Cancer snap-shots: Biochemistry and glycopathology of O-glycans: A review.

Cancer pathogenesis is strongly linked to the qualitative and quantitative alteration of the cell surface glycans, that are glycosidically linked to proteins and lipids. Glycans that are covalently linked to the polypeptide backbone of a protein through nitrogen or oxygen, are known as N-glycans or O-glycans, respectively. Although the role of glycans in the expression, physiology, and communication of cells is well documented, the function of these glycans in tumor biology is not fully elucidated. In this context, current review summarizes biosynthesis, modifications and pathological implications of O-glycans The review also highlights illustrative examples of cancer types modulated by aberrant O-glycosylation. Related O-glycans like Thomsen-nouveau (Tn), Thomsen-Friedenreich (TF), Lewisa/x, Lewisb/y, sialyl Lewisa/x and some other O-glycans are discussed in detail. Since, the overexpression of O-glycans are attributed to the aggressiveness and metastatic behavior of cancer cells, the current review attempts to understand the relation between metastasis and O-glycans.

Purification, characterization of an entomopathogenic fungal lectin from Purpureocillium lilacinum and its involvement in pathogenesis leading to mycotic keratitis.

A lectin PCL, from Purpureocillium lilacinum a saprophytic, filamentous fungus was purified from the crude extract of the mycelia using 70% ammonium sulphate precipitation followed by affinity chromatography on mucin-Sepharose 4 B column. PCL is a monomer with an apparent molecular mass of 18.5 kDa as revealed by SDS-PAGE under both reducing and non-reducing conditions. PCL is a blood group non-specific lectin and has highest affinity towards chitin, mucin, asialomucin, fetuin with a MIC of 0.15 µg/mL and also recognizes L-fucose, galactose, lactose, N-acetyl galactosamine, hyaluronic acid. PCL is stable up to 60 °C and within the pH range 4-8. To understand its role in pathogenesis, effect of PCL was evaluated on human corneal epithelial cells (HCECs). PCL showed strong glycan mediated binding to HCECs and PCL showed proinflammatory response at lower concentrations by stimulating secretion of IL-6, 8. In contrast PCL at higher concentrations revealed opposite effect of HCECs growth inhibition. All these results collectively support the involvement of PCL in mediating host pathogen interactions possibly leading to pathogenesis. In addition, considering the entomopathogenic effect of Purpureocillium lilacinum, PCL may be attributed for this beneficiary effect, which needs to be explored.

Aspergillus niger lectin elicits MyD88 dependent proliferation and apoptosis at lower and higher doses in immortalized human corneal epithelial cells leading to pathogenesis.

An L-fucose lectin, ANL from the corneal smears of a mycotic keratitis patient was reported earlier. Interaction of ANL with immortalized Human Corneal Epithelial Cells (HCECs) was studied in order to assign the role of ANL in pathogenesis. ANL showed strong binding to HCECs which could be blocked by L-fucose and mucin. At concentrations below 0.6 µg/mL ANL showed proliferative effect and highest at 0.07 µg/mL leading to expression of proinflammatory cytokines IL-6 and IL-8. ANL induced proinflammatory response is mediated by TLR-2,-4, MyD88, NFkB and C-Jun dependent signaling. In contrast, ANL at concentrations above 0.6 µg/mL showed growth inhibitory effect at 48 h with an IC50 of 2.75 µg/mL. Western blot analysis revealed that HCECs treated with ANL at lower concentration induced the expression of proinflammatory signaling proteins TLR-2, 4, MyD88, NFkB and C-Jun which maintain high cell proliferating state. At higher concentration ANL induced apoptotic effect in HCECs with an increase in early apoptotic population as demonstrated by Annexin V-PI assay. ANL induced the expression of apoptotic proteins FADD, Caspase 8 and -3 mediated by MyD88. These findings demonstrate implication of ANL in pathogenesis and the findings are of clinical significance in developing strategy for controlling the infection leading to mycotic keratitis.

Sclerotium rolfsii lectin induces opposite effects on normal PBMCs and leukemic Molt-4 cells by recognising TF antigen and its variants as receptors.

