Elucidation of binding specificity of Jacalin toward O-glycosylated peptides: quantitative analysis by frontal affinity chromatography

Jacalin, a lectin from the jackfruit Artocarpus integrifolia, has been known as a valuable tool for specific capturing of O-glycoproteins such as mucins and IgA1. Though its sugar-binding preference for T/Tn-antigens is well established, its detailed specificity has not been elucidated. In this study, we prepared a series of mucin-type glycopeptides using human glycosyltransferases, that is, ST6GalNAc1, Core1Gal-T1 and -T2, beta3Gn-T6, and Core2GnT1, and investigated their binding to immobilized Jacalin by frontal affinity chromatography (FAC). As a result, consistent with the previous observation, Jacalin showed high affinity for T-antigen (Core1) and Tn-antigen (alpha N-acetylgalactosamine)-attached peptides. Furthermore, we here show as novel findings that (1) Jacalin also showed significant affinity for Core3 and sialyl-T (ST)-attached peptides, but (2) Jacalin could not bind to Core2, Core6, and sialyl-Tn (STn)-attached peptides. The results were also confirmed by FAC using p-nitrophenyl (pNP)-derivatized saccharides. In conclusion, Jacalin binds to a GalNAcalpha1-peptide, in which C6-OH of alphaGalNAc is free (i.e., Core1, Tn, Core3, and ST), whereas it cannot recognize a GalNAcalpha1-peptide with a substitution at the C6 position (i.e., Core2, Core6, and STn). These findings provide useful information when applying jacalin for functional analysis of mucin-type glycoproteins and glycopeptides.     

Requirement for Core 2 O-Glycans for Optimal Resistance to Helminth Infection

The migration of lymphocytes to the small intestine is controlled by expression of the integrin α4β7 and the chemokine receptor CCR9. However, the molecules that specifically regulate migration to the large intestine remain unclear. Immunity to infection with the large intestinal helminth parasite Trichuris muris is dependent upon CD4⁺ T cells that migrate to the large intestine. We examine the role of specific chemokine receptors, adhesion molecules and glycosyltransferases in the development of protective immunity to Trichuris. Mice deficient in expression of the chemokine receptors CCR2 or CCR6 were resistant to infection with Trichuris. Similarly, loss of CD34, CD43, CD44 or PSGL-1 had no effect on resistance to infection. In contrast, simultaneous deletion of the Core2 β1,6-N-acetylglucosaminyltransferase (C2GnT) enzymes C2GnT1 and C2Gnt2 resulted in delayed expulsion of worms. These results suggest that C2GnT-dependent modifications may play a role in migration of protective immune cells to the large intestine.     

Enzymes responsible for synthesis of corneal keratan sulfate glycosaminoglycans

Keratan sulfate glycosaminoglycans are among the most abundant carbohydrate components of the cornea and are suggested to play an important role in maintaining corneal extracellular matrix structure. Keratan sulfate carbohydrate chains consist of repeating N-acetyllactosamine disaccharides with sulfation on the 6-O positions of N-acetylglucosamine and galactose. Despite its importance for corneal function, the biosynthetic pathway of the carbohydrate chain and particularly the elongation steps are poorly understood. Here we analyzed enzymatic activity of two glycosyltransferases, beta1,3-N-acetylglucosaminyltansferase-7 (beta3GnT7) and beta1,4-galactosyltransferase-4 (beta4GalT4), in the production of keratan sulfate carbohydrate in vitro. These glycosyltransferases produced only short, elongated carbohydrates when they were reacted with substrate in the absence of a carbohydrate sulfotransferase; however, they produced extended GlcNAc-sulfated poly-N-acetyllactosamine structures with more than four repeats of the GlcNAc-sulfated N-acetyllactosamine unit in the presence of corneal N-acetylglucosamine 6-O sulfotransferase (CGn6ST). Moreover, we detected production of highly sulfated keratan sulfate by a two-step reaction in vitro with a mixture of beta3GnT7/beta4GalT4/CGn6ST followed by keratan sulfate galactose 6-O sulfotransferase treatment. We also observed that production of highly sulfated keratan sulfate in cultured human corneal epithelial cells was dramatically reduced when expression of beta3GnT7 or beta4GalT4 was suppressed by small interfering RNAs, indicating that these glycosyltransferases are responsible for elongation of the keratan sulfate carbohydrate backbone.     

