16 Citations (Scopus)

Abstract

Glycosylation plays a role in regulating many biological activities, including protein folding and cell surface expression of biomolecules. However, the importance of glycosylation for KCC4 function has not previously been demonstrated. Site-directed mutagenesis was performed on the four putative extracellular N-linked glycosylation sites of KCC4 to determine the role of these sites in KCC4 half-life, cell surface expression, and transporter activity, as well as in KCC4-dependent tumor formation. We showed that triple (N312/331/344/Q) and quadruple (N312/331/344/360/Q) mutations of N-linked glycosylation sites disrupt the N-linked glycosylation of KCC4, resulting in the accumulation of KCC4, predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Further investigation indicated that mutations of the central two (N331/344/Q) N-linked glycosylation sites inhibit the membrane trafficking of KCC4. Our data suggest that the glycan moieties at the N331 and N344 sites were Endo H-resistant, complex-form structures, and that the N312 and N360 sites were Endo H-sensitive, high mannose-containing structures. Under hypotonic stress conditions, the ability to adapt to changes in intracellular chloride ion concentrations and RVD (regulatory volume decrease) activities were less efficient in cells containing the deglycosylated form of KCC4 that were not expressed at the cell surface. Deglycosylated forms of KCC4 also demonstrated decreased tumor formation and lung colonization in mouse xenografts. The difference in glycan complexity may account for the differential impact of each branch on the biological effects of KCC4. We propose that glycosylation is essential for the surface expression, stabilization, and bioactivity of KCC4.

Original languageEnglish
Pages (from-to)1133-1146
Number of pages14
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1833
Issue number5
DOIs
Publication statusPublished - 2013 May 1

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Glycosylation
Membranes
Polysaccharides
Mutation
Protein Folding
Osmotic Pressure
Mannose
Site-Directed Mutagenesis
Heterografts
Endoplasmic Reticulum
Half-Life
Chlorides
Neoplasms
Ions
Lung

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

@article{17d81256699045fba989d143e726c16c,
title = "Glycosylation regulates the function and membrane localization of KCC4",
abstract = "Glycosylation plays a role in regulating many biological activities, including protein folding and cell surface expression of biomolecules. However, the importance of glycosylation for KCC4 function has not previously been demonstrated. Site-directed mutagenesis was performed on the four putative extracellular N-linked glycosylation sites of KCC4 to determine the role of these sites in KCC4 half-life, cell surface expression, and transporter activity, as well as in KCC4-dependent tumor formation. We showed that triple (N312/331/344/Q) and quadruple (N312/331/344/360/Q) mutations of N-linked glycosylation sites disrupt the N-linked glycosylation of KCC4, resulting in the accumulation of KCC4, predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Further investigation indicated that mutations of the central two (N331/344/Q) N-linked glycosylation sites inhibit the membrane trafficking of KCC4. Our data suggest that the glycan moieties at the N331 and N344 sites were Endo H-resistant, complex-form structures, and that the N312 and N360 sites were Endo H-sensitive, high mannose-containing structures. Under hypotonic stress conditions, the ability to adapt to changes in intracellular chloride ion concentrations and RVD (regulatory volume decrease) activities were less efficient in cells containing the deglycosylated form of KCC4 that were not expressed at the cell surface. Deglycosylated forms of KCC4 also demonstrated decreased tumor formation and lung colonization in mouse xenografts. The difference in glycan complexity may account for the differential impact of each branch on the biological effects of KCC4. We propose that glycosylation is essential for the surface expression, stabilization, and bioactivity of KCC4.",
author = "Weng, {Tzu Yu} and Wen-Tai Chiu and Hsiao-Sheng Liu and Hung-Chi Cheng and Meng-Ru Shen and Mount, {David B.} and Cheng-Yang Chou",
year = "2013",
month = "5",
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TY - JOUR

T1 - Glycosylation regulates the function and membrane localization of KCC4

AU - Weng, Tzu Yu

AU - Chiu, Wen-Tai

AU - Liu, Hsiao-Sheng

AU - Cheng, Hung-Chi

AU - Shen, Meng-Ru

AU - Mount, David B.

AU - Chou, Cheng-Yang

PY - 2013/5/1

Y1 - 2013/5/1

N2 - Glycosylation plays a role in regulating many biological activities, including protein folding and cell surface expression of biomolecules. However, the importance of glycosylation for KCC4 function has not previously been demonstrated. Site-directed mutagenesis was performed on the four putative extracellular N-linked glycosylation sites of KCC4 to determine the role of these sites in KCC4 half-life, cell surface expression, and transporter activity, as well as in KCC4-dependent tumor formation. We showed that triple (N312/331/344/Q) and quadruple (N312/331/344/360/Q) mutations of N-linked glycosylation sites disrupt the N-linked glycosylation of KCC4, resulting in the accumulation of KCC4, predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Further investigation indicated that mutations of the central two (N331/344/Q) N-linked glycosylation sites inhibit the membrane trafficking of KCC4. Our data suggest that the glycan moieties at the N331 and N344 sites were Endo H-resistant, complex-form structures, and that the N312 and N360 sites were Endo H-sensitive, high mannose-containing structures. Under hypotonic stress conditions, the ability to adapt to changes in intracellular chloride ion concentrations and RVD (regulatory volume decrease) activities were less efficient in cells containing the deglycosylated form of KCC4 that were not expressed at the cell surface. Deglycosylated forms of KCC4 also demonstrated decreased tumor formation and lung colonization in mouse xenografts. The difference in glycan complexity may account for the differential impact of each branch on the biological effects of KCC4. We propose that glycosylation is essential for the surface expression, stabilization, and bioactivity of KCC4.

AB - Glycosylation plays a role in regulating many biological activities, including protein folding and cell surface expression of biomolecules. However, the importance of glycosylation for KCC4 function has not previously been demonstrated. Site-directed mutagenesis was performed on the four putative extracellular N-linked glycosylation sites of KCC4 to determine the role of these sites in KCC4 half-life, cell surface expression, and transporter activity, as well as in KCC4-dependent tumor formation. We showed that triple (N312/331/344/Q) and quadruple (N312/331/344/360/Q) mutations of N-linked glycosylation sites disrupt the N-linked glycosylation of KCC4, resulting in the accumulation of KCC4, predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Further investigation indicated that mutations of the central two (N331/344/Q) N-linked glycosylation sites inhibit the membrane trafficking of KCC4. Our data suggest that the glycan moieties at the N331 and N344 sites were Endo H-resistant, complex-form structures, and that the N312 and N360 sites were Endo H-sensitive, high mannose-containing structures. Under hypotonic stress conditions, the ability to adapt to changes in intracellular chloride ion concentrations and RVD (regulatory volume decrease) activities were less efficient in cells containing the deglycosylated form of KCC4 that were not expressed at the cell surface. Deglycosylated forms of KCC4 also demonstrated decreased tumor formation and lung colonization in mouse xenografts. The difference in glycan complexity may account for the differential impact of each branch on the biological effects of KCC4. We propose that glycosylation is essential for the surface expression, stabilization, and bioactivity of KCC4.

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U2 - 10.1016/j.bbamcr.2013.01.018

DO - 10.1016/j.bbamcr.2013.01.018

M3 - Article

VL - 1833

SP - 1133

EP - 1146

JO - Biochimica et Biophysica Acta - Molecular Cell Research

JF - Biochimica et Biophysica Acta - Molecular Cell Research

SN - 0167-4889

IS - 5

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