Sclerotium rolfsii lectin (SRL) exerts apoptotic effect against various cancer cells and an antitumor activity on mice with colon and breast cancer xenografts. The current study aimed to explore its exquisite carbohydrate specificity on human peripheral blood mononuclear cells (PBMCs) and leukemic T-cells. SRL, showed strong binding (>98%) to resting/activated PBMCs, leukemic Molt-4 and Jurkat cell lines. The glycans mediated binding to these cells was effectively blocked by mucin and fetuin, exhibiting 97% and 94% inhibition respectively. SRL showed mitogenic stimulation of PBMCs at 10 µg/ml as determined by thymidine incorporation assay. In contrast, lectin induced a dose dependent growth inhibition of Molt-4 cells with 58% inhibition at 25 µg/ml. Many common membrane receptors in activated PBMCs, Molt 4 and Jurkat cells were identified by lectin blotting. However, membrane receptors that are recognized by SRL in normal resting PBMCs were totally different and are high molecular weight glycoproteins. Treatment of membrane receptors with glycosidases prior to lectin probing, revealed that fucosylated Thomsen-Friedenreich(TF) antigen glycans are increasingly expressed on transformed Molt-4 leukemic cells compared to other cells. The findings highlight the opposite effects of SRL on transformed and normal hematopoietic cells by recognizing different glycan-receptors. SRL has promising potential for diagnostics and therapeutic applications in leukaemia.

High mannose N-glycan binding lectin from Remusatia vivipara (RVL) limits cell growth, motility and invasiveness of human breast cancer cells.

Breast cancer known for its high metastatic potential is responsible for large mortality rate amongst women; hence it is imperative to search for effective anti-metastatic molecules despite anticancer drugs. The current study describes the potential of Remusatia vivipara lectin (RVL), inducing apoptosis in breast cancer cells there by limiting motility and invasiveness. RVL binds to the cell surface glycans of MDA-MB-468 and MCF-7 cells, exhibiting strong glycan mediated cytotoxic effect, but show marginal effect on non-tumorigenic MCF-10A cells. RVL elicits increased cellular stress, apoptotic vacuoles and nuclear disintegration in both MDA-MB-468 and MCF-7 cells accompanied by depletion of G0/G1, S and G2/M phases. Lectin interaction induced production of reactive oxygen species through altering mitochondrial membrane potential progressing to apoptosis. Further, RVL strongly elicited reproductive cell death in MDA-MB-468 cells and showed strong inhibitory effect on neovascularization demonstrated in chorioallantoic membrane assay. Treatment of MDA-MB-468 cells with RVL, suppress the motility and invasive property as shown by scratch wound heal and Boyden chamber transwell assays respectively. These results provide an insight into significance of interaction of RVL with specific cell surface high mannose N-glycans resulting in curtailing the metastatic ability of cancer cells.

Molecular Cloning, Carbohydrate Specificity and the Crystal Structure of Two Sclerotium rolfsii Lectin Variants.

SRL is a cell wall associated developmental-stage specific lectin secreted by Sclerotium rolfsii, a soil-born pathogenic fungus. SRL displays specificity for TF antigen (Galß1→3GalNAc-α-Ser//Thr) expressed in all cancer types and has tumour suppressing effects in vivo. Considering the immense potential of SRL in cancer research, we have generated two variant gene constructs of SRL and expressed in E. coli to refine the sugar specificity and solubility by altering the surface charge. SSR1 and SSR2 are two different recombinant variants of SRL, both of which recognize TF antigen but only SSR1 binds to Tn antigen (GalNAcα-Ser/Thr). The glycan array analysis of the variants demonstrated that SSR1 recognizes TF antigen and their derivative with high affinity similar to SRL but showed highest affinity towards the sialylated Tn antigen, unlike SRL. The carbohydrate binding property of SSR2 remains unaltered compared to SRL. The crystal structures of the two variants were determined in free form and in complex with N-acetylglucosamine at 1.7 Å and 1.6 Å resolution, respectively. Structural analysis highlighted the structural basis of the fine carbohydrate specificity of the two SRL variants and results are in agreement with glycan array analysis.

