1. Biochemistry and Chemical Biology
  2. Cell Biology
Download icon

Golgi self-correction generates bioequivalent glycans to preserve cellular homeostasis

  1. Haik Mkhikian
  2. Christie-Lynn Mortales
  3. Raymond W Zhou
  4. Khachik Khachikyan
  5. Gang Wu
  6. Stuart M Haslam
  7. Patil Kavarian
  8. Anne Dell
  9. Michael Demetriou  Is a corresponding author
  1. University of California, United States
  2. Imperial College London, United Kingdom
Research Article
  • Cited 25
  • Views 2,091
  • Annotations
Cite this article as: eLife 2016;5:e14814 doi: 10.7554/eLife.14814

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.

https://doi.org/10.7554/eLife.14814.001

eLife digest

Most proteins that are released from cells are modified with sugar molecules that allow the proteins to carry out their role properly. These modifications are called glycans, and are made from sugar subunits joined into chains or branched structures. Investigating how the structure of glycans is linked to their role is complicated by the fact that many different glycans exist, made up of different sugars and arranged into different structures.

Enzymes located in cell compartments known as the endoplasmic reticulum and the Golgi help to build the glycans. For example, the MGAT family of enzymes found in the Golgi generates branched glycans made up of sugar subunits called N-acetyllactosamine (LacNAc). These glycans form part of a molecular mesh on the surface of cells that controls how certain proteins embedded in the cell membrane behave. This is particularly important in immune cells: reducing the number of branches in the glycans weakens the mesh and causes the cells and their membrane proteins to behave inappropriately.

Mkhikian et al. have studied mice that lack specific MGAT enzymes, and so produce LacNAc glycans with drastically fewer branches than normal. Immune cells in these mice had glycans on their surface formed of LacNAc arranged in chains, rather than in short branched structures. These chains turned out to be biologically equivalent to branched LacNAc glycans, containing the same sugar subunits and allowing the immune cells to behave as normal. This suggests that the composition of glycans, rather than their structure, primarily determines their role.

Mkhikian et al. also found that the organization of the enzymes inside the Golgi is likely to be responsible for producing these equivalent glycans. A glycan is built up as it passes through the Golgi, with the branching enzymes located earlier in the Golgi than the extending enzymes. Therefore, if the branching enzymes fail to add LacNAc subunits to the glycan, the extending enzymes can step in later to add the missing components.

Overall, the results presented by Mkhikian et al. indicate that the large number of structurally diverse glycans may be reduced to a much smaller number of glycans with similar roles, based on subunit composition. This will simplify future studies on LacNAc glycans, and further work could focus on defining which other glycan structures share similar roles.

https://doi.org/10.7554/eLife.14814.002

Introduction

Self-correcting mechanisms have evolved to maintain the integrity of critical biological pathways in the face of disruptive insults and stochastic uncertainty. Such mechanisms range from proofreading in DNA replication to functional redundancy of important cellular apparatuses. The galectin-glycoprotein lattice is a dynamic cell surface structure that globally regulates receptor localization and signaling (Demetriou et al., 2001; Partridge et al., 2004; Lau et al., 2007; Dennis et al., 2009; Grigorian et al., 2009). Its importance is underscored by its role in basic cellular processes such as signaling, apoptosis, endocytosis, differentiation, and cell growth, as well as its association with a wide range of diseases including immunity/autoimmunity (Demetriou et al., 2001; Lee et al., 2007; Mkhikian et al., 2011; Li et al., 2013; Wang et al., 2015; Zhou, 2014), cancer (Dennis et al., 1987; Fernandes et al., 1991; Granovsky et al., 2000; Beheshti Zavareh et al., 2012; Croci et al., 2014), and Type 2 diabetes (Ohtsubo et al., 2005; Johswich et al., 2014). However, a mechanism that homeostatically sustains the lattice is not known.

The lattice forms due to the multivalent interactions between extracellular galectins, a family of sugar binding proteins, and the disaccharide N-acetyllactosamine (LacNAc) present on Asn (N)-linked glycans attached to cell surface glycoproteins (Hirabayashi et al., 2002; Brewer et al., 2002; Ahmad et al., 2004). The vast majority of secreted and cell surface proteins are co- or post-translationally modified by the addition of sugars in the ER. As these proteins transit through the ER and Golgi, their glycans undergo dramatic remodeling, generating a vast and heterogeneous array of glycoforms (Kornfeld and Kornfeld, 1985; Schachter, 1991). In the medial Golgi a group of enzymes, MGAT1, 2, 4, and 5, act to produce N-glycans with one, two, three, or four N-acetylglucosamine (GlcNAc) branches (Schachter, 1986). The subsequent addition of galactose by a family of galactosyl transferase enzymes produces the galectin substrate LacNAc (Figure 1—figure supplement 1A). The number of branches depends on the relative activity of the medial Golgi branching enzymes MGAT1, 2, 4, and 5 and the availability of their shared donor substrate UDP-GlcNAc (Lau et al., 2007; Dennis et al., 2009; Grigorian et al., 2007; 2011). Alternating action of β1,3-N-acetylglucosaminyltransferase (B3GNT) and galactosyl transferase enzymes can generate a linear polymer of LacNAc (poly-LacNAc) at any given branch. Although the affinity of galectin binding to a LacNAc monomer is relatively weak, increased LacNAc valency through branching and poly-LacNAc extension can dramatically increase galectin avidity leading to a major impact on cell surface dynamics (Hirabayashi et al., 2002). In T cells for example, galectin - T cell receptor (TCR) interactions directly oppose ligand induced TCR clustering and signaling, thereby negatively regulating T cell development, antigen-dependent T cell growth, and autoimmunity risk.

Glycan analysis of tissues from glycosylation pathway deficient mice has revealed the presence of minor but unusual structures (Stone et al., 2009; Takamatsu et al., 2010; Ismail et al., 2011). The function of these changes is unclear, but some have suggested that the observed structural alterations may reflect production of bioequivalent glycans that are induced by communication between the cell surface and the Golgi (Takamatsu et al., 2010; Dam and Brewer, 2010; Dennis and Brewer, 2013). However, direct evidence supporting this possibility is lacking. Deficiency in the branching enzyme β1,6-N-acetylglucosaminyltransferase V (MGAT5) reduces avidity for galectin, enhancing antigen dependent and independent TCR clustering/signaling, leading to development of spontaneous autoimmune disease (Demetriou et al., 2001; Lee et al., 2007). Based on the current model of the galectin-glycoprotein lattice, more severe reductions in branching should weaken the lattice further and result in greater T cell hyperactivity. Surprisingly, further limiting branching revealed that the Golgi apparatus has a remarkable capacity to buffer challenges to the strength of the galectin-glycoprotein lattice. Our analysis reveals a homeostatic mechanism built into the architecture of the Golgi apparatus that induces bioequivalent poly-LacNAc glycans that act to maintain the function of the galectin-glycoprotein lattice in the face of dysregulated Golgi branching.

Results

Mgat2 deficiency does not increase T cell hyperactivity beyond Mgat5 deficiency

To further investigate the role of branching in T cells, we generated T cell specific Mgat2 deficient mice (Mgat2f/f::Lck-Cre+) (Ye and Marth, 2004). Loss of Mgat2 is expected to limit N-glycans to a single branch, producing hybrid structures; although a second branch via MGAT4 activity is possible (Figure 1—figure supplement 1A). As the branching pathway declines in enzymatic efficiency going from MGAT1 to MGAT5, Mgat2 deficiency also impacts a much greater percentage of cell surface glycans than Mgat5 deletion (Wang et al., 2001). Examination of peripheral T cells from Mgat2f/f::Lck-Cre+ mice indicated loss of Mgat2 in most but not all T cells as assayed by flow cytometry with the plant lectin L-PHA (Phaseolus vulgaris, leukoagglutinin) (Figure 1—figure supplement 1B). β1,6GlcNAc-branched N-glycans produced by MGAT5 specifically bind L-PHA, structures that are also lost following Mgat2 deletion (Demetriou et al., 2001; Cummings and Kornfeld, 1982). Surprisingly, Mgat5 and Mgat2 deficient CD4+ and CD8+ T cells displayed a similar degree of activation and proliferation in response to anti-CD3 (an antibody which induces TCR clustering and signaling) despite the more dramatic reduction in LacNAc branching in Mgat2 deficient T cells (Figure 1A,B,D and E). This suggested that either the β1,6GlcNAc branch produced by the MGAT5 enzyme is uniquely important for regulating T cell activation or that a compensatory mechanism maintains galectin binding when the number of LacNAc branches is reduced. To evaluate for potential differences in total surface LacNAc content between Mgat2 and Mgat5 deficient T cells, galectin-3 binding at the cell surface was measured by flow cytometry. Mgat5 deletion resulted in a significant reduction in the ability of CD4+ and CD8+ T cells to bind galectin-3 (Figure 1C and F), consistent with previously published results (Demetriou et al., 2001). However, Mgat2 deficiency produced no additional decrease in galectin-3 binding (Figure 1C and F), suggesting comparable LacNAc content at the cell surface despite a marked reduction in LacNAc branches in Mgat2 relative to Mgat5 deficient T cells.

Figure 1 with 1 supplement see all
Compensation limits hyperactivity of Mgat2 deficient T cells.

(A, B, D and E) T cells were activated with plate bound anti-CD3 for 24 (A and D) or 72 (B and E) hours. CD4+ (A and B) or CD8+ (D and E) cells were analyzed for CD69 expression (A and D) or 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution (B and E) by flow cytometry, gating on L-PHA- cells where indicated. (C and F) T cells were analyzed for galectin-3 binding by flow cytometry, gating on CD4+ (C) or CD8+ (F) cells and L-PHA- cells where indicated. Normalized geometric mean fluorescence intensity (MFI) is shown. Each mutant was normalized to its control. (G) Thymocytes and splenic T cells were analyzed for L-PHA and LEA binding by flow cytometry. (H) Cells were treated in culture with or without 500 nM SW for 72 hr followed by analysis of LEA binding by flow cytometry. Fold increase in LEA MFI of the SW treated sample compared to the untreated sample is presented. The red line marks one fold or no change. NS, not significant; *p<0.05; **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (C, F and H) and Bonferroni correction (C and F)). Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.

https://doi.org/10.7554/eLife.14814.003

Inhibition of LacNAc branching results in linear extension with poly-LacNAc

Since the branching pathway enzymes act sequentially, we hypothesized that compensatory maintenance of cell surface LacNAc content in Mgat2 deficient T cells would primarily occur by poly-LacNAc extension of the MGAT1 generated branch (Figure 1—figure supplement 1A). To test this prediction, T cells and thymocytes were stained with L-PHA as well as the Lycopersicon Esculentum lectin (LEA). LEA binds to poly-LacNAc structures containing at least three repeating LacNAc units (Kawashima et al., 1990; Merkle and Cummings, 1987). Wild type and Mgat5-/- T cells and thymocytes expressed very low levels of poly-LacNAc. However, in Mgat2f/f::Lck-Cre+ mice, loss of L-PHA staining was accompanied by a ~100 fold increase in LEA staining. The loss of L-PHA binding and the increase in LEA binding appeared to occur concurrently during the double positive stage of thymocyte development, shortly after the Lck promoter driven Cre is first expressed, and were maintained through the single positive stage and in peripheral T cells (Figure 1G). Treatment of various cell types with swainsonine (SW), a mannosidase II inhibitor which blocks N-glycan processing between MGAT1 and MGAT2 (Figure 1—figure supplement 1A), indicated that homeostatic up-regulation of poly-LacNAc was a general feature of many cell types including epithelial, mesenchymal, and hematopoietic cells; with the greatest responses in the latter (Figure 1H).

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).

Figure 2 with 3 supplements see all
Branching deficiency induces poly-LacNAc on N-glycans.

