Structure of O-glycosidically linked oligosaccharides from glycoproteins of Trypanosoma cruzi CL-Brener strain: evidence for the presence of O-linked sialyl-oligosaccharides

Glycoproteins on the cell surface of Trypanosoma cruzi are known to play important roles in the interaction of the parasite with the host cells. We previously determined the structures of the O-glycan chains from the sialoglycoproteins (mucin-like molecules) of the G- and Y-strains and observed significant differences between them. We now report the structures of the sialylated and nonsialylated O-linked oligosaccharides isolated from the cell surface glycoproteins of the myotropic CL-Brener strain grown in the presence of fetal calf serum. The structures of the O-linked oligosaccharide alditols obtained by reductive beta-elimination of the sialoglycoprotein were determined by a combination of methylation analysis, fast atom bombardment-mass spectrometry and nuclear magnetic resonance spectroscopy. The presence of a beta-galactopyranose substituent on the N-acetylglucosamine O-4 position shows that these O-linked oligosaccharides from CL-Brener strain belong to the same family as those isolated from mucins expressed by T. cruzi Y strain, a reticulotropic strain. In addition, novel O-glycans, including alpha2-3 mono-sialylated species are described.     

Leukosialin, a major O-glycan-containing sialoglycoprotein defining leukocyte differentiation and malignancy.

Leukosialin, also called CD43 or sialophorin, is a major sialoglycoprotein expressed widely in various leukocytes (granulocytes, monocytes/macrophages and T-lymphocytes). Leukosialin is heavily glycosylated by O-linked oligosaccharides (70-80 oligosaccharides/molecule) and the structures of those O-glycans are characteristic to each cell lineage and differentiation stage. In particular, the branched hexasaccharide, NeuNAc alpha 2----3Gal beta 1----3(NeuNAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc is specifically expressed in activated T-lymphocytes as well as in thymocytes and T-lymphocytes from patients with leukaemia, and immuno-deficiency syndromes. A portion of these O-glycans are attached to a domain with tandem repeats in the polypeptide of leukosialin. However, the entire translation product, including such tandem repeats, is coded by one exon and a short novel promoter sequence confers the expression of the leukosialin gene. Leukosialin is apparently involved in T-cell-B-cell interaction during immune reaction and binds to ligands on antigen-presenting B-cells. These results imply that leukosialin plays critical roles in immune cell interaction and differences in attached O-glycans most likely influence the interaction of leukosialin with ligands.     

Branched O-linked oligosaccharides ectopically expressed in transgenic mice reduce primary T-cell immune responses.

Core 2 beta-1,6-N-acetylglucosaminyltransferase, C2GnT, is a key enzyme in O-linked oligosaccharide (O-glycan) biosynthesis and the resultant core 2 branch serves as a backbone for additional glycosylation to form oligosaccharide ligands such as sialyl Le(x). Since the expression of C2GnT is highly regulated during T-cell development and increases in pathological conditions such as the Wiskott-Aldrich syndrome, we have generated transgenic mice overexpressing C2GnT in the T-cell lineage. Surprisingly, T lymphocytes in the transgenic mice develop normally, but they exhibit a reduced immune response when assayed by delayed-type hypersensitivity, proliferation upon stimulation and cytokine production. Moreover, T lymphocytes from the transgenic mice adhere much less efficiently to ICAM-1 and fibronectin than do T lymphocytes from non-transgenic mice. These results indicate that overexpression of the core 2 branched O-glycans in T lymphocytes results in reduced immune responses due to impaired cell-cell interaction. Such an impaired immune response may be one of the causes for immunodeficiency in the Wiskott-Aldrich syndrome.     

Protein O-glycosylation analysis

Abstract This review provides an overview on the methods available for analysis of O-glycosylation. Three major themes are addressed: analysis of released O-glycans including different O-glycan liberation, derivatization, and detection methods; analysis of formerly O-glycosylated peptides yielding information on O-glycan attachment sites; analysis of O-glycopeptides, representing by far the most informative but also most challenging approach for O-glycan analysis. Although there are various techniques available for the identification of O-linked oligosaccharides, the focus here is on MS fragmentation techniques such as collision-induced fragmentation, electron capture dissociation, and electron transfer dissociation. Finally, the O-glycan analytical challenges that need to be met will be discussed.     