Glycosyltransferases involved in the synthesis of MUC-associated metastasis-promoting selectin ligands.

The sialyl Lewis a and x (sLe(a/x)) antigens frequently displayed on the surface of tumor cells are involved in metastasis. Their synthesis has been attributed to altered expression of selective glycosyltransferases. Identification of these glycosyltransferases and the glycoproteins that carry these carbohydrate antigens should help advance our understanding of selectin-mediated cancer metastasis. In this study, quantitative real-time polymerase chain reaction analysis coupled with in situ proximity ligation assay and small interference RNA treatment shows involvement of ß3galactosyltransferase-V in the synthesis of MUC16-associated sLe(a) in H292 cells. Also, α3fucosyltransferase-V, which is absent in BEAS-2B human immortalized bronchial epithelial cells and A549 lung carcinoma cells, participates in the synthesis of MUC1-associated sLe(x) in CFT1 human immortalized bronchial epithelial cells and H292 lung carcinoma cells. Neither selectin ligand is found on MUC1 in BEAS-2B and A549 cells. Knockdown of either enzyme suppresses migration, and selectin tethering and rolling properties of H292 cells under dynamic flow as determined by wound healing and parallel plate flow chamber assays, respectively. These results provide insights into how the synthesis of mucin-associated selectin ligands and the metastatic properties of cancer cells can be regulated by selective glycosyltransferases that work on mucins. They may help develop novel anticancer drugs.

Prostatic cell-specific regulation of the synthesis of MUC1-associated sialyl Lewis a.

Sialyl Lewis antigens are selectin ligands involved in leukocyte trafficking and cancer metastasis. Biosynthesis of these selectin ligands occurs by the sequential actions of several glycosyltransferases in the Golgi apparatus following synthesis of the protein backbone in the endoplasmic reticulum. In this study, we examine how the synthesis of sialyl Lewis a (sLe(a)) is regulated in prostatic cells and identify a mucin that carries this glycotope. We treat human prostatic cells including one normal and three cancerous cells with histone deacetylase inhibitors, valproic acid, tricostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA), and then monitor the expression of sLe(a). We have found that SAHA enhances the production of sLe(a) in normal prostatic RWPE-1 cells but not prostatic cancer cells. Employing siRNA technology and co-immunoprecipitation, we show that the sLe(a) is associated with MUC1, which is confirmed by confocal immunofluorescence microscopy and proximity ligation assay. The SAHA-induced production of sLe(a) in RWPE-1 cells is resulted from upregulation of B3GALT1 gene via enhancement of acetylated histone-3 and histone-4. Interestingly, PC3 and LNCaP C-81 cells do not produce detectable amounts of sLe(a) despite expressing high levels of B3GALT1. However, the MUC1-associated sLe(a) is generated in these cells after introduction of MUC1 cDNA. We conclude that the synthesis of sLe(a) is controlled by not only peptide backbone of the glycoprotein but also glycoprotein-specific glycosyltransferases involved in the synthesis of sLe(a). Further, the SAHA induction of this selectin ligand in normal prostatic cells may pose a potentially serious side effect of this drug recently approved by the US Food and Drug Administration.

Golgi phosphoprotein 3 determines cell binding properties under dynamic flow by controlling Golgi localization of core 2 N-acetylglucosaminyltransferase 1.