(A) Thymocytes were analyzed for L-PHA and LEA binding by flow cytometry, gating on CD4+CD8+ double positive cells. (B and C) CHO and Lec1 cells were grown in the presence or absence of 500 nM SW for 3 days followed by analysis for L-PHA (B) or LEA (C) binding by flow cytometry. (D) Resting primary human T cells were treated as indicated for 3 days and analyzed for L-PHA (upper) or LEA (lower) binding by flow cytometry, gating on live, non-blasting CD4+ cells. (E-–G) MALDI-TOF analysis of Sialidase A (E) or Endo-β-galactosidase treated N-glycans from Jurkat T cells treated without (F) or with SW (E and G). Hybrid glycans with extended antennae were observed in (E) at m/z 2897, 3102, 3347, 3551 and 4000, which are highlighted with red arrows. The % intensities of the peaks in (F, G) are shown in the brackets after m/z values. The signals at m/z 518, 722 and 1084 are derived from linear poly-LacNAc antennae and are used to represent the length of antennae. The calculation is shown above the spectrum. In addition, a minority of non-sialylated poly-LacNAc antennae were found to be internally fucosylated yielding GlcNAcβ1,3Galβ1,4(Fucα1,3)GlcNAcβ1,3Gal (m/z 1142) and Galβ1,4GlcNAcβ1,3Galβ1,4(Fucα1,3)GlcNAcβ1,3Gal (m/z 1346) upon digestion. MS/MS analysis of the peaks at m/z 1142 and 1346 also revealed the presence of isobaric pauci-mannose glycans, their relative abundances are indicated on the figure (see Figure 2—figure supplement 3). Ions are in the form of M+Na+. Peaks are annotated with putative structures according to the molecular weight, the glycan biosynthetic pathway and for (E), the MALDI-TOF analysis of the N-glycans before Sialidase A treatment (shown in Figure 2—figure supplement 2). NS, not significant; **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (B–D) and Bonferroni correction (D)). Data show one experiment representative of at least three independent experiments (A–D), except mass spectrometry (E–G), which was performed once. Error bars indicate mean ± s.e.m.

https://doi.org/10.7554/eLife.14814.005

Poly-LacNAc content was also investigated using mass spectrometric glycomic methodologies. SW treated Jurkat cells, rather than T cells from Mgat2f/f::Lck-Cre+ mice, were used for this purpose to provide a sufficient amount of starting material for accurate analysis. MALDI-TOF-MS of N-glycans derived from SW treated Jurkat T cells confirmed the presence of hybrid N-glycans with up to two poly-LacNAc extended branches (m/z 2897, 3102, 3347, 3551, and 4000 in Figure 2E, Figure 2—figure supplement 2). This was qualitatively consistent with the presence of poly-LacNAc following SW treatment, but likely under-represents the length of poly-LacNAc extension due to the limited sensitivity of MALDI-TOF-MS for large poly-LacNAc structures. To better quantitate the change in poly-LacNAc, endo-β-galactosidase digestion was used. This enzyme cuts all internal Galβ1,4 linkages in poly-LacNAc structures, unless the GlcNAc on its reducing side is modified by fucose. Endo-β-galactosidase digestion produced GlcNAcβ1,3Gal (m/z 518) from internal linear poly-LacNAc antennae, together with Galβ1,4GlcNAcβ1,3Gal (m/z 722) and NeuAc-Galβ1,4GlcNAcβ1,3Gal (m/z 1084) from their non-reducing termini (Figure 2F,G and Figure 2—figure supplement 3). The ratio of the internal and terminal glycans examines the relative length of linear poly-LacNAc structures, with a higher ratio signifying longer chains. The non-treated cells had a ratio of 0.5, which increased to 1.1 in the SW treated cells (Figure 2F and G), confirming the presence of longer poly-LacNAc chains after SW treatment. Mgat2 deficient T cells are expected to have a greater increase, given the ~100 fold increase in LEA binding compared to the ~20 fold increase in SW treated Jurkat cells. Together, these data indicate that severe LacNAc branching deficiency increases poly-LacNAc structures on N-glycans.

Homeostatic poly-LacNAc opposes T cell hyperactivity and autoimmunity

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).

Figure 3 with 1 supplement see all
Poly-LacNAc compensation opposes T cell activation and autoimmunity.

(A) Jurkat T cells were treated as indicated for 72 hr in culture and analyzed for galectin-3 binding by flow cytometry. (B and C) Human T cells were pre-treated as indicated for 72 hr in culture without stimulation, then activated with plate bound anti-CD3 for 24 (B) or 72 (C) hours and analyzed for CD69 expression (B) or CFSE dilution (C) by flow cytometry, gating on CD4+ cells. (D) Mouse T cells were analyzed for galectin-3 binding by flow cytometry, gating on CD4+ cells. Where indicated, mice were pre-treated for 3 days with 0.2 mg/ml kifunensine in the drinking water. (E) Mouse T cells were activated for 24 hr with plate bound anti-CD3 and analyzed for CD69 expression by flow cytometry, gating on CD4+ cells. Where indicated, mice were pre-treated for 3 days with 0.2 mg/ml kifunensine in the drinking water followed by 10 μM kifunensine during culture. (F) EAE was induced in age matched female C57BL/6 mice treated with or without kifunensine in the drinking water at 0.2 mg/ml from day -3 to 5, with day 0 indicating the time of immunization (n = 9 per group). (G) On day 30, splenocytes were isolated from representative mice of each EAE group and analyzed for cytokine expression by flow cytometry. NS, not significant; *p<0.05; **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (C, D and G) and Bonferroni correction (C, D and G)). Data shown are one experiment representative of at least three independent experiments (A–E), except EAE (F and G), which was performed once. Error bars indicate mean ± s.e.m.

https://doi.org/10.7554/eLife.14814.009

To further confirm the functional role of homeostatic induction of poly-LacNAc, mouse T cells deficient in both Mgat2 and B3gnt2 were generated. B3GNT2 is one of the major B3GNT enzymes responsible for poly-LacNAc branch extension in mouse T cells and poly-LacNAc up-regulation is expected to be limited in its absence (Togayachi et al., 2007). Indeed, T cells from Mgat2f/f::Lck-Cre+::B3gnt2-/- mice showed significantly reduced galectin-3 binding when compared to Mgat2 deficient T cells, confirming reduced LacNAc content (Figure 3D). Directly comparing T cell activation between these lines showed that both genetic and pharmacological inhibition of homeostatic poly-LacNAc extension resulted in significantly increased T cell hyperactivity (Figure 3E).

Since the galectin lattice has been shown to inhibit autoimmunity, we next sought to determine the functional consequences of glycomic homeostasis in regulating the course and severity of experimental autoimmune encephalomyelitis (EAE), a model that mimics the autoimmune CNS pathology of multiple sclerosis (Grigorian et al., 2011). Mgat2f/f and Mgat2f/f::Lck-Cre+ mice were treated with either vehicle or kifunensine in the drinking water from day -3 to 5, with day 0 indicating time of immunization. As expected, vehicle treated Mgat2f/f::Lck-Cre+ mice displayed a significantly more severe EAE than vehicle treated Mgat2f/f mice. However, kifunensine treatment of Mgat2f/f::Lck-Cre+ mice resulted in a dramatic increase in clinical score and disease incidence early in the disease course, a difference that narrowed later in the absence of kifunensine (Figure 3F and Figure 3—supplement figure 1D). Kifunensine treated mice also had more pro-inflammatory TH1 and TH17 cells compared to vehicle treated mice in vivo and following re-stimulation with MOG 35–55 in vitro (Figure 3G, Figure 3—figure supplement 1E). Taken together these data demonstrate a major role for homeostatic poly-LacNAc extension in controlling T cell growth, differentiation, and self-tolerance.

Homeostatic poly-LacNAc is not induced by alterations in enzyme activity

As the galectin-glycoprotein lattice negatively regulates TCR signaling and TCR signaling promotes lattice strength (Demetriou et al., 2001; Chen et al., 2009), we hypothesized that LacNAc homeostasis may result from a feedback loop linking TCR signaling and cell surface LacNAc content. Such a mechanism, which depends on a cell surface sensor of Golgi activity/branching implies a temporal lag phase during which a defect is detected, a signal is sent, and then the Golgi generates the proper response. With this prediction in mind, Jurkat T cells were treated with SW for various times and analyzed for changes in cell surface glycosylation by L-PHA, LEA, and Concanavalin A (ConA), the latter a plant lectin that binds high-mannose structures increased by SW treatment (Figure 4—figure supplement 1A–C). Although the increase in LEA binding trailed slightly behind an increase in ConA binding, it began to increase within ~1.5 hr of SW treatment, indicating an almost immediate compensatory response. Loss of L-PHA staining exhibited the smallest slope, possibly reflecting the preferential cell surface retention of highly branched glycoproteins (Figure 4—figure supplement 1C).

Since the increase in LEA staining exhibited some delay, the role of TCR signaling in driving poly-LacNAc induction was assessed. Blocking TCR signaling genetically, with TCRβ and Lck deficient Jurkat lines, or pharmacologically, with MAP kinase inhibitors, only partially reduced the magnitude of the poly-LacNAc response induced by SW (Figure 4A and Figure 4—figure supplement 1D). More importantly, directly activating downstream TCR signaling with PMA and ionomycin did not induce poly-LacNAc up-regulation, although it further enhanced poly-LacNAc triggered by SW (Figure 4B). Thus, TCR signaling appears to be neither necessary nor sufficient for the homeostatic poly-LacNAc response, but does contribute to its magnitude in the context of deficient branching.

Since most surface receptors are glycosylated, there are a large number of possible additional cell surface sensors that may alter signaling to drive compensation. However, we reasoned that regardless of the upstream components, a mechanism that acts to increase poly-LacNAc production must do so by either increasing the activity of responsible enzymes or increasing substrate supply (UDP-GlcNAc or glycoprotein) for the reaction. It is unlikely that an increase in mono-antennary glycans could account for the observed compensatory response, as all branches are equally likely to be extended with poly-LacNAc (Antonopoulos et al., 2012; Ishida et al., 2005).

Figure 4 with 1 supplement see all
Increased TCR signaling and UDP-GlcNAc levels are neither necessary nor sufficient for poly-LacNAc induction.

(A) WT, TCRβ-/- and Lck-/- Jurkat cells were treated with SW for the indicated times and analyzed for lectin binding by flow cytometry. (B) Jurkat cells were treated as indicated for 24 hr and analyzed for LEA binding by flow cytometry. (C) Lysates of Jurkat T cells treated with SW as indicated were immune-blotted with anti-B3GNT2 and actin. (D) B3GNT enzyme activity was measured in lysates of Jurkat T cells treated with and without SW for 72 hr. (E–G) Total cellular UDP-GlcNAc levels were measured in mouse T cells (E), or Jurkat T cells treated as indicated (F, G) via mass spectrometry (E and G) or a spectrophotometric method (F). Jurkat cells were treated for the indicated times (F) or for 24 hr (G). NS, not significant; *p<0.05; **p<0.01; ***p<0.001 (unpaired two-tailed t-test with Welch’s (B, D, E and G) and Bonferroni correction (B and G)). Data show one experiment representative of at least three independent experiments. Error bars indicate mean ± s.e.m.

https://doi.org/10.7554/eLife.14814.011

Microarray analysis of purified Mgat2+/+ and Mgat2-/- CD4+ T cells revealed no significant changes in glycosylation genes known to impact poly-LacNAc production or UDP-GlcNAc biosynthesis (Table 1). Gene expression was altered in some genes unrelated to glycosylation, but these are likely related to downstream effects of Mgat2 deficiency (Table 2). Comparing B3GNT2 protein levels in Jurkat T cells treated with or without SW also showed no difference (Figure 4C). Total poly-LacNAc enzyme activity in lysates from control and SW treated Jurkat T cells also revealed no difference (Figure 4D). Thus, increased B3GNT enzyme activity is not responsible for the homeostatic up-regulation of poly-LacNAc induced by branching deficiency.

Table 1

List of N-glycan branching, poly-LacNAc production, and hexosamine pathway genes.