Structure determination by MALDI-IRMPD mass spectrometry and exoglycosidase digestions of O-linked oligosaccharides from Xenopus borealis egg jelly

Differences in the fertilization behavior of Xenopus borealis from X. laevis and X. tropicalis suggest differences in the glycosylation of the egg jellies. To test this assumption, O-linked glycans were chemically released from the egg jelly coat glycoproteins of X. borealis. Over 50 major neutral glycans were observed, and no anionic glycans were detected from the released O-glycan pool. Preliminary structures of ～30 neutral oligosaccharides were determined using matrix-assisted laser desorption/ionization (MALDI) infrared multiphoton dissociation tandem mass spectrometry (MS). The mass fingerprint of a group of peaks for the core-2 structure of O-glycans was conserved in the tandem mass spectra and was instrumental in rapid and efficient structure determination. Among the 29 O-glycans, 22 glycans contain the typical core-2 structure, 3 glycans have the core-1 structure and 2 glycans contained a previously unobserved core structure with hexose at the reducing end. There were seven pairs of structural isomers observed in the major O-linked oligosaccharides. To further elucidate the structures of a dozen O-linked glycans, specific and targeted exoglycosidase digestions were carried out and the products were monitored with MALDI-MS. Reported here are the elucidated structures of O-linked oligosaccharides from glycoproteins of X. borealis egg jelly coats. The structural differences in O-glycans from jelly coats of X. borealis and its close relatives may provide a better understanding of the structure-function relationships and the role of glycans in the fertilization process within Xenopodinae.     

Concepts and Principles of O-Linked Glycosylation

The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational event, is reviewed and compared for the various types of O-glycans. Mucin-type O-glycosylation is initiated by tissue-specific addition of a GalNAc-residue to a serine or a threonine of the fully folded protein. This event is dependent on the primary, secondary, and tertiary structure of the glycoprotein. Further elongation and termination by specific transferases is highly regulated. We also describe some of the physical and biological properties that O-glycosylation confers on the protein to which the sugars are attached. These include providing the basis for rigid conformations and for protein stability. Clustering of O-glycans in Ser/Thr(/Pro)-rich domains allows glycan determinants such as sialyl Lewis X to be presented as multivalent ligands, essential for functional recognition. An additional level of regulation, imposed by exon shuffling and alternative splicing of mRNA, results in the expression of proteins that differ only by the presence or absence of Ser/Thr(/Pro)-rich domains. These domains may serve as protease-resistant spacers in cell surface glycoproteins. Further biological roles for O-glycosylation discussed include the role of isolated mucin-type O-glycans in recognition events (e.g., during fertilization and in the immune response) and in the modulation of the activity of enzymes and signaling molecules. In some cases, the O-linked oligosac-charides are necessary for glycoprotein expression and processing. In contrast to the more common mucin-type O-glycosylation, some specific types of O-glycosylation, such as the O-linked attachment of fucose and glucose, are sequon dependent. The reversible attachment of O-linked GlcNAc to cytoplasmic and nuclear proteins is thought to play a regulatory role in protein function. The recent development of novel technologies for glycan analysis promises to yield new insights in the factors that determine site occupancy, structure-function relationship, and the contribution of O-linked sugars to physiological and pathological processes. These include diseases where one or more of the O-glycan processing enzymes are aberrantly regulated or deficient, such as HEMPAS and cancer.     

Biosynthesis of HNK-1 glycans on O-linked oligosaccharides attached to the neural cell adhesion molecule (NCAM): the requirement for core 2 beta 1,6-N-acetylglucosaminyltransferase and the muscle-specific domain in NCAM

The HNK-1 glycan, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->R, is highly expressed in neuronal cells and apparently plays critical roles in neuronal cell migration and axonal extension. The HNK-1 glycan synthesis is initiated by the addition of beta1,3-linked GlcA to N-acetyllactosamine followed by sulfation of the C-3 position of GlcA. The cDNAs encoding beta1,3-glucuronyltransferase (GlcAT-P) and HNK-1 sulfotransferase (HNK-1ST) have been recently cloned. Among various adhesion molecules, the neural cell adhesion molecule (NCAM) was shown to contain HNK-1 glycan on N-glycans. In the present study, we first demonstrated that NCAM also bears HNK-1 glycan attached to O-glycans when NCAM contains the O-glycan attachment scaffold, muscle-specific domain, and is synthesized in the presence of core 2 beta1,6-N-acetylglucosaminyltransferase, GlcAT-P, and HNK-1ST. Structural analysis of the HNK-1 glycan revealed that the HNK-1 glycan is attached on core 2 branched O-glycans, sulfo-->3GlcAbeta1-->3Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNAc. Using synthetic oligosaccharides as acceptors, we found that GlcAT-P and HNK-1ST almost equally act on oligosaccharides, mimicking N- and O-glycans. By contrast, HNK-1 glycan was much more efficiently added to N-glycans than O-glycans when NCAM was used as an acceptor. These results are consistent with our results showing that HNK-1 glycan is minimally attached to O-glycans of NCAM in fetal brain, heart, and the myoblast cell line, C2C12. These results combined together indicate that HNK-1 glycan can be synthesized on core 2 branched O-glycans but that the HNK-1 glycan is preferentially added on N-glycans over O-glycans of NCAM, probably because N-glycans are extended further than O-glycans attached to NCAM containing the muscle-specific domain.     

