TY - JOUR
AU - Liu,, Fu-Tong
AB - Abstract Galectins are β-galactoside-binding animal lectins primarily found in the cytosol, while their carbohydrate ligands are mainly distributed in the extracellular space. Cytosolic galectins are anticipated to accumulate on damaged endocytic vesicles through binding to glycans initially displayed on the cell surface and subsequently located in the lumen of the vesicles, and this can be followed by cellular responses. To facilitate elucidation of the mechanism underlying this process, we adopted a model system involving induction of endocytic vesicle damage with light that targets the endocytosed amphiphilic photosensitizer disulfonated aluminum phthalocyanine. We demonstrate that the levels of galectins around damaged endosomes are dependent on the composition of carbohydrates recognized by the proteins. By super resolution imaging, galectin-3 and galectin-8 aggregates were found to be distributed in distinct microcompartments. Importantly, galectin accumulation is significantly affected when cell surface glycans are altered. Furthermore, accumulated galectins can direct autophagy adaptor proteins toward damaged endocytic vesicles, which are also significantly affected following alteration of cell surface glycans. We conclude that cytosolic galectins control cellular responses reflect dynamic modifications of cell surface glycans. autophagy, damaged vesicle, galectin-3, galectin-8, glycan alteration Introduction Galectins can be categorized into the following three groups: (a) prototype galectins, which contain one carbohydrate recognition domains (CRD) and can form homodimers, such as galectin-1; (b) tandem repeat type galectins, which contain two different CRDs in tandem connected by a linker region, such as galectin-8 and galectin-9; and (c) chimera-type galectin-3, which contains a short N-terminal region, a proline-rich and glycine-rich region, and one CRD in the C-terminal region (Liu, F.T. and Rabinovich, G.A. 2005). The core structure of carbohydrates recognized by galectins is N-acetyllactosamine (LacNAc); however, there are substantial differences in carbohydrate-binding specificity among different galectins. For example, galectin-3 binds poorly to sialylated LacNAc (Stowell, S.R., Arthur, C.M., et al. 2008a), while the N-terminal CRD of galectin-8 has a preference for sialylated or sulfated carbohydrates (Ideo, H., Matsuzaka, T., et al. 2011). Many extracellular functions of galectins are associated with their binding to extracellular glycoconjugates. However, galectins are largely intracellular proteins and can exert their functions inside the cell, in a manner that may be independent of glycan binding (Liu, F.T., Patterson, R.J., et al. 2002). Recently, several studies have suggested that different galectins, including galectin-3, galectin-8, and galectin-9, are recruited to damaged intracellular vesicles through recognizing glycans (Paz, I., Sachse, M., et al. 2010, Thurston, T.L., Wandel, M.P., et al. 2012). Evidently, glycans initially presenting on the cell surface and subsequently residing in the lumen of these vesicles are exposed to the cytosolic milieu and become readily accessible to cytosolic galectins when the vesicles are damaged. Moreover, this can be followed by cellular responses, such as autophagy. For example, galectin-3 recognizes endocytic vesicles damaged by calcium phosphate precipitates and thereby directs damaged endosomes for autophagy in a manner dependent on the autophagy adapter protein p62 (Chen, X., Khambu, B., et al. 2014). Similarly, galectin-8 is recruited to damaged phagosomes containing Salmonella; it then becomes associated with the autophagy machinery, resulting in autophagy of the damaged bacteria-containing vesicles to eliminate bacterial invasion, in a manner dependent on another autophagy adapter protein, NDP52 (Thurston, T.L., Wandel, M.P., et al. 2012). Interestingly, autophagy adaptor proteins p62 and NDP52 have been reported to form nonoverlapping microdomains surrounding Salmonella and cooperatively promote antibacterial autophagy (Cemma, M., Kim, P.K., et al. 2011). However, the distribution of different galectins on damaged endosomes remains unclear. Importantly, the alteration of cell surface glycan repertoire is commonly observed under a variety of conditions (Arabyan, N., Park, D., et al. 2016, Nita-Lazar, M., Banerjee, A., et al. 2015) and endocytic vesicles are vulnerable to damage (Staring, J., von Castelmur, E., et al. 2017, Thurston, T.L., Wandel, M.P., et al. 2012). The possibility that distinct cytosolic galectins differentially sense these alterations in cell surface glycans, transmitted through the endocytosis process and then are displayed on damaged endocytic vesicles, and coordinately direct cellular responses, is intriguing. Results Galectin-3 accumulates around damaged endocytic vesicles through recognizing glycans To facilitate elucidation of the mechanism underlying galectin accumulation on damaged endosomes, we adopted a model system involving illumination of cells that have taken up photosensitizers, which is known to induce damage of endocytic vesicles in a specific and spatiotemporal manner (Hung, Y.H., Chen, L.M., et al. 2013). We allowed cells to endocytose the amphiphilic photosensitizer disulfonated aluminum phthalocyanine (AlPcS2a) and subsequently illuminated the cells with red light. We used wild-type Chinese hamster ovary (CHO) cells, which display LacNAc-containing N-glycans on their cell surface, as well as CHO cells ectopically expressing various tagged galectins. First, we determined the location of AlPcS2a under our experimental conditions, by incubating CHO cells with the compound along with dextran-fluorescein isothiocyanate (FITC), which is known to localize to endosomes, and the lysotracker DND-99. We observed that most AlPcS2a was located in dextran-FITC positive endosomes (Figure S1A). Then, with galectin-3-enhanced green fluorescent protein (EGFP)-expressing CHO cell loaded with AlPcS2a, we used a red laser to specifically illuminate one spot containing the endocytosed photosensitizers. The illuminated endocytic vesicle immediately lost fluorescence, as indicated by the disappearance of localized red signals, suggesting that the illuminated AlPcS2a was indeed activated by light. Galectin-3 