resulted from overexpression of OGT in keratinocytes. plakoglobin levels were the levels of β-catenin, which also with E-cadherin and α-catenin, were not Increased association of plakoglobin with E-cadherin and α-catenin was in OGT cells increased of adherens junctions. OGT cells demonstrated (i) increased association of plakoglobin with the in plakoglobin from the detergent-soluble (ii) plakoglobin and in the detergent-insoluble and greater membrane localization of proteins plakoglobin, dsg1, desmoplakin, and both adherens junction formation and plakoglobin for of formation (1Lewis J.E. Jensen P.J. Wheelock M.J. Journal of Investigative Dermatology. 1994; 102: 870-877Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 2Lewis J.E. Wahl 3rd, J.K. Sass K.M. Jensen P.J. Johnson K.R. Wheelock M.J. Journal of Cell Biology. 1997; 136: 919-934Crossref PubMed Scopus (215) Google Scholar), stabilization of plakoglobin by O-glycosylation may the formation of adherens junctions, which then formation of desmosomes, in the increase in cell-cell adhesion. We Diaz and for