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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

Inhibition of N-glycan processing or complex N-glycosylation does not preclude NKCC1 plasma membrane localization but impairs its function. ((a) and (d)) Shown are representative immunoblots experiments showing total endogenous expression levels of NKCC1a in COS7 cells incubated with (DMSO, control) 5–10 μg/mL of kifunensine (KIF, (a)) or 1-2 μg/mL swainsonine (SWN, (d)) for 16 h. Note that the expected bands of NKCC1a, that is, ~130 kDa and ~170 kDa, are detected in control and treated cells whereas additional ckNKCC1-immunoreactive bands centered at ~150 kDa are observed only in protein extracts from KIF/SWN-treated cells. As loading control, immunoblots were developed using antibodies directed against β-actin. ((b) and (e)) Shown are representative immunoblots demonstrating expression of EndoH-sensitive hybrid-type N-glycosylated NKCC1a in total protein extracts of COS7 cells treated for 16 hs with KIF (10 μg/mL) or SWN (2 μg/mL). Note the absence of immunoreactive bands corresponding to NKCC1a ~170 kDa in cell extracts obtained from KIF/SWN-treated cells. ((c) and (f)) Shown are representative immunoblots demonstrating expression of NKCC1a in biotinylated plasma membrane fractions of COS7 cells grown under control conditions or treated for 16 h with KIF or SWN. Protein expression of the cytosolic GAPDH was used to assess the purity of the plasma membrane fractions. (g) Densitometry scanning representing the total cellular N-glycan types of NKCC1 in COS7 cells control or treated for 16 h with KIF (10 μg/mL) or SWN (2 μg/mL). (h) Representative densitometry scanning of NKCC1 immunoblots of plasma membrane fractions obtained from COS7 cells control or treated for 16 h with KIF (10 μg/mL) or SWN (2 μg/mL). (i) Cl− uptake into COS7 cells depleted of endogenous Cl− as a function of time (0–60 min). Uptake is represented as nmol/μg protein [mean ± SEM (n = 6)]. Inset: mean Cl− uptake [nmol/μg protein/5 min ± SEM (n = 5)] obtained under isotonic conditions (ISO, black bar) or in the presence of BTD 10 μM (ISO + BTD, white bar). (j) Impact of TUN (2 μg/mL), KIF (10 μg/mL), or SWN (1 μg/mL) treatment (16 hs) on the BTD-sensitive component of Cl− uptake into COS7 cells. Results are expressed as nmol/μg protein/5 min ± SEM (n = 3).
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fig9: Inhibition of N-glycan processing or complex N-glycosylation does not preclude NKCC1 plasma membrane localization but impairs its function. ((a) and (d)) Shown are representative immunoblots experiments showing total endogenous expression levels of NKCC1a in COS7 cells incubated with (DMSO, control) 5–10 μg/mL of kifunensine (KIF, (a)) or 1-2 μg/mL swainsonine (SWN, (d)) for 16 h. Note that the expected bands of NKCC1a, that is, ~130 kDa and ~170 kDa, are detected in control and treated cells whereas additional ckNKCC1-immunoreactive bands centered at ~150 kDa are observed only in protein extracts from KIF/SWN-treated cells. As loading control, immunoblots were developed using antibodies directed against β-actin. ((b) and (e)) Shown are representative immunoblots demonstrating expression of EndoH-sensitive hybrid-type N-glycosylated NKCC1a in total protein extracts of COS7 cells treated for 16 hs with KIF (10 μg/mL) or SWN (2 μg/mL). Note the absence of immunoreactive bands corresponding to NKCC1a ~170 kDa in cell extracts obtained from KIF/SWN-treated cells. ((c) and (f)) Shown are representative immunoblots demonstrating expression of NKCC1a in biotinylated plasma membrane fractions of COS7 cells grown under control conditions or treated for 16 h with KIF or SWN. Protein expression of the cytosolic GAPDH was used to assess the purity of the plasma membrane fractions. (g) Densitometry scanning representing the total cellular N-glycan types of NKCC1 in COS7 cells control or treated for 16 h with KIF (10 μg/mL) or SWN (2 μg/mL). (h) Representative densitometry scanning of NKCC1 immunoblots of plasma membrane fractions obtained from COS7 cells control or treated for 16 h with KIF (10 μg/mL) or SWN (2 μg/mL). (i) Cl− uptake into COS7 cells depleted of endogenous Cl− as a function of time (0–60 min). Uptake is represented as nmol/μg protein [mean ± SEM (n = 6)]. Inset: mean Cl− uptake [nmol/μg protein/5 min ± SEM (n = 5)] obtained under isotonic conditions (ISO, black bar) or in the presence of BTD 10 μM (ISO + BTD, white bar). (j) Impact of TUN (2 μg/mL), KIF (10 μg/mL), or SWN (1 μg/mL) treatment (16 hs) on the BTD-sensitive component of Cl− uptake into COS7 cells. Results are expressed as nmol/μg protein/5 min ± SEM (n = 3).