https://doi.org/10.7554/eLife.14814.013
Gene symbolGene descriptionp ValueFold change
Man1amannosidase 1, alpha0.36021.0628
Man1a2mannosidase, alpha, class 1A, member 20.4885-1.0551
Man1b1mannosidase, alpha, class 1B, member 10.4397-1.0627
Man1c1mannosidase, alpha, class 1C, member 10.40531.0748
Man2a1mannosidase 2, alpha 10.8150-1.0168
Man2a2mannosidase 2, alpha 20.3647-1.0787
Man2b1mannosidase 2, alpha B10.0800-1.1350
Man2b2mannosidase 2, alpha B20.64651.0331
Man2c1mannosidase, alpha, class 2C, member 10.98261.0016
Mgat1mannoside acetylglucosaminyltransferase 10.1005-1.1846
Mgat2mannoside acetylglucosaminyltransferase 20.0000-27.8780
Mgat3mannoside acetylglucosaminyltransferase 30.17571.1377
Mgat4amannoside acetylglucosaminyltransferase 4, isoenzyme A0.1486-1.2068
Mgat4bmannoside acetylglucosaminyltransferase 4, isoenzyme B0.22481.1345
Mgat4cmannosyl (alpha-1,3-)-glycoprotein beta-1,4-N-acetylglucosaminyltransferase, isozyme C (putative)0.56951.0539
Mgat5mannoside acetylglucosaminyltransferase 50.40451.0666
Mgat5bmannoside acetylglucosaminyltransferase 5, isoenzyme B0.14501.1347
B3gnt1UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 10.5597-1.0587
B3gnt2UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 20.15031.1219
B3gnt3UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 30.40311.1072
B3gnt4UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 40.29231.1100
B3gnt5UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 50.17581.1742
B3gnt6UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 6 (core 3 synthase)0.55651.0511
B3gnt7UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 70.80281.0237
B3gnt8UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 80.61571.0443
B3gnt9-psUDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 9, pseudogene0.28411.0856
B3gntl1UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase-like 10.7979-1.0241
B4galt1UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 10.80031.0166
B4galt2UDP-Gal:betaGlcNAc beta 1,4- galactosyltransferase, polypeptide 20.10231.1487
B4galt3UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 30.6938-1.0304
B4galt4UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 40.13781.1624
B4galt5UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 50.83801.0131
B4galt6UDP-Gal:betaGlcNAc beta 1,4-galactosyltransferase, polypeptide 60.0645-1.2196
B4galt7xylosylprotein beta1,4-galactosyltransferase, polypeptide 7 (galactosyltransferase I)0.4116-1.0764
Gckglucokinase0.05481.1856
Hk1hexokinase 10.85931.0129
Hk2hexokinase 20.46881.0771
Hk3hexokinase 30.0236-1.2237
AdpgkADP-dependent glucokinase0.4033-1.0787
Gpi1glucose phosphate isomerase 10.7826-1.0173
Gfpt1glutamine fructose-6-phosphate transaminase 10.2410-1.0941
Gfpt2glutamine fructose-6-phosphate transaminase 20.02891.2116
Gnpda1glucosamine-6-phosphate deaminase 10.6667-1.0364
Gnpda2glucosamine-6-phosphate deaminase 20.94741.0054
Gnpnat1glucosamine-phosphate N-acetyltransferase 10.3646-1.0839
Nat9N-acetyltransferase 9 (GCN5-related, putative)0.22531.0995
Pgm1phosphoglucomutase 10.7697-1.0243
Pgm2phosphoglucomutase 20.7135-1.0360
Pgm2l1phosphoglucomutase 2-like 10.8036-1.0167
Pgm3phosphoglucomutase 30.6032-1.0529
Pgm5phosphoglucomutase 50.23741.1326
Uap1UDP-N-acetylglucosamine pyrophosphorylase 10.65551.0402
Uap1l1UDP-N-acteylglucosamine pyrophosphorylase 1-like 10.2637-1.1092
NagkN-acetylglucosamine kinase0.2907-1.1126
Galegalactose-4-epimerase, UDP0.51961.0579
Gneglucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase0.2860-1.0808
  1. Gene symbol, gene description, p value and fold change of Mgat2 deficient CD4+ T cells relative to control are shown. The microarray confirms loss of Mgat2 expression, highlighted in bold.

Table 2

List of the top 50 differentially expressed genes.

https://doi.org/10.7554/eLife.14814.014
Gene symbolGene descriptionp ValueFold change
Rpl36alribosomal protein L36A-like4.44E-16-15.6258
Mgat2mannoside acetylglucosaminyltransferase 29.43E-14-27.8780
Fn1fibronectin 15.83E-12-7.5845
Cpmcarboxypeptidase M3.54E-103.1233
Zbtb16zinc finger and BTB domain containing 165.45E-10-3.3239
Mmp9matrix metallopeptidase 91.58E-09-3.9658
Serpinb10-psserine (or cysteine) peptidase inhibitor, clade B (ovalbumin), member 10, pseudogene4.39E-09-5.0634
Zfp69zinc finger protein 694.63E-09-2.1283
Cd93CD93 antigen1.04E-08-2.3236
Atp8a2ATPase, aminophospholipid transporter-like, class I, type 8A, member 22.64E-08-2.2711
Gpr141G protein-coupled receptor 1413.83E-08-3.4035
Penkpreproenkephalin5.28E-083.8411
Clec7aC-type lectin domain family 7, member a8.21E-08-2.4336
Lyz1lysozyme 11.06E-07-3.4287
Mpeg1macrophage expressed gene 11.18E-07-4.3408
Klra2killer cell lectin-like receptor, subfamily A, member 21.33E-07-4.4037
Kitkit oncogene2.44E-07-2.3150
Plbd1phospholipase B domain containing 12.47E-07-2.9529
Lyz2lysozyme 22.83E-07-3.6156
Pld4phospholipase D family, member 43.00E-07-4.1375
Aceangiotensin I converting enzyme (peptidyl-dipeptidase A) 13.36E-07-3.3758
Sirpasignal-regulatory protein alpha3.40E-07-3.9975
Clec12aC-type lectin domain family 12, member a3.54E-07-4.4547
Tlr13toll-like receptor 134.15E-07-6.5477
Tnfrsf21tumor necrosis factor receptor superfamily, member 214.86E-07-3.4333
Tgfbitransforming growth factor, beta induced5.57E-07-3.6315
Adrbk2adrenergic receptor kinase, beta 25.98E-07-2.0416
Il12rb2interleukin 12 receptor, beta 27.32E-07-2.6927
Csf1rcolony stimulating factor 1 receptor7.57E-07-5.9870
Xcl1chemokine (C motif) ligand 19.55E-07-2.0969
Mlh1mutL homolog 1 (E. coli)9.66E-07-2.0854
Sulf2sulfatase 21.01E-06-2.9791
Igsf6immunoglobulin superfamily, member 61.04E-06-3.0615
Gpr56G protein-coupled receptor 561.10E-06-2.2352
Fcnaficolin A1.23E-06-4.7706
Clec4a1C-type lectin domain family 4, member a11.29E-06-5.5202
Gm11428predicted gene 114281.33E-06-2.5636
Kcnj16potassium inwardly-rectifying channel, subfamily J, member 161.35E-06-2.5828
Coro2acoronin, actin binding protein 2A1.80E-061.5820
Clec4a3C-type lectin domain family 4, member a131.88E-06-4.7329
Muc13mucin 13, epithelial transmembrane1.91E-06-2.0524
Abcd2ATP-binding cassette, sub-family D (ALD), member 22.49E-06-2.2046
Cd68CD68 antigen2.56E-06-2.6229
Cd34CD34 antigen2.65E-06-1.9429
Cxcr6Sigmachemokine (C-X-C motif) receptor 62.68E-06-3.3244
Mpomyeloperoxidase2.80E-06-15.0742
Car1carbonic anhydrase 12.95E-06-10.5252
Pira11paired-Ig-like receptor A113.03E-06-3.3245
Emr1EGF-like module containing, mucin-like, hormone receptor-like sequence 13.18E-06-6.1010
Pira1paired-Ig-like receptor A13.23E-06-3.6538
  1. Gene symbol, gene description, p value and fold change of Mgat2 deficient CD4+ T cells relative to control are shown. Data are ordered by p value.

Homeostatic poly-LacNAc is not induced by alterations in UDP-GlcNAc levels

Next we investigated whether increased cellular UDP-GlcNAc, the donor substrate for B3GNT enzymes, is triggered by severe branching deficiency. Measurement of total cellular UDP-GlcNAc levels by mass spectrometry and/or a colorimetric assay in cell lysates of purified Mgat2+/+ versus Mgat2-/- T cells as well as Jurkat T cells treated with or without SW and kifunensine revealed no increase from branching deficiency (Figure 4E–G). Supplementing cells with GlcNAc raises cellular UDP-GlcNAc and branching (Lau et al., 2007; Mkhikian et al., 2011; Grigorian et al., 2007; Grigorian et al., 2011). In contrast, treatment of Jurkat T cells with GlcNAc did not increase LEA staining despite a 3–4 fold increase in cellular UDP-GlcNAc concentrations; although it further enhanced SW induced poly-LacNAc extension (Figure 4F,G, and Figure 4—figure supplement 1E). Over-expression of the three UDP-GlcNAc Golgi transporters (SLC35A3, SLC35B4, and SLC35D2) via transfection into Jurkat T cells also did not increase LEA staining (Figure 5F). Thus, much like TCR signaling, increased cellular UDP-GlcNAc levels are neither necessary nor sufficient to induce homeostatic up-regulation of poly-LacNAc. Not surprisingly, poly-LacNAc induction was blocked by 4-Fluoro-GlcNAc, a drug known to inhibit poly-LacNAc production by reducing UDP-GlcNAc biosynthesis (Barthel et al., 2011) (Figure 4—figure supplement 1F–G).

Figure 5 with 1 supplement see all
The UDP-GlcNAc transporters are localized to an earlier Golgi compartment than B3GNT2.

(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).

https://doi.org/10.7554/eLife.14814.015

UDP-GlcNAc redistribution from Cis/Medial to Trans Golgi triggers homeostatic poly-LacNAc

Glycosylation enzymes are organized in order of action from cis to trans along the secretory pathway. The branching enzymes (i.e. MGAT1, 2, 4 and 5) are localized to the medial Golgi, while the galactosyl transferase and B3GNT enzymes reside in the trans Golgi. The CMP-sialic acid transporter has a relatively restricted localization within the Golgi (Zhao et al., 2006), suggesting that sugar-nucleotide donors may be preferentially supplied to specific Golgi compartments. We hypothesized that UDP-GlcNAc supply and associated transporters may be restricted to the cis/medial Golgi compartment, thus preferentially driving branching over extension. Consistent with this hypothesis, raising total cellular UDP-GlcNAc increases branching by the medial Golgi enzymes (Lau et al., 2007; Mkhikian et al., 2011; Grigorian et al., 2007; Grigorian et al., 2011), but does not induce significant up-regulation of poly-LacNAc extension by trans Golgi B3GNT enzymes (Figure 4F,G, and Figure 4—figure supplement 1E). This suggests that cytosolic UDP-GlcNAc lacks direct access to the trans Golgi compartment. To further evaluate this hypothesis, we examined the subcellular localization of the three UDP-GlcNAc Golgi transporters (SLC35A3, SLC35B4, and SLC35D2) and B3GNT2. Jurkat T cells were transfected with DDK or HA tagged versions of these proteins and examined for intra-Golgi localization using confocal microscopy-based line scan analysis. Intra-Golgi localization was determined by co-staining transfected cells with the cis and trans markers GM130 and TGN46, respectively, and determining the peak intensity of fluorescence along a line scan relative to these markers (Dejgaard et al., 2007). All three UDP-GlcNAc transporters localized to the cis and medial Golgi compartments while B3GNT2 localized to a later Golgi compartment (Figure 5A–E). In addition, overexpression of the UDP-GlcNAc transporters did not drive poly-LacNAc production (Figure 5F). These results suggest that the trans Golgi is comparatively deficient in UDP-GlcNAc, thereby limiting B3GNT activity and poly-LacNAc extension under steady state conditions.

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).

To evaluate the effects of reduced branching activity in the medial Golgi, we compared Jurkat T cells treated with and without kifunensine. Consistent with our hypothesis, UDP-GlcNAc levels were elevated in the vesicular but not the cytosolic fractions of kifunensine treated Jurkat T cells (Figure 6A). To examine the Golgi directly, we used antibodies to the trans marker TGN46 to immuno-isolate a Golgi enriched fraction from Jurkat T cell PNS. As expected from the physical connections between Golgi cisterna, western blotting for cis, medial, and trans Golgi markers confirmed that all three cisternal compartments were recovered with anti-TGN46 (Figure 6B). UDP-GlcNAc was elevated in the Golgi of kifunensine treated Jurkat T cells despite no difference in total cellular or cytosolic UDP-GlcNAc levels (Figures 4G,6A,C). Finally, we isolated a fraction enriched for trans-Golgi network (TGN) vesicles from Jurkat T cells by pretreating with Brefeldin A prior to pull down with anti-TGN46. Brefeldin A separates the trans-Golgi network from the rest of the Golgi by fusing the latter with the endoplasmic reticulum (Martínez-Alonso et al., 2013). TGN46 immuno-isolated vesicles from Brefeldin A treated Jurkat T cells were partially depleted for cis and medial Golgi markers relative to trans Golgi markers including B3GNT2, confirming relative enrichment of the trans/TGN compartment (Figure 6D and E). Vesicles from kifunensine treated Jurkat T cells demonstrated increased UDP-GlcNAc relative to control (Figure 6F), indicating that UDP-GlcNAc levels rise in the trans Golgi when medial Golgi branching activity is significantly diminished.

Figure 6 with 1 supplement see all
Intra-Golgi UDP-GlcNAc shifts to later Golgi compartments when use in the medial Golgi is inhibited.

