Limits...
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

Intra-Golgi UDP-GlcNAc shifts to later Golgi compartments when use in the medial Golgi is inhibited.(A–C) LC-MS/MS intensity plots of 1.11 ng/ml UDP-GlcNAc standard (A), and untreated (B) and 0.1% triton-X treated (C) Jurkat T cell vesicular fraction. (D) LC-MS/MS quantitation of UDP-GlcNAc levels in untreated versus 0.1% triton-X or 50 mM UMP treated Jurkat T cell vesicular fractions. (E) Jurkat T cells were treated with the indicated concentrations of pyrrophenone for 45 min, followed by the addition of 500 nM swainsonine for an additional 5 hr, at which time cells were analyzed for LEA and ConA binding by flow cytometry. The y-axis indicates percent reversal of the swainsonine-induced change, where 0 corresponds to lectin binding in untreated cells and 100 to swainsonine treatment alone. (F–H) Jurkat T cells were treated with the indicated concentrations of pyrrophenone for 45 min, followed by the addition of 500 nM swainsonine where indicated for an additional 5 hr, after which cells were analyzed for LEA, ConA, and L-PHA binding by flow cytometry. NS, not significant; **p<0.01; ***p<0.001; (unpaired two-tailed t-test with Welch’s (D and F–H) and Bonferroni correction (F–H). Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.DOI:http://dx.doi.org/10.7554/eLife.14814.018
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4940165&req=5

fig6s1: Intra-Golgi UDP-GlcNAc shifts to later Golgi compartments when use in the medial Golgi is inhibited.(A–C) LC-MS/MS intensity plots of 1.11 ng/ml UDP-GlcNAc standard (A), and untreated (B) and 0.1% triton-X treated (C) Jurkat T cell vesicular fraction. (D) LC-MS/MS quantitation of UDP-GlcNAc levels in untreated versus 0.1% triton-X or 50 mM UMP treated Jurkat T cell vesicular fractions. (E) Jurkat T cells were treated with the indicated concentrations of pyrrophenone for 45 min, followed by the addition of 500 nM swainsonine for an additional 5 hr, at which time cells were analyzed for LEA and ConA binding by flow cytometry. The y-axis indicates percent reversal of the swainsonine-induced change, where 0 corresponds to lectin binding in untreated cells and 100 to swainsonine treatment alone. (F–H) Jurkat T cells were treated with the indicated concentrations of pyrrophenone for 45 min, followed by the addition of 500 nM swainsonine where indicated for an additional 5 hr, after which cells were analyzed for LEA, ConA, and L-PHA binding by flow cytometry. NS, not significant; **p<0.01; ***p<0.001; (unpaired two-tailed t-test with Welch’s (D and F–H) and Bonferroni correction (F–H). Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.DOI:http://dx.doi.org/10.7554/eLife.14814.018

Mentions: As B3GNT requires UDP-GlcNAc to generate poly-LacNAc, we reasoned that these two factors must co-localize to drive homeostatic up-regulation of poly-LacNAc following disruption of branching activity. Co-localization may arise from movement of UDP-GlcNAc transporters to the trans Golgi and/or movement of B3GNT to the medial Golgi. However, SW treatment did not alter the Golgi localization of the three UDP-GlcNAc transporters or B3GNT2, arguing against this possibility (Figure 5E and Figure 5—figure supplement 1A–D). Alternatively, UDP-GlcNAc may directly shift from the medial to trans Golgi via the inter-cisternal transport system. Significant loss of activity of medial Golgi branching enzymes should acutely raise UDP-GlcNAc levels within the medial Golgi, with excess UDP-GlcNAc subsequently moving forward to the trans Golgi via inter-cisternal diffusion. Measuring UDP-GlcNAc by LC-MS/MS in a whole cell vesicular fraction isolated from post nuclear supernatant revealed significant levels of UDP-GlcNAc (Figure 6—figure supplement 1A–B). To ensure that UDP-GlcNAc was indeed within the vesicles as opposed to merely associated with the outer membrane, the post nuclear supernatant (PNS) was treated with either 0.1% Triton-X or 50 mM uridine monophosphate (UMP) for 15 min prior to isolation of the vesicular fraction by ultra-centrifugation. Both treatments significantly reduced the amount of UDP-GlcNAc in the vesicular fraction, confirming UDP-GlcNAc was located in vesicles containing UDP-GlcNAc/UMP anti-porters (Figure 6—figure supplement 1B–D).


Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis
Intra-Golgi UDP-GlcNAc shifts to later Golgi compartments when use in the medial Golgi is inhibited.(A–C) LC-MS/MS intensity plots of 1.11 ng/ml UDP-GlcNAc standard (A), and untreated (B) and 0.1% triton-X treated (C) Jurkat T cell vesicular fraction. (D) LC-MS/MS quantitation of UDP-GlcNAc levels in untreated versus 0.1% triton-X or 50 mM UMP treated Jurkat T cell vesicular fractions. (E) Jurkat T cells were treated with the indicated concentrations of pyrrophenone for 45 min, followed by the addition of 500 nM swainsonine for an additional 5 hr, at which time cells were analyzed for LEA and ConA binding by flow cytometry. The y-axis indicates percent reversal of the swainsonine-induced change, where 0 corresponds to lectin binding in untreated cells and 100 to swainsonine treatment alone. (F–H) Jurkat T cells were treated with the indicated concentrations of pyrrophenone for 45 min, followed by the addition of 500 nM swainsonine where indicated for an additional 5 hr, after which cells were analyzed for LEA, ConA, and L-PHA binding by flow cytometry. NS, not significant; **p<0.01; ***p<0.001; (unpaired two-tailed t-test with Welch’s (D and F–H) and Bonferroni correction (F–H). Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.DOI:http://dx.doi.org/10.7554/eLife.14814.018
© Copyright Policy
Related In: Results  -  Collection

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

fig6s1: Intra-Golgi UDP-GlcNAc shifts to later Golgi compartments when use in the medial Golgi is inhibited.(A–C) LC-MS/MS intensity plots of 1.11 ng/ml UDP-GlcNAc standard (A), and untreated (B) and 0.1% triton-X treated (C) Jurkat T cell vesicular fraction. (D) LC-MS/MS quantitation of UDP-GlcNAc levels in untreated versus 0.1% triton-X or 50 mM UMP treated Jurkat T cell vesicular fractions. (E) Jurkat T cells were treated with the indicated concentrations of pyrrophenone for 45 min, followed by the addition of 500 nM swainsonine for an additional 5 hr, at which time cells were analyzed for LEA and ConA binding by flow cytometry. The y-axis indicates percent reversal of the swainsonine-induced change, where 0 corresponds to lectin binding in untreated cells and 100 to swainsonine treatment alone. (F–H) Jurkat T cells were treated with the indicated concentrations of pyrrophenone for 45 min, followed by the addition of 500 nM swainsonine where indicated for an additional 5 hr, after which cells were analyzed for LEA, ConA, and L-PHA binding by flow cytometry. NS, not significant; **p<0.01; ***p<0.001; (unpaired two-tailed t-test with Welch’s (D and F–H) and Bonferroni correction (F–H). Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.DOI:http://dx.doi.org/10.7554/eLife.14814.018
Mentions: As B3GNT requires UDP-GlcNAc to generate poly-LacNAc, we reasoned that these two factors must co-localize to drive homeostatic up-regulation of poly-LacNAc following disruption of branching activity. Co-localization may arise from movement of UDP-GlcNAc transporters to the trans Golgi and/or movement of B3GNT to the medial Golgi. However, SW treatment did not alter the Golgi localization of the three UDP-GlcNAc transporters or B3GNT2, arguing against this possibility (Figure 5E and Figure 5—figure supplement 1A–D). Alternatively, UDP-GlcNAc may directly shift from the medial to trans Golgi via the inter-cisternal transport system. Significant loss of activity of medial Golgi branching enzymes should acutely raise UDP-GlcNAc levels within the medial Golgi, with excess UDP-GlcNAc subsequently moving forward to the trans Golgi via inter-cisternal diffusion. Measuring UDP-GlcNAc by LC-MS/MS in a whole cell vesicular fraction isolated from post nuclear supernatant revealed significant levels of UDP-GlcNAc (Figure 6—figure supplement 1A–B). To ensure that UDP-GlcNAc was indeed within the vesicles as opposed to merely associated with the outer membrane, the post nuclear supernatant (PNS) was treated with either 0.1% Triton-X or 50 mM uridine monophosphate (UMP) for 15 min prior to isolation of the vesicular fraction by ultra-centrifugation. Both treatments significantly reduced the amount of UDP-GlcNAc in the vesicular fraction, confirming UDP-GlcNAc was located in vesicles containing UDP-GlcNAc/UMP anti-porters (Figure 6—figure supplement 1B–D).

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