aggregated at the illuminated site in a gradual and specific manner, as indicated by the appearance of localized galectin-3-EGFP green signals (Movie S1). This suggests that galectin-3 accumulates around the damaged endocytic vesicle. We also illuminated the whole field of photosensitizer-containing cells with red light. Galectin-3-EGFP-expressing cells loaded with photosensitizers, but not illuminated, or illuminated without endocytosed photosensitizers, did not form galectin-3 aggregates. Those cells treated with photosensitizers followed by red light illumination resulted in the formation of a large number of intracellular galectin-3 puncta in most cells (Figure 1A). Figure 1 Open in new tabDownload slide Galectin-3 accumulates around damaged endocytic vesicles through recognizing glycans (A) Microscope images of galectin-3-EGFP (Gal3-EGFP)-expressing CHO cells in the absence or presence of AlPcS2a, before or after illumination. (B) Electron microscope images of galectin-3-Flag-APEX2 (Gal3-Flag-APEX2)-expressing CHO cells before and after illumination. The boxed region is magnified and shown in the upper right panel. The dark region within the damaged vesicle represents aggregated Gal3-Flag-APEX2. In the upper left panel, the membrane of the depicted endocytic vesicle is marked by solid or dashed lines to represent intact (arrows) and damaged (arrow heads) membranes, respectively. Aggregated Gal3-Flag-APEX2 is highlighted in light blue, and more densely aggregated Gal3-Flag-APEX2 is highlighted in deep blue. (C) Microscope images of illuminated Gal3-EGFP-expressing CHO cells stained with Hoechst and AAL. Figure 1 Open in new tabDownload slide Galectin-3 accumulates around damaged endocytic vesicles through recognizing glycans (A) Microscope images of galectin-3-EGFP (Gal3-EGFP)-expressing CHO cells in the absence or presence of AlPcS2a, before or after illumination. (B) Electron microscope images of galectin-3-Flag-APEX2 (Gal3-Flag-APEX2)-expressing CHO cells before and after illumination. The boxed region is magnified and shown in the upper right panel. The dark region within the damaged vesicle represents aggregated Gal3-Flag-APEX2. In the upper left panel, the membrane of the depicted endocytic vesicle is marked by solid or dashed lines to represent intact (arrows) and damaged (arrow heads) membranes, respectively. Aggregated Gal3-Flag-APEX2 is highlighted in light blue, and more densely aggregated Gal3-Flag-APEX2 is highlighted in deep blue. (C) Microscope images of illuminated Gal3-EGFP-expressing CHO cells stained with Hoechst and AAL. We conducted similar experiments with CHO cells expressing galectin-3-Flag-enhanced ascorbate peroxidase 2 (APEX2), and electron microscope images suggested that galectin-3 aggregates (appearing as dark-colored regions) were associated with endocytic vesicles exhibiting damaged membrane structures (Figure 1B). In other experiments, we illuminated galectin-3-EGFP-expressing CHO cells containing AlPcS2a, followed by staining the cells with a plant lectin, Aleuria aurantia lectin (AAL), which binds preferentially to fucose-linked glycans. We found that in most cells, most of galectin-3-EGFP aggregates were stained positively with this lectin (Figure 1C), suggesting that galectin-3 was associated with glycans. We repeated the experiments with cells incubated in the presence of lactose, which is a noncell permeable galectin inhibitor that can inhibit the binding of galectin-3 to CHO cells. This did not obviously inhibit galectin-3 aggregation in most cells (Figure S1B and S1C). The results suggest that the aggregates detected are formed intracellularly. Accumulation of galectin-3 on damaged endosomes reflects cell surface carbohydrate composition To determine which part of galectin-3 contributes to its aggregation, we generated CHO cells expressing galectin-3-N1-EGFP, which expresses the N-terminal region of galectin-3 but lacks the carbohydrate-binding C-terminal region. In contrast to full-length galectin-3, galectin-3-N1-EGFP did not form intracellular puncta under similar conditions. This supports the requirement for the carbohydrate-binding C-terminal region of galectin-3 in this process (Figure 2A and2B). To further elucidate whether the carbohydrate-binding activity of galectin-3 contributes to the accumulation of this protein, TD139, a high-affinity and cell-permeable inhibitor of the galectin-3 carbohydrate-binding domain (Delaine, T., Collins, P., et al. 2016), was utilized. We observed that pretreatment with TD139 did not interfere the uptake of AlPcS2a but blocked the aggregation of intracellular galectin-3 following illumination, indicating that the carbohydrate-binding activity of galectin-3 is essential for this process (Figure 2C, 2D, and S2A). Therefore, we conclude that internalized cell surface glycans and the CRD of galectin-3 contribute to the observed intracellular aggregation of galectin-3. Figure 2 Open in new tabDownload slide Accumulation of galectin-3 on damaged endosomes reflects cell surface carbohydrate composition (A)(B) Microscope images of illuminated Gal3-EGFP- and galectin-3-N1-EGFP (Gal3N1-EGFP)-expressing CHO cells and quantification of Gal3-EGFP or Gal3N1-EGFP puncta. (C)(D) Microscope images of illuminated Gal3-EGFP-expressing CHO cells and TD139-pretreated cells, and quantification of Gal3-EGFP puncta. (E)(F) Microscope images of illuminated Gal3-EGFP-expressing CHO and Lec1 cells, and quantification of Gal3-EGFP puncta. (G)(H) Microscope images of illuminated Gal3-EGFP-expressing CHO and kifunensine-treated cells, and quantification of Gal3-EGFP puncta. Data from three independent experiments are presented as mean ± SEM, *P < 0.05. Figure 2 Open in new tabDownload slide Accumulation of galectin-3 on damaged endosomes reflects cell surface carbohydrate composition (A)(B) Microscope images of illuminated Gal3-EGFP- and galectin-3-N1-EGFP (Gal3N1-EGFP)-expressing CHO cells and quantification of Gal3-EGFP or Gal3N1-EGFP puncta. (C)(D) Microscope images of illuminated Gal3-EGFP-expressing CHO cells and TD139-pretreated cells, and quantification of Gal3-EGFP puncta. (E)(F) Microscope images of illuminated Gal3-EGFP-expressing CHO and Lec1 cells, and quantification of Gal3-EGFP puncta. (G)(H) Microscope images of illuminated Gal3-EGFP-expressing CHO and kifunensine-treated