Core 2 N-acetylglucosaminyltransferase 1 (C2GnT1) is a key enzyme participating in the synthesis of core 2-associated sialyl Lewis x (C2-O-sLe(x)), a ligand involved in selectin-mediated leukocyte trafficking and cancer metastasis. To accomplish that, C2GnT1 needs to be localized to the Golgi and this step requires interaction of its cytoplasmic tail (CT) with a protein that has not been identified. Employing C2GnT1 CT as the bait to perform a yeast two-hybrid screen, we have identified Golgi phosphoprotein 3 (GOLPH3) as a principal candidate protein that interacts with C2GnT1 and demonstrated that C2GnT1 binds to GOLPH3 via the LLRRR(9) sequence in the CT. Confocal fluorescence microscopic analysis shows substantial Golgi co-localization of C2GnT1 and GOLPH3. Upon GOLPH3 knockdown, C2GnT1 is found mainly in the endoplasmic reticulum and decorated with complex-type N-glycans, indicating that the enzyme has been transported to the Golgi but is not retained. Also, we have found that a recombinant protein consisting of C2GnT1 CT(1-16)-Leu(17-32)-Gly(33-42)-GFP is localized to the Golgi although the same construct with mutated CT (AAAAA(9)) is not. The data demonstrate that the C2GnT1 CT is necessary and sufficient for Golgi localization of C2GnT1. Furthermore, GOLPH3 knockdown results in reduced synthesis of C2-O-sLe(x) associated with P-selectin glycoprotein ligand-1, reduced cell tethering to and rolling on immobilized P- or E-selectin, and compromised E-selectin-induced activation of spleen tyrosine kinase and cell adhesion to intercellular adhesion molecule-1 under dynamic flow. Our results reveal that GOLPH3 can regulate cell-cell interaction by controlling Golgi retention of C2GnT1.

Glycosylation potential of human prostate cancer cell lines.

Altered glycosylation is a universal feature of cancer cells and altered glycans can help cancer cells escape immune surveillance, facilitate tumor invasion, and increase malignancy. The goal of this study was to identify specific glycoenzymes, which could distinguish prostate cancer cells from normal prostatic cells. We investigated enzymatic activities and gene expression levels of key glycosyl- and sulfotransferases responsible for the assembly of O- and N-glycans in several prostatic cells. These cells included immortalized RWPE-1 cells derived from normal prostatic tissues, and prostate cancer cells derived from metastasis in bone (PC-3), brain (DU145), lymph node (LNCaP), and vertebra (VCaP). We found that all cells were capable of synthesizing complex N-glycans and O-glycans with the core 1 structure, and each cell line had characteristic biosynthetic pathways to modify these structures. The in vitro measured activities corresponded well to the mRNA levels of glycosyltransferases and sulfotransferases. Lectin and antibody binding to whole cells supported these results, which form the basis for the development of tumor cell-specific targeting strategies.

The TF-antigen binding lectin from Sclerotium rolfsii inhibits growth of human colon cancer cells by inducing apoptosis in vitro and suppresses tumor growth in vivo.

Glycan array analysis of Sclerotium rolfsii lectin (SRL) revealed its exquisite binding specificity to the oncofetal Thomsen-Friedenreich (Galß1-3GalNAcα-O-Ser/Thr, T or TF) antigen and its derivatives. This study shows that SRL strongly inhibits the growth of human colon cancer HT29 and DLD-1 cells by binding to cell surface glycans and induction of apoptosis through both the caspase-8 and -9 mediated signaling. SRL showed no or very weak binding to normal human colon tissues but strong binding to cancerous and metastatic tissues. Intratumor injection of SRL at subtoxic concentrations in NOD-SCID mice bearing HT29 xenografts resulted in total tumor regression in 9 days and no subsequent tumor recurrence. As the increased expression of TF-associated glycans is commonly seen in human cancers, SRL has the potential to be developed as a therapeutic agent for cancer.

Exquisite binding specificity of Sclerotium rolfsii lectin toward TF-related O-linked mucin-type glycans.

Sclerotium rolfsii lectin (SRL), a secretory protein from the soil borne phytopathogenic fungus Sclerotium rolfsii, has shown in our previous studies to bind strongly to the oncofetal Thomson-Friedenreich carbohydrate (Galß1-3GalNAc-ser/thr, T or TF) antigen. TF antigen is widely expressed in many types of human cancers and the strong binding of SRL toward such a cancer-associated carbohydrate structure led us to characterize the carbohydrate binding specificity of SRL. Glycan array analysis, which included 285 glycans, shows exclusive binding of SRL to the O-linked mucin type but not N-linked glycans and amongst the mucin type O-glycans, lectin recognizes only mucin core 1, core 2 and weakly core 8 but not to other mucin core structures. It binds with high specificity to "α-anomers" but not the "ß-anomers" of the TF structure. The axial C4-OH group of GalNAc and C2-OH group of Gal is both essential for SRL interaction with TF disaccharide, and substitution on C3 of galactose by sulfate or sialic acid or N-acetylglucosamine, significantly enhances the avidity of the lectin. SRL differs in its binding to TF structures compared to other known TF-binding lectins such as the Arachis hypogea (peanut) agglutinin, Agaricus bisporus (mushroom) lectin, Jackfruit, Artocarpus integrifolia (jacalin) and Amaranthus caudatus (Amaranthin) lectin. Thus, SRL has unique carbohydrate-binding specificity toward TF-related O-linked carbohydrate structures. Such a binding specificity will make this lectin a very useful tool in future structural as well as functional analysis of the cellular glycans in cancer studies.

