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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.


NKCC1a transcripts are expressed in COS7 cells. (a) Representation of the last nine exons (boxes) of the human SLC12A2 gene. Alternative spliced exon 21 is indicated as a grey box in NKCC1a (NM_001046). Introns are represented with V-shaped lines. White boxes represent 3′-UTRs. Underneath, shown are the primer sets used to coamplify NKCC1a and NKCC1b mRNAs (represented as opposite arrowheads connected by a line). These primers are labeled according to the size in bp of the expected amplicons (see text). (b) Shown is a representative 2% agarose gel where bands of predicted sizes for NKCC1a are detected (608 bp and 516 bp). (c) In human brain, bands of predicted sizes for NKCC1a and NKCC1b are coamplified: 608 bp + 560 bp and 516 bp + 468 bp, respectively. As positive control, RT-PCR reactions amplified GAPDH (555 bp). As negative control, RT-PCR reactions were performed using H2O instead of total RNA and GAPDH-555 primer sets. (d) Representative DNA sequence chromatogram corresponding to amplicons obtained with primer set NKCC1-608a/560b encompassing exons 20, 21, and 22 in NKCC1 mRNAs. The sequences obtained from COS7 cells were aligned against chimpanzee (XM_526998) and human NKCC1 transcripts of reference. The sequence difference found in that region is highlighted.
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fig1: NKCC1a transcripts are expressed in COS7 cells. (a) Representation of the last nine exons (boxes) of the human SLC12A2 gene. Alternative spliced exon 21 is indicated as a grey box in NKCC1a (NM_001046). Introns are represented with V-shaped lines. White boxes represent 3′-UTRs. Underneath, shown are the primer sets used to coamplify NKCC1a and NKCC1b mRNAs (represented as opposite arrowheads connected by a line). These primers are labeled according to the size in bp of the expected amplicons (see text). (b) Shown is a representative 2% agarose gel where bands of predicted sizes for NKCC1a are detected (608 bp and 516 bp). (c) In human brain, bands of predicted sizes for NKCC1a and NKCC1b are coamplified: 608 bp + 560 bp and 516 bp + 468 bp, respectively. As positive control, RT-PCR reactions amplified GAPDH (555 bp). As negative control, RT-PCR reactions were performed using H2O instead of total RNA and GAPDH-555 primer sets. (d) Representative DNA sequence chromatogram corresponding to amplicons obtained with primer set NKCC1-608a/560b encompassing exons 20, 21, and 22 in NKCC1 mRNAs. The sequences obtained from COS7 cells were aligned against chimpanzee (XM_526998) and human NKCC1 transcripts of reference. The sequence difference found in that region is highlighted.

Mentions: First-strand cDNA synthesis was initiated with ~1 μg of total RNA, 250 ng of random hexamers, 500 μM of dNTPs, 10 mM of DTT, 40 units of RNase-OUT, and 200 units of SuperScript-III reverse transcriptase (RT) in a final volume of 20 μL at 50°C for 50 min. The thermostable polymerase reaction (PCR) was subsequently performed as described in detail elsewhere [10]. Simultaneous screening of NKCC1a and NKCC1b transcripts in COS7 cells was performed following the strategy developed by Mao et al. [27] and adapted to our cell model. PCR oligonucleotide primer sets were designed using human NKCC1 transcript sequences of reference (RefSeq accession numbers NM_001046 and NM_001256461) as templates. The following sets of primers were used (from 5′ to 3′): NKCC1-516a/468b sense: ATG GAG TAG TGG TTA TTC GCC TAA AAG AAG, NKCC1-516a/468b antisense: TGA TAT CAG AAA AGT CTA TCC GGA ACT TGC; NKCC1-608a/560b sense: ACA TAC AAT ATG GAG TAG TGG TTA TTC GCC, NKCC1-608a/560b antisense: ATG AAG TCT GTA TGG CTC AAT GAT TTC CTC (seeFigure 1(a), for a graphic representation of them). Control RT-PCR reactions were performed by using validated primers for human glyceraldehyde phosphate dehydrogenase (GAPDH, RefSeq accession number NM_002046): GAPDH-555 sense: GTG AAG GTC GGA GTC AAC GGA TTT, GAPDH-555 antisense: CAC AGT CTT CTG GGT GGC AGT GAT [28]. NKCC1 primer design and in silico analysis of sequences obtained were performed using the software package Geneious R7 (Biomatters, Auckland, New Zealand). The nucleotide sequence identity of DNA fragments present in PCR reaction tubes was determined after in situ treatment with ExoSAP-it (Affimetrix/USB, Cleveland, OH) to eliminate excess nucleotides and single stranded DNA. These noncloned but purified amplicons were sequenced by PCR using the same primer sets used to generate them (Beckman Coulter Genomics, Beverly, MA).