cells, and quantification of Gal3-EGFP puncta. Data from three independent experiments are presented as mean ± SEM, *P < 0.05. Next, we investigated the effects of altered glycan composition. We employed Lec1 cells; these are CHO glycosylation mutants deficient in β1,2-N-acetylglucosaminyltransferase I, which only express high-mannose N-glycans instead of LacNAc-containing N-glycans on the cell surface. We found that parental cells and glycan-modified cells endocytosed similar amounts of AlPcS2a (Figure S2B–S2E). In contrast to the results observed with CHO cells, only a few galectin-3 puncta were detected in Lec1 cells. This shows that intracellular galectin-3 can sense genetically mediated alterations in carbohydrate composition in its accumulation on damaged endocytic vesicles (Figure 2E and2F). Glycosylation can also be modified by inhibiting enzymes involved in the biosynthetic pathways. For example, treatment with the alkaloid kifunensine, an α-mannosidase I inhibitor isolated from Kitasatosporia kifunense, results in cell surface sugars being enriched in high-mannose N-glycans instead of LacNAc-containing N-glycans. As shown in Figure 2G and2H, unlike untreated CHO cells, few puncta were observed in kifunensine-treated cells. Galectin-8 accumulates around damaged endocytic vesicles through recognizing glycans We generated CHO cells expressing galectin-8-EGFP and conducted similar experiments. The red signal indicating localized AlPcS2a disappeared after specific illumination of the region containing endocytosed photosensitizers, and a green punctum representing galectin-8-EGFP aggregates formed immediately (Movie S2). This suggests that galectin-8 accumulates at impaired vesicles more quickly than galectin-3, when the vesicles are damaged. Cells loaded with photosensitizers, but not illuminated, or illuminated without endocytosed photosensitizers, did not form obvious galectin-8 aggregates. Illumination with red light of the whole field of cells containing endocytosed photosensitizers resulted in formation of a large number of galectin-8 aggregates in most cells (Figure 3A). Figure 3 Open in new tabDownload slide Galectin-8 accumulates around damaged endocytic vesicles through recognizing glycans (A) Microscope images of galectin-8-EGFP (Gal8-EGFP)-expressing CHO cells in the absence or presence of AlPcS2a, before or after illumination. (B) Electron microscope images of galectin-8-Flag-APEX2 (Gal8-Flag-APEX2)-expressing CHO cells before and after illumination. The boxed region is magnified and shown in the upper right panel. The dark region within the damaged vesicle represents aggregated Gal8-Flag-APEX2. In the upper left panel, the membrane of the depicted endocytic vesicle is marked by solid or dashed lines to represent intact (arrows) and damaged (arrow heads) membranes, respectively. Aggregated Gal8-Flag-APEX2 is highlighted in light blue. (C) Microscope images of illuminated Gal8-EGFP-expressing CHO cells stained with Hoechst and AAL. Figure 3 Open in new tabDownload slide Galectin-8 accumulates around damaged endocytic vesicles through recognizing glycans (A) Microscope images of galectin-8-EGFP (Gal8-EGFP)-expressing CHO cells in the absence or presence of AlPcS2a, before or after illumination. (B) Electron microscope images of galectin-8-Flag-APEX2 (Gal8-Flag-APEX2)-expressing CHO cells before and after illumination. The boxed region is magnified and shown in the upper right panel. The dark region within the damaged vesicle represents aggregated Gal8-Flag-APEX2. In the upper left panel, the membrane of the depicted endocytic vesicle is marked by solid or dashed lines to represent intact (arrows) and damaged (arrow heads) membranes, respectively. Aggregated Gal8-Flag-APEX2 is highlighted in light blue. (C) Microscope images of illuminated Gal8-EGFP-expressing CHO cells stained with Hoechst and AAL. In addition, we conducted experiments with CHO cells expressing galectin-8-Flag-APEX2. Electron microscope images also indicated that galectin-8 aggregates (appearing as dark-colored regions) were associated with damaged membrane structures of endocytic vesicles (Figure 3B). We illuminated galectin-8-EGFP-expressing CHO cells containing AlPcS2a, followed by staining the cells with AAL, and confirmed that galectin-8 aggregates colocalized with carbohydrates, since they were stained positively with this lectin in most cells (Figure 3C). Accumulation of galectin-8 on damaged endosomes reflects cell surface carbohydrate composition In contrast to galectin-3, which only carries one CRD specific for LacNAc (Patnaik, S.K., Potvin, B., et al. 2006), galectin-8 possesses two distinct CRDs; the N-terminal CRD recognizes sialylated or sulfated glycoconjugates (Ideo, H., Matsuzaka, T., et al. 2011), while the C-terminal CRD recognizes LacNAc (Stowell, S.R., Arthur, C.M., et al. 2008b). Unlike galectin-3, we found that galectin-8 still formed a few intracellular aggregates in galectin-8-EGFP-expressing Lec1 cells (Figure 4A and4B). This suggests that glycans other than those containing LacNAc may be involved in mediating intracellular galectin-8 aggregation. Figure 4 Open in new tabDownload slide Accumulation of galectin-8 on damaged endosomes reflects cell surface carbohydrate composition (A)(B) Microscope images of illuminated Gal8-EGFP-expressing CHO and Lec1 cells, and quantification of Gal8-EGFP puncta. (C)(D) Microscope images of illuminated Gal8-EGFP-expressing CHO and Lec2 cells, and quantification of Gal8-EGFP puncta. (E)(F) Microscope images of illuminated Gal8-EGFP-expressing CHO and Lec3.2.8.1 cells, and quantification of Gal8-EGFP puncta. Data from three independent experiments are presented as mean ± SEM, *P < 0.05. Figure 4 Open in new tabDownload slide Accumulation of galectin-8 on damaged endosomes reflects cell surface carbohydrate composition (A)(B) Microscope images of illuminated Gal8-EGFP-expressing CHO and Lec1 cells, and quantification of Gal8-EGFP puncta. (C)(D) Microscope images of illuminated Gal8-EGFP-expressing CHO and Lec2 cells, and quantification of Gal8-EGFP puncta. (E)(F) Microscope images of illuminated Gal8-EGFP-expressing CHO and Lec3.2.8.1 cells, and quantification of Gal8-EGFP puncta. Data from three independent experiments are presented as mean ± SEM, *P < 0.05. Next, we investigated