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.


Related in: MedlinePlus

NKCC1 localizes in the ER of COS7 cells. ((a)-(b)) Representative image of COS7 cells immunolabeled against NKCC1 using T4 (a) or calreticulin (CRT, (b)) antibodies and developed using fluorescently labeled secondary antibodies: Cy3 (NKCC1, red) and FITC (CRT, green). (c) Overlay of (a) and (b). Pictures were superimposed to obtain an image where colocalization could be digitally estimated as yellow pixels (red + green = yellow). Scale bar represents 10 μm. (d) Semiquantitation of red, green, and yellow pixels corresponding to NKCC1, CRT, and NKCC1 + CRT, respectively. Shown are the results obtained from at least 10 cells and represented as mean fluorescence intensity in arbitrary units ± SEM. (e) Colocalization heat-map of the squared cell in (c) computed by using NIH ImageJ. ((f)-(g)) Shown are representative images of COS7 cells grown in the presence of cycloheximide (1 μg/mL) to acutely inhibit protein synthesis and deplete endogenous NKCC1 from the ER. The presence of NKCC1 (f) or CRT (g) was codetected by using the relevant primary antibodies and developed using secondary antibodies labeled with Cy3 (red) or FITC (green) fluorophores. (h) Superimposition of (f) and (g) images to estimate colocalization as heat-maps, as shown in (i).
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4553341&req=5

fig5: NKCC1 localizes in the ER of COS7 cells. ((a)-(b)) Representative image of COS7 cells immunolabeled against NKCC1 using T4 (a) or calreticulin (CRT, (b)) antibodies and developed using fluorescently labeled secondary antibodies: Cy3 (NKCC1, red) and FITC (CRT, green). (c) Overlay of (a) and (b). Pictures were superimposed to obtain an image where colocalization could be digitally estimated as yellow pixels (red + green = yellow). Scale bar represents 10 μm. (d) Semiquantitation of red, green, and yellow pixels corresponding to NKCC1, CRT, and NKCC1 + CRT, respectively. Shown are the results obtained from at least 10 cells and represented as mean fluorescence intensity in arbitrary units ± SEM. (e) Colocalization heat-map of the squared cell in (c) computed by using NIH ImageJ. ((f)-(g)) Shown are representative images of COS7 cells grown in the presence of cycloheximide (1 μg/mL) to acutely inhibit protein synthesis and deplete endogenous NKCC1 from the ER. The presence of NKCC1 (f) or CRT (g) was codetected by using the relevant primary antibodies and developed using secondary antibodies labeled with Cy3 (red) or FITC (green) fluorophores. (h) Superimposition of (f) and (g) images to estimate colocalization as heat-maps, as shown in (i).

Mentions: If half of endogenous NKCC1 is core/high-mannose N-glycosylated (Figure 3), then the transporter locates in the endoplasmic reticulum (ER) in a similar proportion. To determine the immunolocalization pattern of NKCC1 in COS7 cells, we used immunofluorescence microscopy coupled to antibodies directed against calreticulin (CRT), a validated ER marker [41]. As shown in Figures 5(a)–5(c), immunoreactive NKCC1 locates in CRT-positive compartments, confirming ER location of the transporter. To estimate the extent of NKCC1 ER localization, the spatial overlap between NKCC1 (red) and CRT (green) signals in immunofluorescence images was quantified in silico. As shown in Figure 5(d), ~50% of total NKCC1-related signal overlaps with that of CRT, a proportion that correlates the EndoH-sensitive NKCC1 signal obtained in immunoblots (seeFigure 3(b)). From the immunofluorescence data presented in Figures 5(a)–5(c), it is evident that NKCC1 also locates in CRT-negative compartments. To visualize the extrareticular component of NKCC1 localization in COS7 cells, ongoing NKCC1 biosynthesis in the ER was inhibited with a 2 h pulse of cycloheximide (CHX); then, cells were fixed and immunolabeled against CRT. As shown in Figures 5(f)–5(i), inhibition of protein synthesis depletes NKCC1 from CRT-positive compartments evidencing a vesicular, post-ER cytoplasmic pattern of NKCC1 expression in these cells.