Glycosyltransferase Function in Core 2-Type Protein O Glycosylation

Three glycosyltransferases have been identified in mammals that can initiate core 2 protein O glycosylation. Core 2 O-glycans are abundant among glycoproteins but, to date, few functions for these structures have been identified. To investigate the biological roles of core 2 O-glycans, we produced and characterized mice deficient in one or more of the three known glycosyltransferases that generate core 2 O-glycans (C2GnT1, C2GnT2, and C2GnT3). A role for C2GnT1 in selectin ligand formation has been described. We now report that C2GnT2 deficiency impaired the mucosal barrier and increased susceptibility to colitis. C2GnT2 deficiency also reduced immunoglobulin abundance and resulted in the loss of all core 4 O-glycan biosynthetic activity. In contrast, the absence of C2GnT3 altered behavior linked to reduced thyroxine levels in circulation. Remarkably, elimination of all three C2GnTs was permissive of viability and fertility. Core 2 O-glycan structures were reduced among tissues from individual C2GnT deficiencies and completely absent from triply deficient mice. C2GnT deficiency also induced alterations in I-branching, core 1 O-glycan formation, and O mannosylation. Although the absence of C2GnT and C4GnT activities is tolerable in vivo, core 2 O glycosylation exerts a significant influence on O-glycan biosynthesis and is important in multiple physiological processes.Protein O glycosylation is a posttranslational modification implicated in a wide range of physiological processes, including cell adhesion and trafficking, T-cell apoptosis, cell signaling, endocytosis and pathogen-host interaction (1, 6, 27, 30, 54, 61, 71). Core-type protein O glycosylation is initiated in the secretory pathway by the covalent addition of a N-acetylgalactosamine (GalNAc) to the hydroxyl group of serine or threonine residues by one of multiple polypeptide GalNAc transferases (ppGalNAcTs) (20, 44, 57, 58). After linkage of the GalNAc monosaccharide to serine or threonine, other glycosyltransferases sequentially and sometimes competitively elaborate the repertoire of O-glycan structures to include different core subtypes (31, 42, 48, 49).The core 2 β1,6-N-acetylglucosaminyltransferases (C2GnTs) and the Core 2 O-glycans they generate are widely expressed among cells of mammalian species. The C2GnTs act after the core 1 β-1,3-galactosyltransferase adds a galactose in a β1,3-linkage to the GalNAc-Ser/Thr generating the initial core 1 O-glycan disaccharide structure (26). Then, one of the three C2GnTs (C2GnT1, C2GnT2, and C2GnT3) can add an N-acetylglucosamine (GlcNAc) in a β1,6-linkage to the GalNAc to initiate what is known as the core 2 O-glycan branch (Fig. ​(Fig.1a)1a) (7, 50, 51, 69). In a distinct pathway, core 3 β-1,3-N-acetylglucosaminyltransferase (C3GnT) can add a GlcNAc to the unmodified GalNAc to generate a core 3 O-glycan (24). In this case, C2GnT2 can add a GlcNAc in β1,6-linkage to the GalNAc of the core 3 O-glycan disaccharide to initiate the formation of a core 4 O-glycan (Fig. ​(Fig.1b)1b) (50, 69). In addition, both C2GnT2 and the I β-1,6-N-acetylglucosaminyltransferase (IGnT) are independently capable of forming branched polylactosamine structures (I-branches) from otherwise linear polylactosamine glycan chains (Fig. ​(Fig.1c)1c) (69).Open in a separate windowFIG. 1.Activity and expression of C2GnTs. (a to c) Monosaccharides are depicted as geometric shapes, with GalNAc as a yellow square, galactose as a yellow circle, and GlcNAc as a blue square. In addition, the vertical arrows indicate that each branch can be further elaborated by additional saccharide linkages. (a) Biantennary core 2 O-glycans are generated when any of the three C2GnTs acts on the core 1 O-glycan disaccharide. (b) C2GnT2 can generate core 4 O-glycans from core 3 O-glycans by adding a GlcNAc to the initiating GalNAc. (c) C2GnT2, in addition to IGnT, also has the ability to generate branched polylactosamine repeats from linear polylactosamine repeats. The figure depicts distal I-branching as the GlcNAc is transferred to the predistal galactose, the preferential I-branching activity of C2GnT2. However, IGnT preferentially has central I-branching activity that adds GlcNAc on the internal galactose in Galβ1→4GlcNAcβ1→3Gal-R (69). (d) RNA expression of murine Gcnt3 (left panel) and Gcnt4 (right panel), which code for C2GnT2 and C2GnT3, respectively, as determined by qPCR. The data on single animals are graphed relative to testes expression. All values are means ± the standard errors of the mean (SEM).C2GnT1-deficient mice have been shown to have an unexpected phenotype first observed as leukocytosis reflecting neutrophilia (14). This appears to be due to a severe but selective defect in selectin ligand biosynthesis among myeloid cells, leading to decreased recruitment of neutrophils that attenuates inflammation and vascular disease pathogenesis (14, 64). C2GnT1-deficient mice also exhibit a partial reduction in L-selectin ligand biosynthesis on high endothelial venules, resulting in reduced B-cell homing and colonization of peripheral lymph nodes (18, 21). Furthermore, thymic progenitors from C2GnT1-deficient mice have a reduced ability to home to the thymus due to the loss of P-selectin ligands on these cells (46). However, as of yet, C2GnT2 and C2GnT3 have not been similarly investigated, and their biological functions remain to be elucidated. To further investigate why multiple glycosyltransferases capable of core 2 O-glycan formation have been conserved, we have generated mice singly and multiply deficient in the three known C2GnTs and characterized the resulting physiology and alterations to the glycome.     