whether sialic acid contributes to the galectin-8 aggregation using Lec2 cells, which are deficient in the cytidine 5′-monophosphate (CMP)-sialic acid transporter and possess few sialic acid residues on glycoconjugates. We observed fewer puncta in galectin-8-EGFP-expressing Lec2 cells compared with CHO cells, indicating that sialic acid contributes to galectin-8 aggregation (Figure 4C and4D). We also used galectin-8-EGFP-expressing Lec3.2.8.1 cells, which are deficient in both LacNAc and sialic acid. Galectin-8-EGFP did not form puncta in these cells (Figure 4E and4F), suggesting that both LacNAc and sialic acid contribute to galectin-8 aggregation. Collectively, our data indicate that galectin-3 and galectin-8 differentially recognize glycans on impaired endocytic vesicles. Galectin-3 and galectin-8 aggregates are distributed in distinct microdomains To study the distribution of galectin-3 and galectin-8 aggregates, we employed structured illumination microscopy (SIM), a super resolution technique. CHO cells expressing both TagRFP-galectin-3 and galectin-8-EGFP were allowed to endocytose photosensitizers and then illuminated with red light. We observed that galectin-3 and galectin-8 aggregates reside in distinct microdomains: galectin-3 was mainly located in the outer region, while galectin-8 was mainly located in the inner space (Figure 5). Figure 5 Open in new tabDownload slide Galectin-3 and galectin-8 aggregates are located in different microdomains SIM images of TagRFP-galectin-3- and Gal8-EGFP-expressing CHO cells after illumination. The boxed regions are magnified and shown in the right panels. Scale bars: 500 nm. Figure 5 Open in new tabDownload slide Galectin-3 and galectin-8 aggregates are located in different microdomains SIM images of TagRFP-galectin-3- and Gal8-EGFP-expressing CHO cells after illumination. The boxed regions are magnified and shown in the right panels. Scale bars: 500 nm. Accumulation of galectins reflects glycosidase-induced changes in cell surface glycans The host carbohydrate repertoire on the cell surface is frequently and directly modulated by extracellular glycosidases derived from microbes during infection and under other conditions, such as inflammation and tumor metastasis (Dube, D.H. and Bertozzi, C.R. 2005, Yang, W.H., Heithoff, D.M., et al. 2017). Therefore, we investigated whether intracellular galectins could detect changes in cell surface carbohydrates induced by a microbial glycosidase, namely PNGase F, which is an endoglycosidase secreted by Flavobacterium meningosepticum that hydrolyzes N-linked glycans (Hall, M.K., Weidner, D.A., et al. 2014). As shown in Figure 6A and6B, few puncta were observed in PNGase F-treated CHO cells that had endocytosed the photosensitizers and subsequently illuminated, suggesting that galectin-3 is an intracellular sensor that detects pathogen-induced changes in cell surface glycans. Figure 6 Open in new tabDownload slide Accumulation of galectins reflects glycosidase-mediated changes in cell surface glycans (A)(B) Microscope images of illuminated Gal3-EGFP-expressing CHO and PNGase F-treated cells, and quantification of Gal3-EGFP puncta. (C)(D) Microscope images of illuminated A549 cells and neuraminidase (Neu)-treated cells stained with galectin-8 antibody, and quantification of galectin-8 puncta. (E)(F) Microscope images of illuminated galectin-8-deficient and galectin-3-EGFP-expressing (Gal8KD/Gal3-EGFP) A549 cells and Neu-treated cells, and quantification of Gal3-EGFP puncta in Gal8KD/Gal3-EGFP A549 cells and Neu-treated cells. Data from three independent experiments are presented as mean ± SEM, *P < 0.05. Figure 6 Open in new tabDownload slide Accumulation of galectins reflects glycosidase-mediated changes in cell surface glycans (A)(B) Microscope images of illuminated Gal3-EGFP-expressing CHO and PNGase F-treated cells, and quantification of Gal3-EGFP puncta. (C)(D) Microscope images of illuminated A549 cells and neuraminidase (Neu)-treated cells stained with galectin-8 antibody, and quantification of galectin-8 puncta. (E)(F) Microscope images of illuminated galectin-8-deficient and galectin-3-EGFP-expressing (Gal8KD/Gal3-EGFP) A549 cells and Neu-treated cells, and quantification of Gal3-EGFP puncta in Gal8KD/Gal3-EGFP A549 cells and Neu-treated cells. Data from three independent experiments are presented as mean ± SEM, *P < 0.05. Microbes also express neuraminidases, which cleave sialic acids presented on host glycoproteins to obtain nutrients, or for cellular entry during infection (Arabyan, N., Park, D., et al. 2016). Significantly fewer galectin-8 puncta were formed in lung epithelial A549 cells containing endocytosed photosensitizers after illumination, when cells were first treated with neuraminidase before allowing to endocytose the photosensitizers (Figure 6C and6D). This illustrates that intracellular galectin-8 surveys the repertoire of cell surface glycans affected by desialylation. We also generated galectin-8-deficient and galectin-3-EGFP-expressing A549 cells and found more galectin-3-EGFP puncta in neuraminidase-treated cells compared with nontreated cells, indicating that intracellular galectin-3 also detects alteration in cell surface sialylation (Figure 6E and6F). Importantly, while cell surface desialylation leads to reduced galectin-8 aggregation, it promotes galectin-3 aggregation around damaged endosomes. Alteration in cell surface sialylation down regulates galectin-8-mediated autophagic activation Galectin-8 physically interacts with the autophagy adapter protein NDP52, and accumulated galectin-8 attracts NDP52 and the autophagy marker LC3 to Salmonella damaged vesicles, leading to autophagy (Thurston, T.L., Wandel, M.P., et al. 2012). Next, we studied whether desialylation of cell surface carbohydrates results in altered recruitment of NDP52 and LC3 to damaged endosomes. We showed that galectin-8 effectively recruited NDP52 and LC3, since it colocalized with these molecules in A549 cells treated to induce endosomal damage. However, fewer galectin-8, NDP52, and LC3 aggregates were detected in A549 cells, when the cells were first treated with neuraminidase (Figure 7A–D). We conclude that cell surface sialylation contributes to galectin-8 accumulation as well as galectin-8-mediated autophagic activation. Desialylation of cell