O-linked glycan expression during Drosophila development

Mucin-type O-linked glycosylation is an evolutionarily conserved protein modification that is essential for viability in Drosophila melanogaster. However, the exact role of O-glycans and the identity of the crucial apoproteins modified with O-linked N-acetylgalactosamine (O-GalNAc) remain unknown. In an effort to elucidate the O-linked glycans expressed during Drosophila development, we have employed fluorescent confocal microscopy using a battery of lectins and an antibody specific for the GalNAcalpha-Ser/Thr structure (Tn antigen). Confocal microscopy provides high-resolution images of the diversity of glycans expressed in many developing organ systems. In particular, O-glycans are highly expressed on a number of ectodermally derived tissues such as the salivary glands, developing gut, and the tracheal system, suggesting a role for O-glycans in cell polarity and tube formation common to these organs. Additionally, O-glycans are found in the developing nervous system and within subregions of developing tissues known to be active in cell signaling events. This study provides us with temporal and spatial information regarding O-glycan expression as well as a set of reagents for the isolation of glycoproteins from specific developmental stages and organ systems. This information will aid us in identifying the in vivo substrates of the UDP-GalNAc: polypeptide N-acetylgalactosaminyltranferases, in a continuing effort to define the biological role of O-linked glycoproteins during development.     

Glycoprotein biosynthesis in porcine aortic endothelial cells and changes in the apoptotic cell population

Porcine aortic endothelial cells (PAECs) produce glycoproteins with important biological functions, such as the control of cell adhesion, blood clotting, blood pressure, the immune system, and apoptosis. Cell surface glycoproteins play important roles in these biological activities. To understand the control of cell surface glycosylation, we elucidated biosynthetic pathways leading to N- and O-glycans in PAECs. Based on the enzyme activities, PAECs should be rich in complex biantennary N-glycans. In addition, the enzymes synthesizing complex O-glycans with core 1 and 2 structures are present in PAECs. The first enzyme of the O-glycosylation pathway, polypeptide GalNAc-transferase, was particularly active. Its specificity toward synthetic peptide substrates was found to be similar to that of purified bovine colostrum enzyme T1. A significant fraction of PAECs treated with tumour necrosis factor alpha or human serum detached from the culture plate, and most of these cells were apoptotic. The apoptotic cell population exhibited decreased core 2 beta 6-GlcNAc-transferase activity. In contrast, the activities of core 1 beta 3-Gal-transferase, which synthesizes O-glycan core 1, and of alpha 3-sialyltransferase (O), which sialylates core 1, were increased in apoptotic PAECs. Thus, apoptotic PAECs are predicted to have fewer complex O-glycans and a higher proportion of short, sialylated core 1 chains.     

Multiple Novel Functions of Henipavirus O-glycans: The First O-glycan Functions Identified in the Paramyxovirus Family

O-linked glycosylation is a ubiquitous protein modification in organisms belonging to several kingdoms. Both microbial and host protein glycans are used by many pathogens for host invasion and immune evasion, yet little is known about the roles of O-glycans in viral pathogenesis. Reportedly, there is no single function attributed to O-glycans for the significant paramyxovirus family. The paramyxovirus family includes many important pathogens, such as measles, mumps, parainfluenza, metapneumo- and the deadly Henipaviruses Nipah (NiV) and Hendra (HeV) viruses. Paramyxoviral cell entry requires the coordinated actions of two viral membrane glycoproteins: the attachment (HN/H/G) and fusion (F) glycoproteins. O-glycan sites in HeV G were recently identified, facilitating use of the attachment protein of this deadly paramyxovirus as a model to study O-glycan functions. We mutated the identified HeV G O-glycosylation sites and found mutants with altered cell-cell fusion, G conformation, G/F association, viral entry in a pseudotyped viral system, and, quite unexpectedly, pseudotyped viral F protein incorporation and processing phenotypes. These are all important functions of viral glycoproteins. These phenotypes were broadly conserved for equivalent NiV mutants. Thus our results identify multiple novel and pathologically important functions of paramyxoviral O-glycans, paving the way to study O-glycan functions in other paramyxoviruses and enveloped viruses.     

