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Galectin-3 is crucial to many physiological and pathological processes. The generally accepted dogma is that galectins function extracellularly by binding specifically to β(1→4)-galactoside epitopes on cell surface glycoconjugates. Here, we used crystallography and NMR spectroscopy to demonstrate that negatively charged homogalacturonans (HG, linear polysaccharides of α(1→4)-linked-D-galacturonate (GalA)) bind to the galectin-3 carbohydrate recognition domain. The HG carboxylates at the C6 positions in GalA rings mandate that this saccharide bind galectin-3 in an unconventional, “topsy-turvy” orientation that is flipped by about 180o relative to that of the canonical β-galactoside lactose. In this binding mode, the reducing end GalA β-anomer of HGs takes the position of the nonreducing end galactose residue in lactose. This novel orientation maintains interactions with the conserved tryptophan and seven of the most crucial lactose-binding residues, albeit with different H-bonding interactions. Nevertheless, the HG molecular orientation and new interactions have essentially the same thermodynamic binding parameters as lactose. Overall, our study provides structural details for a new type of galectin–sugar interaction that broadens glycospace for ligand binding to Gal-3 and suggests how the lectin may recognize other negatively charged polysaccharides like glycoaminoglycans (e.g. heparan sulfate) on the cell surface. This discovery impacts on our understanding of galectin-mediated biological function.

Galectin-13 (Gal-13) plays numerous roles in regulating the relationship between maternal and fetal tissues. Low expression levels or mutations of the lectin can result in pre-eclampsia. The previous crystal structure and gel filtration data show that Gal-13 dimerizes via formation of two disulfide bonds formed by Cys136 and Cys138. In the present study, we mutated them to serine (C136S, C138S and C136S/C138S), crystalized the variants and solved their crystal structures. All variants crystallized as monomers. In the C136S structure, Cys138 formed a disulfide bond with Cys19, indicating that Cys19 is important for regulation of reversible disulfide bond formation in this lectin. Hemagglutination assays demonstrated that all variants are inactive at inducing erythrocyte agglutination, even though gel filtration profiles indicate that C136S and C138S could still form dimers, suggesting that these dimers do not exhibit the same activity as wild-type (WT) Gal-13. In HeLa cells, the three variants were found to be distributed the same as with WT Gal-13. However, a Gal-13 variant (delT221) truncated at T221 could not be transported into the nucleus, possibly explaining why women having this variant get pre-eclampsia. Considering the normally high concentration of glutathione in cells, WT Gal-13 should exist mostly as a monomer in cytoplasm, consistent with the monomeric variant C136S/C138S, which has a similar ability to interact with HOXA1 as WT Gal-13.

Galectin-3 (Gal-3) has a long, aperiodic, and dynamic proline-rich N-terminal tail (NT). The functional role of the NT with its numerous prolines has remained enigmatic since its discovery. To provide some resolution to this puzzle, we individually mutated all 14 NT prolines over the first 68 residues and assessed their effects on various Gal-3–mediated functions. Our findings show that mutation of any single proline (especially P37A, P55A, P60A, P64A/H, and P67A) dramatically and differentially inhibits Gal-3–mediated cellular activities (i.e., cell migration, activation, endocytosis, and hemagglutination). For mechanistic insight, we investigated the role of prolines in mediating Gal-3 oligomerization, a fundamental process required for these cell activities. We showed that Gal-3 oligomerization triggered by binding to glycoproteins is a dynamic process analogous to liquid–liquid phase separation (LLPS). The composition of these heterooligomers is dependent on the concentration of Gal-3 as well as on the concentration and type of glycoprotein. LLPS-like Gal-3 oligomerization/condensation was also observed on the plasma membrane and disrupted endomembranes. Molecular- and cell-based assays indicate that glycan binding–triggered Gal-3 LLPS (or LLPS-like) is driven mainly by dynamic intermolecular interactions between the Gal-3 NT and the carbohydrate recognition domain (CRD) F-face, although NT–NT interactions appear to contribute to a lesser extent. Mutation of each proline within the NT differentially controls NT–CRD interactions, consequently affecting glycan binding, LLPS, and cellular activities. Our results unveil the role of proline polymorphisms (e.g., at P64) associated with many diseases and suggest that the function of glycosylated cell surface receptors is dynamically regulated by Gal-3.

12 p.-8 fig., Calix[4]arene PTX008 is an angiostatic agent that inhibits tumor growth in mice by binding to galectin‐1, a β‐galactoside‐binding lectin. To assess the affinity profile of PTX008 for galectins, we used 15N,1H HSQC NMR spectroscopy to show that PTX008 also binds to galectin‐3 (Gal‐3), albeit more weakly. We identified the contact site for PTX008 on the F‐face of the Gal‐3 carbohydrate recognition domain. STD NMR revealed that the hydrophobic phenyl ring crown of the calixarene is the binding epitope. With this information, we performed molecular modeling of the complex to assist in improving the rather low affinity of PTX008 for Gal‐3. By removing the N‐dimethyl alkyl chain amide groups, we produced PTX013 whose reduced alkyl chain length and polar character led to an approximately eightfold stronger binding than PTX008. PTX013 also binds Gal‐1 more strongly than PTX008, whereas neither interacts strongly, if at all, with Gal‐7. In addition, PTX013, like PTX008, is an allosteric inhibitor of galectin binding to the canonical ligand lactose. This study broadens the scope for galectin targeting by calixarene‐based compounds and opens the perspective for selective galectin blocking., K.H.M. is grateful to the Ludwigs-Maximillians-Universität (LMU) Center for Advanced Study, as well as to the Alexander von Humboldt Stiftung, for financial support during his sabbatical stay at LMU in Munich, Germany. K.H.M. also holds a van der Laar Visiting Professorship in Structural Biology at Maastricht University,Netherlands. K.H.M. acknowledges that NMR instrumentation was provided with funds from the National Science Foundation(BIR-961477), the University of Minnesota Medical School, and the Minnesota Medical Foundation.