Perspectives on the significance of altered glycosylation of glycoproteins in cancer

No abstract Abbreviations: Sia, sialic acid, type unspecified; Tn antigen, GalNAcα 1-O-Ser/Thr; T antigen, Galβ1-3GalNAcα-O-Ser/Thr; Sialyl LewisX, Siaα2-3Galβ1-4(Fucα1-3)GlcNAc; Sialyl Lewisa, Siaα2-3Galβ1-3(Fucα1-4)GlcNAc; Sialyl-Tn antigen, Siaα2-6GalNAcα1-O-Ser/Thr; FucT, fucosyltransferase; ST, sialyltransferase. This revised version was published online in November 2006 with corrections to the Cover Date.     

Expression of core 2 beta-1,6-N-acetylglucosaminyltransferase in a human pancreatic cancer cell line results in altered expression of MUC1 tumor-associated epitopes.

Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 beta-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcalpha2,6(Galbeta1,3)GalNAcalpha-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcalpha2,3Galbeta1,4(Fucalpha1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O-glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes.     

Sclerotium rolfsii Lectin Induces Stronger Inhibition of Proliferation in Human Breast Cancer Cells than Normal Human Mammary Epithelial Cells by Induction of Cell Apoptosis

Sclerotium rolfsii lectin (SRL) isolated from the phytopathogenic fungus Sclerotium rolfsii has exquisite binding specificity towards O-linked, Thomsen-Freidenreich (Galβ1-3GalNAcα1-Ser/Thr, TF) associated glycans. This study investigated the influence of SRL on proliferation of human breast cancer cells (MCF-7 and ZR-75), non-tumorigenic breast epithelial cells (MCF-10A) and normal mammary epithelial cells (HMECs). SRL caused marked, dose-dependent, inhibition of proliferation of MCF-7 and ZR-75 cells but only weak inhibition of proliferation of non-tumorigenic MCF-10A and HMEC cells. The inhibitory effect of SRL on cancer cell proliferation was shown to be a consequence of SRL cell surface binding and subsequent induction of cellular apoptosis, an effect that was largely prevented by the presence of inhibitors against caspases -3, -8, or -9. Lectin histochemistry using biotin-labelled SRL showed little binding of SRL to normal human breast tissue but intense binding to cancerous tissues. In conclusion, SRL inhibits the growth of human breast cancer cells via induction of cell apoptosis but has substantially less effect on normal epithelial cells. As a lectin that binds specifically to a cancer-associated glycan, has potential to be developed as an anti-cancer agent.     

