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Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis

View Article: PubMed Central - PubMed

ABSTRACT

Essential biological systems employ self-correcting mechanisms to maintain cellular homeostasis. Mammalian cell function is dynamically regulated by the interaction of cell surface galectins with branched N-glycans. Here we report that N-glycan branching deficiency triggers the Golgi to generate bioequivalent N-glycans that preserve galectin-glycoprotein interactions and cellular homeostasis. Galectins bind N-acetyllactosamine (LacNAc) units within N-glycans initiated from UDP-GlcNAc by the medial-Golgi branching enzymes as well as the trans-Golgi poly-LacNAc extension enzyme β1,3-N-acetylglucosaminyltransferase (B3GNT). Marginally reducing LacNAc content by limiting N-glycans to three branches results in T-cell hyperactivity and autoimmunity; yet further restricting branching does not produce a more hyperactive state. Rather, new poly-LacNAc extension by B3GNT maintains galectin binding and immune homeostasis. Poly-LacNAc extension is triggered by redistribution of unused UDP-GlcNAc from the medial to trans-Golgi via inter-cisternal tubules. These data demonstrate the functional equivalency of structurally dissimilar N-glycans and suggest a self-correcting feature of the Golgi that sustains cellular homeostasis.

Doi:: http://dx.doi.org/10.7554/eLife.14814.001

No MeSH data available.


Branching deficiency does not cause relocalization of the UDP-GlcNAc transporters or B3GNT2.(A–E) Jurkat cells were transfected with plasmids containing B3GNT2-HA (A), SLC35A3-DDK (B), SLC35B4-DDK (C), or SLC35D2-DDK (D) and cultured for 24 hr with (A–D) or without swainsonine (E). Cells were then fixed and stained for GM130 (green), TGN46 (blue) and either anti-HA (A), anti-DDK (B–D), or anti-Mannosidase II (E) in red. Shown are representative confocal slices from analyzed cells. The white arrows demonstrate areas deemed suitable for line scan analysis and the histograms (far right) demonstrate signal intensities along the line scans. DAPI staining is shown in grey scale.DOI:http://dx.doi.org/10.7554/eLife.14814.016
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fig5s1: Branching deficiency does not cause relocalization of the UDP-GlcNAc transporters or B3GNT2.(A–E) Jurkat cells were transfected with plasmids containing B3GNT2-HA (A), SLC35A3-DDK (B), SLC35B4-DDK (C), or SLC35D2-DDK (D) and cultured for 24 hr with (A–D) or without swainsonine (E). Cells were then fixed and stained for GM130 (green), TGN46 (blue) and either anti-HA (A), anti-DDK (B–D), or anti-Mannosidase II (E) in red. Shown are representative confocal slices from analyzed cells. The white arrows demonstrate areas deemed suitable for line scan analysis and the histograms (far right) demonstrate signal intensities along the line scans. DAPI staining is shown in grey scale.DOI:http://dx.doi.org/10.7554/eLife.14814.016

Mentions: (A–D) Jurkat T cells were transfected with plasmids containing B3GNT2-HA (A), SLC35A3-DDK (B), SLC35B4-DDK (C), or SLC35D2-DDK (D), cultured for 24 hr and then stained for GM130 (green), TGN46 (blue) and either anti-HA (A) or anti-DDK (B–D) in red. Shown are representative confocal slices from transfected cells. The white arrows demonstrate areas deemed suitable for line scan analysis and the histograms (far right) demonstrate signal intensities along the line scans. DAPI staining is shown in grey scale. (E) Average localizations of the indicated Golgi proteins in Jurkat T cells treated with and without swainsonine (see Figure 5—figure supplement 1) relative to GM130 (cis) and TGN46 (trans) markers. Error bars show standard deviation. (F) Jurkat cells were transfected with mVenus alone or in combination with the four constructs of interest. After 48 hr, cells were analyzed for LEA binding by flow cytometry gating on mVenus- and mVenus+ cells as indicated. NS, not significant; *p<0.05; **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (E and F) and Bonferroni correction (E)). Data show images and histograms from representative cells (A–D), or pooled analysis from 30–40 cells per condition (E). Error bars indicate mean ± S.D. (E) or mean ± s.e.m (F).


Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis
Branching deficiency does not cause relocalization of the UDP-GlcNAc transporters or B3GNT2.(A–E) Jurkat cells were transfected with plasmids containing B3GNT2-HA (A), SLC35A3-DDK (B), SLC35B4-DDK (C), or SLC35D2-DDK (D) and cultured for 24 hr with (A–D) or without swainsonine (E). Cells were then fixed and stained for GM130 (green), TGN46 (blue) and either anti-HA (A), anti-DDK (B–D), or anti-Mannosidase II (E) in red. Shown are representative confocal slices from analyzed cells. The white arrows demonstrate areas deemed suitable for line scan analysis and the histograms (far right) demonstrate signal intensities along the line scans. DAPI staining is shown in grey scale.DOI:http://dx.doi.org/10.7554/eLife.14814.016
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4940165&req=5

fig5s1: Branching deficiency does not cause relocalization of the UDP-GlcNAc transporters or B3GNT2.(A–E) Jurkat cells were transfected with plasmids containing B3GNT2-HA (A), SLC35A3-DDK (B), SLC35B4-DDK (C), or SLC35D2-DDK (D) and cultured for 24 hr with (A–D) or without swainsonine (E). Cells were then fixed and stained for GM130 (green), TGN46 (blue) and either anti-HA (A), anti-DDK (B–D), or anti-Mannosidase II (E) in red. Shown are representative confocal slices from analyzed cells. The white arrows demonstrate areas deemed suitable for line scan analysis and the histograms (far right) demonstrate signal intensities along the line scans. DAPI staining is shown in grey scale.DOI:http://dx.doi.org/10.7554/eLife.14814.016
Mentions: (A–D) Jurkat T cells were transfected with plasmids containing B3GNT2-HA (A), SLC35A3-DDK (B), SLC35B4-DDK (C), or SLC35D2-DDK (D), cultured for 24 hr and then stained for GM130 (green), TGN46 (blue) and either anti-HA (A) or anti-DDK (B–D) in red. Shown are representative confocal slices from transfected cells. The white arrows demonstrate areas deemed suitable for line scan analysis and the histograms (far right) demonstrate signal intensities along the line scans. DAPI staining is shown in grey scale. (E) Average localizations of the indicated Golgi proteins in Jurkat T cells treated with and without swainsonine (see Figure 5—figure supplement 1) relative to GM130 (cis) and TGN46 (trans) markers. Error bars show standard deviation. (F) Jurkat cells were transfected with mVenus alone or in combination with the four constructs of interest. After 48 hr, cells were analyzed for LEA binding by flow cytometry gating on mVenus- and mVenus+ cells as indicated. NS, not significant; *p<0.05; **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (E and F) and Bonferroni correction (E)). Data show images and histograms from representative cells (A–D), or pooled analysis from 30–40 cells per condition (E). Error bars indicate mean ± S.D. (E) or mean ± s.e.m (F).

View Article: PubMed Central - PubMed

ABSTRACT

Essential biological systems employ self-correcting mechanisms to maintain cellular homeostasis. Mammalian cell function is dynamically regulated by the interaction of cell surface galectins with branched N-glycans. Here we report that N-glycan branching deficiency triggers the Golgi to generate bioequivalent N-glycans that preserve galectin-glycoprotein interactions and cellular homeostasis. Galectins bind N-acetyllactosamine (LacNAc) units within N-glycans initiated from UDP-GlcNAc by the medial-Golgi branching enzymes as well as the trans-Golgi poly-LacNAc extension enzyme &beta;1,3-N-acetylglucosaminyltransferase (B3GNT). Marginally reducing LacNAc content by limiting N-glycans to three branches results in T-cell hyperactivity and autoimmunity; yet further restricting branching does not produce a more hyperactive state. Rather, new poly-LacNAc extension by B3GNT maintains galectin binding and immune homeostasis. Poly-LacNAc extension is triggered by redistribution of unused UDP-GlcNAc from the medial to trans-Golgi via inter-cisternal tubules. These data demonstrate the functional equivalency of structurally dissimilar N-glycans and suggest a self-correcting feature of the Golgi that sustains cellular homeostasis.

Doi:: http://dx.doi.org/10.7554/eLife.14814.001

No MeSH data available.