Background The structure of human galectin-16 (Gal-16) has yet to be solved, and its function has remained elusive. Methods X-ray crystallography was used to determine the atomic structures of Gal-16 and two of its mutants. The Gal-16 oligomer state was investigated by gel filtration, its hemagglutination activity was determined along with its ability to bind lactose using ITC. The cellular distribution of EGFP-tagged Gal-16 in various cell lines was also investigated, and the interaction between Gal-16 and c-Rel was assessed by pull-down studies, microscale thermophoresis and immunofluorescence. Results Unlike other galectins, Gal-16 lacks the ability to bind the β-galactoside lactose. Lactose binding could be regained by replacing an arginine (Arg55) with asparagine, as shown in the crystal structures of two lactose-loaded Gal-16 mutants (R55N and R55N/H57R). Gal-16 was also shown to be monomeric by gel filtration, as well as in crystal structures. Thus, this galectin could not induce erythrocyte agglutination. EGFP-tagged Gal-16 was found to be localized mostly in the nucleus of various cell types, and can interact with c-Rel, a member of NF-κB family. Conclusions Gal-16 exists as a monomer and its ligand binding is significantly different from that of other prototype galectins, suggesting that it has a novel function(s). The interaction between Gal-16 and c-Rel indicates that Gal-16 may regulate signal transduction pathways via the c-Rel hub in B or T cells at the maternal-fetal interface. General significance The present study lays the foundation for further studies into the cellular and physiological functions of Gal-16.

Galactans are linear polysaccharides of β(1→4)-linked galactose residues. Although they can antagonize galectin function, the nature of their binding to galectins needs to be better defined to develop them as drugs. Here, we investigated interactions between galectin-3 (Gal-3) and a series of galactans ranging in weight average molecular weight from 670 to 7550 Da. 15N-1H HSQC NMR studies with 15N-labeled Gal-3 carbohydrate recognition domain (CRD) indicate that each of these galactans interacts primarily with residues in β-strands 4, 5 and 6 on the canonical, β-galactoside sugar binding S-face. Although these galactans also bind to full length Gal-3 (CRD plus N-terminal tail) to the same extent, it appears that binding to the S-face attenuates interactions between the CRD F-face and N-terminal tail, making interpretation of site-specific binding unclear. Following assignment of galactan 13C and 1H resonances using HSQC, HMBC and TOCSY experiments, we used 13C-1H HSQC data to demonstrate that the Gal-3 CRD binds to the terminal, non-reducing end of these galactans, regardless of their size, but with binding affinity increasing as the galactan chain length increases. Overall, our findings increase understanding as to how galactans interact with Gal-3 at the non-reducing, terminal end of galactose-containing polysaccharides as found on the cell surface.

Heat-treated pectin is known to have anti-tumor activity. The present study was designed to identify its active components and mechanism of action. Here, we investigated the anti-tumor effect of heat-treated Helianthus annuus L. pectin (HT-HAP), alkali-inactivated HT-HAP representing the pectin fragment fraction (HT-HAP-P), and heat-treated galacturonic acid representing the small molecule fraction (HT-HAP-S). Our results demonstrate that HT-HAP induces colon cancer CT26 cells apoptosis and inhibits CT26 tumor growth like 5-fluorouracil, whereas it shows no spleen and thymus toxicity in contrast to 5-fluorouracil. Mechanistically, HT-HAP attenuates Akt activation in tumors, and enhances it in the spleen and thymus. Interestingly, HT-HAP-S inhibited tumor growth, but promoted immunity mildly, whereas HT-HAP-P had little effect on tumor growth, but effectively promoted immunity. Overall, we attribute the anti-tumor effect from HT-HAP to the action of small molecules and the immune enhancement primarily to pectin fragments, with Akt activation being the underlying mechanism of action.

Dextran is a biocompatible glucose-based polymer that is widely used clinically as a plasma volume expander, anti-thrombotic agent, macromolecular carrier, peripheral blood flow enhancer, and artificial tears promoter. Because dextran has polydisperse molecular weights and tends to produce innumerable multi-charged precursor ions in the ion source, it is difficult to analysis this polymer using conventional liquid chromatography mass spectrometry. In this assay, all ion fragmentation strategy is used to solve this problem by allowing all dextran precursor ions generated in the ion source to enter the collision cell. Then serial dextran-specific fragments can be effectively generated from all precursor ions by higher energy collision dissociation and scanned by Orbitrap detector. These high resolution fragment ions provide high specificity and sensitivity for the quantitation of dextran in rat plasma. Here, we report a new quantitative method using size exclusion chromatography combined with Q Exactive mass spectrometry based on an all ion fragmentation strategy. Assay validation showed that this method is linear over the concentration range from 3 to 300 μg/mL. Our approach has been successfully applied to a pharmacokinetic study of dextran in rat and will promote the development of studies with other polysaccharides.

Here, we investigated the role of the cell membrane protein CD146 in galectin-3-mediated endothelial cell migration at the molecular level. Our results show that knocking down CD146 significantly attenuates galectin-3-mediated cell migration. Pull-down assays, gel filtration, and biolayer interferometry further demonstrate that galectin-3 binds to the CD146 ectodomain (eFL) with a KD of ~1.1 μm. To identify the galectin-3-binding site, we used mass spectrometry to show that CD146 eFL has four N-glycosites, with PNGase F treatment indicating that N-glycans define the binding epitope. Galectin-3 likely interacts with Domain 5 on CD146 eFL, because it contains poly-N-acetyllactosamine sites, and deletion of this domain significantly reduces binding. Overall, our findings provide a better understanding of how galectin-3 interacts with cell membrane receptors to mediate endothelial cell migration.

A novel polysaccharide AAMP-A70 (5.6kDa) has been purified from the fruiting bodies of Amillariella mellea. Compositional analysis and 1H and 13C NMR spectra indicate that AAMP-A70 is a branched β-glucan with a main chain that consists of β-d-(1→6) linked Glcρ residues substituted at O-3 by β-Glcρ or α-d-(1→6)-linked Galρ side chains. AAMP-A70 increases macrophage phagocytosis and secretion of NO, ROS, TNF-α, IL-6 and IL-1β. Mechanistically, AAMP-A70 promotes degradation of IκB-α and nuclear translocation of the NF-κB p65 subunit, and enhances phosphorylation of MAPKs. In particular, the function blocking antibody to TLR2 substantially suppresses TNF-α and IL-6 production. Our data demonstrate that AAMP-A70 activates macrophages via NF-κB/MAPK signaling pathways and the TLR2 receptor. Overall, AAMP-A70 may serve as a good food supplement to enhance immunity.