5-Aza-2'-deoxycytidine increases sialyl Lewis X on MUC1 by stimulating ß-galactoside:α2,3-sialyltransferase 6 gene.

Sialyl Lewis X is a tumor-associated antigen frequently found in the advanced cancers. However, the mechanism for the production of this cancer antigen is not entirely clear. The objective of this study is to examine whether epigenetics is involved in the regulation of the formation of this antigen. We observed an increase of sialyl Lewis X in HCT15 cells, a colon cancer cell line, treated with 5-Aza-2'-deoxycytidine. This treatment enhanced the expression of ß-galactoside:α2,3-sialyltransferase 6 gene and sialyl Lewis X on MUC1, and the adherence of these cells to E-selectin under dynamic flow conditions. In addition, 5-Aza-2'-deoxycytidine treatment inhibited methylation of ß-galactoside:α2,3-sialyltransferase 6 gene and siRNA knockdown of this gene drastically reduced sialyl Lewis X without affecting MUC1 expression. We conclude that 5-Aza-2'-deoxycytidine treatment increases sialyl Lewis X on MUC1 by stimulating the ß-galactoside:α2,3-sialyltransferase 6 gene via inhibition of DNA methylation. Increased sialyl Lewis X by 5-Aza-2'-deoxycytidine raises a concern about the safety of this chemotherapeutic drug. In addition, ß-galactoside:α2,3-sialyltransferase 6 gene may be a potential therapeutic target for suppressing tumorigenicity of colon cancer.

Purification and characterization of a mitogenic lectin from cephalosporium, a pathogenic fungus causing mycotic keratitis.

Ophthalmic mycoses caused by infectious fungi are being recognized as a serious concern since they lead to total blindness. Cephalosporium is one amongst several opportunistic fungal species implicated in ophthalmic infections leading to mycotic keratitis. A mitogenic lectin has been purified from the mycelia of fungus Cephalosporium, isolated from the corneal smears of a keratitis patient. Cephalosporium lectin (CSL) is a tetramer with subunit mass of 14 kDa, agglutinates human A, B, and O erythrocytes, and exhibits high affinity for mucin compared to fetuin and asialofetuin but does not bind to simple sugars indicating its complex sugar specificity. CSL showed strong binding to normal human peripheral blood mononuclear cells (PBMCs) to elicit mitogenic activity. The sugar specificity of the lectin and its interaction with PBMCs to exhibit mitogenic effect indicate its possible role in adhesion and infection process of Cephalosporium.

Rhizoctonia bataticola lectin (RBL) induces mitogenesis and cytokine production in human PBMC via p38 MAPK and STAT-5 signaling pathways.