surface glycoconjugates causes a reduction in these responses. Our data provide evidence that cell surface desialylation results in reduced galectin-8-mediated autophagic activation. Figure 7 Open in new tabDownload slide Alteration in cell surface sialylation down regulates galectin-8-mediated autophagic activation (A–D) Microscope images of A549 cells and Neu-treated cells stained with galectin-8, NDP52, and LC3 antibodies 30 minutes after illumination, and quantification of galectin-8, NDP52, and LC3 puncta. Data from three independent experiments are presented as mean ± SEM, *P < 0.05. Figure 7 Open in new tabDownload slide Alteration in cell surface sialylation down regulates galectin-8-mediated autophagic activation (A–D) Microscope images of A549 cells and Neu-treated cells stained with galectin-8, NDP52, and LC3 antibodies 30 minutes after illumination, and quantification of galectin-8, NDP52, and LC3 puncta. Data from three independent experiments are presented as mean ± SEM, *P < 0.05. Alteration in cell surface sialylation promotes galectin-3-mediated autophagic activation A previous study showed that galectin-3 recognizes endocytic vesicles damaged by calcium phosphate precipitates and thereby directs damaged endosomes for autophagy in a manner dependent on the autophagy adapter protein p62 (Chen, X., Khambu, B., et al. 2014). We studied galectin-3 in a similar manner and demonstrated colocalization of galectin-3-EGFP, p62, and LC3, in galectin-8-deficient and galectin-3-EGFP-expressing A549 cells (Figure 8A). Treatment with neuraminidase also significantly increased the number of galectin-3 puncta, supporting that desialylation contributes to augmented galectin-3 accumulation around damaged endosomes (Figure 8A and8B). Moreover, this treatment resulted in more p62 and LC3 molecules colocalizing with galectin-3 aggregates (Figure 8A, 8C, and8D). Thus, exaggerated galectin-3 accumulation results in the recruitment of higher levels of p62 and LC3. Figure 8 Open in new tabDownload slide Alteration in cell surface sialylation promotes galectin-3-mediated autophagic activation (A–D) Microscope images of Gal8KD/Gal3-EGFP A549 cells and Neu-treated cells stained with p62 and LC3 antibodies 30 minutes after illumination, and quantification of Gal3-EGFP puncta, Gal3-EGFP-positive p62 puncta, and Gal3-EGFP-positive LC3 puncta. Data from three independent experiments are presented as mean ± SEM, *P < 0.05. Figure 8 Open in new tabDownload slide Alteration in cell surface sialylation promotes galectin-3-mediated autophagic activation (A–D) Microscope images of Gal8KD/Gal3-EGFP A549 cells and Neu-treated cells stained with p62 and LC3 antibodies 30 minutes after illumination, and quantification of Gal3-EGFP puncta, Gal3-EGFP-positive p62 puncta, and Gal3-EGFP-positive LC3 puncta. Data from three independent experiments are presented as mean ± SEM, *P < 0.05. Discussion Glycosylation is a ubiquitous modification of membrane-anchored proteins and lipids, and is dynamically and diversely modulated by endogenous and exogenous glycosyltransferases and glycosidases. For example, influenza virus and pneumococcus directly desialylate cell surface carbohydrates in airway epithelial cells (Ju, X., Yan, Y., et al. 2015, Nita-Lazar, M., Banerjee, A., et al. 2015). How host cells respond to changes in cell surface carbohydrates during pathogen infections and challenges by environmental insults is intriguing. Here, we illustrate the internalization of cell surface glycans and their consequent exposure to the cytosolic milieu is a fundamental process that mediates the intracellular accumulation of galectins on damaged endosomes. We show that both galectin-3 and galectin-8 accumulate on damaged endosomes, and both can sense changes in cell surface glycan composition. Importantly, changes in carbohydrates have contrasting effects on the accumulation of the two different galectins; however, similar autophagic consequences are mediated by these galectins, suggesting that they share a complementary role. Similarly, our research group recently found that cytosolic galectin-3 and galectin-8 could detect cell surface carbohydrate alterations in macrophages subjected to Listeria monocytogenes infection (Weng, I.C., Chen, H.L., et al. 2018). Overall, our data indicate that changes in cell surface glycans can be detected intracellularly and that intracellular galectins are novel mediators that act in combination to direct impaired endocytic vesicles for autophagy. Importantly, as the composition of cellular surface glycans can be modified by various means, our data also suggest that one can control intracellular galectin-guided cellular responses, such as autophagic activation for pathogen clearance, by modification of cell surface glycans. Figure 9 Open in new tabDownload slide Intracellular galectins detect cell surface carbohydrate alteration and control cellular responses The AlPcS2a is endocytosed along with cell surface glycoconjugates into the lumen of endocytic vesicles. Red light illumination activates endocytosed AlPcS2a and triggers damage of endocytic vesicles. Cell surface carbohydrate composition is constantly altered under various circumstances, for example, neuraminidase produced by various microbes directly desialylates cell surface carbohydrates. Intracellular galectin-8 accumulates around damaged vesicles by preferentially binding to intracellularly exposed sialylated carbohydrates, while galectin-3 does so by preferentially binding to intracellularly exposed nonsialylated carbohydrates. Both then recruit autophagy adapter proteins and direct damaged vesicles for autophagy. Collectively, intracellular galectins are novel intracellular sensors that decrypt sophisticated information from distinct cell surface sugar codes and together direct damaged vesicles for autophagy. Figure 9 Open in new tabDownload slide Intracellular galectins detect cell surface carbohydrate alteration and control cellular responses The AlPcS2a is endocytosed along with cell surface glycoconjugates into the lumen of endocytic vesicles. Red light illumination activates endocytosed AlPcS2a and triggers damage of endocytic vesicles. Cell surface carbohydrate composition is constantly altered under various circumstances, for example, neuraminidase produced by various