Our previous paper reported the structure of ginseng pectic polysaccharides related to the cell migration inhibitory effects, but the underlying mechanisms are poorly understood. In this manuscript, rhamnogalacturonan I (RGI)-rich pectins prepared from ginseng pectin were investigated for their effect on cell migration. The results indicated that the combination of homogalacturonan (HG) and RGI-rich pectins exerted stronger effects than either HG- or RGI-rich pectin alone. Further studies revealed that the effects of HG- and RGI-rich pectins were dependent on pretreatment, which caused alterations in cell morphologies such as cell size and shape, focal adhesion, and the organization of actin filaments, suggesting that HG and RGI pectins exert synergistic effects on cell migration, likely through different ways. Morphological data and quantitative cell adhesion and spreading assays showed that HG- and RGI-rich pectin treatment decreased cell adhesion and cell spreading on the substratum, suggesting that HG- and RGI-rich pectins may exert their effects on cell migration via decreasing cell adhesion and cell spreading. Additionally, we showed that L-929 cells expressed little galectin-3 (Gal-3) and that lactose, an inhibitor of Gal-3 did not block the activities of HG- and RGI-rich pectins, implicating that cell migration inhibited by pectin did not correlate to Gal-3.

Galectin-10 (Gal-10) which forms Charcot-Leyden crystals in vivo, is crucial to regulating lymph cell function. Here, we solved the crystal structures of Gal-10 and eight variants at resolutions of 1.55-2.00 Å. Structural analysis and size exclusion chromatography demonstrated that Gal-10 dimerizes with a novel global shape that is different from that of other prototype galectins (e.g., Gal-1, -2 and -7). In the Gal-10 dimer, Glu33 from one subunit modifies the carbohydrate-binding site of another, essentially inhibiting disaccharide binding. Nevertheless, glycerol (and possibly other small hydroxylated molecules) can interact with residues at the ligand binding site, with His53 being the most crucial for binding. Alanine substitution of the conserved Trp residue (Trp72) that is crucial to saccharide binding in other galectins, actually leads to enhanced erythrocyte agglutination, suggesting that Trp72 negatively regulates Gal-10 ligand binding. Overall, our crystallographic and biochemical results provide insight into Gal-10 ligand binding specificity.

Interactions between galectins and polysaccharides are crucial to many biological processes, and yet these are some of the least understood, usually being limited to studies with small saccharides and short oligosaccharides. The present study is focused on human galectin-3 (Gal-3) interactions with a 60 kDa rhamnogalacturonan RG-I-4 that we use as a model to garner information as to how galectins interact with large polysaccharides, as well as to develop this agent as a therapeutic against human disease. Gal-3 is unique among galectins, because as the only chimera-type, it has a long N-terminal tail (NT) that has long puzzled investigators due to its dynamic, disordered nature and presence of numerous prolines. Here, we use 15N–1H heteronuclear single quantum coherence NMR spectroscopy to demonstrate that multiple sites on RG-I-4 provide epitopes for binding to three sites on 15N-labeled Gal-3, two within its carbohydrate recognition domain (CRD) and one at a novel site within the NT encompassing the first 40 residues that are highly conserved among all species of Gal-3. Moreover, strong binding of RG-I-4 to the Gal-3 NT occurs on a very slow time scale, suggesting that it may be mediated by cis–trans proline isomerization, a well-recognized modulator of many biological activities. The NT binding epitope within RG-I-4 appears to reside primarily in the side chains of the polysaccharide, some of which are galactans. Our results provide new insight into the role of the NT in Gal-3 function.

// Huiting Xue 1 , Lu Liu 1 , Zihan Zhao 1 , Zhongyu Zhang 1 , Yuan Guan 1 , Hairong Cheng 1 , Yifa Zhou 1 and Guihua Tai 1 1 School of Life Sciences, Northeast Normal University, Changchun, China Correspondence to: Guihua Tai, email: taigh477@nenu.edu.cn Keywords: galectin-3, apoptosis, ERK, ROS, truncated protein Received: September 12, 2016 Accepted: April 24, 2017 Published: May 10, 2017 ABSTRACT Galectin-3 is a galectin with a unique flexible N-terminal tail (NT) connected to the conserved carbohydrate recognition domain (CRD). Galectin-3 is associated with tumor immune tolerance and exhibits an ability to induce T cell apoptosis. We used Jurkat, Jurkat E6-1 and CEM T-cell lines and human peripheral blood mononuclear cells (PBMCs) to investigate the specific roles of the CRD and NT in inducing T cell apoptosis. Galectin-3 triggered sustained extracellular signal-regulated kinase (ERK) phosphorylation that induced apoptosis. ERK was situated upstream of caspase-9 and was independently activated by reactive oxygen species (ROS) and protein kinase C (PKC). The first twelve NT residues had no role in the apoptosis. Residues 13-68 were essential for activating ROS, but did not activate PKC. However, residues 69-110 were required for activation of PKC. An NT fragment and a NT-specific antibody antagonized the apoptosis triggered by full-length galectin-3 further supporting our findings. These findings indicate the CRD and NT play important roles during induction of T cell apoptosis, which suggests their potential as therapeutic targets for reversing cancer immune tolerance.

Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is a cytokine that selectively induces apoptosis in many tumor cells while leaving normal cells intact and is thus an attractive candidate for antitumor therapies. This paper reports that the combination of tunicamycin plus TRAIL produced a strong synergistic effect in TRAIL-sensitive human colon cancer HCT116 cells and TRAIL-resistant HT-29 cells. On a cellular mechanistic level, tunicamycin-enhanced TRAIL-induced apoptosis by death receptor (DR) 5 upregulation and DR4 deglycosylation. Knockdown of DR5 but not DR4 expression by specific shRNAs or siRNAs significantly increased tunicamycin-mediated and TRAIL-mediated cell viability. DR5 induction was regulated by C/EBP homologous protein (CHOP) and JNK as CHOP siRNA or JNK inhibitor SP600125 considerably abolished the DR5 induction. In addition, tunicamycin inhibited epidermal growth factor receptor glycosylation and the downstream signaling pathways, Akt and extracellular signal-regulated kinases activation, which might also be required for TRAIL sensitization by tunicamycin. In summary, tunicamycin effectively enhanced TRAIL-induced apoptosis might through JNK-CHOP-mediated DR5 upregulation and the inhibition of the epidermal growth factor receptor pathway.