Molecular Cloning of Pigeon UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase and UDP-galactose:��-d-Galactoside ��1,4-Galactosyltransferase, Two Novel Enzymes Catalyzing the Formation of Gal��1�C4Gal��1�C4Gal��1�C4GlcNAc Sequence

We previously found that pigeon IgG possesses unique N-glycan structures that contain the Galα1–4Galβ1–4Galβ1–4GlcNAc sequence at their nonreducing termini. This sequence is most likely produced by putative α1,4- and β1,4-galactosyltransferases (GalTs), which are responsible for the biosynthesis of the Galα1–4Gal and Galβ1–4Gal sequences on the N-glycans, respectively. Because no such glycan structures have been found in mammalian glycoproteins, the biosynthetic enzymes that produce these glycans are likely to have distinct substrate specificities from the known mammalian GalTs. To study these enzymes, we cloned the pigeon liver cDNAs encoding α4GalT and β4GalT by expression cloning and characterized these enzymes using the recombinant proteins. The deduced amino acid sequence of pigeon α4GalT has 58.2% identity to human α4GalT and 68.0 and 66.6% identity to putative α4GalTs from chicken and zebra finch, respectively. Unlike human and putative chicken α4GalTs, which possess globotriosylceramide synthase activity, pigeon α4GalT preferred to catalyze formation of the Galα1–4Gal sequence on glycoproteins. In contrast, the sequence of pigeon β4GalT revealed a type II transmembrane protein consisting of 438 amino acid residues, with no significant homology to the glycosyltransferases so far identified from mammals and chicken. However, hypothetical proteins from zebra finch (78.8% identity), frogs (58.9–60.4%), zebrafish (37.1–43.0%), and spotted green pufferfish (43.3%) were similar to pigeon β4GalT, suggesting that the pigeon β4GalT gene was inherited from the common ancestors of these vertebrates. The sequence analysis revealed that pigeon β4GalT and its homologs form a new family of glycosyltransferases.     

Trimeric Tn Antigen on Syndecan 1 Produced by ppGalNAc-T13 Enhances Cancer Metastasis via a Complex Formation with Integrin α5β1 and Matrix Metalloproteinase 9

We demonstrated previously that ppGalNAc-T13 (T13), identified as an up-regulated gene with increased metastasis in a DNA microarray, generated trimeric Tn (tTn) antigen (GalNAcα1-Ser/Thr)₃ on Syndecan 1 in highly metastatic sublines of Lewis lung cancer. However, it is not known how tTn antigen regulates cancer metastasis. Here, we analyzed the roles of tTn antigen in cancer properties. tTn antigen on Syndecan 1 increased cell adhesion to fibronectin in an integrin-dependent manner. Furthermore, cell adhesion to fibronectin induced phosphorylation of focal adhesion kinase and paxillin in T13-transfectant cells. In the search of Syndecan 1-interacting molecules, it was demonstrated that tTn antigen-carrying Syndecan 1 interacted with integrin α5β1 and matrix metalloproteinase 9 and that these molecules shifted to a glycolipid-enriched microdomain/rafts along with increased metastatic potential in T13-transfectant cells. We also identified a tTn substitution site on Syndecan 1, demonstrating that tTn on Syndecan 1 is essential for the interaction with integrin α5β1 as well as for the reaction with mAb MLS128. These data suggest that high expression of the ppGalNAc-T13 gene generates tTn antigen on Syndecan 1 under reduced expression of GM1, leading to enhanced invasion and metastasis via the formation of a molecular complex consisting of integrin α5β1, Syndecan 1, and MMP-9 in the glycolipid-enriched microdomain/rafts.     

Identification of the Drosophila core 1 beta1,3-galactosyltransferase gene that synthesizes T antigen in the embryonic central nervous system and hemocytes

T antigen (Galbeta1-3GalNAcalpha1-Ser/Thr), the well-known tumor-associated antigen, is a core 1 mucin-type O-glycan structure that is synthesized by core 1 beta1,3-galactosyltransferase (C1beta3GalT), which transfers Gal from UDP-Gal to Tn antigen (GalNAcalpha1-Ser/Thr). Three putative C1beta3GalTs have been identified in Drosophila. However, although all three are expressed in embryos, their roles during embryogenesis have not yet been clarified. In this study, we used P-element inserted mutants to show that CG9520, one of the three putative C1beta3GalTs, synthesizes T antigen expressed on the central nervous system (CNS) during embryogenesis. We also found that T antigen was expressed on a subset of the embryonic hemocytes. CG9520 mutant embryos showed the loss of T antigens on the CNS and on a subset of hemocytes. Then, the loss of T antigens was rescued by precise excision of the P-element inserted into the CG9520 gene. Our data demonstrate that T antigens expressed on the CNS and on a subset of hemocytes are synthesized by CG9520 in the Drosophila embryo. In addition, we found that the number of circulating hemocytes was reduced in third instar larvae of CG9520 mutant. We, therefore, named the CG9520 gene Drosophila core 1 beta1,3-galactosyltransferase 1 because it is responsible for the synthesis and function of T antigen in vivo.     

