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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

Induction of poly-LacNAc structures occurs preferentially on N-glycans.(A–D) T cells isolated from mice of the indicated genotypes were treated for 4 hr with or without 2500 units of PNGase F and analyzed for L-PHA (A and B) or LEA (C and D) binding by flow cytometry, gating on CD4+ cells. ***p<0.001 (unpaired two-tailed t-test with Welch’s (B and D) and Bonferroni correction (D)). 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.14814.006
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fig2s1: Induction of poly-LacNAc structures occurs preferentially on N-glycans.(A–D) T cells isolated from mice of the indicated genotypes were treated for 4 hr with or without 2500 units of PNGase F and analyzed for L-PHA (A and B) or LEA (C and D) binding by flow cytometry, gating on CD4+ cells. ***p<0.001 (unpaired two-tailed t-test with Welch’s (B and D) and Bonferroni correction (D)). 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.14814.006

Mentions: Poly-LacNAc may occur on N-glycans as well as O-glycans and glycolipids (Fukuda et al., 1986; Watanabe et al., 1979). Furthermore, LEA has been reported to bind to high-mannose structures in addition to poly-LacNAc (Oguri, 2005). To investigate the structural basis for the increase in LEA staining, Mgat2 deficient T cells were treated with PNGase F, an amidase which specifically cleaves N –glycans (Maley et al., 1989). PNGase F treatment of live cells incompletely removes N-glycans, with a four hour treatment of Mgat2f/f T cells reducing cell surface L-PHA binding by ~50% (Figure 2—figure supplement 1A–B). Nevertheless, the same treatment resulted in a >80% reduction in LEA binding in Mgat2 deficient T cells, suggesting that the vast majority of LEA staining was due to cell surface N-glycans (Figure 2—figure supplement 1C–D). To further evaluate this question, we directly compared thymocytes derived from Mgat2f/f::Lck-Cre+ and Mgat1f/f::Lck-Cre+ mice (Zhou, 2014). Mgat1 deficiency blocks all branching and poly-LacNAc extension in N-glycans, but not O-glycans or glycolipids. Indeed, unlike Mgat2 deficiency, Mgat1 deficient thymocytes do not show an increase in LEA staining concurrent with the loss in L-PHA staining (Figure 2A). Similarly, SW increases LEA staining in CHO cells but not Mgat1 deficient CHO (Lec1) cells (Figure 2B and C). Furthermore, increased LEA staining induced by SW treatment of T cells was reversed by the mannosidase I inhibitors deoxymannojirimycin (DMN) and kifunensine (kif), which block the N-glycan pathway prior to MGAT1 (Figure 2D).10.7554/eLife.14814.005Figure 2.Branching deficiency induces poly-LacNAc on N-glycans.


Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis
Induction of poly-LacNAc structures occurs preferentially on N-glycans.(A–D) T cells isolated from mice of the indicated genotypes were treated for 4 hr with or without 2500 units of PNGase F and analyzed for L-PHA (A and B) or LEA (C and D) binding by flow cytometry, gating on CD4+ cells. ***p<0.001 (unpaired two-tailed t-test with Welch’s (B and D) and Bonferroni correction (D)). 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.14814.006
© Copyright Policy
Related In: Results  -  Collection

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

fig2s1: Induction of poly-LacNAc structures occurs preferentially on N-glycans.(A–D) T cells isolated from mice of the indicated genotypes were treated for 4 hr with or without 2500 units of PNGase F and analyzed for L-PHA (A and B) or LEA (C and D) binding by flow cytometry, gating on CD4+ cells. ***p<0.001 (unpaired two-tailed t-test with Welch’s (B and D) and Bonferroni correction (D)). 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.14814.006
Mentions: Poly-LacNAc may occur on N-glycans as well as O-glycans and glycolipids (Fukuda et al., 1986; Watanabe et al., 1979). Furthermore, LEA has been reported to bind to high-mannose structures in addition to poly-LacNAc (Oguri, 2005). To investigate the structural basis for the increase in LEA staining, Mgat2 deficient T cells were treated with PNGase F, an amidase which specifically cleaves N –glycans (Maley et al., 1989). PNGase F treatment of live cells incompletely removes N-glycans, with a four hour treatment of Mgat2f/f T cells reducing cell surface L-PHA binding by ~50% (Figure 2—figure supplement 1A–B). Nevertheless, the same treatment resulted in a >80% reduction in LEA binding in Mgat2 deficient T cells, suggesting that the vast majority of LEA staining was due to cell surface N-glycans (Figure 2—figure supplement 1C–D). To further evaluate this question, we directly compared thymocytes derived from Mgat2f/f::Lck-Cre+ and Mgat1f/f::Lck-Cre+ mice (Zhou, 2014). Mgat1 deficiency blocks all branching and poly-LacNAc extension in N-glycans, but not O-glycans or glycolipids. Indeed, unlike Mgat2 deficiency, Mgat1 deficient thymocytes do not show an increase in LEA staining concurrent with the loss in L-PHA staining (Figure 2A). Similarly, SW increases LEA staining in CHO cells but not Mgat1 deficient CHO (Lec1) cells (Figure 2B and C). Furthermore, increased LEA staining induced by SW treatment of T cells was reversed by the mannosidase I inhibitors deoxymannojirimycin (DMN) and kifunensine (kif), which block the N-glycan pathway prior to MGAT1 (Figure 2D).10.7554/eLife.14814.005Figure 2.Branching deficiency induces poly-LacNAc on N-glycans.

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