The expression of prototype galectin-14 (Gal-14) in human placenta is higher than any other galectin, suggesting that it may play a role in fetal development and regulation of immune tolerance during pregnancy. Here, we solved the crystal structure of dimeric Gal-14 and found that its global fold is significantly different from that of other galectins with two β-strands (S5 and S6) extending from one monomer and contributing to the carbohydrate-binding domain of the other. The hemagglutination assay showed that this lectin could induce agglutination of chicken erythrocytes, even though lactose could not inhibit Gal-14-induced agglutination activity. Calorimetry indicates that lactose does not interact with this lectin. Compared to galectin-1, galectin-3, and galectin-8, Gal-14 has two key amino acids (a histidine and an arginine) in the normally conserved, canonical sugar-binding site, which are substituted by glutamine (Gln53) and histidine (His57), thus likely explaining why lactose binding to this lectin is very weak. Lactose was observed in the ligand-binding site of one Gal-14 structure, most likely because ligand binding is weak and crystals were allowed to grow over a long period of time in the presence of lactose. We also found that EGFP-tagged Gal-14 is primarily localized within the nucleus of different cell types. In addition, Gal-14 colocalized with c-Rel (a member of NF-κB family) in HeLa cells. These findings indicate that Gal-14 might regulate signal transduction pathways through NF-κB hubs. Overall, the present study provides impetus for further research into the function of Gal-14 in embryology.

Galectin-3 (Gal-3) can induce T-cell activation and apoptosis and plays a role in tumor immune tolerance. Here, we demonstrate that ginseng pectins selectively inhibit Gal-3-induced T-cell apoptosis, while not affecting T-cell activation. This finding stands in contrast to that from the use of modified citrus pectin (MCP) and potato galactan (P-galactan) that inhibit both. Whereas PKC/ERK and ROS/ERK pathways are involved in both T-cell activation and apoptosis, the Ras/PI3K/Akt pathway is unique to T-cell activation. Ginseng pectins selectively inhibit the ROS/ERK pathway. Using the Sarcomar-180 mouse model in which Gal-3 expression is increased, we found that ginseng pectins (but not MCP or P-galactan) significantly promote T-cell proliferation and IL-2 expression, and inhibit tumor growth by 45%. These in vivo data correlate well with selective effects of pectins on Gal-3-mediated T-cell apoptosis and activation. Our study suggests a novel approach for the development of polysaccharide-based agents that target Gal-3 function.

Heat-modified citrus pectin, a water-soluble indigestible polysaccharide fiber derived from citrus fruits and modified by temperature treatment, has been reported to exhibit anticancer effects. However, the bioactive fractions and their mechanisms remain unclear. In this current study, we isolated an active compound, trans-4,5-dihydroxy-2-cyclopentene-l-one (DHCP), from heat-treated citrus pectin, and found that is induces cell death in colon cancer cells via induction of mitochondrial ROS. On the molecular level, DHCP triggers ROS production by inhibiting the activity of succinate ubiquinone reductase (SQR) in mitochondrial complex II. Furthermore, cytotoxicity, apoptotic activity, and activation of caspase cascades were determined in HCT116 and HT-29 cell-based systems, the results indicated that DHCP enhances the sensitivity of cancer cells to tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), with DHCP-induced ROS accounting for the synergistic effect between DHCP and TRAIL. Furthermore, the combination of DHCP and TRAIL inhibits the growth of HCT116 and HT-29 xenografts synergistically. ROS significantly increases the expression of TRAIL death receptor 5 (DR5) via the p53 and C/EBP homologous protein pathways. Collectively, our findings indicate that DHCP has a favorable toxicity profile and is a new TRAIL sensitizer that shows promise in the development of pectin-based pharmaceuticals, nutraceuticals, and dietary agents aimed at combating human colon cancer.

Galectin-2 (Gal-2) plays a role in cancer, myocardial infarction, immune response, and gastrointestinal tract diseases. The only reported crystal structure of Gal-2 shows that it is a dimer in which the monomer subunits have almost identical structures, each binding with one molecule of lactose. In this study, we crystallized Gal-2 under new conditions that produced three crystal structures. In each Gal-2 dimer structure, lactose was shown to be bound to only one of the carbohydrate recognition domain subunits. In solution studies, the thermal shift assay demonstrated that inequivalent monomer subunits in the Gal-2 dimer become equivalent upon ligand binding. In addition, galectin-mediated erythrocyte agglutination assays using lactose and larger complex polysaccharides as inhibitors showed the structural differences between Gal-1 and Gal-2. Overall, our results reveal some novel aspects to the structural differentiation in Gal-2 and expand the potential for different types of molecular interactions that may be specific to this lectin.

Citrus pectin is widely used as a gelling/thickening agent and stabilizer in the food and pharmaceutical industries. The usefulness of citrus pectin, however, has been limited due to its large size, an obstacle that can be partially overcome with pH-modified citrus pectin (MCP). In the present study, we fractionated MCP into four fractions (MCP-1, −2, −3, and −4) and then sub-fractions of type-I rhamnogalacturonan-rich pectins (RG-I: MCP-1a, MCP-2a, MCP-3Sa and MCP-4a) and homogalacturonan-rich pectins (HG: MCP-2b, MCP-3Sb, MCP-3P, MCP-4b). Upon analysis of their chemical structures, we found that the RG-I sub-fractions contain (1 → 4)-linked β- D -galactan and (1 → 5)-linked α- L -arabinans or type-I arabinogalactan side-chains. Because parent MCP is known to inhibit hemagglutination mediated by galectin-3 (a lectin associated with cancer progression and metastasis), we used this assay to define which MCP fraction best promotes anti-galectin-3 activity. Our results demonstrate that MCP-2 was the most inhibitory with a MIC of 0.06 μg/ml (ten-fold more potent than parent MCP), and that RG-I-rich pectins with (1 → 4)-linked β- D -galactan side-chains were more active than the other RG-I-rich and HG-rich pectins. These findings provide new insight into structure-activity relationships of MCP and provide impetus towards the development of MCP-based food stabilizers and galectin-3 targeted therapeutics for use in the clinical setting.