Unique gangliosides synthesized in vitro by sialyltransferases from marine bacteria and their characterization: ganglioside synthesis by bacterial sialyltransferases

On the basis of the results outlined in our previous report, bacterial sialyltransferases (ST) from marine sources were further characterized using glycosphingolipids (GSL), especially ganglio-series GSLs, based on the enzymatic characteristics and kinetic parameters obtained by Line weaver-Burk plots. Among them, GA1 and GA2 were found to be good substrates for these unique STs. Thus, new gangliosides synthesized by α2-3 and α2-6STs were structurally characterized by several analytical procedures. The ganglioside generated by the catalytic activity of α2-3ST was identified as GM1b. On the other hand, when enzyme reactions by α2-6STs were performed using substrates GA2 and GA1, very unique gangliosides were generated. The structures were identified as NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer and NeuAcα2-6Galβ1-3GalNAcβ1-4Galβ1-4Glcβ-Cer, respectively. The synthesized ganglioside NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer showed binding activity to the influenza A virus {A/Panama/2007/99 (H3N2)} at a similar level to purified sialyl(α2-3)paragloboside (S2-3PG) and sialyl(α2-6)paragloboside (S2-6PG) from mammalian sources. The evidence suggests that these STs have unique features, including substrate specificities restricted not only to lacto-series but also to ganglio-series GSLs, as well as catalytic potentials for ganglioside synthesis. This evidence demonstrates that effective in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing sialic acid (Sia) modifications, thereby preparing large-scale gangliosides and permitting the exploration of unknown functions.     

Glycosyltransferase-specific Golgi-targeting Mechanisms

Glycosylation of secreted and membrane-bound mucins is carried out by glycosyltransferases localized to specific Golgi compartments according to the step in which each enzyme participates. However, the Golgi-targeting mechanisms of these enzymes are not clear. Herein, we investigate the Golgi-targeting mechanisms of core 1 β3 galactosyltransferase (C1GalT1) and core 2 β1,6-N-acetylglucosaminyltransferase-2 or mucus type (C2GnT-M), which participate in the early O-glycosylation steps. siRNAs, co-immunoprecipitation, and confocal fluorescence microscopy were employed to identify the golgins involved in the Golgi docking of vesicular complexes (VCs) that carry these two enzymes. We have found that these VCs use different golgins for docking: C2GnT-M-carrying VC (C2GnT-M-VC) utilizes Giantin, whereas C1GalT1-VC employs GM130-GRASP65 complex. However, in the absence of GRASP65, C1GalT1-VC utilizes GM130-Giantin complex. Also, we have found that these VCs are 1.1–1.2 μm in diameter, specific for each enzyme, and independent of coat protein complex II and I (COPII and COPI). These two fluorescently tagged enzymes exhibit different fluorescence recovery times in the Golgi after photobleaching. Thus, novel enzyme-specific Golgi-targeting mechanisms are employed by glycosyltransferases, and multiple Golgi docking strategies are utilized by C1GalT1.     

Different glycosyltransferases are differentially processed for secretion, dimerization, and autoglycosylation