Mentions: Like in the case of NKCC2 engineered to lack consensus N-glycosylation sites [21–26], precluding N-glycosylation with TUN impairs N-glycan maturation and therefore the role of the latter in plasma membrane targeting cannot be addressed with these kinds of experiments. To determine the role of N-glycan maturation in plasma membrane targeting of endogenous NKCC1, cells were treated with kifunensine (KIF, 5–10 μg/mL), an inhibitor of ER-located mannosidase-I and complex N-glycosylation [51–53]. As shown in Figure 9(a), KIF treatment results in increased expression levels of core/high-mannose and hybrid-type N-glycosylated NKCC1 (Figure 9(g), green trace), as expected for inhibition of ER-mannosidase-I [52, 54]. To confirm the hybrid N-glycan nature of KIF-treated NKCC1, protein extracts obtained from KIF-treated COS7 cells were subjected to enzymatic digestion with EndoH. As shown in Figure 9(b), EndoH treatment results in a nearly complete elimination of NKCC1 bands heavier than ~130 kDa, indicating that KIF impairs NKCC1 complex N-glycosylation. At the plasma membrane level, KIF treatment did not impact core/high-mannose NKCC1 (~130 kDa) expression levels but resulted in almost undetectable complex N-glycosylated NKCC1 and a parallel increase in the hybrid-type form (Figures 9(c) and 9(h), green trace). Therefore, these results suggest that inhibition of complex-type N-glycosylation does not impact plasma membrane location of NKCC1. To further substantiate these findings, expression of the transporter was determined in COS7 cells treated with swainsonine (SWN, 1-2 μg/mL), an inhibitor of medial-Golgi Man2, the enzyme involved in the first biosynthetic step of complex N-glycosylation [55, 56]. As shown in Figure 9(d), SWN increases precursor core/high-mannose and hybrid-type N-glycosylated NKCC1 expression (Figure 9(g), red trace), as expected. Further, as shown in Figure 9(e), EndoH digestion of SWN-treated samples resulted in complete elimination of NKCC1 bands higher than ~130 kDa, but not in control samples where bands of ~170 kDa complex N-glycosylated, EndoH-resistant NKCC1 proteins are observed suggesting complete inhibition of complex N-glycosylation of NKCC1 by SWN. At the plasma membrane level, SWN also eliminated complex N-glycosylated NKCC1 (Figures 9(f) and 9(h)). However, SWN treatment increased the levels of core/high-mannose and hybrid-type N-glycosylated NKCC1 paralleling the increase in their total expression levels. Together, these results confirm that plasma membrane targeting of NKCC1 is independent of 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)

Inhibition of N-glycan processing or complex N-glycosylation does not preclude NKCC1 plasma membrane localization but impairs its function. ((a) and (d)) Shown are representative immunoblots experiments showing total endogenous expression levels of NKCC1a in COS7 cells incubated with (DMSO, control) 5–10 μg/mL of kifunensine (KIF, (a)) or 1-2 μg/mL swainsonine (SWN, (d)) for 16 h. Note that the expected bands of NKCC1a, that is, ~130 kDa and ~170 kDa, are detected in control and treated cells whereas additional ckNKCC1-immunoreactive bands centered at ~150 kDa are observed only in protein extracts from KIF/SWN-treated cells. As loading control, immunoblots were developed using antibodies directed against β-actin. ((b) and (e)) Shown are representative immunoblots demonstrating expression of EndoH-sensitive hybrid-type N-glycosylated NKCC1a in total protein extracts of COS7 cells treated for 16 hs with KIF (10 μg/mL) or SWN (2 μg/mL). Note the absence of immunoreactive bands corresponding to NKCC1a ~170 kDa in cell extracts obtained from KIF/SWN-treated cells. ((c) and (f)) Shown are representative immunoblots demonstrating expression of NKCC1a in biotinylated plasma membrane fractions of COS7 cells grown under control conditions or treated for 16 h with KIF or SWN. Protein expression of the cytosolic GAPDH was used to assess the purity of the plasma membrane fractions. (g) Densitometry scanning representing the total cellular N-glycan types of NKCC1 in COS7 cells control or treated for 16 h with KIF (10 μg/mL) or SWN (2 μg/mL). (h) Representative densitometry scanning of NKCC1 immunoblots of plasma membrane fractions obtained from COS7 cells control or treated for 16 h with KIF (10 μg/mL) or SWN (2 μg/mL). (i) Cl− uptake into COS7 cells depleted of endogenous Cl− as a function of time (0–60 min). Uptake is represented as nmol/μg protein [mean ± SEM (n = 6)]. Inset: mean Cl− uptake [nmol/μg protein/5 min ± SEM (n = 5)] obtained under isotonic conditions (ISO, black bar) or in the presence of BTD 10 μM (ISO + BTD, white bar). (j) Impact of TUN (2 μg/mL), KIF (10 μg/mL), or SWN (1 μg/mL) treatment (16 hs) on the BTD-sensitive component of Cl− uptake into COS7 cells. Results are expressed as nmol/μg protein/5 min ± SEM (n = 3).