(A) LC-MS/MS quantitation of UDP-GlcNAc in post-nuclear supernatants (PNS), vesicular, and cytosolic fractions of kifunensine treated and untreated Jurkat T cells. (B) PNS (input) from kifunensine treated and untreated Jurkat T cells was used for Golgi enrichment via anti-TGN46 immuno-isolation followed by blotting for the Golgi compartment markers GM130 (cis), GS27 (medial/trans), and Syntaxin6 (trans). (C) LC-MS/MS quantitation of UDP-GlcNAc in anti-TGN46 bound and unbound fractions from kifunensine treated and untreated Jurkat T cell PNS. (D) PNS of Jurkat T cells treated for 0, 15, and 30 min with the Golgi disruptor Brefeldin A were used for anti-TGN46 immuno-isolation followed by blotting for B3GNT2 and the GM130 (cis), GS28 (cis/medial), and TGN46 (trans) Golgi markers. (E) Quantitation of D, (F) LC-MS/MS quantitation of UDP-GlcNAc in anti-TGN46 immuno-isolates from Jurkat cells treated with Brefeldin A for 15 min. (G) LEA flow cytometric analysis of Jurkat T cells pre-treated with nocodazole where indicated for 45 min, followed by swainsonine where indicated for 5 hr. (H) Jurkat T cells that were treated with nocodazole +/- pyrrophenone as indicated for 45 min were treated with or without swainsonine for 5 hr and then analyzed for LEA and ConA binding by flow cytometry. Shown is the ratio of LEA MFI to ConA MFI for each condition. NS, not significant; *p<0.05; **p<0.01; ***p<0.001; (unpaired one-tailed (A, C, F and G) or two-tailed (H) t-test with Welch’s correction and Bonferroni correction (H)). Data show one experiment representative of at least three independent experiments except F which shows combined data from two independent experiments. Error bars indicate mean ± s.e.m.

https://doi.org/10.7554/eLife.14814.017

Although the mechanism for cargo transport between Golgi cisterna is incompletely understood, the prevailing model is cisternal maturation, where entire cisterna move forward with their cargo, while Golgi enzymes/transporters are pulled back to earlier cisterna by vesicular transport (Glick and Luini, 2011). However, this model has recently been expanded by data suggesting that small cargo transits the Golgi by diffusion via inter-cisternal tubules that vertically connect the cis, medial, and trans compartments (Martínez-Alonso et al., 2013; Beznoussenko et al., 2014; Trucco et al., 2004). Indeed, at least in CHO cells, there is evidence for functional continuity throughout sub-compartments of the Golgi (Kim et al., 2001). Golgi stacks, consisting of the cis, medial, and trans compartments, are also connected longitudinally to other Golgi stacks by tubules. Nocodazole, which interrupts longitudinal but not vertical Golgi tubules, had no effect on poly-LacNAc induction by SW in Jurkat T cells (Figure 6G). In contrast, pyrrophenone disrupts both longitudinal and inter-cisternal vertical tubules via inhibition of cytosolic phospholipase A2-α (San Pietro et al., 2009). Pyrrophenone significantly blocked SW induced up-regulation of poly-LacNAc in Jurkat T cells in a dose dependent manner, as indicated by comparing its effects on LEA and ConA staining; the latter controlling for any changes in Golgi transport (Figure 6H and Figure 6—figure supplement 1E). In contrast, pyrrophenone had no effect on ConA, LEA, or L-PHA levels in control Jurkat T cells lacking SW (Figure 6—supplement figure 1F–H). We conclude that when branching enzymes under-utilize UDP-GlcNAc in the medial Golgi, UDP-GlcNAc accumulates and then shifts by diffusion to the trans Golgi via inter-cisternal tubules, thereby increasing substrate supply to B3GNT and triggering poly-LacNAc extension (Figure 7). However, our data do not exclude additional transfer of UDP-GlcNAc via vesicles or by cisternal maturation.

Model of an inherent Golgi self-correcting mechanism to maintain LacNAc homeostasis.

The majority of UDP-GlcNAc entering the Golgi is supplied to its early compartments by the UDP-GlcNAc/UMP antiporters, which are preferentially localized to the cis/medial Golgi. Under branching proficient conditions (A), UDP-GlcNAc is used by the branching enzymes MGAT1, 2, 4, and 5, with little unused UDP-GlcNAc supplying the poly-LacNAc synthesizing B3GNT enzymes. The resulting array of N-glycans produced thus contains more LacNAc branches than linear LacNAc polymers (A). When the branching pathway is disrupted (B), or presumably the Golgi is otherwise stressed, leading to reduced UDP-GlcNAc usage in the medial Golgi, UDP-GlcNAc is driven forward (at least partially through intercisternal tubules) and promotes production of bioequivalent poly-LacNAc containing glycans by the trans Golgi-resident B3GNT family of enzymes. Under this scenario, loss of LacNAc branches is balanced by increased production of linear LacNAc polymers, a self-correcting ability that serves to maintain cell surface LacNAc density and thus the galectin-glycoprotein lattice (A). In the context of T cells, this homeostatic mechanism acts to curtail catastrophic T cell hyperactivity and promotes self-tolerance.

https://doi.org/10.7554/eLife.14814.019

Discussion

Our data demonstrates that structurally disparate glycans are functionally equivalent based on LacNAc content and suggests a novel self-correcting mechanism in the Golgi that sustains cellular homeostasis by ensuring LacNAc content is maintained at a minimal level within N-glycans. The complexity of glycan structures provides tremendous challenges for discerning the functional role of glycans in biology. Although N-glycan structures are highly diverse, redundancy of structural motifs within individual glycans has previously received little consideration as a means to collapse complexity and decipher glycan information. Deficiency of glycosylation pathway genes often give rise to new and unusual structures, triggering some to suggest that these may represent compensatory structures (Stone et al., 2009; Takamatsu et al., 2010; Ismail et al., 2011; Dam and Brewer, 2010; Dennis and Brewer, 2013). However, proof of functional equivalency has largely been lacking. The similar degree of hyperactivity between Mgat5 and Mgat2 deficient T cells indicates that poly-LacNAc extended hybrid structures are functionally equivalent to complex tri-antennary structures. Importantly, blockade of mannosidase II and Mgat2 deficiency produced a similar phenotype, which was blocked/reversed by both mannosidase I inhibition and Mgat1 deficiency, despite each targeting distinct biochemical steps and resulting in distinct structures. Thus, our data demonstrate that structurally diverse glycans are biologically equivalent if they share a similar amount of LacNAc. The overall glycan structure sets the total LacNAc content, thereby largely determining avidity of binding for galectins. It should be noted however, that although these differing glycoforms are clearly overlapping, they might not be strictly identical due to distinct geometries of LacNAc presentation or modification by fucose or sialic acid.

Our data support a model of N-glycosylation that emphasizes the significance of functional units within an N-glycan, such as LacNAc groups, rather than uniqueness of the overall glycan structure in determining biological function. The National Academy of Sciences position paper on Glycoscience has proposed a major focus on glycan structural determination (National Academy of Sciences, 2012). Our data suggests that glycan diversity may be collapsed into a much smaller group of bioequivalent structures based on the number of structural subunits within the overall glycan that bind to a given animal lectin. Indeed, it may be possible that through further studies exploring bioequivalence among glycans, a glycan code can be deciphered based on interaction with animal lectins. Such an approach may greatly alleviate the complexity in determining structure-function relationships and relevance to human health.

Essential biological systems employ homeostatic mechanisms to maintain cellular integrity. N-glycosylation is an essential biosynthetic pathway necessary for cellular homeostasis and mammalian development (Dennis et al., 2009), yet lacks a known self-correcting mechanism. Our data suggest that homeostatic control of LacNAc content does not result from changes in enzyme levels/activity or metabolic production of UDP-GlcNAc substrate; but rather appears to arise from the structure of the Golgi. A defining feature of the Golgi is its polarized compartmentalization, with a cis to trans organization. Glycosylation enzymes are arranged along the Golgi roughly in the order in which they act. Why the Golgi has evolved this organization remains an open question. Our data indicate that the UDP-GlcNAc transporters preferentially supply UDP-GlcNAc to the cis/medial over trans Golgi, thus prioritizing branching over extension. Despite the presence of continuities between Golgi cisterna and previous work arguing that the Golgi is functionally interconnected (Kim et al., 2001), our data suggest that the segmented organization of enzymes and transporters allows for local depletion of substrate prior to diffusion to other compartments. In this manner, the evolutionary placement of the B3GNT poly-LacNAc extension enzymes in the trans Golgi provides a backup mechanism that captures unused UDP-GlcNAc from the medial Golgi shunted via inter-cisternal tubules. This backup system may also explain the lack of evolutionary pressure to produce genetic redundancy in the Golgi branching enzymes in mammals and the dramatically more severe phenotype seen with Mgat1 deficiency, which blocks all production of N-glycan LacNAc, compared to other Mgat genes (Ioffe and Stanley, 1994; Metzler et al., 1994).

The Golgi is charged with maintaining the integrity of the glycome/lattice or risking disease. Thus, fidelity of glycan biosynthesis must be maintained under a variety of cellular states and be active virtually at all times. High protein transit rates present a significant challenge to the Golgi and have the potential to reduce the medial Golgi branching efficiency by decreasing the time available for branching reactions. Under this scenario, having a back-up system that provides poly-LacNAc extension in the trans Golgi by capturing unused UDP-GlcNAc from the medial Golgi would be critical for the maintenance of the lattice at a minimal essential level. Even in an unstressed system, a second step in the assembly line would act to counteract the moment to moment variability and stochastic uncertainty of glycan synthesis. Severe branching deficiency (induced by SW treatment or Mgat2 deletion) uncovers this continual process. Such a mechanism is akin to DNA repair, which is occurring all of the time but made more apparent in the context of physiological or external stress.

A clear understanding of the network of interacting factors that coalesce to determine the state of the galectin-glycoprotein lattice is required for the successful exploitation of its therapeutic potential. Interventions must be able to overcome or at least take into account the homeostatic cellular response to the initial disturbance. For example, the effectiveness of SW as an anti-cancer therapeutic is likely limited by compensatory poly-LacNAc production (Goss et al., 1994; 1997). Thus, therapeutic strategies aimed at disrupting the lattice should block branching and extension simultaneously, or target a more proximal regulator such as UDP-GlcNAc metabolism or transport. In light of these considerations, the lattice approach to cancer treatment may merit re-visitation. Conversely, a deeper knowledge of the central regulatory machinery that determines total LacNAc content will also be required for therapeutic approaches that seek to strengthen the lattice.

The presence of functional groups (LacNAc) as subcomponents of glycans, combined with our current understanding of Golgi transport, may also explain the curious use of three different nucleotides to charge the array of sugars used as substrate donors for glycosylation. These sugar-nucleotides are transported into the Golgi via an anti-port mechanism where cytosolic sugar-nucleotides are exchanged for Golgi nucleotides generated by the action of glycosyltransferase enzymes. Increased availability and use of UDP-GlcNAc in the trans Golgi would increase uridine-monophosphate (UMP) levels, thereby driving anti-port of UDP-Galactose into the trans Golgi to promote further extension with poly-LacNAc rather than capping by sialic acid. Were sialic acid also charged with UDP rather than cytidine-monophosphate (CMP), UDP-GlcNAc usage in the trans Golgi would drive import of UDP-Sialic acid and capping equally with UDP-Galactose and extension.

Materials and methods

Galectin-3 binding

Request a detailed protocol

Recombinant mouse galectin-3 (R&D Systems, Minneapolis, MN) was labeled using an Alexa Fluor 488 protein labeling kit (ThermoFisher Scientific, Waltham, MA). Cells were stained for flow cytometry using 3 μg labeled galectin-3 per test. Staining was carried out for 30 min at room temperature followed by one wash and fixation with paraformaldehyde. For the lactose control samples, 50 mM lactose was included in all steps.

PNGase F treatment of live cells

Request a detailed protocol

Purified mouse T cells were washed twice in HBSS and resuspended in 100 ul of G7 reaction buffer. 2500 units of glycerol free PNGase F (New England Biolabs, Ipswich, MA) was added to the cells and the reaction was allowed to proceed for up to four hours at 37 degrees Celsius. Cells were then washed with HBSS and immediately stained for flow cytometry.

MALDI-TOF mass spectrometry

Request a detailed protocol

Control and SW treated Jurkat T cells were homogenized by sonication in 25 mM Tris, 150 mM NaCl, 5 mM EDTA, and 1% CHAPS, pH 7.4, dialyzed, reduced, and carboxymethylated, digested with trypsin to generate glycopeptides and treated with PNGase F (Roche, Basel, Switzerland) to release N-glycans. N-glycans were digested by endo-β-galactosidase (AMS Biotechnology) or Sialidase A (Prozyme, Hayward, CA) prior to permethylation and mass spectrometry analysis.

Cell lines

Request a detailed protocol

Human Jurkat cell line E6-1 and its derivative cell lines with deficiency in TCRβ (J.RT3-T3.5), and Lck (J.CaM1.6) were purchased from ATCC. Two separate lots of E6-1 Jurkat cells were purchase throughout the study and found to be equivalent in LEA increase in response to SW treatment. Cells were cultured, expanded and frozen down to create low passage stocks. These were subsequently thawed and used for experiments, limiting usage to a maximum of ten passages. They were grown in RPMI 1640 medium with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin and 2 μM 2-mercaptoethanol. CFPAC-1, CHO, Lec1, Raji, RPMI 8226, RS4; 11 and Kasumi-1 cell lines were also purchased from the ATCC. BJ, HT-29 and HEK-293 cells were a kind gift from Bogi Andersen. MCF-7 and Hela cells were a kind gift from Marian Waterman. Mouse postnatal day 1 neural stem cells were a kind gift from Thomas Lane. HUVEC cells were a kind gift from Christopher Hughes, and K562 cells were a kind gift from David Fruman. No routine mycoplasma testing or identity authentication was performed.