Modification of Golgi glycosyltransferases, such as formation of disulfide-bonded dimers and proteolytical release from cells as a soluble form, are important processes to regulate the activity of glycosyltransferases. To better understand these processes, six glycosyltransferases were selected on the basis of the donor sugars, including two N-acetylglucosaminyltransferases, core 1 beta1,3-N-acetylglucosaminyltransferase (C1-beta3GnT) and core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT-I); two fucosyltransferases, alpha1,2-fucosyltransferase-I (FucT-I) and alpha1,3-fucosyltransferase-VII (FucT-VII); and two sialyltransferases, alpha2,3-sialyltransferase-I (ST3Gal-I) and alpha2,6-sialyltransferase-I (ST6Gal-I). These enzymes were fused with enhanced green fluorescence protein and stably expressed in Chinese hamster ovary cells. Spectrofluorimetric detection and immunoblotting analyses showed that all of these glycosyltransferases except FucT-VII were secreted in the medium. By examining dimers formed in cells and culture media, we found that all of the enzymes, except ST3Gal-I, form a combination of monomers and dimers in cells, whereas the molecules released in the media are either exclusively monomers (C2GnT-I and ST6Gal-I), dimers (FucT-I) or a mixture of both (C1-beta3GnT). These results indicate that dimerization does not always lead to Golgi retention. Analysis of the N-glycosylation status of the enzymes revealed that the secreted proteins are generally more heavily N-glycosylated and sialylated than their membrane-associated counterparts, suggesting that the proteolytic cleavage occurs before the glycosylation is completed. Using FucT-I and ST6Gal-I as a model, we also show that these glycosyltransferases are able to perform autoglycosylation in the dimeric forms. These results indicate that different glycosyltranferases differ significantly in dimerization, proteolytic digestion and secretion, and autoglycosylation. These results strongly suggest that disulfide-bonded dimerization and secretion differentially plays a role in the processing and function of different glycosyltransferases in the Golgi apparatus.     

Remodeling of Marrow Hematopoietic Stem and Progenitor Cells by Non-self ST6Gal-1 Sialyltransferase

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin−c-Kit+ and Lin−Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin−Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.     

The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn,Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell and in close proximity to extended glycan T (Galβ1,3GalNAc) the addition of Gal to the GalNAc being catalysed by the T synthase. The presence of active T synthase suggests that loss of the private chaperone for T synthase, COSMC, does not explain the expression of Tn and STn in breast cancer cells. We show that MUC1 carrying both Tn or STn can bind to the C-type lectin MGL and using atomic force microscopy show that they bind to MGL with a similar deadadhesion force. Tumour associated STn is associated with poor prognosis and resistance to chemotherapy in breast carcinomas, inhibition of DC maturation, DC apoptosis and inhibition of NK activity. As engagement of MGL in the absence of TLR triggering may lead to anergy, the binding of MUC1-STn to MGL may be in part responsible for some of the characteristics of STn expressing tumours.     

Differential Expression Patterns of N-Acetylglucosaminyl Transferases and Polylactosamines in Uterine Lesions

Polylactosamine (polyLacNAc) is a fundamental structure in glycoconjugates and it is expressed in specific cells/tissues associated with the development and carcinogenesis. β1,3-N-acetylglucosaminyl transferases ((β3GnTs) play an important role in polyLacNAc synthesis, however the roles of these glycosyltransferases and their products in cancer progression are still unclear. In this sense, this work aimed to evaluate differential expression pattern of the N-acetylglucosaminyl transferases and polylactosamines in invasive and premalignant lesions of the uterus cervix. The expression of β3GnT2 and β3GnT3 were evaluated in normal (n=10) and uterine cervix lesions (n=120), both malignant [squamous carcinoma (SC)] and premalignant [cervical intraepithelial neoplasia (CIN), grades 1, 2 and 3] using immunohistochemistry. Besides, lectin histochemistry with Phytolacca americana lectin (PWM) and Wheat germ agglutinin (WGA) was also carried out to observe the presence of polyLacNAc chains and N-acetylglucosamine (GlcNAc), respectively. The β3GnT3 was expressed in almost all samples (99%) and β3GnT2 was higher expressed in disease samples mainly in CIN 3, when compared with normal (P=0.002), CIN 1 (P=0.009) and CIN 2 (P=0.03). The expression of polyLacNAc was higher is SC samples, when compared with normal (P=0.03), CIN 1 (P=0.02) and CIN 3 (P=0.004), and was observed only nuclear expression in nearly 50% of the SC samples, showing a statistically significant when compared with normal (P=0.01), CIN 1 (P=0.002), CIN 2 (P=0.007) and CIN 3 (P=0.04). Deferring from transferases and polyLacNAc chains, GlcNAc (WGA ligand) reveals a gradual staining pattern decrease with the increase of the lesion degree, being more expressed in CIN 1 lesions when compared with normal (P<0.0001), CIN 2 (P<0.0001), SC (P<0.0001) and CIN 3 (P=0.0003). Our data reveal that β3GnT2 and polyLacNAc may be involved in the progression of the pre-malignant lesions of the human uterine cervix. In addition, polyLacNAc expression only in the nucleus can be associated a poor prognostic in uterine lesions.