microbes directly desialylates cell surface carbohydrates. Intracellular galectin-8 accumulates around damaged vesicles by preferentially binding to intracellularly exposed sialylated carbohydrates, while galectin-3 does so by preferentially binding to intracellularly exposed nonsialylated carbohydrates. Both then recruit autophagy adapter proteins and direct damaged vesicles for autophagy. Collectively, intracellular galectins are novel intracellular sensors that decrypt sophisticated information from distinct cell surface sugar codes and together direct damaged vesicles for autophagy. In this study, we applied a model system to demonstrate that intracellular galectins can decipher sophisticated sugar codes on the cell surface through differential accumulation to damaged endocytic vesicles. Recent studies indicated that galectins accumulated on damaged endosomes or lysosomes under several scenarios, such as microbe infection (Staring, J., von Castelmur, E., et al. 2017, Thurston, T.L., Wandel, M.P., et al. 2012), the exposure of cells to aggregated α-synuclein (Freeman, D., Cedillos, R., et al. 2013) or amyloid beta peptides (Oku, Y., Murakami, K., et al. 2017), and the uptake of silica or monosodium urate (Maejima, I., Takahashi, A., et al. 2013). These findings suggest that galectins have important roles in regulating cellular responses in diverse physiological and pathological processes associated with intracellular organelle damage, and our study suggests that our conclusions may be generalized to a variety of conditions. In the meantime, while we have established a system in which endosomes are damaged, earlier studies indicate that under different conditions, lysosomes are damaged in cells that have taken up the same photosensitizers and then illuminated (Hung, Y.H., Chen, L.M., et al. 2013). Thus, our systems may be extended to study the biological responses associated with lysosomal damage. In this study, we found that galectin-8 immediately accumulates, while galectin-3 gradually accumulates around damaged endosome after specific red-light illumination of endocytosed photosensitizers (Movie S1 and S2). Similarly, SIM revealed that galectin-3 and galectin-8 localize in distinct microdomains of damaged endosomes: galectin-3 mainly distributes in the outer region, while galectin-8 mainly accumulates in the inner region around damaged endosomes (Figure 5). Because these two galectins preferentially recognize different glycans, the results suggest that different microdomains on damaged endosomes have distinct glycan compositions. In addition to galectin-3 and galectin-8, previous studies have shown that other galectins, including galectin-1, galectin-4, and galectin-9, also targeted towards impaired endocytic vesicles (Mansilla Pareja, M.E., Bongiovanni, A., et al. 2017). As these galectins prefer specific carbohydrates and differentially recognize glycans displayed on cells (Patnaik, S.K., Potvin, B., et al. 2006, Rabinovich, G.A. and Toscano, M.A. 2009), further study is needed to determine whether changes in cell surface carbohydrates spatially and temporally contribute to different galectin accumulation patterns around impaired organelles and further differentially regulate versatile downstream cellular responses. Previously studies indicate that both galectin-3 and galectin-8 contribute to directing damaged vesicles for autophagy (Chen, X., Khambu, B., et al. 2014, Thurston, T.L., Wandel, M.P., et al. 2012). We found that desialylation diminishes galectin-8 accumulation followed by reduced NDP52 and LC3 accumulation in A549 cells (Figure 7). Moreover, we observed that less NDP52 and LC3 accumulated to damaged endosomal vesicles in galectin-8-deficient A549 cells compared with control cells (data not shown), supporting that galectin-8 mediates NDP52 and LC3 and direct endosomal vesicles for autophagy in A549 cells. In addition, we observed p62 and LC3 accumulation in galectin-8-deficient and galectin-3-EGFP-expressing A549 cells. Furthermore, desialylation promotes galectin-3 accumulation, followed by enhanced p62 and LC3 accumulation, indicating that galectin-3 leads to p62 and LC3 accumulation at damaged vesicles in those cells (Figure 8). These findings support that both galectin-3 and galectin-8 contribute to directing damaged vesicles for autophagy in our model system. The accumulation and dissociation of galectins around damaged vesicles seem to be a dynamic process. We loaded cells with the photosensitizer and spot-illuminated photosensitizer-containing endosomes in TagRFP-galectin-3- and EGFP2-p62-expressing CHO cells. Through a time-lapse confocal microscopic study, we found that galectin-3 and p62 did not accumulate around targeted endosomes before spot illumination in selected cells. After illumination, we observed that galectin-3 accumulated at the illuminated spot first at the 5-minute time point. Later, p62 gradually accumulated around galectin-3-positive area at the 20-minute time point. At the 30-minute time point, galectin-3 signal gradually diminished (Figure S6A and Figure S6B). We also performed field illumination of cells containing photosensitizers and immunostaining of galectins, autophagy adapters, and markers at different time points after endosomal damage. We found that galectins formed aggregates after field illumination of these cells. We noticed that the number of galectin-8 puncta reached a maximum at 15-minute and slightly declined at the 20- and 30-minute time points. However, the number of galectin-3 puncta decreased substantially at the 20-minute time point in galectin-8-deficient- and galectin-3-EGFP-expressing A549 cells. In both cases, the number of puncta composed of autophagy adapters and markers gradually increased during the 30-minute observation period. These data suggest that galectin-3 and galectin-8 have different dissociation kinetics after binding to the glycans following endosomal damage (Figure S7A and S7B). Our findings suggest that galectins may eventually dissociate from the damaged vesicles, resulting in some puncta composed of autophagy adapters or markers and being devoid of galectins, as observed in Figure 7 and Figure 8. In addition to selective autophagy, recent studies have shown that galectin accumulation may contribute to other cellular responses. For example, galectin-3 