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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

A small proportion of total NKCC1 locates in the plasma membrane. (a) Representative NKCC1 immunoblot of equal amounts of total protein extracts and plasma membrane biotinylated fractions obtained from COS7 cells growing under normal conditions. Shown are bands of expected sizes of NKCC1, that is, ~170 kDa and ~130 kDa, in total cellular extracts and in biotinylated plasma membrane fractions. Protein expression of cytosolic GAPDH was used as a loading control for total protein lysates and to assess the purity of biotinylated plasma membrane fractions. (b) Densitometry analysis of immunoblots showing estimated levels of the two main immunobands of NKCC1: core/high-mannose ~130 kDa (green bars) and complex/hybrid ~170 kDa (red bars) in total lysates or biotinylated plasma membrane fractions. Data is expressed as the mean ± SEM (n = 3). (c) Pie-chart representation of the estimated, relative contribution of total NKCC1 to the plasma membrane.
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fig4: A small proportion of total NKCC1 locates in the plasma membrane. (a) Representative NKCC1 immunoblot of equal amounts of total protein extracts and plasma membrane biotinylated fractions obtained from COS7 cells growing under normal conditions. Shown are bands of expected sizes of NKCC1, that is, ~170 kDa and ~130 kDa, in total cellular extracts and in biotinylated plasma membrane fractions. Protein expression of cytosolic GAPDH was used as a loading control for total protein lysates and to assess the purity of biotinylated plasma membrane fractions. (b) Densitometry analysis of immunoblots showing estimated levels of the two main immunobands of NKCC1: core/high-mannose ~130 kDa (green bars) and complex/hybrid ~170 kDa (red bars) in total lysates or biotinylated plasma membrane fractions. Data is expressed as the mean ± SEM (n = 3). (c) Pie-chart representation of the estimated, relative contribution of total NKCC1 to the plasma membrane.

Mentions: It is generally accepted that complex N-glycosylation of NKCCs is necessary for plasma membrane insertion [16, 21, 23–26]. Therefore, the results presented in Figure 3 predict that at least a quarter of total NKCC1 locates in the plasma membrane. To corroborate this prediction, the immunological presence of NKCC1 was determined in total protein extracts and in biotinylated plasma membrane fractions purified from COS7 cells growing under normal conditions. As shown in Figure 4(a), a small relative proportion of NKCC1 bands of ~130 kDa and ~170 kDa were found in the biotinylated fraction indicating that core/high-mannose and hybrid/complex-type N-glycosylated NKCC1 reach the plasma membrane. Of note, the facts that (i) intact cells were biotinylated, collected, lysed, and then affinity-purified with streptavidin-coated agarose beads and (ii) cytosolic GAPDH is minimally detected in blots of purified biotinylated fractions all indicate that NKCC1 of ~130 kDa represents plasma membrane located NKCC1 rather than a contamination from potentially biotinylated intracellular compartments. To estimate the relative contribution of these N-glycans to plasma membrane NKCC1, we performed densitometry analysis of immunoblots loaded with equivalent amounts of protein. As shown in Figures 4(b) and 4(c), ~10% of total NKCC1 is found in the plasma membrane, mostly corresponding to the core/high-mannose or hybrid forms of the transporter. Therefore, these results suggest that the overall N-glycan nature of NKCC1 does not determine its plasma membrane location.


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)

A small proportion of total NKCC1 locates in the plasma membrane. (a) Representative NKCC1 immunoblot of equal amounts of total protein extracts and plasma membrane biotinylated fractions obtained from COS7 cells growing under normal conditions. Shown are bands of expected sizes of NKCC1, that is, ~170 kDa and ~130 kDa, in total cellular extracts and in biotinylated plasma membrane fractions. Protein expression of cytosolic GAPDH was used as a loading control for total protein lysates and to assess the purity of biotinylated plasma membrane fractions. (b) Densitometry analysis of immunoblots showing estimated levels of the two main immunobands of NKCC1: core/high-mannose ~130 kDa (green bars) and complex/hybrid ~170 kDa (red bars) in total lysates or biotinylated plasma membrane fractions. Data is expressed as the mean ± SEM (n = 3). (c) Pie-chart representation of the estimated, relative contribution of total NKCC1 to the plasma membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: A small proportion of total NKCC1 locates in the plasma membrane. (a) Representative NKCC1 immunoblot of equal amounts of total protein extracts and plasma membrane biotinylated fractions obtained from COS7 cells growing under normal conditions. Shown are bands of expected sizes of NKCC1, that is, ~170 kDa and ~130 kDa, in total cellular extracts and in biotinylated plasma membrane fractions. Protein expression of cytosolic GAPDH was used as a loading control for total protein lysates and to assess the purity of biotinylated plasma membrane fractions. (b) Densitometry analysis of immunoblots showing estimated levels of the two main immunobands of NKCC1: core/high-mannose ~130 kDa (green bars) and complex/hybrid ~170 kDa (red bars) in total lysates or biotinylated plasma membrane fractions. Data is expressed as the mean ± SEM (n = 3). (c) Pie-chart representation of the estimated, relative contribution of total NKCC1 to the plasma membrane.
Mentions: It is generally accepted that complex N-glycosylation of NKCCs is necessary for plasma membrane insertion [16, 21, 23–26]. Therefore, the results presented in Figure 3 predict that at least a quarter of total NKCC1 locates in the plasma membrane. To corroborate this prediction, the immunological presence of NKCC1 was determined in total protein extracts and in biotinylated plasma membrane fractions purified from COS7 cells growing under normal conditions. As shown in Figure 4(a), a small relative proportion of NKCC1 bands of ~130 kDa and ~170 kDa were found in the biotinylated fraction indicating that core/high-mannose and hybrid/complex-type N-glycosylated NKCC1 reach the plasma membrane. Of note, the facts that (i) intact cells were biotinylated, collected, lysed, and then affinity-purified with streptavidin-coated agarose beads and (ii) cytosolic GAPDH is minimally detected in blots of purified biotinylated fractions all indicate that NKCC1 of ~130 kDa represents plasma membrane located NKCC1 rather than a contamination from potentially biotinylated intracellular compartments. To estimate the relative contribution of these N-glycans to plasma membrane NKCC1, we performed densitometry analysis of immunoblots loaded with equivalent amounts of protein. As shown in Figures 4(b) and 4(c), ~10% of total NKCC1 is found in the plasma membrane, mostly corresponding to the core/high-mannose or hybrid forms of the transporter. Therefore, these results suggest that the overall N-glycan nature of NKCC1 does not determine its plasma membrane location.

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