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)

NKCC1a transcripts are expressed in COS7 cells. (a) Representation of the last nine exons (boxes) of the human SLC12A2 gene. Alternative spliced exon 21 is indicated as a grey box in NKCC1a (NM_001046). Introns are represented with V-shaped lines. White boxes represent 3′-UTRs. Underneath, shown are the primer sets used to coamplify NKCC1a and NKCC1b mRNAs (represented as opposite arrowheads connected by a line). These primers are labeled according to the size in bp of the expected amplicons (see text). (b) Shown is a representative 2% agarose gel where bands of predicted sizes for NKCC1a are detected (608 bp and 516 bp). (c) In human brain, bands of predicted sizes for NKCC1a and NKCC1b are coamplified: 608 bp + 560 bp and 516 bp + 468 bp, respectively. As positive control, RT-PCR reactions amplified GAPDH (555 bp). As negative control, RT-PCR reactions were performed using H2O instead of total RNA and GAPDH-555 primer sets. (d) Representative DNA sequence chromatogram corresponding to amplicons obtained with primer set NKCC1-608a/560b encompassing exons 20, 21, and 22 in NKCC1 mRNAs. The sequences obtained from COS7 cells were aligned against chimpanzee (XM_526998) and human NKCC1 transcripts of reference. The sequence difference found in that region is highlighted.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig1: NKCC1a transcripts are expressed in COS7 cells. (a) Representation of the last nine exons (boxes) of the human SLC12A2 gene. Alternative spliced exon 21 is indicated as a grey box in NKCC1a (NM_001046). Introns are represented with V-shaped lines. White boxes represent 3′-UTRs. Underneath, shown are the primer sets used to coamplify NKCC1a and NKCC1b mRNAs (represented as opposite arrowheads connected by a line). These primers are labeled according to the size in bp of the expected amplicons (see text). (b) Shown is a representative 2% agarose gel where bands of predicted sizes for NKCC1a are detected (608 bp and 516 bp). (c) In human brain, bands of predicted sizes for NKCC1a and NKCC1b are coamplified: 608 bp + 560 bp and 516 bp + 468 bp, respectively. As positive control, RT-PCR reactions amplified GAPDH (555 bp). As negative control, RT-PCR reactions were performed using H2O instead of total RNA and GAPDH-555 primer sets. (d) Representative DNA sequence chromatogram corresponding to amplicons obtained with primer set NKCC1-608a/560b encompassing exons 20, 21, and 22 in NKCC1 mRNAs. The sequences obtained from COS7 cells were aligned against chimpanzee (XM_526998) and human NKCC1 transcripts of reference. The sequence difference found in that region is highlighted.
Mentions: First-strand cDNA synthesis was initiated with ~1 μg of total RNA, 250 ng of random hexamers, 500 μM of dNTPs, 10 mM of DTT, 40 units of RNase-OUT, and 200 units of SuperScript-III reverse transcriptase (RT) in a final volume of 20 μL at 50°C for 50 min. The thermostable polymerase reaction (PCR) was subsequently performed as described in detail elsewhere [10]. Simultaneous screening of NKCC1a and NKCC1b transcripts in COS7 cells was performed following the strategy developed by Mao et al. [27] and adapted to our cell model. PCR oligonucleotide primer sets were designed using human NKCC1 transcript sequences of reference (RefSeq accession numbers NM_001046 and NM_001256461) as templates. The following sets of primers were used (from 5′ to 3′): NKCC1-516a/468b sense: ATG GAG TAG TGG TTA TTC GCC TAA AAG AAG, NKCC1-516a/468b antisense: TGA TAT CAG AAA AGT CTA TCC GGA ACT TGC; NKCC1-608a/560b sense: ACA TAC AAT ATG GAG TAG TGG TTA TTC GCC, NKCC1-608a/560b antisense: ATG AAG TCT GTA TGG CTC AAT GAT TTC CTC (seeFigure 1(a), for a graphic representation of them). Control RT-PCR reactions were performed by using validated primers for human glyceraldehyde phosphate dehydrogenase (GAPDH, RefSeq accession number NM_002046): GAPDH-555 sense: GTG AAG GTC GGA GTC AAC GGA TTT, GAPDH-555 antisense: CAC AGT CTT CTG GGT GGC AGT GAT [28]. NKCC1 primer design and in silico analysis of sequences obtained were performed using the software package Geneious R7 (Biomatters, Auckland, New Zealand). The nucleotide sequence identity of DNA fragments present in PCR reaction tubes was determined after in situ treatment with ExoSAP-it (Affimetrix/USB, Cleveland, OH) to eliminate excess nucleotides and single stranded DNA. These noncloned but purified amplicons were sequenced by PCR using the same primer sets used to generate them (Beckman Coulter Genomics, Beverly, MA).

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.