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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.


Related in: MedlinePlus

Poly-LacNAc compensation opposes T cell activation and autoimmunity.(A and B) Human T cells were treated as indicated for 72 hr in culture without stimulation to establish baseline differences in glycan expression. Cells were analyzed for L-PHA (top) and LEA (middle) binding by flow cytometry, gating on CD4+ cells (A) or CD8+ cells (B). Cells treated in parallel were then activated with plate bound anti-CD3 for 24 hr and analyzed for CD69 expression (bottom) by flow cytometry, gating on CD4+ cells (A) or CD8+ cells (B). (C) Human T cells were treated as indicated for 72 hr in culture without stimulation to establish baseline differences in glycan expression. Cells were then activated with plate bound anti-CD3 for 24 hr and analyzed for CD69 expression by flow cytometry, gating on CD8+ cells. (D) Disease incidence of EAE in each group. Mice were scored as positive for EAE only after two consecutive days of clinical score ≥1. See also Figure 3F (E) At EAE day 30, splenocytes were isolated from representative mice of each group and activated in vitro for 5 days with 20 μg/ml MOG 35–55 peptide, followed by analysis of cytokine expression by flow cytometry to assay recall responses. **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (E) and Bonferroni correction (E)). Data show one experiment representative of at least three independent experiments (A–C), except EAE (D–E), which was performed once. Error bars indicate mean ± s.e.m.DOI:http://dx.doi.org/10.7554/eLife.14814.010
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fig3s1: Poly-LacNAc compensation opposes T cell activation and autoimmunity.(A and B) Human T cells were treated as indicated for 72 hr in culture without stimulation to establish baseline differences in glycan expression. Cells were analyzed for L-PHA (top) and LEA (middle) binding by flow cytometry, gating on CD4+ cells (A) or CD8+ cells (B). Cells treated in parallel were then activated with plate bound anti-CD3 for 24 hr and analyzed for CD69 expression (bottom) by flow cytometry, gating on CD4+ cells (A) or CD8+ cells (B). (C) Human T cells were treated as indicated for 72 hr in culture without stimulation to establish baseline differences in glycan expression. Cells were then activated with plate bound anti-CD3 for 24 hr and analyzed for CD69 expression by flow cytometry, gating on CD8+ cells. (D) Disease incidence of EAE in each group. Mice were scored as positive for EAE only after two consecutive days of clinical score ≥1. See also Figure 3F (E) At EAE day 30, splenocytes were isolated from representative mice of each group and activated in vitro for 5 days with 20 μg/ml MOG 35–55 peptide, followed by analysis of cytokine expression by flow cytometry to assay recall responses. **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (E) and Bonferroni correction (E)). Data show one experiment representative of at least three independent experiments (A–C), except EAE (D–E), which was performed once. Error bars indicate mean ± s.e.m.DOI:http://dx.doi.org/10.7554/eLife.14814.010

Mentions: To assess the functional consequences of poly-LacNAc up-regulation, we first reversed SW induced poly-LacNAc by blocking all branching using the mannosidase I inhibitors deoxymannojirimycin (DMN) or kifunensine. Whereas SW treatment alone moderately reduced galectin-3 binding, the addition of kifunensine dramatically reduced galectin-3 binding of Jurkat T cells (Figure 3A). As previously shown, SW treatment alone caused significant increases in both anti-CD3 induced activation and proliferation of primary human T cells (Figure 3B,C and Figure 3—figure supplement 1). However, the addition of kifunensine or DMN resulted in much greater hyperactivity, particularly at lower doses of anti-CD3. Careful titration of kifunensine in the presence of SW did not further reduce L-PHA binding, yet caused a dose dependent decrease in LEA binding and increase in CD69 induction, indicating that poly-LacNAc extension dose dependently regulates T cell activation thresholds (Figure 3—figure supplement 1A–B).10.7554/eLife.14814.009Figure 3.Poly-LacNAc compensation opposes T cell activation and autoimmunity.


Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis
Poly-LacNAc compensation opposes T cell activation and autoimmunity.(A and B) Human T cells were treated as indicated for 72 hr in culture without stimulation to establish baseline differences in glycan expression. Cells were analyzed for L-PHA (top) and LEA (middle) binding by flow cytometry, gating on CD4+ cells (A) or CD8+ cells (B). Cells treated in parallel were then activated with plate bound anti-CD3 for 24 hr and analyzed for CD69 expression (bottom) by flow cytometry, gating on CD4+ cells (A) or CD8+ cells (B). (C) Human T cells were treated as indicated for 72 hr in culture without stimulation to establish baseline differences in glycan expression. Cells were then activated with plate bound anti-CD3 for 24 hr and analyzed for CD69 expression by flow cytometry, gating on CD8+ cells. (D) Disease incidence of EAE in each group. Mice were scored as positive for EAE only after two consecutive days of clinical score ≥1. See also Figure 3F (E) At EAE day 30, splenocytes were isolated from representative mice of each group and activated in vitro for 5 days with 20 μg/ml MOG 35–55 peptide, followed by analysis of cytokine expression by flow cytometry to assay recall responses. **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (E) and Bonferroni correction (E)). Data show one experiment representative of at least three independent experiments (A–C), except EAE (D–E), which was performed once. Error bars indicate mean ± s.e.m.DOI:http://dx.doi.org/10.7554/eLife.14814.010
© Copyright Policy
Related In: Results  -  Collection

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

fig3s1: Poly-LacNAc compensation opposes T cell activation and autoimmunity.(A and B) Human T cells were treated as indicated for 72 hr in culture without stimulation to establish baseline differences in glycan expression. Cells were analyzed for L-PHA (top) and LEA (middle) binding by flow cytometry, gating on CD4+ cells (A) or CD8+ cells (B). Cells treated in parallel were then activated with plate bound anti-CD3 for 24 hr and analyzed for CD69 expression (bottom) by flow cytometry, gating on CD4+ cells (A) or CD8+ cells (B). (C) Human T cells were treated as indicated for 72 hr in culture without stimulation to establish baseline differences in glycan expression. Cells were then activated with plate bound anti-CD3 for 24 hr and analyzed for CD69 expression by flow cytometry, gating on CD8+ cells. (D) Disease incidence of EAE in each group. Mice were scored as positive for EAE only after two consecutive days of clinical score ≥1. See also Figure 3F (E) At EAE day 30, splenocytes were isolated from representative mice of each group and activated in vitro for 5 days with 20 μg/ml MOG 35–55 peptide, followed by analysis of cytokine expression by flow cytometry to assay recall responses. **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (E) and Bonferroni correction (E)). Data show one experiment representative of at least three independent experiments (A–C), except EAE (D–E), which was performed once. Error bars indicate mean ± s.e.m.DOI:http://dx.doi.org/10.7554/eLife.14814.010
Mentions: To assess the functional consequences of poly-LacNAc up-regulation, we first reversed SW induced poly-LacNAc by blocking all branching using the mannosidase I inhibitors deoxymannojirimycin (DMN) or kifunensine. Whereas SW treatment alone moderately reduced galectin-3 binding, the addition of kifunensine dramatically reduced galectin-3 binding of Jurkat T cells (Figure 3A). As previously shown, SW treatment alone caused significant increases in both anti-CD3 induced activation and proliferation of primary human T cells (Figure 3B,C and Figure 3—figure supplement 1). However, the addition of kifunensine or DMN resulted in much greater hyperactivity, particularly at lower doses of anti-CD3. Careful titration of kifunensine in the presence of SW did not further reduce L-PHA binding, yet caused a dose dependent decrease in LEA binding and increase in CD69 induction, indicating that poly-LacNAc extension dose dependently regulates T cell activation thresholds (Figure 3—figure supplement 1A–B).10.7554/eLife.14814.009Figure 3.Poly-LacNAc compensation opposes T cell activation and autoimmunity.

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.


Related in: MedlinePlus