Reagents, mice and flow cytometry

Request a detailed protocol

Isolated human CD3+ T cells purified by negative selection (RosetteSep, StemCell Technologies, Vancouver, Canada) were stimulated with plate-bound anti-CD3 (OKT3, eBioscience, San Diego, CA). Procedures with human subjects were approved by the Institutional Review Board of the University of California, Irvine. Mgat2f/f (006892), Mgat1f/f (006891) and Lck-Cre (003802) mice were obtained from Jackson Laboratory. B3gnt2+/- (11605) embryos were from the MMRRC and were rederived by the UC Irvine Transgenic Mouse Facility. Inter-breeding generated all other mice. Mice were selected randomly for experiments and approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. Human cells were stained with anti-CD4, anti-CD69 (eBioscience) and/or L-PHA-FITC (Phaseolus Vulgaris Leukoagglutinin, Vector Labs, Burlingame, CA). Mouse cells were stained with anti-CD4 (RM 4–5), anti-CD8 (53–6.7), and anti-CD69 (H1.2F3) (eBioscience). Proliferation was assessed by 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE; ThermoFisher Scientific) at 1 uM in PBS (20 min., 4°C) and stimulated with plate-bound anti-CD3e. Cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum, 2 μM L-glutamine, 100 U/ml penicillin/streptomycin and 2 μM 2-mercaptoethanol. Where indicated, 40 mM GlcNAc (Ultimate Glucosamine, Wellesley Therapeutics Inc., Toronto, Canada) and 10 mM uridine (Sigma-Aldrich, St Louis, MO) were added to cells in culture at time 0. For flow cytometric analysis of glycan expression, cells were stained with 2 μg/ml L-PHA-FITC, ConA-FITC, LEA-FITC or biotinylated versions of these lectins followed by DyLight649 labeled streptavidin (Vector Labs). Staining was carried out as previously described (Lee et al., 2007; Grigorian et al., 2007). Flow cytometry experiments were performed with the BD FACSCalibur, LSR II, or Attune Acoustic Focusing Cytometer. Data analysis was performed using FlowJo software.

RNA isolation and microarray

Request a detailed protocol

CD4+ T cells isolated from Mgat2f/f mice and L-PHA-CD4+ T cells isolated from Mgat2f/f::Lck-Cre+ mice were used for analysis. The RNeasy mini kit (Qiagen, Valencia, CA) was used for RNA extraction. Gene expression was assessed using the Affymetrix Mouse Gene 1.0 ST arrays in triplicate. Array data were quantified with Expression Console version 1.1 software (Affymetrix, Santa Clara, CA) using the PLIER Algorithm default values. Expression values were then filtered as present/absent at expression 100. The Cyber-T web server was used for data analysis and to compare samples.

Enzymatic assays

Request a detailed protocol

B3GNT enzyme activity was measured using a glycosyltransferase activity kit (R&D systems). Five million Jurkat cells grown with or without SW were washed three times with Tris buffered saline and lysed with 300 μl of lysis buffer (10 mM Tris pH 7.5, 2 mM MnCl2, 4 mM CaCl2, 0.5% Triton-X, with protease inhibitors). The lysate was cleared of insoluble material by centrifugation and dialyzed overnight in an identical solution to remove cellular phosphate. Enzyme activity was measured following the instructions of the kit. Briefly, 25 μl of lysate was mixed with 25 μl of reaction mixture to give a final concentration of 6 mM N-acetyllactosamine, 5 mM UDP-GlcNAc, and 6 ng/μl coupling phosphatase. The reaction was allowed to proceed for two hours at 37°C, followed by visualization of released phosphate by a malachite green reagent as indicated in the kit. Absorbance at 620 nm was determined using a plate reader and converted using a phosphate standard run in parallel. Background was determined and subtracted by parallel reactions that lacked the specific acceptor N-acetyllactosamine but contained UDP-GlcNAc and coupling phosphatase.

Experimental autoimmune encephalomyelitis

Request a detailed protocol

EAE was induced by subcutaneous immunization of randomly selected female mice at days 0 with 100 μg of bovine MOG35-55 (AnaSpec, Fremont, CA) emulsified in Complete Freund’s Adjuvant containing 4 mg/ml heat-inactivated Mycobacterium tuberculosis (H37RA; Difco). On days 0 and 2, mice received 150 ng of pertussis toxin (List Biological Laboratories, Campbell, CA) by intraperitoneal injection. Mice were examined daily for clinical signs of EAE over the next 30 days with observer blinded to treatment conditions. Mice were scored daily as follows: 0, no disease; 1, loss of tail tone; 2, hindlimb weakness; 3, hindlimb paralysis; 4, forelimb weakness or paralysis and hindlimb paralysis; 5, moribund or dead. Kifunensine was given orally via the drinking water at 0.2 mg/ml for 8 days starting 3 days before immunization. All mice were housed with 12 hr light/dark cycles.

Spectrophotometric measurement of UDP-GlcNAc

Request a detailed protocol

UDP-GlcNAc was measured spectrophotometrically as previously described (Barthel et al., 2011). Briefly, Jurkat cells were grown in various conditions, washed thoroughly with PBS and pelleted at 50 × 106 cells per 1.5 ml tube. Cells were lysed by addition of 200 μl of chloroform/water (1:1), vortexed for 2 min and centrifuged at 15,000 × g for 20 min at 4°C. The aqueous phase was transferred to a fresh tube and 10 μl of 1 N HCl was added to hydrolyze UDP-GlcNAc to GlcNAc. After heating for 20 min at 80°C, the sample was neutralized with 10 μl 1 N KOH. Next, 50 μl of 200 mM potassium tetraborate (Sigma-Aldrich) was added, and the sample was heated at 80°C for 25 min and then cooled on ice for 5 min. 150 μl of Ehrlich's reagent (Sigma-Aldrich) (diluted 1:2 in acetic acid) was added to the sample, mixed, and incubated for 20 min at 37°C. The sample was centrifuged at 15,000 × g for 20 min, 200 μl were added to a 96-well plate, and the absorbance was measured at 595 nm. Absorbance was converted to UDP-GlcNAc concentration by comparing to a GlcNAc standard curve run simultaneously.

UDP-GlcNAc measurement by LC-MS/MS

Request a detailed protocol

Jurkat and mouse T cells were treated as described in the text, then harvested, washed twice with ice-cold 1x PBS and counted. After normalizing for cell number, cells were pelleted in 1.5 ml Eppendorf tubes by centrifuging at 350 g for 7 min at 4°C. Metabolites were extracted from the pellets by the addition of 1 ml of ice-cold solution of 40% acetonitrile, 40% methanol, and 20% water. The tubes were vortexed for 1 hr at 4°C and spun down at 14000 rpm for 10 min at 4°C (Eppendorf, Hamburg, Germany). The supernatant was transferred to fresh tubes and evaporated to dryness in an Eppendorf Vacufuge at 30°C (Eppendorf). The dry samples were stored at -80°C until analysis at which point they were reconstituted in 100 μl of LC-MS grade water and centrifuged again at 14000 rpm for 10 min at 4°C. The supernatants were carefully transferred to fresh tubes to be analyzed by mass spectrometry. The instrument used was a Waters Quattro Premier XE LC-MS/MS (Manchester, UK) equipped with ultra-performance liquid chromatography (UPLC) and a refrigerated autosampler. The separation was performed on BEH C18 reversed phase column (Waters, Manchester, UK) with a mobile phase A containing LC-MS grade water (JT Baker) modified with 0.2% Acetic Acid, 2% Acetonitrile and 5 mM Ammonium Acetate while mobile B containing LC-MS grade Acetonitrile (JT Baker) modified with 0.2% Acetic Acid and 5 mM ammonium Acetate. The gradient was performed with a 1 min ramp from 2% mobile B to 90%, which was then kept at 90% for 1 more minute and ramped back to 2% for column equilibration. The mass spectrometry measurement was performed in negative mode where the parent ion and fragment ion were measured simultaneously to be used for quantitation of the metabolites. A standard curve was generated using UDP-GlcNAc (Sigma-Aldrich), which was used for the quantitation of the metabolites produced by the cells.

Cellular subfractionation and Golgi immunoisolation

Request a detailed protocol

Jurkat cells were grown with or without kifunensine for four days, or until cultures reached a density between 1.5–2 x 106 cells/ml. Cells used for Golgi compartment disruption were treated with 1000x Brefeldin A (eBioscience 00-4506-51) for up to 30 min. Cells were washed with HEPES buffered saline (145 mM NaCl, 100 μM HEPES, pH 7.4) and incubated in an ice-cold hypotonic solution (30 mM KCl, 3 mM MgOAc, 2 mM DTT, 20 mM HEPES, pH 7.5) for 5 min on ice. The volume of the hypotonic solution used was 2.5 times the volume of the cell pellet. The cell solution was then passed through a 26.5 gauge syringe five times before a 1/10 volume of ice-cold hypertonic solution (375 mM KCl, 22.5 mM MgOAc, 1 mM DTT, 220 mM HEPES, pH 7.5) was added. The solution was centrifuged twice in succession at 1000x g for 5 min each to pellet cells and nuclei. The post-nuclear supernatant (PNS) was carefully transferred to a new tube and processed further. Ultracentrifugation of the PNS at 100,000 g for 30 min yielded a vesicular fraction (the pellet) and a cytosolic fraction (the supernatant). For immunoisolation experiments, PNS was incubated with anti-TNG46 antibody (Sigma-Aldrich T7576-200UL) previously conjugated to Protein G Dynabeads (ThermoFisher Scientific) for 30 min at 4°C with tumbling. Supernatant was removed, and immunoisolated Golgi was washed with HEPES buffered saline before processing for mass spectrometry or immunoblot analysis.

Plasmids, transfection and microscopy

Request a detailed protocol

DNA encoding mVenus and human B3GNT2 (HA tagged at the C terminus) were codon-optimized for human expression, synthesized, and cloned into the pmax Cloning vector (Lonza, Basel, Switzerland). Myc-DDK tagged human SLC35A3 (RC215108), SLC35B4 (RC203263), and SLC35D2 (RC218472) cDNA clones were purchased from Origene (Rockville, MD) in pCMV6 expression vectors. Cells were transfected with each construct individually (for microscopy) or in combination with mVenus to label transfected cells (for flow cytometry). 106 cells were transfected with 4–8 μg of DNA using an Amaxa Nucleofector IIb set to program G-10. Cells were immediately transferred to pre-equilibrated media in 6 well plates and placed in the tissue culture incubator. After 5 hr, media was changed and pharmacological treatments were started. For localization studies, cells were transferred to poly-L-lysine coated chamber slides (Falcon) after 24–48 hr of treatment. Cells were incubated in the chamber slides for 15 min to allow attachment to the slides and then fixed for 1 hr with 4% paraformaldehyde in PBS. Standard immunocytochemical staining was performed. Permeabilization was achieved with 0.1% saponin (Sigma-Aldrich). Primary antibodies used were to GM130 (Clone 35, BD Biosciences, Franklin Lakes, NJ), TGN46 (AbD Serotec, Hercules, CA), Mannosidase II (a kind gift from Dr. Christine Suetterlin), HA (ab9110, Abcam, Cambridge, UK), and DDK (Clone 4C5, Origene). Slides were stained with the appropriate secondary antibodies conjugated to Alexa Fluor (AF) 488, 555 and 647 and mounted with Prolong Gold Antifade reagent with DAPI (ThermoFisher Scientific). GM130 and TGN46 were always visualized with AF 488 and 647 respectively, while the protein of interest was visualized with AF 555. Slides were imaged on a Zeiss LSM 780 confocal microscope using a Zeiss plan-apochromat 100x oil objective with numerical aperture of 1.4. Localization of the proteins of interest was determined relative to GM130 and TGN46 using a line-scan method essentially identical to a previously published report (Dejgaard et al., 2007). Briefly, 30–50 transfected cells were imaged per experimental condition. Files were coded to blind the analyzer to treatment and protein of interest. Images were imported into the Zeiss Zen software (blue edition) with the AF 555 channel disabled. Line-scans (8–10 pixels wide) were drawn perpendicular to the long axis on areas of the Golgi showing maximal separation of GM130 and TGN46. Line-scans were used if they resulted in a single clearly discernable peak in all three channels. The relative distance of the peak intensity of the protein of interest compared to the peak intensities of GM130 and TGN46 was then used to determine intra-Golgi localization.

Immunoblotting

Request a detailed protocol

Immunoblotting was performed as described (Demetriou et al., 2001; Lee et al., 2007; Grigorian et al., 2007). Antibodies against GM130 (610822), GS27 (611034), GS28 (611184), and STX6 (610635) were from BD Biosciences. The TGN46 antibodies used for immunoisolation (T7576) and immunoblotting (SAB4200235) were from Sigma, and the antibody against B3GNT2 was from Origene (TA505283).

Statistical analysis

Request a detailed protocol

Statistics were calculated with Prism software (GraphPad). P values were from two- or one-tailed unpaired t-tests (with Welch's correction). The Bonferroni correction was applied to all multiple comparisons. No statistical method was used to predetermine sample size. The experiments were not randomized.

