Limits...
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.


NKCC1 localizes in cis/medial-Golgi cisternae. ((a)–(c)) Shown are representative images of COS7 cells grown under control conditions or treated for 16 h with 1 μg/mL of brefeldin A (BFA, (d)–(f)). Immunoreactive α-mannosidase II (Man2, (a) and (d)) and NKCC1 ((b) and (e)) were detected by using primary antibodies directed against these proteins and developed using secondary antibodies labeled with FITC (green) and Cy3 (red), respectively. ((c) and (f)) Shown are superimposed images to obtain an overlay representing colocalization of NKCC1 and Man2. The scale bar represents 10 μm. ((g)-(h)) Heat-maps of the squared cells in (c) and (f), respectively, computed by using NIH ImageJ.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4553341&req=5

fig7: NKCC1 localizes in cis/medial-Golgi cisternae. ((a)–(c)) Shown are representative images of COS7 cells grown under control conditions or treated for 16 h with 1 μg/mL of brefeldin A (BFA, (d)–(f)). Immunoreactive α-mannosidase II (Man2, (a) and (d)) and NKCC1 ((b) and (e)) were detected by using primary antibodies directed against these proteins and developed using secondary antibodies labeled with FITC (green) and Cy3 (red), respectively. ((c) and (f)) Shown are superimposed images to obtain an overlay representing colocalization of NKCC1 and Man2. The scale bar represents 10 μm. ((g)-(h)) Heat-maps of the squared cells in (c) and (f), respectively, computed by using NIH ImageJ.

Mentions: To identify these additional CRT-negative intracellular compartments, NKCC1 was coimmunolabeled with Rab11, a small GTPase considered a steady-state marker of trans-Golgi network, secretory granules, constitutive exocytic vesicles, and recycling/sorting endosomes [42, 43]. As shown in Figures 6(a)–6(d), ~90% of endogenous NKCC1 locates in Rab11-positive compartments consistent with the notion that NKCC1 is largely confined to the intracellular space. Moreover, these observations indicate that NKCC1 may constitutively traffic between these Rab11-positive compartments including recycling endosomes, ER and Golgi. Therefore, we tested whether NKCC1 is present in Golgi structures. To this end, we used Man2, a marker of cis/medial-Golgi cisternae [44, 45]. As shown in Figures 7(a)–7(c), NKCC1 appears concentrated in structures immunolabeled with Man2 demonstrating that NKCC1 locates in the cis/medial-Golgi. To add support to this conclusion, Golgi cisternae were collapsed into the ER by using brefeldin A (BFA) [46, 47]. As shown in Figures 7(d)–7(f), BFA treatment resulted in redistribution of NKCC1 and Man2 to the perinuclear region of the cells. Therefore, altogether, these results indicate that the intracellular location of endogenous core/high-mannose and hybrid/complex-type N-glycosylated NKCC1 in ER, Rab11-positive vesicles, and cis/medial-Golgi cisternae reflects its biosynthetic pathway.


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 localizes in cis/medial-Golgi cisternae. ((a)–(c)) Shown are representative images of COS7 cells grown under control conditions or treated for 16 h with 1 μg/mL of brefeldin A (BFA, (d)–(f)). Immunoreactive α-mannosidase II (Man2, (a) and (d)) and NKCC1 ((b) and (e)) were detected by using primary antibodies directed against these proteins and developed using secondary antibodies labeled with FITC (green) and Cy3 (red), respectively. ((c) and (f)) Shown are superimposed images to obtain an overlay representing colocalization of NKCC1 and Man2. The scale bar represents 10 μm. ((g)-(h)) Heat-maps of the squared cells in (c) and (f), respectively, computed by using NIH ImageJ.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4553341&req=5

fig7: NKCC1 localizes in cis/medial-Golgi cisternae. ((a)–(c)) Shown are representative images of COS7 cells grown under control conditions or treated for 16 h with 1 μg/mL of brefeldin A (BFA, (d)–(f)). Immunoreactive α-mannosidase II (Man2, (a) and (d)) and NKCC1 ((b) and (e)) were detected by using primary antibodies directed against these proteins and developed using secondary antibodies labeled with FITC (green) and Cy3 (red), respectively. ((c) and (f)) Shown are superimposed images to obtain an overlay representing colocalization of NKCC1 and Man2. The scale bar represents 10 μm. ((g)-(h)) Heat-maps of the squared cells in (c) and (f), respectively, computed by using NIH ImageJ.
Mentions: To identify these additional CRT-negative intracellular compartments, NKCC1 was coimmunolabeled with Rab11, a small GTPase considered a steady-state marker of trans-Golgi network, secretory granules, constitutive exocytic vesicles, and recycling/sorting endosomes [42, 43]. As shown in Figures 6(a)–6(d), ~90% of endogenous NKCC1 locates in Rab11-positive compartments consistent with the notion that NKCC1 is largely confined to the intracellular space. Moreover, these observations indicate that NKCC1 may constitutively traffic between these Rab11-positive compartments including recycling endosomes, ER and Golgi. Therefore, we tested whether NKCC1 is present in Golgi structures. To this end, we used Man2, a marker of cis/medial-Golgi cisternae [44, 45]. As shown in Figures 7(a)–7(c), NKCC1 appears concentrated in structures immunolabeled with Man2 demonstrating that NKCC1 locates in the cis/medial-Golgi. To add support to this conclusion, Golgi cisternae were collapsed into the ER by using brefeldin A (BFA) [46, 47]. As shown in Figures 7(d)–7(f), BFA treatment resulted in redistribution of NKCC1 and Man2 to the perinuclear region of the cells. Therefore, altogether, these results indicate that the intracellular location of endogenous core/high-mannose and hybrid/complex-type N-glycosylated NKCC1 in ER, Rab11-positive vesicles, and cis/medial-Golgi cisternae reflects its biosynthetic pathway.

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.