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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.


Related in: MedlinePlus

NKCC1 is expressed in COS7 cells as a core/high-mannose, hybrid- and complex-type N-glycosylated protein. ((a)-(b)) Representative immunoblots of protein extracts from COS7 cells grown under basal conditions. Untreated protein extracts (control) or enzymatically digested with PNGaseF (a) or EndoH (b) were immunoblotted and developed using ckNKCC1 coupled to HRP-conjugated secondary antibodies. As loading control, membranes were probed with antibodies against β-actin. (c) Densitometry analysis of immunoblots of proteins control or treated with PNGaseF or EndoH. Shown are the estimated levels of the two main bands of NKCC1, that is, ~170 kDa (red bars) and ~130 kDa (green bars), before and after enzymatic deglycosylation. Data are expressed as means ± SEM (n = 5). (d) Pie-chart representing the approximate relative contribution of core/high-mannose, hybrid- and complex-type N-glycans to total endogenous NKCC1 in COS7 cells. Results are calculated based on the percent of increase in the densitometry signal of bands of ~130 kDa after enzymatic treatment.
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fig3: NKCC1 is expressed in COS7 cells as a core/high-mannose, hybrid- and complex-type N-glycosylated protein. ((a)-(b)) Representative immunoblots of protein extracts from COS7 cells grown under basal conditions. Untreated protein extracts (control) or enzymatically digested with PNGaseF (a) or EndoH (b) were immunoblotted and developed using ckNKCC1 coupled to HRP-conjugated secondary antibodies. As loading control, membranes were probed with antibodies against β-actin. (c) Densitometry analysis of immunoblots of proteins control or treated with PNGaseF or EndoH. Shown are the estimated levels of the two main bands of NKCC1, that is, ~170 kDa (red bars) and ~130 kDa (green bars), before and after enzymatic deglycosylation. Data are expressed as means ± SEM (n = 5). (d) Pie-chart representing the approximate relative contribution of core/high-mannose, hybrid- and complex-type N-glycans to total endogenous NKCC1 in COS7 cells. Results are calculated based on the percent of increase in the densitometry signal of bands of ~130 kDa after enzymatic treatment.

Mentions: Enzymatic deglycosylation coupled to immunoblotting was used to determine the basal N-glycosylation state and global N-glycan nature of endogenous NKCC1 expressed in COS7 cells. To this end, protein lysates were treated with PNGaseF or EndoH, enzymes that cleave all N-linked glycans or high-mannose/hybrid-type, respectively. As shown in Figure 3(a), treatment with PNGaseF resulted in a significant, nearly complete elimination of the ~170 kDa band and a parallel ~2-fold increase in core NKCC1 levels (~130 kDa). These results suggest that at least half of total NKCC1 expressed in COS7 cells is core/high-mannose N-glycosylated. Further, as shown in Figure 3(b), treatment of COS7 cells protein extracts with EndoH resulted in ~50% decrease of the ~170 kDa bands of NKCC1 indicating that half of the total N-glycan composition of NKCC1 is of the core/high-mannose/hybrid-type. To visualize these changes, immunoblots were subjected to densitometry analysis. Figures 3(c) and 3(d) show the estimated relative contribution of core and hybrid- and complex-type N-glycans to the total pool of endogenous NKCC1, a parameter calculated as percentage densitometry increase in the bands of ~130 kDa after deglycosylation. To interpret these results, we applied the following reasoning: if the % densitometry signal of ~130 kDa bands increased twice after PNGaseF treatment, then 50% of total NKCC1 is N-glycosylated. Similarly, if the % densitometry signal of ~170 kDa bands after EndoH treatment was half, then ~25% of total NKCC1 is hybrid-type N-glycosylated. Therefore, our results suggest that ~25% of total NKCC1 expressed in COS7 cells is complex N-glycosylated.


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)

NKCC1 is expressed in COS7 cells as a core/high-mannose, hybrid- and complex-type N-glycosylated protein. ((a)-(b)) Representative immunoblots of protein extracts from COS7 cells grown under basal conditions. Untreated protein extracts (control) or enzymatically digested with PNGaseF (a) or EndoH (b) were immunoblotted and developed using ckNKCC1 coupled to HRP-conjugated secondary antibodies. As loading control, membranes were probed with antibodies against β-actin. (c) Densitometry analysis of immunoblots of proteins control or treated with PNGaseF or EndoH. Shown are the estimated levels of the two main bands of NKCC1, that is, ~170 kDa (red bars) and ~130 kDa (green bars), before and after enzymatic deglycosylation. Data are expressed as means ± SEM (n = 5). (d) Pie-chart representing the approximate relative contribution of core/high-mannose, hybrid- and complex-type N-glycans to total endogenous NKCC1 in COS7 cells. Results are calculated based on the percent of increase in the densitometry signal of bands of ~130 kDa after enzymatic treatment.
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Related In: Results  -  Collection

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fig3: NKCC1 is expressed in COS7 cells as a core/high-mannose, hybrid- and complex-type N-glycosylated protein. ((a)-(b)) Representative immunoblots of protein extracts from COS7 cells grown under basal conditions. Untreated protein extracts (control) or enzymatically digested with PNGaseF (a) or EndoH (b) were immunoblotted and developed using ckNKCC1 coupled to HRP-conjugated secondary antibodies. As loading control, membranes were probed with antibodies against β-actin. (c) Densitometry analysis of immunoblots of proteins control or treated with PNGaseF or EndoH. Shown are the estimated levels of the two main bands of NKCC1, that is, ~170 kDa (red bars) and ~130 kDa (green bars), before and after enzymatic deglycosylation. Data are expressed as means ± SEM (n = 5). (d) Pie-chart representing the approximate relative contribution of core/high-mannose, hybrid- and complex-type N-glycans to total endogenous NKCC1 in COS7 cells. Results are calculated based on the percent of increase in the densitometry signal of bands of ~130 kDa after enzymatic treatment.
Mentions: Enzymatic deglycosylation coupled to immunoblotting was used to determine the basal N-glycosylation state and global N-glycan nature of endogenous NKCC1 expressed in COS7 cells. To this end, protein lysates were treated with PNGaseF or EndoH, enzymes that cleave all N-linked glycans or high-mannose/hybrid-type, respectively. As shown in Figure 3(a), treatment with PNGaseF resulted in a significant, nearly complete elimination of the ~170 kDa band and a parallel ~2-fold increase in core NKCC1 levels (~130 kDa). These results suggest that at least half of total NKCC1 expressed in COS7 cells is core/high-mannose N-glycosylated. Further, as shown in Figure 3(b), treatment of COS7 cells protein extracts with EndoH resulted in ~50% decrease of the ~170 kDa bands of NKCC1 indicating that half of the total N-glycan composition of NKCC1 is of the core/high-mannose/hybrid-type. To visualize these changes, immunoblots were subjected to densitometry analysis. Figures 3(c) and 3(d) show the estimated relative contribution of core and hybrid- and complex-type N-glycans to the total pool of endogenous NKCC1, a parameter calculated as percentage densitometry increase in the bands of ~130 kDa after deglycosylation. To interpret these results, we applied the following reasoning: if the % densitometry signal of ~130 kDa bands increased twice after PNGaseF treatment, then 50% of total NKCC1 is N-glycosylated. Similarly, if the % densitometry signal of ~170 kDa bands after EndoH treatment was half, then ~25% of total NKCC1 is hybrid-type N-glycosylated. Therefore, our results suggest that ~25% of total NKCC1 expressed in COS7 cells is complex N-glycosylated.

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.


Related in: MedlinePlus