Tumor-associated macrophages (TAMs) with an M2-like phenotype have been linked to immunosuppression and resistance to chemotherapies of cancer, thus targeting TAMs has been an attractive therapeutic strategy to cancer immunotherapy. We have reported that the β-D-(1→6) glucan (AAMP-A70) isolated from Amillariella Mellea could promote macrophage activation. The present study showed that the β-1,6-glucan could promote the transformation of M2-like macrophages to M1-like phenotype and inhibit the viability of colon cancer cells in vitro and in vivo. On a cellular mechanistic level, the β-1,6-glucan reset tumor-promoting M2-like macrophages to tumor-inhibiting M1-like phenotype via increasing the phosphorylation of Akt/NF-κB and MAPK. Further, TLR2 was identified as the receptor of β-1,6-glucan in the transformation effect. In addition, a very similar β-1,6-glucan with side chains of β-Glc or α-Galρ which was purified from Lentinus edodes showed same activities with those from Amillariella Mellea. Our findings shed light on the action mode of β-1,6-glucan in cancer immunotherapy.

Charcot-Leyden crystal protein/Gal-10, abundantly expressed in eosinophils and basophils, is related to several immune diseases. Recently, crystallographic and biochemical studies showed that Gal-10 cannot bind lactose, because a glutamate residue (Glu33) from another monomer blocks the binding site. Moreover, Gal-10 actually forms a novel dimeric structure compared to other galectins. To investigate the role that Glu33 plays in inhibiting lactose binding, we mutated this residue to glutamine, aspartate, and alanine. The structure of E33A shows that Gal-10 can now bind lactose. In the hemagglutination assay, lactose could inhibit E33A from inducing chicken erythrocyte agglutination. Furthermore, we identified a tryptophan residue (Trp127) at the interface of homodimer that is crucial for Gal-10 dimerization. The variant W127A, which exists as a monomer, exhibited higher hemagglutination activity than wild type Gal-10. The solid phase assay also showed that W127A could bind to lactose-modified sepharose-6B, whereas wild type Gal-10 could not. This indicates that the open carbohydrate-binding site of the W127A monomer can bind to lactose. In addition, the distribution of EGFP-tagged Gal-10 and its variants in HeLa cells was investigated. Because Trp72 is the highly conserved in the ligand binding sites of galectins, we used EGFP-tagged W72A to show that Gal-10 could not be transported into the nucleus, indicating that Trp72 is crucial for Gal-10 transport into that organelle.

Galectin-3 (Gal-3) binds to cell adhesion glycoprotein CD146 to promote cytokine secretion and mediate endothelial cell migration. Here, we used Nuclear Magnetic Resonance (NMR) 15N-Heteronuclear Single Quantum Coherence (HSQC) spectroscopy to investigate binding between 15N-labeled Gal-3 and the extracellular domain (eFL) of purified CD146 (five Ig-like ectodomains D1–D5) and a shorter, D5-deleted version of CD146 (D1–D4). Binding of Gal-3 and its carbohydrate recognition domain (CRD) to CD146 D1–D4 is greatly reduced vis-à-vis CD146 eFL, supporting the proposal of a larger number of glycosylation sites on D5. Even though the canonical sugar-binding β-sheet S-face (β-strands 1, 10, 3, 4, 5, 6) of the Gal-3 β-sandwich is involved in interactions with CD146 (e.g. N-linked glycosylation sites), equivalent HSQC spectral perturbations at residues on the opposing Gal-3 F-face β-sheet (β-strands 11, 2, 7, 8, 9) indicate involvement of the Gal-3 F-face in binding CD146. This is supported by the observation that addition of lactose, while significantly attenuating Gal-3 binding (primarily with the S-face) to CD146 eFL, does not abolish it. Bio-Layer Interferometry studies with Gal-3 F-face mutants yield KD values to demonstrate a significant decrease (L203A) or increase (V204A, L218A, T243A) in net binding to CD146 eFL compared to wild type Gal-3. However, HSQC lactose titrations show no highly significant effects on sugar binding to the Gal-3 CRD S-face. Overall, our findings indicate that Gal-3 binding to CD146 is more involved than simple interactions with β-galactoside epitopes on the cell receptor, and that there is a direct role for the lectin’s CRD F-face in the CD146 binding process.

Tumor-associated macrophages (TAMs) with an M2-like phenotype have been linked to the proliferation, invasion and metastasis of tumor cells. Resetting tumor-associated macrophages represents an attractive target for an effective cancer immunotherapy. WCCP-N-b, a novel linear 3-O-methylated galactan, isolated from Cantharellus cibarius, can convert tumor-promoting M2-like macrophages to tumor-inhibiting M1-like phenotype. On a cellular mechanistic level, WCCP-N-b inhibited M2-like macrophages polarization through suppression of STAT6 activation. Furthermore, WCCP-N-b increased the phosphorylation of mitogen-activated protein kinases (MAPKs) and degradation of IκB-α through targeting Toll-like receptor 2 (TLR2). The activation of MAPKs and degradation of IκB-α were responsible for converting M2-like macrophages to M1-like macrophages. Importantly, cell culture supernatants of WCCP-N-b-treated M2-like macrophages could inhibit the cell viability of B16F1 and B16F10. Our findings provide a potential natural and harmless polysaccharide for macrophage-based tumor immunotherapy.