References

  1. 1
  2. 2
  3. 3
  4. 4
  5. 5
  6. 6
  7. 7
  8. 8
  9. 9
    Characterization of the structural determinants required for the high affinity interaction of asparagine-linked oligosaccharides with immobilized Phaseolus vulgaris leukoagglutinating and erythroagglutinating lectins
    1. RD Cummings
    2. S Kornfeld
    (1982)
    Journal of Biological Chemistry 257:11230–11234.
  10. 10
  11. 11
  12. 12
  13. 13
  14. 14
  15. 15
  16. 16
    Beta 1-6 branched oligosaccharides as a marker of tumor progression in human breast and colon neoplasia
    1. B Fernandes
    2. U Sagman
    3. M Auger
    4. M Demetrio
    5. JW Dennis
    (1991)
    Cancer Research 51:718–723.
  17. 17
    Structures of O-linked oligosaccharides isolated from normal granulocytes, chronic myelogenous leukemia cells, and acute myelogenous leukemia cells
    1. M Fukuda
    2. SR Carlsson
    3. JC Klock
    4. A Dell
    (1986)
    Journal of Biological Chemistry 261:12796–12806.
  18. 18
  19. 19
  20. 20
    Phase IB clinical trial of the oligosaccharide processing inhibitor swainsonine in patients with advanced malignancies
    1. PE Goss
    2. CL Reid
    3. D Bailey
    4. JW Dennis
    (1997)
    Clinical Cancer Research 3:1077–1086.
  21. 21
  22. 22
  23. 23
  24. 24
  25. 25
  26. 26
  27. 27
  28. 28
  29. 29
  30. 30
  31. 31
  32. 32
  33. 33
  34. 34
  35. 35
  36. 36
  37. 37
  38. 38
    Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains
    1. RK Merkle
    2. RD Cummings
    (1987)
    Journal of Biological Chemistry 262:8179–8189.
  39. 39
    Complex asparagine-linked oligosaccharides are required for morphogenic events during post-implantation development
    1. M Metzler
    2. A Gertz
    3. M Sarkar
    4. H Schachter
    5. JW Schrader
    6. JD Marth
    (1994)
    The EMBO Journal 13:2056–2065.
  40. 40
  41. 41
    Transforming Glycoscience: A Roadmap for the Future
    1. National Academy of Sciences
    (2012)
    Washington, DC: National Academies Press.
  42. 42
  43. 43
  44. 44
  45. 45
  46. 46
  47. 47
  48. 48
  49. 49
  50. 50
  51. 51
  52. 52
  53. 53
  54. 54
    Characterization of a blood group I-active ganglioside. Structural requirements for I and i specificities
    1. K Watanabe
    2. SI Hakomori
    3. RA Childs
    4. T Feizi
    (1979)
    Journal of Biological Chemistry 254:3221–3228.
  55. 55
  56. 56
  57. 57

Decision letter

  1. Benjamin S Glick
    Reviewing Editor; The University of Chicago, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Golgi proofreading homeostatically controls cell growth and differentiation" for consideration by eLife. Your article has been favorably evaluated by Vivek Malhotra (Senior editor) and four reviewers, one of whom, Benjamin Glick, is a member of our Board of Reviewing Editors. One of the reviewers has agreed to reveal his identity: Hudson Freeze.

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The four reviewers are in agreement that your manuscript is interesting, thorough, and of high quality and potentially of broad interest.

Our major concern is that there is no evidence that the phenomena you have described reflect a physiologically relevant homeostatic proofreading mechanism. This speculative aspect of the study needs to be toned down.

The main insight is bio-equivalence of the different LacNAc structures. This conclusion should be the main emphasis, and the title should be revised accordingly. One reviewer noted that "bio-equivalent glycans can be seen in many places – for instance man-6-P on various structures of high mannose glycans on lysosomal enzymes, or the MECA79 antigen structures as extended Core 1 structures rather than exclusively Core2." Therefore, your results should be placed in a more general context.

Essential revisions:

As indicated above, the emphasis of the paper needs to be adjusted to reflect the strong evidence for glycan bio-equivalence rather than the more speculative evidence for a homeostatic proofreading mechanism.

For the revision, please evaluate the detailed suggestions according to your best judgment, and make changes that you are confident will improve the paper.

Reviewer #1:

This manuscript by Haik Mkhikian et al. describes a mechanism by which the Golgi regulates the type of N-glycans it produces. With this mechanism, N-glycans are converted from highly branched to single-branched elongated LacNac structures. Both types of structures have high avidity for the secreted protein Galectin-3. This galectin forms a Galectin-glycoprotein lattice at the cell surface that regulates cell proliferation and differentiation of T-cells.

The paper proposes that upon perturbation of N-glycan branching by either genetic or chemical perturbation, cells react by increasing the amount of elongated LacNac structures through increasing the substrate required for LacNac, UDP-GlcNac, in the trans cisternae of the Golgi. GlcNac is usually present only in cis-medial Golgi, but lack of use by the branching enzymes is proposed to lead to its diffusion to the trans cisternae.

Overall, this manuscript and the data presented are of high quality and very exhaustive. The question tackled, the regulation of protein glycosylation, is not easy and in need of this type of analysis. The methods used by the authors are varied and cutting-edge, for instance the use of mass spec analysis for glycome analysis.

In terms of results, the increase in LacNac structures as detected by LEA staining is particularly striking and is reproducible across multiple systems.

However, I am not convinced by the notion of proof-reading for the following reasons:

The correction mechanism seems to be kicking in only after an extensive perturbation and is not completely correcting: the loss of Mgat5 or Mgat2 does not lead to compensation of Galectin biding to the wild-type levels. In fact, loss of Mgat5 does not induce any significant increase in LacNac branches. This is at odds with the authors model, since levels of UDP-GlcNac would be expected to increase in trans cisternae. In addition, this result suggests a very sloppy "proof-reading" system.

The perturbations used by the authors are artificial: genetic depletion of Mgat2 or treatment with swainsonine (SW). It is not clear what kind of naturally occurring problems are being mimicked by these treatments. Proof-reading implies that a system is sensing a defect and reacting to correct it. However, in this case, it is not the state of the lattice that is being sensed (or very mildly as per the authors own admission) but the level of use of sugar precursors (UDP-GlcNac) in the cis/medial Golgi. The compensatory mechanism is relatively indirect: other perturbations in the levels of UDP-GlcNac (for instance a change in the amount of cargo being traffic and glycosylated) would presumably lead to change in glycosylation that are unrelated to the status of the lattice.

Therefore, while the study is interesting and very well executed, at this stage, it is difficult to ascertain whether the phenomenon observed, the reality of which I do not doubt, is a compensatory mechanism occurring only in experimental conditions or whether it has a real biological function in a physiological context.

Reviewer #2:

The authors present a novel perspective for maintaining glycosylation homeostasis. They argue that "bio-equivalent" structures can substitute for certain functional glycans whose structure has been shown to be critical in determining cancer metastasis and autoimmunity. This lab and others have shown that the presence of poly-N-acetyllactosamine on the N-glycan branch created by an N-acetyl-Glucosaminyltransferse (GNT-V, encoded by MgatV) is essential for creation of lattices (or aggregate) composed of protein-bound glycans and multivalent lectins called galectins. This was a novel perspective when it was introduced 15 years ago. Evidence favoring it has grown in time and its functionality has been documented in deletion of GNT-V (MgatV) that leads to the loss of poly-N-acetyllactosamine on that branch, loss of galectin binding and dramatic decrease in tumor metastasis and increase in autoimmunity. Normally the lattice appears to prevent the inappropriate activation of the T-cell receptor. With the lattice gone, the TCR is able to recruit other cell surface molecules to mount T-cell response.

In this paper, the authors ask what would happen if two additional N-glycan branches were removed? Presumably, this would further reduce lectin binding and lattice formation and lead to more severe functional response. This did not occur. Instead they found a compensatory functional response in which the poly-N-acetyllactosamine repeats are added to another branch that normally would not contain them. To explain this, they propose a mechanism based on donor substrate localization to account for the compensation and float the hypothesis that generating bioequivalent structures may be a common way to maintain homeostasis in response to imbalances or insults. This is a novel perspective.

It is known that the terminal sugars of both N and O-linked glycans, especially repeating units, have a preferential site of addition, but it is generally not restricted to a single site.

In the Abstract, the word "transport" is used to refer to the movement of already transported substrates from the medial to the trans Golgi compartment. It is much more likely that it is simply diffusion from the medial to the trans Golgi. The existence of both vertical and horizontal inter-cisternal tubules has been known for some time. An important, but obscure and often overlooked paper 15 years ago (PMID:12376727) shows seven glycosyltransferases from different Golgi stacks and 3 nucleotide sugar transporters are functionally co-localized in a single continuous compartment that is accessible to any of the transported donors. They are freely diffusible within the Golgi and do not depend on any vesicular transport. This reference was not included, but must be acknowledged.

I think the authors adapt their results into a rationale for its existence. I hesitate to call it a proofreading mechanism to sustain homeostasis, but a much simpler explanation is that if a donor substrate is not used in a preferred location and it has access to a similar reaction in another accessible location, this is almost an inevitable outcome.

The study is thorough and very well done, using novel approaches and sophisticated techniques to make their arguments.

In Figure 2, the authors say there are no changes in Lec1 cells, yet it rates "**" significant differences. Need to reconcile those conflicting statements.

The MALDI results in Figure 2F and G as well as similar ones in the supplemental figures are impossible for a reader to quantitatively assemble and make conclusions. The increased ratio of 0.5 to 1.1 in the presence of SW may be true, but the authors need to show this via color-coding or another way to indicate which peaks are to be in the numerators and denominators. This is especially important when isobaric masses have different structures. It was very confusing.

The authors need to more precisely indicate which figure corresponds to which statement in the text because they are often confusing when multiple figures are indicated and statement refers to figures with many panels. For instance, the comment about a 3-4 fold increase in UDP-GlcNAc is enhanced SW induced poly-LacNAc extension in Figure 4F, 4G and Figure 4—figure supplement 1. Too much searching needed.

The statement in the Results section – "This suggests that cytosolic UDP-GlcNAc lacks direct access to the trans Golgi compartment" is not necessarily true. It may have been transported into a different compartment, but based on PMID:12376727, it likely has access to all compartments.

Likewise suggesting that the trans Golgi is deficient in UDP-GlcNAc, thereby limiting β3GnT activity and poly-LacNAc extension under steady state conditions is not necessarily true because the assumption relies on discreet non-interconnected compartments.

Reviewer #3:

In this manuscript the authors propose "that the cis to trans-Golgi organization permits a self-correcting mechanism that serves to preserve cellular homeostasis by maintaining galectin-glycoprotein interactions." And that "new poly-LacNAc extension by β3GnT maintains galectin binding and immune homeostasis. Poly-LacNAc extension is triggered by transport of unused UDP-GlcNAc from the medial to trans-Golgi via inter-cisternal tubules." This hypothesis is novel and the data presented in support of it are of high quality. The following points should be addressed in revision:

1) Some of the authors of this manuscript have previously published nice evidence of glycan elongation as a compensatory mechanism in glycoproteins from core 2 knockout mice. These and other results on this topic should be clearly described in the Introduction and relevant publications quoted.

2) The authors emphasize that a large drop in poly-LacNAc occurs in glycoproteins from Mgat2[-/-] compared to Mgat5[-/-] cells. However, based on the MS data, MGAT4 is operational in Mgat2[-/-] cells and thus the drop is merely from 3 to 2 branches. Going from 3 to 2 branches seems to be the threshold that stimulates the synthesis of LEA binding glycans.

3) The authors use mixed sugar symbols for MS spectra, the pathway diagram and Figure 7. It is strongly recommended that the symbols used on the MS spectra be used in all figures in the manuscript.

4) A key question that is not addressed is how much LacNAc is on each branch of N-glycans from WT vs. Mgat2 KO vs. Mgat5 KO T cells? The authors should note why they confined MS experiments to Jurkat cells. Given the large increase in LEA binding to Jurkat cells treated with SW, it is surprising that Figure 2E and 2F show a maximum of only 3 LacNAcs on 2 branches, but that peak is extremely small. By far the predominate species with a LacNAc extension has only 1 LacNAc (m/z 3102). This is not poly-LacNAc. The authors should discuss this disconnect.

5) The evidence that UDP-GlcNAc moves to the Trans Golgi when use in the medial Golgi decreases is largely indirect but reasonable, although many differences are small and there is a reliance on inhibitors that give modest effects and whose specificity is not investigated. The authors should discuss qualifications and potential caveats in the Discussion.

6) Bioequivalence is a fine concept but should be viewed as one function of surface glycans that is distinct from but complementary with biospecificity. It is not helpful to so dogmatically put all eggs in a bioequivalence basket.

