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Cell surface N-glycans influence the level of functional E-cadherin at the cell-cell border.

Hall MK, Weidner DA, Dayal S, Schwalbe RA - FEBS Open Bio (2014)

Bottom Line: While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell-cell border.Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell-cell border, and consequently reduced the strength of cell-cell interactions.We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell-cell border and thereby control E-cadherin mediated cell-cell adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27834, USA.

ABSTRACT
E-cadherin is crucial for adhesion of cells to each other and thereby development and maintenance of tissue. While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell-cell border. Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell-cell border, and consequently reduced the strength of cell-cell interactions. We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell-cell border and thereby control E-cadherin mediated cell-cell adhesion.

No MeSH data available.


Related in: MedlinePlus

Blots of glycoproteins from cultured cells treated with an endoglycosidase. Lectin blots of whole cell lysates (∼25 μg) from E-cadherin transfected Pro-5 CHO cells treated without (−) and with (+) PNGase F prior to (A) and after (B) formation of cell–cell contacts. Blots were probed with L-PHA (∼25 μg/mL). Gels loaded with similar amounts of electrophoresed proteins were also stained with Coomassie blue (A and B). Horizontal lines denote the Kaleidoscope markers (in kDa) from top to bottom (150, 100, 75, and 50). Western blots of E-cadherin from total membranes from E-cadherin transfected Pro-5 CHO cells treated without (−) and with (+) PNGase F prior to and after formation of cell–cell contacts, as indicated (C). Total membranes of E-cadherin transfected Pro-5 CHO cells were also digested with PNGase F (test tube) to denote the electrophoretic mobility of unglycosylated E-cadherin (aglycoform). Black and gray arrows point to the fully and partially glycosylated E-cadherin proteins, respectively. Similar findings were observed on at least three separate blots.
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f0010: Blots of glycoproteins from cultured cells treated with an endoglycosidase. Lectin blots of whole cell lysates (∼25 μg) from E-cadherin transfected Pro-5 CHO cells treated without (−) and with (+) PNGase F prior to (A) and after (B) formation of cell–cell contacts. Blots were probed with L-PHA (∼25 μg/mL). Gels loaded with similar amounts of electrophoresed proteins were also stained with Coomassie blue (A and B). Horizontal lines denote the Kaleidoscope markers (in kDa) from top to bottom (150, 100, 75, and 50). Western blots of E-cadherin from total membranes from E-cadherin transfected Pro-5 CHO cells treated without (−) and with (+) PNGase F prior to and after formation of cell–cell contacts, as indicated (C). Total membranes of E-cadherin transfected Pro-5 CHO cells were also digested with PNGase F (test tube) to denote the electrophoretic mobility of unglycosylated E-cadherin (aglycoform). Black and gray arrows point to the fully and partially glycosylated E-cadherin proteins, respectively. Similar findings were observed on at least three separate blots.

Mentions: Whole cell lysates from E-cadherin transfected Pro-5 cells that underwent treatment in the absence (−) and presence (+) of PNGase F were used for lectin blotting. PNGase F was added to cultured cells either before (Fig. 2A) or after (Fig. 2B) the establishment of cell–cell contacts. This allowed us to ascertain whether cell surface N-glycans controlled the recruitment and/or retention of E-cadherin at the cell–cell border. Phaseolus vulgaris Leucoagglutinin (L-PHA) was the lectin employed for blotting glycoproteins since the far majority of N-glycans from Pro-5 cells are of complex type [20,21]. L-PHA interactions with glycoproteins from transfected Pro-5 cells treated with PNGase F prior to and after cell–cell contact were weaker than those from cells treated without PNGase F. In both cases, SDS-gels were stained with Coomassie blue to show that similar amounts of proteins were loaded.


Cell surface N-glycans influence the level of functional E-cadherin at the cell-cell border.

Hall MK, Weidner DA, Dayal S, Schwalbe RA - FEBS Open Bio (2014)

Blots of glycoproteins from cultured cells treated with an endoglycosidase. Lectin blots of whole cell lysates (∼25 μg) from E-cadherin transfected Pro-5 CHO cells treated without (−) and with (+) PNGase F prior to (A) and after (B) formation of cell–cell contacts. Blots were probed with L-PHA (∼25 μg/mL). Gels loaded with similar amounts of electrophoresed proteins were also stained with Coomassie blue (A and B). Horizontal lines denote the Kaleidoscope markers (in kDa) from top to bottom (150, 100, 75, and 50). Western blots of E-cadherin from total membranes from E-cadherin transfected Pro-5 CHO cells treated without (−) and with (+) PNGase F prior to and after formation of cell–cell contacts, as indicated (C). Total membranes of E-cadherin transfected Pro-5 CHO cells were also digested with PNGase F (test tube) to denote the electrophoretic mobility of unglycosylated E-cadherin (aglycoform). Black and gray arrows point to the fully and partially glycosylated E-cadherin proteins, respectively. Similar findings were observed on at least three separate blots.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215119&req=5

f0010: Blots of glycoproteins from cultured cells treated with an endoglycosidase. Lectin blots of whole cell lysates (∼25 μg) from E-cadherin transfected Pro-5 CHO cells treated without (−) and with (+) PNGase F prior to (A) and after (B) formation of cell–cell contacts. Blots were probed with L-PHA (∼25 μg/mL). Gels loaded with similar amounts of electrophoresed proteins were also stained with Coomassie blue (A and B). Horizontal lines denote the Kaleidoscope markers (in kDa) from top to bottom (150, 100, 75, and 50). Western blots of E-cadherin from total membranes from E-cadherin transfected Pro-5 CHO cells treated without (−) and with (+) PNGase F prior to and after formation of cell–cell contacts, as indicated (C). Total membranes of E-cadherin transfected Pro-5 CHO cells were also digested with PNGase F (test tube) to denote the electrophoretic mobility of unglycosylated E-cadherin (aglycoform). Black and gray arrows point to the fully and partially glycosylated E-cadherin proteins, respectively. Similar findings were observed on at least three separate blots.
Mentions: Whole cell lysates from E-cadherin transfected Pro-5 cells that underwent treatment in the absence (−) and presence (+) of PNGase F were used for lectin blotting. PNGase F was added to cultured cells either before (Fig. 2A) or after (Fig. 2B) the establishment of cell–cell contacts. This allowed us to ascertain whether cell surface N-glycans controlled the recruitment and/or retention of E-cadherin at the cell–cell border. Phaseolus vulgaris Leucoagglutinin (L-PHA) was the lectin employed for blotting glycoproteins since the far majority of N-glycans from Pro-5 cells are of complex type [20,21]. L-PHA interactions with glycoproteins from transfected Pro-5 cells treated with PNGase F prior to and after cell–cell contact were weaker than those from cells treated without PNGase F. In both cases, SDS-gels were stained with Coomassie blue to show that similar amounts of proteins were loaded.

Bottom Line: While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell-cell border.Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell-cell border, and consequently reduced the strength of cell-cell interactions.We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell-cell border and thereby control E-cadherin mediated cell-cell adhesion.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Brody School of Medicine at East Carolina University, Greenville, NC 27834, USA.

ABSTRACT
E-cadherin is crucial for adhesion of cells to each other and thereby development and maintenance of tissue. While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell-cell border. Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell-cell border, and consequently reduced the strength of cell-cell interactions. We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell-cell border and thereby control E-cadherin mediated cell-cell adhesion.

No MeSH data available.


Related in: MedlinePlus