Placental protein 13/galectin-13 (Gal-13) is highly expressed in placenta, where its lower expression is related to pre-eclampsia. Recently, the crystal structures of wild-type Gal-13 and its variant R53H at high resolution were solved. The crystallographic and biochemical results showed that Gal-13 and R53H could not bind lactose. Here, we used site-directed mutagenesis to re-engineer the ligand binding site of wild-type Gal-13, so that it could bind lactose. Of six newly engineered mutants, we were able to solve the crystal structures of four of them. Three variants (R53HH57R, R53HH57RD33G and R53HR55NH57RD33G had the same two mutations (R53 to H, and H57 to R) and were able to bind lactose in the crystal, indicating that these mutations were sufficient for recovering the ability of Gal-13 to bind lactose. Moreover, the structures of R53H and R53HR55N show that these variants could co-crystallize with a molecule of Tris. Surprisingly, although these variants, as well as wild-type Gal-13, could all induce hemagglutination, high concentrations of lactose could not inhibit agglutination, nor could they bind to lactose-modified Sepharose 6b beads. Overall, our results indicate that Gal-3 is not a normal galectin, which could not bind to β-galactosides. Lastly, the distribution of EGFP-tagged wild-type Gal-13 and its variants in HeLa cells showed that they are concentrated in the nucleus and could be co-localized within filamentary materials, possibly actin.

Galactans exhibit many biological activities, including modulation of human gut microbiota. By optimizing hydrolysis conditions and fractionation protocols, we purified a series of galacto-oligosaccharides from potato galactan, with degrees of polymerization (DP) up to 15. These oligosaccharides were characterized by using HPAEC, MALDI-TOF, 1H13C-NMR HSQC and HMBC experiments. Analysis of NMR data allowed chemical shift assignments to be made for oligos up to DP15. We used these NMR data to calculate intensity ratios of resonances from non-terminal to terminal residues, and correlated them with galactan chain length. Using this approach, we were able to estimate the length of galactan side chains in complex rhamnogalacturonan-I polysaccharides. Furthermore, we demonstrate here that prebiotic effects on Lactobacillus bacteria are related to galactan chain length, suggesting a potential application in the study of the human gut microbiome.

// Zhongyu Zhang 1 , Yuan Guan 1 , Xiyao Yu 1 , Hairong Cheng 1 , Yifa Zhou 1 and Guihua Tai 1 1 School of Life Sciences, Northeast Normal University, Changchun, PR China Correspondence to: Guihua Tai, email: taigh477@nenu.edu.cn Keywords : galectin-3; CRD; migration; HMEC-1; β1 integrin Received: August 18, 2017 Accepted: January 02, 2018 Epub: January 11, 2018 Abstract Galectin-3 (Gal-3) is composed of a long N-terminal tail (NT) connected to its conserved carbohydrate recognition domain (CRD). The NT is degraded by matrix metalloproteinases and prostate-specific antigen. Here, we demonstrate that NT-truncated Gal-3 variants with deletion of the first 12 or 68 residues, or the entire 110-residues NT, triggers migration of human microvascular endothelial cells (HMEC- 1). Using mass spectrometry and pull-down analysis, we show that the Gal-3 CRD binds to and activates cell surface β1 integrin promoting phosphorylation of FAK. Antibody blocking assays suggested that α2, α4, and α6 integrins are involved, with α2 and β1 forming a heterodimer. shRNA knockdown or antibody-mediated inhibition of β1 integrin function inhibited CRD-induced cell migration and attenuated FAK phosphorylation. FAK-specific inhibitors also inhibited Gal-3 CRD-induced cell migration. Interestingly, full-length Gal-3 and its CRD promoted similar effects. These findings highlight the need to reconsider Gal-3 NT cleavage in formulating strategies to address tumor angiogenesis.

ManNAc analogues are important chemical tools for probing sialylation dynamically via metabolic oligosaccharide engineering (MOE). The size of N-acyl and the nature of the chemical handle are two determinants of metabolic incorporation efficiency. We demonstrated a minimal, stable, bioorthogonal, and reactive N-Cp (N-(cycloprop-2-ene-1-ylcarbonyl)) group and the imaging of sialylated glycans using Ac4ManNCp in vitro and in vivo. The results revealed that the Cp group can efficiently be incorporated into the cellular sialic acid and detected rapidly by the reaction with FITC-Tz in different cells. The metabolic incorporation efficiency of non-cytotoxic Ac4ManNCp is not only superior to Ac4ManNMCp, but also superior to the widely-used Ac4ManNAz in some cell lines. Moreover, when Ac4ManNCp was administered to mice, a rapid and intense labelling of splenocytes as well as glycoproteins of sera and organs was observed. This is the first reported metabolic labelling of cyclopropene-modified sugars in vivo. Therefore, Ac4ManNCp is a powerful probe for efficient and rapid MOE and it may find wide applications in the labelling of glycans.

Twelve novel N-dialkylphosphoryliminosugar derivatives were synthesized and their immunosuppressive activities were evaluated on the proliferation of the mouse splenocytes and the secretion of IFN-γ and IL-4. The experimental data demonstrated that the iminosugars with the double long alkyl chains exhibited better inhibitory effects than those with the single long alkyl chain, and the iminosugars with the 10-carbon linear alkyl chain exhibited the strongest immunosuppressive activities. The assay of the cytokine secretion showed that the introduction of dialkyl chains on iminosugars could regulate the polarization of immune inhibition by varying the length of the alkyl chains. The disclosure of the structure-activity relationships may benefit the structural modifications of iminosugars to find new types of immunosuppressive agents.

We isolated and purified a new polysaccharide (WFVP-N-b1) with a molecular weight of 20 kDa from Flammulina velutipes. Results showed that WFVP-N-b1 is composed of an α (1 → 6)-linked D-galactan backbone and branched at the O-2 of its Galp residues by an α-D-(1 → 6)-linked Manp attached to t-β-D-Glcp or t-α-D-Fucp side chains. WFVP-N-b1 can significantly induce cytokines secretion and release of toxic molecules. On a cellular level, WFVP-N-b1 is recognized by Toll-like receptor 4 (TLR4). Thereby, the hetero-galactan increased the phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt, promoted degradation of IκB-α and the nuclear translocation of the NF-κB p65 subunit. Importantly, our results indicate that WFVP-N-b1 activated macrophage is mediated by autophagy, as blockade of WFVP-N-b1-induced autophagy by Baf-A1 significantly decreases macrophage activation. This is the first report that hetero-galactan-induced macrophage activation is mediated by autophagy. Collectively, WFVP-N-b1 activated RAW264.7 cells through MAPKs, autophagy, and Akt/NF-κB signaling pathways via TLR4 receptor.