Reviewer #4:

The galectin-glycoprotein lattice is a dynamic extracellular structure important for receptor localization and function. This paper focuses on quality control of the N-acetyllactosamine (LacNAc) component, which binds galectin. The hypothesis is that cells employ a homeostatic regulatory mechanism to ensure a suitable level of LacNAc in their extracellular N-glycans. Results presented here suggest that the Golgi has a built-in self-correction mechanism in which a deficiency in synthesis of LacNAc branches by cis/medial-Golgi Mgat enzymes is compensated for by enhanced synthesis of linear poly-LacNAc chains by the β-1,3-N-acetylglucosaminyltransferase (B3GnT) enzyme in the trans-Golgi. This compensation is proposed to occur when reduced branching enzyme activity results in elevated levels of the UDP-GlcNAc substrate becoming available in the trans-Golgi for linear poly-LacNAc synthesis.

The conclusions of this paper have two broad implications. First, it seems that the branched and linear LacNAc components are functionally interchangeable with regard to galectin binding. If so, the "glycan code" may be much less complex than has been assumed. Second, therapeutic approaches that target the galectin-glycoprotein lattice can be made more effective by taking into account functional redundancy in the glycan linkages. Both of these insights could be quite significant, making the paper potentially suitable for eLife.

This paper is a dense read but is well written, logical, and rigorously controlled. Although I would defer to other referees for evaluating the immunology, the glycobiology and cell biology aspects strike me as largely persuasive, with the following caveats.

1) All of the experiments employ artificial manipulations such as gene knockouts and glycosylation inhibitors. Is there any justification for assuming that activity of the Mgat branching enzymes can be perturbed under conditions that prevail in an organism? In other words, is this a physiologically relevant phenomenon? I'm not sure if this question can be answered definitively, but it would be nice to see an explanation of how such a perturbation might occur.

2) Is it clear that B3GnT is normally limited by the levels of UDP-GlcNAc in the trans-Golgi? If so, why doesn't addition of GlcNAc promote poly-LacNAc formation in the absence of swainsonine? It seems unlikely that all of the excess UDP-GlcNAc generated by GlcNAc addition would be consumed by increased branching, so there should be more UDP-GlcNAc available for B3GnT.

3) While reading the paper, I wondered from the beginning why sialic acid capping would not block LacNAc extension. This issue was not mentioned until the very end of the Discussion. Is it possible that regulation of poly-LacNAc synthesis occurs at the level of sialic acid capping? If so, an entire dimension may be missing from the story. For example, returning to #2 above, perhaps B3GnT is actually limited not by UDP-ClcNAc levels but rather by sialic acid capping.

https://doi.org/10.7554/eLife.14814.020

Author response

[…] The main insight is bio-equivalence of the different LacNAc structures. This conclusion should be the main emphasis, and the title should be revised accordingly. One reviewer noted that "bio-equivalent glycans can be seen in many places – for instance man-6-P on various structures of high mannose glycans on lysosomal enzymes, or the MECA79 antigen structures as extended Core 1 structures rather than exclusively Core2." Therefore, your results should be placed in a more general context.

Essential revisions:

As indicated above, the emphasis of the paper needs to be adjusted to reflect the strong evidence for glycan bio-equivalence rather than the more speculative evidence for a homeostatic proofreading mechanism.

For the revision, please evaluate the detailed suggestions according to your best judgment, and make changes that you are confident will improve the paper.

We would like to thank all of the editors and reviewers for their time and efforts in service of our manuscript. We cannot help but be pleased by the refreshingly well-reasoned responses and the expeditious overall process at eLife. We feel that the issues raised have led to valuable discussion and analysis among the authors and contributed considerably to an improved manuscript, which we submit along with this response.

The major points made by the reviewers are well taken. We have also previously debated the relative merits of emphasizing the bio-equivalence versus the proofreading aspects of the story. Given the fresh perspective of the expert panel, we are now also in agreement that the emphasis of the paper should be adjusted to highlight glycan bio-equivalence. We have re-worked the paper to better emphasize bio-equivalence. For example, we have removed the term “proof-reading” from the title and text, and describe this now as self-correction. We have also re-organized the paper to focus on bio-equivalence. Although we think it likely that a proof-reading like mechanism is truly at play, we recognize that the positive data directly supporting this hypothesis are limited. This is due in no small part to the fact that well established tools to analyze and track sugar nucleotides at sub-Golgi resolution currently do not exist. However, we are encouraged by the interest and discussion of our colleagues to pursue the issue further in future work.

Reviewer #1:

[…] Overall, this manuscript and the data presented are of high quality and very exhaustive. The question tackled, the regulation of protein glycosylation, is not easy and in need of this type of analysis. The methods used by the authors are varied and cutting-edge, for instance the use of mass spec analysis for glycome analysis.

In terms of results, the increase in LacNac structures as detected by LEA staining is particularly striking and is reproducible across multiple systems.

However, I am not convinced by the notion of proof-reading for the following reasons:

The correction mechanism seems to be kicking in only after an extensive perturbation and is not completely correcting: the loss of Mgat5 or Mgat2 does not lead to compensation of Galectin biding to the wild-type levels. In fact, loss of Mgat5 does not induce any significant increase in LacNac branches. This is at odds with the authors model, since levels of UDP-GlcNac would be expected to increase in trans cisternae. In addition, this result suggests a very sloppy "proof-reading" system.

These are great points. At the cell surface, mono- and bi-antennary glycans greatly outnumber tri- and tetra-antennary glycans, particularly in low branching cell types such as T cells. This is also apparent when we consider the relative affinity of the MGAT enzymes for UDP-GlcNAc. MGAT1 has the best affinity with Km ~0.04mM. The affinities decrease sequentially for MGAT2 (~0.9mM), MGAT4 (~4mM) and MGAT5 (~11mM). In other words, relative UDP-GlcNAc usage markedly decreases from MGAT1 to MGAT5. Therefore, according to our model, inhibition or loss of MGAT5 is expected to result in very little increase in UDP-GlcNAc availability to B3GNT2, as enzymes with much greater activity (i.e. MGAT1 and 2) will utilize the excess supply. In comparison, MGAT2 loss or inhibition would be expected to result in a much larger rise in UDP-GlcNAc levels in the trans Golgi. Note that B3GNT has a Km for UDP-GlcNAc that is comparable to MGAT2.

The perturbations used by the authors are artificial: genetic depletion of Mgat2 or treatment with swainsonine (SW). It is not clear what kind of naturally occurring problems are being mimicked by these treatments.

Our thinking is that this is a mechanism that is constantly at work to counter the uncertainty of a loosely directed and “template-less” synthetic process. In contrast, the synthesis of DNA, RNA, and protein, being template driven is guided by very specific instructions. To use the names of the processes literally, it is as if the assignment is to copy or transcribe or translate (word for word) an assigned text. Predicting the outcome is straightforward given the quality of the template and the competence of “the staff”. Glycan synthesis by comparison is akin to asking many more players to produce a finger painting under a specific rule set (you can only use certain fingers (monosaccarides) in specific orders and patterns). The “instruction” is specified by the enzymatic and chemical restrictions, but the resulting product cannot be predicted with anything near the exactness of template driven processes. In addition, the integrity of the genome is maintained in part by restricting DNA replication to a highly controlled context and defined period of time. Glycan biosynthesis by contrast must function in a variety of cellular states and be active virtually at all times. Nevertheless, the Golgi system is charged with maintaining the integrity of the glycome/lattice or risk disease. Just as you point out, the most obvious/likely situations in which such an assembly-line corrective mechanism might function is in response to changes in protein synthesis, metabolism, and cargo transit rates. It is a reasonable hypothesis that high protein transit rates through the medial Golgi would reduce branching efficiency by decreasing the time available for branching reactions. Under this scenario, having a back-up system that provides poly-LacNAc extension in the trans Golgi by capturing unused UDP-GlcNAc from the medial Golgi would be critical for the maintenance of the lattice at a minimal essential level. Even in an unstressed system, a second step in the assembly line would act to counteract the moment to moment variability and stochastic uncertainty of glycan synthesis. We believe that severe branching deficiency (induced by SW treatment or Mgat2 deletion) uncovers this continual process which is difficult to appreciate at baseline due to technical limitations. This would be akin to DNA repair mechanisms, which are occurring all of the time but made more apparent in the context of super-physiological stress.

However, physiological relevance may also arise from environmental factors. For example, swainsonine is a natural compound found in plants (locoweed) and ingestion causes disease in livestock. Given the large number of genes involved in glycan biosynthesis, the number of naturally occurring substances which may interfere with these pathways is likely underappreciated. Corrective mechanisms that can buffer such environmental insults to glycan biosynthesis may play a physiological role in these situations.

Proof-reading implies that a system is sensing a defect and reacting to correct it. However, in this case, it is not the state of the lattice that is being sensed (or very mildly as per the authors own admission) but the level of use of sugar precursors (UDP-GlcNac) in the cis/medial Golgi. The compensatory mechanism is relatively indirect: other perturbations in the levels of UDP-GlcNac (for instance a change in the amount of cargo being traffic and glycosylated) would presumably lead to change in glycosylation that are unrelated to the status of the lattice.

We have removed the term “proof-reading” from the text and substituted it with more appropriate terminology such as “self-correction.”

Proofreading in its exact publishing meaning (we had to look it up) refers to comparing typeset with copy prior to mass production. In this sense it is restricted to situations when two texts are compared and assessed for typesetting errors, and therefore has strict analogy to template driven synthetic processes. We had used the term in its broader current usage, where it further applies to reading for grammar, syntax, spelling, flow, logic, internal consistency etc. (the strict term for which is copy editing). We agree with your assessment that what is being sensed is UDP-GlcNAc concentration, and thus reduced UDP-GlcNAc usage by the prior compartment. We are not suggesting that B3GNT2 is a proofreading enzyme specifically, but rather that the entire glycan biosynthetic system with its directional intra-Golgi organization contains a sequential assembly-line self-correcting capability. This function is important for preserving the galectin-glycoprotein lattice.

Therefore, while the study is interesting and very well executed, at this stage, it is difficult to ascertain whether the phenomenon observed, the reality of which I do not doubt, is a compensatory mechanism occurring only in experimental conditions or whether it has a real biological function in a physiological context.

We are excited that others share our interest in these issues. We are also intrigued by this very question of the physiologic role of this mechanism, and despite our arguments for its likely role, we acknowledge that direct evidence is currently lacking. We plan to actively explore these questions in the future.

Reviewer #2:

[…] In the Abstract, the word "transport" is used to refer to the movement of already transported substrates from the medial to the trans Golgi compartment. It is much more likely that it is simply diffusion from the medial to the trans Golgi. The existence of both vertical and horizontal inter-cisternal tubules has been known for some time. An important, but obscure and often overlooked paper 15 years ago (PMID:12376727) shows seven glycosyltransferases from different Golgi stacks and 3 nucleotide sugar transporters are functionally co-localized in a single continuous compartment that is accessible to any of the transported donors. They are freely diffusible within the Golgi and do not depend on any vesicular transport. This reference was not included, but must be acknowledged.

These are great points. We agree that UDP-GlcNAc likely diffuses or is carried forward by cisternal maturation. Thank you for pointing out the potential confusion with the word “transport.” The manuscript has been revised to replace “transport” with clearer wording. We also thank the reviewer for bringing to our attention this relevant reference, and we acknowledge that the Golgi is functionally interconnected and continuous via vertical and horizontal tubules. However, our data are consistent with a Golgi model that though continuous, is organizationally segmented such that enzymes can locally deplete substrate. A discussion of these points is now included in the revised manuscript.

I think the authors adapt their results into a rationale for its existence. I hesitate to call it a proofreading mechanism to sustain homeostasis, but a much simpler explanation is that if a donor substrate is not used in a preferred location and it has access to a similar reaction in another accessible location, this is almost an inevitable outcome.

This view assumes that the Golgi is a one-pot system, which our data argue against. Our view is that the organization of the Golgi (with its sub-compartments and specific set of particularly localized resident enzymes and transporters) has arisen due to evolutionary pressures that provide advantages over other arrangements. For example, our data indicate that the trans Golgi lacks direct access to UDP-GlcNAc via transporters and relies on diffusion from more proximal compartments, with the rate of diffusion limited by the utilization of UDP-GlcNAc in more proximal compartments. This is the result of the evolutionary driven placement of enzymes and transporters in different sub-compartments and therefore differs from a one-pot system.

The study is thorough and very well done, using novel approaches and sophisticated techniques to make their arguments.

In Figure 2, the authors say there are no changes in Lec1 cells, yet it rates "**" significant differences. Need to reconcile those conflicting statements.

Thank you for pointing this out. These asterisks were present in error and have been removed.

The MALDI results in Figure 2F and G as well as similar ones in the supplemental figures are impossible for a reader to quantitatively assemble and make conclusions. The increased ratio of 0.5 to 1.1 in the presence of SW may be true, but the authors need to show this via color-coding or another way to indicate which peaks are to be in the numerators and denominators. This is especially important when isobaric masses have different structures. It was very confusing.