colocalizes with components of the inflammasome complex, including Nalp3, Ipaf, ASC, and caspase-1, suggesting that accumulated galectin-3 may participate in inflammasome activation (Chen, Y.J., Wang, S.F., et al. 2018, Dupont, N., Lacas-Gervais, S., et al. 2009). Moreover, galectin-8 colocalizes with the SNARE protein TRIM16 to mediate IL-1β secretion through secretory autophagy (Kimura, T., Jia, J., et al. 2017). In addition, galectin-8 interacts with Ser/Thr protein kinase mTOR apparatus and inhibits its activity, whereas galectin-9 interacts with transforming growth factor-β activating kinase 1 (TAK1) and promotes AMP-activated protein kinase activity (Jia, J., Abudu, Y.P., et al. 2018). As changes in cell surface carbohydrates modulate differential galectin accumulation, further investigation is needed to determine whether the modulation of the glycan composition of glycoconjugates affects inflammasome activation, secretory autophagy, or mTOR regulation through galectins. Our findings indicate that both intracellular galectin-3 and galectin-8 accumulate on damaged endosomes through binding to glycans, thus contributing to the integrated processing of impaired endocytic vesicles for autophagy. Importantly, galectin-3 and galectin-8 distribute in different regions and moreover show different patterns of aggregation in response to cell surface glycan alterations. We conclude that galectins are novel intracellular sensors that decrypt sophisticated information from distinct cell surface sugar codes and together direct damaged vesicles for autophagy (Figure 9). Materials and methods Reagents The AlPcS2a was purchased from Frontier Scientific. Kifunensine was obtained from Sigma, PNGase F was obtained from New England Biolabs, and neuraminidases were purchased from Sigma and Roche. The synthesis of TD139 was originally described in U.S. Patent Application Publication (No. 2014/0011765 A1) and the material used for this study was synthesized in the laboratory of Dr. Chun-Hung Lin (Hsieh, T.J., Lin, H.Y., et al. 2016). Cell culture CHO, Lec1, Lec2, and A549 cells were obtained from the American Type Culture Collection, and Lec3.2.8.1 cells were obtained from Dr. Pamela Stanley (Albert Einstein College of Medicine, Bronx, NY). CHO and Lec cells were grown in minimum essential medium alpha supplemented with 10% fetal bovine serum and A549 cells were grown in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2. Cells were transfected with galectin-expressing plasmids using Lipofectamine 2000 transfection reagent (Invitrogen). This was followed by fluorescence-activated cell sorting using a FACSJazz Cell Sorter (BD Biosciences) and Geneticin selection to obtain galectin-EGFP-expressing cells. Cells were transfected with galectin-Flag-APEX2-expressing plasmids similarly followed by puromycin selection to obtain galectin-3-Flag-APEX2- and galectin-8-Flag-APEX2-expressing cells. Cells were pretreated with the reagents at the following concentrations and for the following periods before illumination: TD139: 25 μM in culture medium, 3 hours; kifunensine: 20 μM in culture medium, 48 hours; PNGase F, 50,000 U/mL in serum free medium, 1 hour; and neuraminidases, 1:1 0.3 U/mL of each neuraminidase in serum-free medium, 1 hour. To identify the location of endocytosed AlPcS2a, lysosomes were first tagged by preloading cells with 200 nM lysotracker DND-99 for 2 hours. Cells were then loaded with 0.25 mg/mL: dextran-FITC to tag endosomes, in combination with AlPcS2a for 15 minutes. Cells were fixed in 4% paraformaldehyde in PBS for 10 minutes and were then stained with 0.2 μg/mL Hoechst 33342 (Invitrogen) for 15 minutes. To quantify the amount of endocytosed AlPcS2a, AlPcS2a-loaded cells were fixed in 4% paraformaldehyde in PBS for 10 minutes and analyzed using an Attune NxT flow cytometer (Invitrogen) (Figure S2A–E). To examine endogenous galectin-8, autophagy adapter proteins NDP52 or p62, and autophagy maker protein LC3, cells were blocked and permeabilized with 0.05% saponin in 1% bovine serum albumin (BSA) containing PBS for 30 minutes at 4°C. Cells were stained with primary antibodies overnight at 4°C and Alexa Fluor-conjugated secondary antibodies for 1 hour at 4°C. Cells were then stained with 0.2 μg/mL Hoechst 33342 (Invitrogen) for 15 minutes at room temperature, and images were acquired and analyzed. To examine carbohydrates on the cell-surface and damaged vesicles, galectin-expressing cells were illuminated, blocked with PBS containing 1% BSA, and permeabilized with 0.05% saponin in PBS with 1% BSA for 30 minutes at 4°C. Cells were stained with biotinylated 2 μg/mL AAL for 1 hour at 4°C and further stained with 0.5 μg/mL Alexa Fluor-conjugated streptavidin for 1 hour at 4°C. Then, cells were stained with 0.2 μg/mL Hoechst 33342 (Invitrogen) for 15 minutes at room temperature, and images were acquired and analyzed. To examine cell surface glycans, cells were washed in PBS containing 2% BSA and incubated for 1 hour at 4°C with 5 μg/mL FITC-conjugated recombinant galectin-3, 10 μg/mL FITC-conjugated recombinant galectin-8, or 5 μg/mL biotinylated Maackia amurensis lectin followed by 0.5 μg/mL fluorescent-conjugated streptavidin staining for 1 hour at 4°C. Images were acquired and analyzed (Figure S3A–F). Plasmids Galectin-3-EGFP-expressing, galectin-3-N1-EGFP, and TagRFP-galectin-3-expressing plasmids were constructed as previously described (Chu, Y.P., Hung, Y.H., et al. 2017, Wang, S.F., Tsao, C.H., et al. 2014). We observed the amount of ectopically expressed galectin-3-EGFP was much higher than endogenous galectin-3 in galectin-EGFP-expressing CHO cells (Figure S4A and S4B). In addition, we found the fluorescence intensities of galectin-3-EGFP and galectin-3-N1-EGFP were similar in CHO cells expressing those two constructs, suggesting that the amounts of these proteins were comparable in those cells (Figure S4C). Galectin-8 cDNA was cloned into pEGFP-N1 vector (Clontech) to generate a galectin-8-EGFP-expressing plasmid. Galectin-3, galectin-8, Flag tag, and APEX2 cDNA were cloned into pLAS5w.Ppuro vector (National RNAi Core Facility, Academia Sinica) to generate galectin-3-Flag-APEX2- and galectin-8-Flag-APEX2-expressing plasmids. EGFP2-p62 expressing plasmid was constructed as previously described (Hung, Y.H., Chen, L.M., et al. 