Although pectin-derived polysaccharides can antagonize galectin function in various pathological disorders, the nature of their binding interactions needs to be better defined for developing them as drugs. Moreover, given their relatively large size and complexity, pectin-derived polysaccharides are also useful as model systems to assess inter-polysaccharide and protein–polysaccharide interactions. Here, we investigated interactions between galectin-3 (Gal-3) and pectin-derived polysaccharides: a rhamnogalacturonan (RG) and two homogalacturonans (HGs). BioLayer Interferometry and fluorescence-linked immunosorbent assays indicate that these polysaccharides bind Gal-3 with macroscopic or apparent KD values of 49 nM, 46 µM, and 138 µM, respectively. 15N-1H heteronuclear single quantum coherence (HSQC) NMR studies reveal that these polysaccharides interact primarily with the F-face of the Gal-3 carbohydrate recognition domain. Even though their binding to Gal-3 does not inhibit Gal-3-mediated T-cell apoptosis and only weakly attenuates hemagglutination, their combination in specific proportions increases activity synergistically along with avidity for Gal-3. This suggests that RG and HG polysaccharides act in concert, a proposal supported by polysaccharide particle size measurements and 13C-1H HSQC data. Our model has HG interacting with RG to promote increased avidity of RG for Gal-3, likely by exposing additional lectin-binding sites on the RG. Overall, the present study contributes to our understanding of how complex HG and RG polysaccharides interact with Gal-3.

This study was aimed at investigating the immunomodulating activity of Hericium erinaceus polysaccharide (HEP) in mice, by assessing splenic lymphocyte proliferation (cell-mediated immunity), serum hemolysin levels (humoral immunity), phagocytic capacity of peritoneal cavity phagocytes (macrophage phagocytosis), and NK cell activity. ELISA of immunoglobulin A (SIgA) in the lamina propria, and western blotting of small intestinal proteins were also performed to gain insight into the mechanism by which HEP affects the intestinal immune system. Here, we report that HEP improves immune function by functionally enhancing cell-mediated and humoral immunity, macrophage phagocytosis, and NK cell activity. In addition, HEP was found to upregulate the secretion of SIgA and activate the MAPK and AKT cellular signaling pathways in the intestine. In conclusion, all these results allow us to postulate that the immunomodulatory effects of HEP are most likely attributed to the effective regulation of intestinal mucosal immune activity.

Iminosugars have gained a remarkable importance as new therapeutic agents since 1966. In this study, compounds A and B, two iminosugar analogs synthesized previously, showed an inhibition of the growth of K562 cells. They allowed cell cycle arrested at the G0/G1 phase, promoted apoptotic activities and also lowered the mitochondrial membrane potential. Further exploration of the apoptosis mechanism revealed that compound B significantly suppressed the expression of Hsp70, which is a major anti-apoptotic molecular chaperone. Significant decrease was also found in the expression of Akt, a serine/threonine-specific protein kinase with anti-apoptosis activities also known as protein kinase B (PKB). At mitochondria level in comparison with compound A, compound B brought a better promotion in the expression of pro-apoptotic protein Bad in Bcl-2 family. As a result of the promotion, the expression of anti-apoptotic protein Bcl-xL was down-regulated. Cytochrome c was released, activating the intrinsic signaling pathways of caspase and resulting in the occurrence of cascade reaction. In addition, compound B stimulated autophagy effectively by up-regulating Beclin 1, thus causing the conversion of LC3-I to LC3-II through Akt/mTOR signaling pathway. In summary, these results indicated that compounds A and B induced cell death through multiple pathways. The disclosed results not only provide an evidence of antitumor activity of iminosugars as a foundation for further studies, but also may find potential applications in chronic myeloid leukemia therapy as new heat shock protein inhibitors and autophagy inducer.

The presence and level of circulating galectin-3 (Gal-3), a member of the galectin family, is associated with diverse diseases ranging from heart failure, immune disorders to cancer metastasis and serves as a biomarker of diagnosis and treatment response. However, the mechanisms by which exogenous Gal-3 affects pathobiology events remain elusive. In the current study, we found that exogenous Gal-3 slightly delays, while prolonging tyrosine phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in HeLa cells through a calcium-sensitive and PKC-dependent signaling pathway. The activation was dependent on the sugar-binding properties of Gal-3, since the antagonist lactose could inhibit it. The sugar-binding motif of Gal-3 was required for the activation of ERK1/2. The activation of ERK1/2 was necessary for the initiation and induction of cell migration associated with the phosphorylation of paxillin. All the results presented in this study suggest a novel calcium-sensitive and PKC-dependent pathway through which circulating Gal-3 promotes cell migration and activating the ERK1/2. Taken together, the data depicted here propose a biological function and a target for the diseases' associated circulating Gal-3.

The fungus Penicillum oxalicum can selectively metabolize the major 20(S)-protopanaxadiol ginsenosides Rb1, Rb2, and Rc using extracellular glycosidases yielding a series of bioactive metabolites. A β-glucosidase GH1 was purified from the culture of P. oxalicum with a yield of 9.5% and a specific activity of 3.9 × 103 U/mg. GH1 was a tetramer with a native molecular weight of 484 kDa and its pI value was pH 4.2. GH1 specifically cleaved the β-(1-6)-glucosidic linkage at C-20 site of ginsenoside Rb1 to give the sole product Rd. The optimum conditions were established to be pH 4.5, 55°C, and 0.25 U/ml purified enzyme at 2 mg/ml ginsenoside Rb1. GH1 could be used in the pharmaceutical industry.