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 the ER of COS7 cells. ((a)-(b)) Representative image of COS7 cells immunolabeled against NKCC1 using T4 (a) or calreticulin (CRT, (b)) antibodies and developed using fluorescently labeled secondary antibodies: Cy3 (NKCC1, red) and FITC (CRT, green). (c) Overlay of (a) and (b). Pictures were superimposed to obtain an image where colocalization could be digitally estimated as yellow pixels (red + green = yellow). Scale bar represents 10 μm. (d) Semiquantitation of red, green, and yellow pixels corresponding to NKCC1, CRT, and NKCC1 + CRT, respectively. Shown are the results obtained from at least 10 cells and represented as mean fluorescence intensity in arbitrary units ± SEM. (e) Colocalization heat-map of the squared cell in (c) computed by using NIH ImageJ. ((f)-(g)) Shown are representative images of COS7 cells grown in the presence of cycloheximide (1 μg/mL) to acutely inhibit protein synthesis and deplete endogenous NKCC1 from the ER. The presence of NKCC1 (f) or CRT (g) was codetected by using the relevant primary antibodies and developed using secondary antibodies labeled with Cy3 (red) or FITC (green) fluorophores. (h) Superimposition of (f) and (g) images to estimate colocalization as heat-maps, as shown in (i).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig5: NKCC1 localizes in the ER of COS7 cells. ((a)-(b)) Representative image of COS7 cells immunolabeled against NKCC1 using T4 (a) or calreticulin (CRT, (b)) antibodies and developed using fluorescently labeled secondary antibodies: Cy3 (NKCC1, red) and FITC (CRT, green). (c) Overlay of (a) and (b). Pictures were superimposed to obtain an image where colocalization could be digitally estimated as yellow pixels (red + green = yellow). Scale bar represents 10 μm. (d) Semiquantitation of red, green, and yellow pixels corresponding to NKCC1, CRT, and NKCC1 + CRT, respectively. Shown are the results obtained from at least 10 cells and represented as mean fluorescence intensity in arbitrary units ± SEM. (e) Colocalization heat-map of the squared cell in (c) computed by using NIH ImageJ. ((f)-(g)) Shown are representative images of COS7 cells grown in the presence of cycloheximide (1 μg/mL) to acutely inhibit protein synthesis and deplete endogenous NKCC1 from the ER. The presence of NKCC1 (f) or CRT (g) was codetected by using the relevant primary antibodies and developed using secondary antibodies labeled with Cy3 (red) or FITC (green) fluorophores. (h) Superimposition of (f) and (g) images to estimate colocalization as heat-maps, as shown in (i).
Mentions: If half of endogenous NKCC1 is core/high-mannose N-glycosylated (Figure 3), then the transporter locates in the endoplasmic reticulum (ER) in a similar proportion. To determine the immunolocalization pattern of NKCC1 in COS7 cells, we used immunofluorescence microscopy coupled to antibodies directed against calreticulin (CRT), a validated ER marker [41]. As shown in Figures 5(a)–5(c), immunoreactive NKCC1 locates in CRT-positive compartments, confirming ER location of the transporter. To estimate the extent of NKCC1 ER localization, the spatial overlap between NKCC1 (red) and CRT (green) signals in immunofluorescence images was quantified in silico. As shown in Figure 5(d), ~50% of total NKCC1-related signal overlaps with that of CRT, a proportion that correlates the EndoH-sensitive NKCC1 signal obtained in immunoblots (seeFigure 3(b)). From the immunofluorescence data presented in Figures 5(a)–5(c), it is evident that NKCC1 also locates in CRT-negative compartments. To visualize the extrareticular component of NKCC1 localization in COS7 cells, ongoing NKCC1 biosynthesis in the ER was inhibited with a 2 h pulse of cycloheximide (CHX); then, cells were fixed and immunolabeled against CRT. As shown in Figures 5(f)–5(i), inhibition of protein synthesis depletes NKCC1 from CRT-positive compartments evidencing a vesicular, post-ER cytoplasmic pattern of NKCC1 expression in these cells.

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