2013). RNA interference A549 cells were infected with lentivirus containing shRNAs against luciferase or galectin-8. The shRNA sequences used were as follows: control shRNA targeting the luciferase gene: 5′-GCGGTTGCCAAGAGGTTCCAT-3′; and galectin-8 shRNA: 5′-TATTACCTCTTTCCCATTTAG-3′. Cells were subjected to puromycin selection and the level of galectin-8 was quantified by immunostaining with galectin-8 antibody (Figure S1D). Binding analysis of galectin-tag proteins to β-galactosides Cells expressing galectin-EGFP, TagRFP-galectin, or galectin-Flag-APEX2 were collected and lysed in RIPA lysis buffer (Millipore) containing protease inhibitor cocktail. Cell lysate was centrifuged to remove cell debris. One-twentieth volume of the cell lysate was loaded as input control. The same amount of the cell lysate was incubated with lactose-conjugated Sepharose beads for 1 hour at 4°C. After centrifugation, the supernatant was collected and loaded as nonbound fraction. After washing, the bound material was eluted from the beads with sodium dodecyl sulfate (SDS) sample buffer. Samples were analyzed by SDS-PAGE and immunoblotting using anti-EGFP, anti-TagRFP, or anti-Flag antibodies (Figure S5A–E). Antibodies, recombinant galectins, lectins, and streptavidin Antibodies were obtained from R&D Systems (galectin-8, AF1305), Abcam (NDP52, ab68588; p62, ab56416; EGFP, ab184601), MBL (LC3, M152-3, and PM036), Evrogen (TagRFP, AB233), Sigma (FLAG, A8592), and Invitrogen (Alexa Fluor-conjugated secondary antibodies, A31571, A10037, A10042, A31573, and A11055). Recombinant human galectin-3 was prepared by Miss Chi-Chun Huang at the Institute of Biomedical Sciences (IBMS), Academia Sinica (AS), and recombinant human galectin-8 was provided by Professor Kuo-I Lin at the Genomics Research Center, AS, and Dr. Hsien-Ya Lin at the Institute of Biological Chemistry, AS. Biotinylated AAL, B-1395, and biotinylated Maackia amurensis lectin II, B-1265 were obtained from Vector Labs. Streptavidin was obtained from Invitrogen (Alexa Fluor-conjugated streptavidin, S11226). Light-induced damage to endocytic vesicles Cells were loaded with 1 mM AlPcS2a in phenol red free medium for 15 minutes and then washed three times with PBS. Cells were cultured in phenol red free medium and illuminated with a red laser using LSM 510 confocal microscope (Carl Zeiss) with a ×100 (1.3 NA) oil immersion objective or the LED red light (Thorlabs) to induce endosomal damage. Microscopy Cells were grown in a 60-mm culture dish with culture inserts (iBidi) for one spot laser illumination, continuous confocal microscope observations, and analysis using LSM 510 confocal microscope (Carl Zeiss) with a ×100 (1.3 NA) oil immersion objective. Cells were grown in CellCarrier 96 polystyrene microplates (PerkinElmer) for whole field illumination. Treated cells were washed with PBS and fixed in 4% paraformaldehyde in PBS for 10 minutes at room temperature, followed by 0.2 μg/mL Hoechst 33342 (Invitrogen) staining for 15 minutes at room temperature. Images were automatically acquired and analyzed using ImageXpress Micro system (Molecular Devices) with a plan apochromatic ×40 (0.95 NA) air objective, and the numbers of aggregates and the fluorescence intensity of galectin-EGFP in cells were determined with the custom module of MetaXpress software (Molecular Devices). Electron Microscopy Galectin-3-Flag-APEX2- and galectin-8-Flag-APEX2-expressing cells were grown on an electron microscopy (EM) membrane in a 60-mm culture dish. Cells were loaded with the photosensitizers, followed by whole field illumination; treated cells were further processed for 3,3-diaminobenzidine staining and EM imaging (Martell, J.D., Deerinck, T.J., et al. 2017). Structured Illumination Microscopy Structured illumination microscope images were acquired using a Zeiss ELYRA PS.1 LSM780 system with a plan apochromatic ×63 (1.4 NA) oil immersion DIC M27 objective (Carl Zeiss). The raw images were reconstructed using ZEN 2012 (Carl Zeiss) under the default parameters, and the reconstructed images were corrected for chromatic aberration in the x, y, and z directions using multicolor beads (100 nm). Statistical Analysis Data from three independent experiments are presented as mean ± SEM and analyzed using unpaired two-tailed Student’s t test. P < 0.05 was considered statistically significant. Funding This work was supported by grants from Ministry of Science and Technology (MOST 104-0210-01-09-02, MOST 105-0210-01-13-01 and MOST 106-0210-01-15-02), and Academia Sinica Thematic Project (AS-105-TP-B08). Acknowledgements We thank Professor Pamela Stanley at the Albert Einstein College of Medicine for providing Lec3.2.8.1 cells; Miss Chi-Chun Huang at the Institute of Biomedical Sciences (IBMS), Academia Sinica (AS), for preparation of recombinant human galectin-3; Professor Kuo-I Lin at the Genomics Research Center, AS, and Dr. Hsien-Ya Lin at the Institute of Biological Chemistry, AS, for providing recombinant human galectin-8; and Mr. Ting-Jui Tu at the IBMS, AS, for designing and cloning the galectin-Flag-APEX2 constructs. We thank Dr. Chia-Lin Ho at Molecular Devices and the common equipment core facility of IBMS (Core Facility Project AS-CFII-108-113 and AS-CFII-108-115) and the Institute of Molecular Biology, AS, for assisting in DNA sequencing, image acquisition and flow cytometry analysis; the EM core facility of IBMS and ICOB, AS, for assisting in sample preparation and EM graph acquisition (Core Facility Project AS-CFII-108-119); the National RNAi Core Facility, AS, for providing shRNA reagents and plasmids. We thank Professor Chi-Yu Fu and Mr. Ferdie Fatiga at the Institute of Cellular and Organismic Biology (ICOB), AS, for their advice on APEX2-3,3-diaminobenzidine staining for electron microscopy (EM), and Dr. Jane M. Liu at Pomona College for reading and commenting on the manuscript. Conflict of interest statement None declared. 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TI - Intracellular galectins control cellular responses commensurate with cell surface carbohydrate composition
JF - Glycobiology
DO - 10.1093/glycob/cwz075
DA - 2020-01-01
UR - https://www.deepdyve.com/lp/oxford-university-press/intracellular-galectins-control-cellular-responses-commensurate-with-G04hjRayzo
SP - 49
VL - 30
IS - 1
DP - DeepDyve
ER -