BACKGROUND: Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus Rhizoctonia bataticola is highly mitogenic towards human peripheral blood mononuclear cells (PBMC). The lectin has sugar specificity towards N-glycans and binds to glycoproteins containing complex N-glycans (Nagre et al., Glycoconj J. 2010). In this study, we investigated the role of Mitogen Activated Protein Kinase (MAPK) and Signal Transducers and Activators of Transcription (STAT)-5 signaling in RBL-induced proliferation and production of Th1/Th2 cytokines. METHODS: Human PBMC were stimulated with RBL and proliferation was determined by tritiated thymidine incorporation assay, cytokine profiles by ELISA and activation of MAPK and STAT-5 by western blotting. RBL binding was monitored by immunofluorescence staining. Expression of IL-2Rα (CD25) was measured by flow cytometry. RESULTS: The binding and mitogenic activities of RBL were inhibited by glycoproteins- mucin, asialofetuin and fetuin. RBL stimulated expression of IL-2Rα and production of Th1/Th2 cytokines- IL-2, IFN-γ, IL-4 and IL-10. RBL-induced phosphorylation of ERK1/2 and p38 MAPK was detected at 1h and 3h respectively. Significant phosphorylation of STAT-5 (tyr(694)) was observed at 12h. Pharmacological inhibitors of p38 MAPK (SB203580) and JAK/STAT (AG490) but not ERK (PD98059) abrogated proliferation. RBL-induced expression of IL-2Rα and secretion of cytokines were drastically inhibited by SB203580 and AG490. CONCLUSIONS: RBL-induced proliferation and production of Th1/Th2 cytokines are mediated via p38 MAPK and STAT-5 signaling. GENERAL SIGNIFICANCE: RBL, a lectin with complex sugar specificity, is strongly mitogenic to human PBMC and stimulates the production of Th1 and Th2 cytokines. The results identified the signaling mechanism underlying the immunostimulatory activity of RBL.

A potent mitogenic lectin from the mycelia of a phytopathogenic fungus, Rhizoctonia bataticola, with complex sugar specificity and cytotoxic effect on human ovarian cancer cells.

A lectin with strong mitogenic activity towards human peripheral blood mononuclear cells (PBMCs) and cytotoxic effect on human ovarian cancer cells has been purified from the mycelium of a phytopathogenic fungus, Rhizoctonia bataticola, using ion exchange chromatography and affinity chromatography on asialofetuin-Sepharose. The lectin, termed RBL, is a tetramer of 11-kDa subunits and has unique amino acid sequence at its blocked N-terminus. The purified RBL was blood group nonspecific and its hemagglutination activity was inhibited by mucin (porcine stomach), fetuin (fetal calf serum) and asialofetuin. Glycan array analysis revealed high affinity binding of RBL towards N-glycans and also the glycoproteins containing complex N-glycan chains. Interestingly, the lectin showed high affinity for glycans which are part of ovarian cancer marker CA125, a high molecular weight mucin containing high mannose and complex bisecting type N-linked glycans as well core 1 and 2 type O-glycans. RBL bound to human PBMCs eliciting strong mitogenic response, which could be blocked by mucin, fetuin and asialofetuin demonstrating the carbohydrate-mediated interaction with the cells. Analysis of the kinetics of binding of RBL to PBMCs revealed a delayed mitogenic response indicating a different signaling pathway compared to phytohemagglutinin-L. RBL had a significant cytotoxic effect on human ovarian cancer cell line, PA-1.

Structural basis for the carbohydrate recognition of the Sclerotium rolfsii lectin.

The crystal structure of a novel fungal lectin from Sclerotium rolfsii (SRL) in its free form and in complex with N-acetyl-d-galactosamine (GalNAc) and N-acetyl- d -glucosamine (GlcNAc) has been determined at 1.1 A, 2.0 A, and 1.7 A resolution, respectively. The protein structure is composed of two beta-sheets, which consist of four and six beta-strands, connected by two alpha-helices. Sequence and structural comparisons reveal that SRL is the third member of a newly identified family of fungal lectins, which includes lectins from Agaricus bisporus and Xerocomus chrysenteron that share a high degree of structural similarity and carbohydrate specificity. The data for the free SRL are the highest resolution data for any protein of this family. The crystal structures of the SRL in complex with two carbohydrates, GalNAc and GlcNAc, which differ only in the configuration of a single epimeric hydroxyl group, provide the structural basis for its carbohydrate specificity. SRL has two distinct carbohydrate-binding sites, a primary and a secondary. GalNAc binds at the primary site, whereas GlcNAc binds only at the secondary site. Thus, SRL has the ability to recognize and probably bind at the same time two different carbohydrate structures. Structural comparison to Agaricus bisporus lectin-carbohydrate complexes reveals that the primary site is also able to bind the Thomsen-Friedenreich antigen (Galbeta1-->3GalNAc-alpha- glycan structures) whereas the secondary site cannot. The features of the molecular recognition at the two sites are described in detail.