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Impact of Hybrid and Complex N-Glycans on Cell Surface Targeting of the Endogenous Chloride Cotransporter Slc12a2.

Singh R, Almutairi MM, Pacheco-Andrade R, Almiahuob MY, Di Fulvio M - Int J Cell Biol (2015)

Bottom Line: In addition, inhibition of the first step of N-glycan biosynthesis with tunicamycin decreases total and plasma membrane located NKCC1 resulting in almost undetectable cotransport function.Moreover, inhibition of N-glycan maturation with swainsonine or kifunensine increased core/hybrid-type NKCC1 expression but eliminated plasma membrane complex N-glycosylated NKCC1 and transport function.Together, these results suggest that (i) NKCC1 is delivered to the plasma membrane of COS7 cells independently of its N-glycan nature, (ii) most of NKCC1 in the plasma membrane is core/hybrid-type N-glycosylated, and (iii) the minimal proportion of complex N-glycosylated NKCC1 is functionally active.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Boonshoft School of Medicine, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA.

ABSTRACT
The Na(+)K(+)2Cl(-) cotransporter-1 (Slc12a2, NKCC1) is widely distributed and involved in cell volume/ion regulation. Functional NKCC1 locates in the plasma membrane of all cells studied, particularly in the basolateral membrane of most polarized cells. Although the mechanisms involved in plasma membrane sorting of NKCC1 are poorly understood, it is assumed that N-glycosylation is necessary. Here, we characterize expression, N-glycosylation, and distribution of NKCC1 in COS7 cells. We show that ~25% of NKCC1 is complex N-glycosylated whereas the rest of it corresponds to core/high-mannose and hybrid-type N-glycosylated forms. Further, ~10% of NKCC1 reaches the plasma membrane, mostly as core/high-mannose type, whereas ~90% of NKCC1 is distributed in defined intracellular compartments. In addition, inhibition of the first step of N-glycan biosynthesis with tunicamycin decreases total and plasma membrane located NKCC1 resulting in almost undetectable cotransport function. Moreover, inhibition of N-glycan maturation with swainsonine or kifunensine increased core/hybrid-type NKCC1 expression but eliminated plasma membrane complex N-glycosylated NKCC1 and transport function. Together, these results suggest that (i) NKCC1 is delivered to the plasma membrane of COS7 cells independently of its N-glycan nature, (ii) most of NKCC1 in the plasma membrane is core/hybrid-type N-glycosylated, and (iii) the minimal proportion of complex N-glycosylated NKCC1 is functionally active.

No MeSH data available.


The first step of N-glycan biosynthesis is required for NKCC1 protein expression. ((a)-(b)) Shown are representative immunofluorescence microscopy images of COS7 cells grown under control conditions (a) or treated for 16 h with 2 μg/mL tunicamycin (TUN, (b)). NKCC1 immunolocalization was analyzed with T4 and FITC-labeled secondary antibodies (green). (c) Representative immunoblot with ckNKCC1 demonstrating expression of NKCC1 in the biotinylated plasma membrane fraction purified from COS7 cells control or treated with TUN. Protein expression of cytosolic GAPDH was used to assess the purity of biotinylated plasma membrane fractions. (d) Densitometry scanning of the immunoblot in (c) representing the extent to which TUN (red trace) decreases plasma membrane located NKCC1 (black trace). (e) Representative immunoblot showing the expression pattern of total NKCC1 in COS7 cells in response to TUN 2 μg/mL during the indicated periods of time. As loading control, immunoblots were probed against tubulin.
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fig8: The first step of N-glycan biosynthesis is required for NKCC1 protein expression. ((a)-(b)) Shown are representative immunofluorescence microscopy images of COS7 cells grown under control conditions (a) or treated for 16 h with 2 μg/mL tunicamycin (TUN, (b)). NKCC1 immunolocalization was analyzed with T4 and FITC-labeled secondary antibodies (green). (c) Representative immunoblot with ckNKCC1 demonstrating expression of NKCC1 in the biotinylated plasma membrane fraction purified from COS7 cells control or treated with TUN. Protein expression of cytosolic GAPDH was used to assess the purity of biotinylated plasma membrane fractions. (d) Densitometry scanning of the immunoblot in (c) representing the extent to which TUN (red trace) decreases plasma membrane located NKCC1 (black trace). (e) Representative immunoblot showing the expression pattern of total NKCC1 in COS7 cells in response to TUN 2 μg/mL during the indicated periods of time. As loading control, immunoblots were probed against tubulin.

Mentions: To study the role of N-glycosylation on endogenous NKCC1 trafficking and localization, we first treated cells with tunicamycin (TUN, 2 μg/mL for 16 h), an inhibitor of the first step of N-glycan biosynthesis. As shown in Figures 8(a) and 8(b), TUN significantly decreases total immunoreactive NKCC1 protein expression levels consistent with the notion that core N-glycosylation of proteins plays a key role in protein stability and quality control in the ER [48–50]. To correlate these results at the plasma membrane level, TUN-treated cells were biotinylated and plasma membrane fractions were purified and subjected to immunoblotting. As shown in Figures 8(c) and 8(d), TUN decreased expression of plasma membrane located NKCC1. Since TUN also decreased total expression levels of NKCC1, as seen in Figures 8(d) and 8(e), these results suggest that the extent of plasma membrane located NKCC1 reflects its biosynthesis levels rather than its N-glycosylation state.