Pectin has been shown to inhibit the actions of galectin-3, a β-galactoside-binding protein associated with cancer progression. The structural features of pectin involved in this activity remain unclear. We investigated the effects of different ginseng pectins on galectin-3 action. The rhamnogalacturonan I-rich pectin fragment, RG-I-4, potently inhibited galectin-3-mediated hemagglutination, cancer cell adhesion and homotypic aggregation, and binding of galectin-3 to T-cells. RG-I-4 specifically bound to the carbohydrate recognition domain of galectin-3 with a dissociation constant of 22.2 nm, which was determined by surface plasmon resonance analysis. The structure-activity relationship of RG-I-4 was investigated by modifying the structure through various enzymatic and chemical methods followed by activity tests. The results showed that (a) galactan side chains were essential to the activity of RG-I-4, whereas arabinan side chains positively or negatively regulated the activity depending on their location within the RG-I-4 molecule. (b) The activity of galactan chain was proportional to its length up to 4 Gal residues and largely unchanged thereafter. (c) The majority of galactan side chains in RG-I-4 were short with low activities. (d) The high activity of RG-I-4 resulted from the cooperative action of these side chains. (e) The backbone of the molecule was very important to RG-I-4 activity, possibly by maintaining a structural conformation of the whole molecule. (f) The isolated backbone could bind galectin-3, which was insensitive to lactose treatment. The novel discovery that the side chains and backbone play distinct roles in regulating RG-I-4 activity is valuable for producing highly active pectin-based galectin-3 inhibitors.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a potent cancer cell-specific apoptosis-inducing cytokine with little toxicity to most normal cells. Here, we report that gefitinib and TRAIL in combination produce a potent synergistic effect on TRAIL-sensitive human colon cancer HCT116 cells and an additive effect on TRAIL-resistant HT-29 cells. Interestingly, gefitinib increases the expression of cell surface receptors DR4 and DR5, possibly explaining the synergistic effect. Knockdown of DR4 and DR5 by siRNA significantly decreases gefitinib- and TRAIL-mediated cell apoptosis, supporting this idea. Because the inhibition of gefitinib-induced autophagy by 3-MA significantly decreases DR4 and DR5 upregulation, as well as reduces gefitinib- and TRAIL-induced apoptosis, we conclude that death receptor upregulation is autophagy mediated. Furthermore, our results indicate that death receptor expression may also be regulated by JNK activation, because pre-treatment of cells with JNK inhibitor SP600125 significantly decreases gefitinib-induced death receptor upregulation. Interestingly, SP600125 also inhibits the expression CHOP, yet CHOP has no impact on death receptor expressions. We also find here that phosphorylation of Akt and ERK might also be required for TRAIL sensitization. In summary, our results indicate that gefitinib effectively enhances TRAIL-induced apoptosis, likely via autophagy and JNK- mediated death receptor expression and phosphorylation of Akt and ERK.

Galectin-3 is an adhesion/growth-regulatory protein with a modular design comprising an N-terminal tail ( NT, residues 1-111) and the conserved carbohydrate recognition domain ( CRD, residues 112-250). The chimera-type galectin interacts with both glycan and peptide motifs. Complete C-13/N-15-assignment of the human protein makes NMR-based analysis of its structure beyond the CRD possible. Using two synthetic NT polypeptides covering residues 1-50 and 51-107, evidence for transient secondary structure was found with helical conformation from residues 5 to 15 as well as proline-mediated, multi-turn structure from residues 18 to 32 and around PGAYP repeats. Intramolecular interactions occur between the CRD F-face ( the 5-stranded beta-sheet behind the canonical carbohydrate-binding 6-stranded beta-sheet of the S-face) and NT in full-length galectin-3, with the sequence P(23)GAW(26)... P(37)GASYPGAY(45) defining the primary binding epitope within the NT. Work with designed peptides indicates that the PGAX motif is crucial for self-interactions between NT/CRD. Phosphorylation at position Ser6 ( and Ser12) ( a physiological modification) and the influence of ligand binding have minimal effect on this interaction. Finally, galectin-3 molecules can interact weakly with each other via the F-faces of their CRDs, an interaction that appears to be assisted by their NTs. Overall, our results add insight to defining binding sites on galectin-3 beyond the canonical contact area for beta-galactosides.

Although several approaches have been used to evaluate binding of carbohydrates to lectins, results are not always comparable, especially with larger polysaccharides. Here, we quantitatively assessed and compared binding of pectin-derived polysaccharides to galectin-3 (Gal-3) using five methods: surface plasmon resonance (SPR), bio-layer interferometry (BLI), fluorescence polarization (FP), competitive fluorescence-linked immunosorbance (cFLISA), and the well-known cell-based hemagglutination assay (G3H). Our studies revealed that whereas Gal-3-pectin binding parameters determined by SPR and BLI were comparable and correlated with inhibitory potencies from the G3H assay, results using FP and cFLISA assays were highly variable and depended greatly on the probe and mass of the polysaccharide. In the cFLISA assay, for example, pectins showed no inhibition when using the DTAF-labeled asialofetuin probe, but did when using a DTAF-labeled pectin probe. And the FP approach with the DTAF-lactose probe did not work on polysaccharides and large galactan chains, although it did work well with smaller galactans. Nevertheless, even though results derived from all of these methods are in general agreement, derived KD, IC50, and MIC values do differ. Our results reflect the variability using various techniques and therefore will be useful to investigators who are developing pectin-derived Gal-3 antagonists as anti-cancer agents.

As a member of the galectin family of animal lectins, galectin-12 is preferentially expressed in adipocytes and leukocytes. In adipocytes, galectin-12 is associated with lipid droplets and regulates lipid metabolism and energy balance, whereas its role in leukocytes is not clear. Analysis of galectin-12 expression in a public data set of acute myeloid leukemia (AML) samples revealed that it is selectively overexpressed in the M3 subtype, which is also known as acute promyelocytic leukemia (APL). To investigate the role of galectin-12 in APL cells, we manipulated its expression in the APL cell line, NB4, and measured resultant effects on all-trans-retinoic acid (ATRA)–induced granulocytic differentiation. With a doxycycline-inducible gene knockdown system, we found that suppression of galectin-12 promoted ATRA-induced neutrophil differentiation but inhibited lipid droplet formation. Our results indicate that overexpression of galectin-12 contributes to a differentiation block in APL cells, and suppression of galectin-12 facilitates granulocytic differentiation. Furthermore, these data suggest that lipogenesis and other aspects of myeloid differentiation can be differentially regulated. Taken together, these findings suggest that galectin-12 may be a target for treatment of the ATRA-resistant subset of APL.