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fig9: Inhibition of N-glycan processing or complex N-glycosylation does not preclude NKCC1 plasma membrane localization but impairs its function. ((a) and (d)) Shown are representative immunoblots experiments showing total endogenous expression levels of NKCC1a in COS7 cells incubated with (DMSO, control) 5–10 μg/mL of kifunensine (KIF, (a)) or 1-2 μg/mL swainsonine (SWN, (d)) for 16 h. Note that the expected bands of NKCC1a, that is, ~130 kDa and ~170 kDa, are detected in control and treated cells whereas additional ckNKCC1-immunoreactive bands centered at ~150 kDa are observed only in protein extracts from KIF/SWN-treated cells. As loading control, immunoblots were developed using antibodies directed against β-actin. ((b) and (e)) Shown are representative immunoblots demonstrating expression of EndoH-sensitive hybrid-type N-glycosylated NKCC1a in total protein extracts of COS7 cells treated for 16 hs with KIF (10 μg/mL) or SWN (2 μg/mL). Note the absence of immunoreactive bands corresponding to NKCC1a ~170 kDa in cell extracts obtained from KIF/SWN-treated cells. ((c) and (f)) Shown are representative immunoblots demonstrating expression of NKCC1a in biotinylated plasma membrane fractions of COS7 cells grown under control conditions or treated for 16 h with KIF or SWN. Protein expression of the cytosolic GAPDH was used to assess the purity of the plasma membrane fractions. (g) Densitometry scanning representing the total cellular N-glycan types of NKCC1 in COS7 cells control or treated for 16 h with KIF (10 μg/mL) or SWN (2 μg/mL). (h) Representative densitometry scanning of NKCC1 immunoblots of plasma membrane fractions obtained from COS7 cells control or treated for 16 h with KIF (10 μg/mL) or SWN (2 μg/mL). (i) Cl− uptake into COS7 cells depleted of endogenous Cl− as a function of time (0–60 min). Uptake is represented as nmol/μg protein [mean ± SEM (n = 6)]. Inset: mean Cl− uptake [nmol/μg protein/5 min ± SEM (n = 5)] obtained under isotonic conditions (ISO, black bar) or in the presence of BTD 10 μM (ISO + BTD, white bar). (j) Impact of TUN (2 μg/mL), KIF (10 μg/mL), or SWN (1 μg/mL) treatment (16 hs) on the BTD-sensitive component of Cl− uptake into COS7 cells. Results are expressed as nmol/μg protein/5 min ± SEM (n = 3).
Mentions: Like in the case of NKCC2 engineered to lack consensus N-glycosylation sites [21–26], precluding N-glycosylation with TUN impairs N-glycan maturation and therefore the role of the latter in plasma membrane targeting cannot be addressed with these kinds of experiments. To determine the role of N-glycan maturation in plasma membrane targeting of endogenous NKCC1, cells were treated with kifunensine (KIF, 5–10 μg/mL), an inhibitor of ER-located mannosidase-I and complex N-glycosylation [51–53]. As shown in Figure 9(a), KIF treatment results in increased expression levels of core/high-mannose and hybrid-type N-glycosylated NKCC1 (Figure 9(g), green trace), as expected for inhibition of ER-mannosidase-I [52, 54]. To confirm the hybrid N-glycan nature of KIF-treated NKCC1, protein extracts obtained from KIF-treated COS7 cells were subjected to enzymatic digestion with EndoH. As shown in Figure 9(b), EndoH treatment results in a nearly complete elimination of NKCC1 bands heavier than ~130 kDa, indicating that KIF impairs NKCC1 complex N-glycosylation. At the plasma membrane level, KIF treatment did not impact core/high-mannose NKCC1 (~130 kDa) expression levels but resulted in almost undetectable complex N-glycosylated NKCC1 and a parallel increase in the hybrid-type form (Figures 9(c) and 9(h), green trace). Therefore, these results suggest that inhibition of complex-type N-glycosylation does not impact plasma membrane location of NKCC1. To further substantiate these findings, expression of the transporter was determined in COS7 cells treated with swainsonine (SWN, 1-2 μg/mL), an inhibitor of medial-Golgi Man2, the enzyme involved in the first biosynthetic step of complex N-glycosylation [55, 56]. As shown in Figure 9(d), SWN increases precursor core/high-mannose and hybrid-type N-glycosylated NKCC1 expression (Figure 9(g), red trace), as expected. Further, as shown in Figure 9(e), EndoH digestion of SWN-treated samples resulted in complete elimination of NKCC1 bands higher than ~130 kDa, but not in control samples where bands of ~170 kDa complex N-glycosylated, EndoH-resistant NKCC1 proteins are observed suggesting complete inhibition of complex N-glycosylation of NKCC1 by SWN. At the plasma membrane level, SWN also eliminated complex N-glycosylated NKCC1 (Figures 9(f) and 9(h)). However, SWN treatment increased the levels of core/high-mannose and hybrid-type N-glycosylated NKCC1 paralleling the increase in their total expression levels. Together, these results confirm that plasma membrane targeting of NKCC1 is independent of 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.


Related in: MedlinePlus