Impact of Hybrid and Complex N-Glycans on Cell Surface Targeting of the Endogenous Chloride Cotransporter Slc12a2.

Singh R, Almutairi MM, Pacheco-Andrade R, Almiahuob MY, Di Fulvio M - Int J Cell Biol (2015)

The first step of N-glycan biosynthesis is required for NKCC1 protein expression. ((a)-(b)) Shown are representative immunofluorescence microscopy images of COS7 cells grown under control conditions (a) or treated for 16 h with 2 μg/mL tunicamycin (TUN, (b)). NKCC1 immunolocalization was analyzed with T4 and FITC-labeled secondary antibodies (green). (c) Representative immunoblot with ckNKCC1 demonstrating expression of NKCC1 in the biotinylated plasma membrane fraction purified from COS7 cells control or treated with TUN. Protein expression of cytosolic GAPDH was used to assess the purity of biotinylated plasma membrane fractions. (d) Densitometry scanning of the immunoblot in (c) representing the extent to which TUN (red trace) decreases plasma membrane located NKCC1 (black trace). (e) Representative immunoblot showing the expression pattern of total NKCC1 in COS7 cells in response to TUN 2 μg/mL during the indicated periods of time. As loading control, immunoblots were probed against tubulin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig8: The first step of N-glycan biosynthesis is required for NKCC1 protein expression. ((a)-(b)) Shown are representative immunofluorescence microscopy images of COS7 cells grown under control conditions (a) or treated for 16 h with 2 μg/mL tunicamycin (TUN, (b)). NKCC1 immunolocalization was analyzed with T4 and FITC-labeled secondary antibodies (green). (c) Representative immunoblot with ckNKCC1 demonstrating expression of NKCC1 in the biotinylated plasma membrane fraction purified from COS7 cells control or treated with TUN. Protein expression of cytosolic GAPDH was used to assess the purity of biotinylated plasma membrane fractions. (d) Densitometry scanning of the immunoblot in (c) representing the extent to which TUN (red trace) decreases plasma membrane located NKCC1 (black trace). (e) Representative immunoblot showing the expression pattern of total NKCC1 in COS7 cells in response to TUN 2 μg/mL during the indicated periods of time. As loading control, immunoblots were probed against tubulin.
Mentions: To study the role of N-glycosylation on endogenous NKCC1 trafficking and localization, we first treated cells with tunicamycin (TUN, 2 μg/mL for 16 h), an inhibitor of the first step of N-glycan biosynthesis. As shown in Figures 8(a) and 8(b), TUN significantly decreases total immunoreactive NKCC1 protein expression levels consistent with the notion that core N-glycosylation of proteins plays a key role in protein stability and quality control in the ER [48–50]. To correlate these results at the plasma membrane level, TUN-treated cells were biotinylated and plasma membrane fractions were purified and subjected to immunoblotting. As shown in Figures 8(c) and 8(d), TUN decreased expression of plasma membrane located NKCC1. Since TUN also decreased total expression levels of NKCC1, as seen in Figures 8(d) and 8(e), these results suggest that the extent of plasma membrane located NKCC1 reflects its biosynthesis levels rather than its N-glycosylation state.

Bottom Line: In addition, inhibition of the first step of N-glycan biosynthesis with tunicamycin decreases total and plasma membrane located NKCC1 resulting in almost undetectable cotransport function.Moreover, inhibition of N-glycan maturation with swainsonine or kifunensine increased core/hybrid-type NKCC1 expression but eliminated plasma membrane complex N-glycosylated NKCC1 and transport function.Together, these results suggest that (i) NKCC1 is delivered to the plasma membrane of COS7 cells independently of its N-glycan nature, (ii) most of NKCC1 in the plasma membrane is core/hybrid-type N-glycosylated, and (iii) the minimal proportion of complex N-glycosylated NKCC1 is functionally active.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Boonshoft School of Medicine, Wright State University, 3640 Colonel Glenn Highway, Dayton, OH 45435, USA.

ABSTRACT
The Na(+)K(+)2Cl(-) cotransporter-1 (Slc12a2, NKCC1) is widely distributed and involved in cell volume/ion regulation. Functional NKCC1 locates in the plasma membrane of all cells studied, particularly in the basolateral membrane of most polarized cells. Although the mechanisms involved in plasma membrane sorting of NKCC1 are poorly understood, it is assumed that N-glycosylation is necessary. Here, we characterize expression, N-glycosylation, and distribution of NKCC1 in COS7 cells. We show that ~25% of NKCC1 is complex N-glycosylated whereas the rest of it corresponds to core/high-mannose and hybrid-type N-glycosylated forms. Further, ~10% of NKCC1 reaches the plasma membrane, mostly as core/high-mannose type, whereas ~90% of NKCC1 is distributed in defined intracellular compartments. In addition, inhibition of the first step of N-glycan biosynthesis with tunicamycin decreases total and plasma membrane located NKCC1 resulting in almost undetectable cotransport function. Moreover, inhibition of N-glycan maturation with swainsonine or kifunensine increased core/hybrid-type NKCC1 expression but eliminated plasma membrane complex N-glycosylated NKCC1 and transport function. Together, these results suggest that (i) NKCC1 is delivered to the plasma membrane of COS7 cells independently of its N-glycan nature, (ii) most of NKCC1 in the plasma membrane is core/hybrid-type N-glycosylated, and (iii) the minimal proportion of complex N-glycosylated NKCC1 is functionally active.

No MeSH data available.