Please see the response to Reviewer 1 above.

The authors need to more precisely indicate which figure corresponds to which statement in the text because they are often confusing when multiple figures are indicated and statement refers to figures with many panels. For instance, the comment about a 3-4 fold increase in UDP-GlcNAc is enhanced SW induced poly-LacNAc extension in Figure 4F, 4G and Figure 4—figure supplement 1. Too much searching needed.

This point is well taken. We have revised the manuscript to contain reference to specific lettered panels in the figure supplements where appropriate.

The statement in the Results section – "This suggests that cytosolic UDP-GlcNAc lacks direct access to the trans Golgi compartment" is not necessarily true. It may have been transported into a different compartment, but based on PMID:12376727, it likely has access to all compartments.

Likewise suggesting that the trans Golgi is deficient in UDP-GlcNAc, thereby limiting β3GnT activity and poly-LacNAc extension under steady state conditions is not necessarily true because the assumption relies on discreet non-interconnected compartments.

Again, we agree that the Golgi compartments are interconnected. However, based on our data, we suggest that the segmented organization of enzymes and transporters allows for local depletion of substrate prior to diffusion to other compartments. As we attempt to illustrate in our model in Figure 7, if UDP-GlcNAc is transported to the cis/medial Golgi, rapid use by the branching enzymes will limit diffusion to the later compartments despite intra-Golgi continuities.

Reviewer #3:

In this manuscript the authors propose "that the cis to trans-Golgi organization permits a self-correcting mechanism that serves to preserve cellular homeostasis by maintaining galectin-glycoprotein interactions." And that "new poly-LacNAc extension by β3GnT maintains galectin binding and immune homeostasis. Poly-LacNAc extension is triggered by transport of unused UDP-GlcNAc from the medial to trans-Golgi via inter-cisternal tubules." This hypothesis is novel and the data presented in support of it are of high quality. The following points should be addressed in revision:

1) Some of the authors of this manuscript have previously published nice evidence of glycan elongation as a compensatory mechanism in glycoproteins from core 2 knockout mice. These and other results on this topic should be clearly described in the Introduction and relevant publications quoted.

While some of our authors have published data on unusual structures induced in glycan deficient mice, the possibility that these represent a functional compensation mechanism were largely proposed in reviews by others. Importantly, to our knowledge no one has previously directly proven that the induced structures were bio-equivalent and able to functionally compensate for the loss of other glycans. We now describe this issue in the Introduction.

2) The authors emphasize that a large drop in poly-LacNAc occurs in glycoproteins from Mgat2[-/-] compared to Mgat5[-/-] cells. However, based on the MS data, MGAT4 is operational in Mgat2[-/-] cells and thus the drop is merely from 3 to 2 branches. Going from 3 to 2 branches seems to be the threshold that stimulates the synthesis of LEA binding glycans.

These are important points. We state that there is a significant drop in LacNAc branching/content not poly-LacNAc (we assume this is a typo in your comment) between Mgat5 and Mgat2 deficient cells. Also, as you point out below, the MS data are from SW treated Jurkat cells not Mgat2 deficient cells. Thus, it is possible that MGAT4 is not active in Mgat2 deficient T cells. You are correct that the MGAT4 branch is present in the SW treated Jurkat cells. As noted above in the comments to reviewer 1, MGAT2 has a higher affinity for UDP-GlcNAc than MGAT4. Based on this we would predict that loss of MGAT2 activity or inhibition by SW (despite an operational MGAT4) may result in greater UDP-GlcNAc availability for poly-LacNAc extension than loss of MGAT4/MGAT5, despite both scenarios resulting in a maximum of two branches. In other words, going from 3 to 2 branches by combined loss of MGAT4/MGAT5 may not be equivalent to MGAT2 loss or SW treatment. Our model would suggest that UDP-GlcNAc usage by the branching enzymes rather than the number of branches maximally produced dictates extent of poly-LacNAc compensation. This is a subtle but important distinction.

3) The authors use mixed sugar symbols for MS spectra, the pathway diagram and Figure 7. It is strongly recommended that the symbols used on the MS spectra be used in all figures in the manuscript.

This point is well taken. The discordant symbols have been replaced with a uniform set.

4) A key question that is not addressed is how much LacNAc is on each branch of N-glycans from WT vs. Mgat2 KO vs. Mgat5 KO T cells? The authors should note why they confined MS experiments to Jurkat cells. Given the large increase in LEA binding to Jurkat cells treated with SW, it is surprising that Figure 2E and 2F show a maximum of only 3 LacNAcs on 2 branches, but that peak is extremely small. By far the predominate species with a LacNAc extension has only 1 LacNAc (m/z 3102). This is not poly-LacNAc. The authors should discuss this disconnect.

These are excellent points. Indeed, the Mgat2 deficient cells exhibited far greater fold LEA increase (~100 fold) than the Jurkat cells (~20 fold). We therefore made several attempts to perform MS experiments on Mgat2 KO vs. WT T-cells. However, the analyses revealed that we were unable to isolate enough biological material to generate meaningful spectra (we only detected high mannose structures), despite pooling cells from many mice. This was due in part to the fact that we are dealing with a T cell specific knockout, which is inefficiently deleted, and mouse T cells yield relatively low amounts of glycan. We therefore resorted to the SW treated Jurkat cells as a reasonable alternative. As far as the extent to which poly-LacNAc is represented on the spectra, these have to be interpreted cautiously because they are not quantitative, particularly for large poly-LacNAc structures. For that reason we performed the endo-β-galactosidase digestion analysis, which revealed increased poly-LacNAc in the Jurkat sample.

5) The evidence that UDP-GlcNAc moves to the Trans Golgi when use in the medial Golgi decreases is largely indirect but reasonable, although many differences are small and there is a reliance on inhibitors that give modest effects and whose specificity is not investigated. The authors should discuss qualifications and potential caveats in the Discussion.

Some of the differences noted are indeed small. We suspect that this is because we are achieving mild relative enrichment of the late Golgi compartments, rather than isolating a pure fraction. This dilutes the true difference. As noted above, we have toned down our discussion on this subject by focusing the paper more on bio-equivalence than proof-reading.

6) Bioequivalence is a fine concept but should be viewed as one function of surface glycans that is distinct from but complementary with biospecificity. It is not helpful to so dogmatically put all eggs in a bioequivalence basket.

We agree about bio-specificity; however galectin bio-specificity is for LacNAc, rather than the entire glycan structure. The overall glycan structure sets the total LacNAc content, thereby largely determining avidity of binding without affecting affinity of binding (assuming other modifications that may impact binding such as fucose and sialic acid are held constant). Thus, we are not arguing that there is a change in bio-specificity of galectin-glycan interactions, just a recognition that the number of binding sites in the overall glycan (i.e. LacNAc content) is a major driver of bio-equivalence in a manner that does not affect bio-specificity.

Reviewer #4:

[…] This paper is a dense read but is well written, logical, and rigorously controlled. Although I would defer to other referees for evaluating the immunology, the glycobiology and cell biology aspects strike me as largely persuasive, with the following caveats.

1) All of the experiments employ artificial manipulations such as gene knockouts and glycosylation inhibitors. Is there any justification for assuming that activity of the Mgat branching enzymes can be perturbed under conditions that prevail in an organism? In other words, is this a physiologically relevant phenomenon? I'm not sure if this question can be answered definitively, but it would be nice to see an explanation of how such a perturbation might occur.

Please see above comment to reviewer 1.

2) Is it clear that B3GnT is normally limited by the levels of UDP-GlcNAc in the trans-Golgi? If so, why doesn't addition of GlcNAc promote poly-LacNAc formation in the absence of swainsonine? It seems unlikely that all of the excess UDP-GlcNAc generated by GlcNAc addition would be consumed by increased branching, so there should be more UDP-GlcNAc available for B3GnT.

GlcNAc treatment markedly increases total cellular UDP-GlcNAc and branching without substantial changes in LEA binding in the absence of swainsonine. Coupled with our data indicating that UDP-GlcNAc transporters are absent from the trans Golgi, we interpret this to mean that excess UDP-GlcNAc generated by GlcNAc addition is indeed consumed by the medial Golgi branching enzymes. In the presence of SW, GlcNAc treatment does substantially increase LEA binding, implying that UDP-GlcNAc is limiting for poly-LacNAc production but only becomes available to B3GNT when the branching enzymes are inhibited. It should be noted that an increase in total cellular UDP-GlcNAc may not necessarily result in one for one increase in Golgi UDP-GlcNAc. The apparent Km of the transporters is measured at 1-10 uM, which is lower than the cytosolic UDP-GlcNAc concentration. Therefore it is likely that increased cytosolic UDP-GlcNAc levels are not fully translated to the Golgi levels.

3) While reading the paper, I wondered from the beginning why sialic acid capping would not block LacNAc extension. This issue was not mentioned until the very end of the Discussion. Is it possible that regulation of poly-LacNAc synthesis occurs at the level of sialic acid capping? If so, an entire dimension may be missing from the story. For example, returning to #2 above, perhaps B3GnT is actually limited not by UDP-ClcNAc levels but rather by sialic acid capping.

Another excellent point. Sialic acid capping could be limiting poly-LacNAc length. However, we did not see significant changes in sialyltransferase transcript levels in our microarray experiment. Moreover, in our mass spec data there appears to be more sialylation of the poly-LacNAc antennae in the Swainsonine treated sample (compare m/z 722 and 1084 in Figure 2F and 2G), arguing against reduced sialic acid capping activity resulting from SW treatment.

https://doi.org/10.7554/eLife.14814.021

Article and author information

Author details

  1. Haik Mkhikian

    Department of Microbiology and Molecular Genetics, University of California, Irvine, United States
    Contribution
    HM, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  2. Christie-Lynn Mortales

    Department of Microbiology and Molecular Genetics, University of California, Irvine, United States
    Contribution
    C-LM, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  3. Raymond W Zhou

    Department of Neurology and Institute for Immunology, University of California, Irvine, United States
    Contribution
    RWZ, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  4. Khachik Khachikyan

    Department of Microbiology and Molecular Genetics, University of California, Irvine, United States
    Contribution
    KK, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  5. Gang Wu

    Department of Life Sciences, Imperial College London, London, United Kingdom
    Contribution
    GW, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  6. Stuart M Haslam

    Department of Life Sciences, Imperial College London, London, United Kingdom
    Contribution
    SMH, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  7. Patil Kavarian

    Department of Microbiology and Molecular Genetics, University of California, Irvine, United States
    Contribution
    PK, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  8. Anne Dell

    Department of Life Sciences, Imperial College London, London, United Kingdom
    Contribution
    AD, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  9. Michael Demetriou

    1. Department of Microbiology and Molecular Genetics, University of California, Irvine, United States
    2. Department of Neurology and Institute for Immunology, University of California, Irvine, United States
    Contribution
    MD, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    mdemetri@uci.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8547-5774

Funding

National Heart, Lung, and Blood Institute (F30HL108451)

  • Haik Mkhikian

Biotechnology and Biological Sciences Research Council (BB/K016164/1)

  • Stuart M Haslam
  • Anne Dell

Wellcome Trust

  • Anne Dell

National Institute of Allergy and Infectious Diseases (R01AI053331)

  • Michael Demetriou

National Center for Complementary and Integrative Health (R01AT007452)

  • Michael Demetriou

National Institute of Allergy and Infectious Diseases (R01AI108917)

  • Michael Demetriou

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

Research was supported by the National Institute of Allergy and Infectious Diseases (R01AI053331, R01AI108917) and National Center for Complementary and Integrative Health (R01AT007452) to MD. HM was supported by National Heart, Lung, and Blood Institute of the National Institutes of Health under award number F30HL108451. Mass spectroscopy work was supported by the Biotechnology and Biological Sciences Research Council grant BB/K016164/1 (to AD and SMH). AD is a Wellcome Trust Senior Investigator. We thank Jesse Rodriguez for artistic assistance and Adeela Syed, John Greaves, Beniam Berhane and the UCI Genomics High-Throughput Facility for technical assistance.

Ethics

Human subjects: Informed consent was obtained from human subjects to obtain peripheral blood for isolation of T cells and that resulting publications and/or presentations will not contain identifiable information. This was approved by the University of California Irvine Institutional Review board (HS#2001-2075).

Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols (#2001-2305) of the University of California, Irvine.

Reviewing Editor

  1. Benjamin S Glick, The University of Chicago, United States

Publication history

  1. Received: January 29, 2016
  2. Accepted: June 7, 2016
  3. Accepted Manuscript published: June 8, 2016 (version 1)
  4. Version of Record published: July 11, 2016 (version 2)

Copyright

© 2016, Mkhikian et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,091
    Page views
  • 505
    Downloads
  • 25
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Biochemistry and Chemical Biology
    2. Evolutionary Biology
    Corinne von Känel et al.
    Research Article
    1. Biochemistry and Chemical Biology
    Jennifer L Fribourgh et al.
    Research Article