Alpha1,4GlcNAc-capped mucin-type O-glycan inhibits cholesterol alpha-glucosyltransferase from Helicobacter pylori and suppresses H. pylori growth

Helicobacter pylori infects over half of the world's population and is thought to be a leading cause of gastric ulcer, gastric carcinoma, and gastric malignant lymphoma of mucosa-associated lymphoid tissue type. Previously, we reported that a gland mucin (MUC6) present in the lower portion of the gastric mucosa containing alpha1,4-N-acetylglucosamine (alpha1,4GlcNAc)-capped core 2-branched O-glycans suppresses H. pylori growth by inhibiting the synthesis of alpha-glucosyl cholesterol, a major constituent of the H. pylori cell wall (Kawakubo et al. 2004. Science. 305:1003-1006). Therefore, we cloned the genomic DNA encoding cholesterol alpha-glucosyltransferase (HP0421) and expressed its soluble form in Escherichia coli. Using this soluble HP0421, we show herein that HP0421 sequentially acts on uridine diphosphoglucose and cholesterol in an ordered Bi-Bi manner. We found that competitive inhibition of HP0421 by alpha1,4GlcNAc-capped core 2-branched O-glycan is much more efficient than noncompetitive inhibition by newly synthesized alpha-glucosyl cholesterol. Utilizing synthetic oligosaccharides, alpha-glucosyl cholesterol, and monosaccharides, we found that alpha1,4GlcNAc-capped core 2-branched O-glycan most efficiently inhibits H. pylori growth. These findings together indicate that alpha1,4GlcNAc-capped O-glycans suppress H. pylori growth by inhibiting HP0421, and that alpha1,4GlcNAc-capped core 2 O-glycans may be useful to treat patients infected with H. pylori.     

Poly-N-acetyllactosamine extension in N-glycans and core 2- and core 4-branched O-glycans is differentially controlled by i-extension enzyme and different members of the beta 1,4-galactosyltransferase gene family

Poly-N-acetyllactosamines are attached to N-glycans, O-glycans, and glycolipids and serve as underlying glycans that provide functional oligosaccharides such as sialyl Lewis(X). Poly-N-acetyllactosaminyl repeats are synthesized by the alternate addition of beta1,3-linked GlcNAc and beta1,4-linked Gal by i-extension enzyme (iGnT) and a member of the beta1,4-galactosyltransferase (beta4Gal-T) gene family. In the present study, we first found that poly-N-acetyllactosamines in N-glycans are most efficiently synthesized by beta4Gal-TI and iGnT. We also found that iGnT acts less efficiently on acceptors containing increasing numbers of N-acetyllactosamine repeats, in contrast to beta4Gal-TI, which exhibits no significant change. In O-glycan biosynthesis, N-acetyllactosamine extension of core 4 branches was found to be synthesized most efficiently by iGnT and beta4Gal-TI, in contrast to core 2 branch synthesis, which requires iGnT and beta4Gal-TIV. Poly-N-acetyllactosamine extension of core 4 branches is, however, less efficient than that of N-glycans or core 2 branches. Such inefficiency is apparently due to competition between a donor substrate and acceptor in both galactosylation and N-acetylglucosaminylation, since a core 4-branched acceptor contains both Gal and GlcNAc terminals. These results, taken together, indicate that poly-N-acetyllactosamine synthesis in N-glycans and core 2- and core 4-branched O-glycans is achieved by iGnT and distinct members of the beta4Gal-T gene family. The results also exemplify intricate interactions between acceptors and specific glycosyltransferases, which play important roles in how poly-N-acetyllactosamines are synthesized in different acceptor molecules.     

Galectin-1 binds different CD43 glycoforms to cluster CD43 and regulate T cell death

Galectin-1 kills immature thymocytes and activated peripheral T cells by binding to glycans on T cell glycoproteins including CD7, CD45, and CD43. Although roles for CD7 and CD45 in regulating galectin-1-induced death have been described, the requirement for CD43 remains unknown. We describe a novel role for CD43 in galectin-1-induced death, and the effects of O-glycan modification on galectin-1 binding to CD43. Loss of CD43 expression reduced galectin-1 death of murine thymocytes and human T lymphoblastoid cells, indicating that CD43 is required for maximal T cell susceptibility to galectin-1. CD43, which is heavily O-glycosylated, contributes a significant fraction of galectin-1 binding sites on T cells, as T cells lacking CD43 bound approximately 50% less galectin-1 than T cells expressing CD43. Although core 2 modification of O-glycans on other glycoprotein receptors is critical for galectin-1-induced cross-linking and T cell death, galectin-1 bound to CD43 fusion proteins modified with either unbranched core 1 or branched core 2 O-glycans and expression of core 2 O-glycans did not enhance galectin-1 binding to CD43 on T cells. Moreover, galectin-1 binding clustered CD43 modified with either core 1 or core 2 O-glycans on the T cell surface. Thus, CD43 bearing either core 1 or core 2 O-glycans can positively regulate T cell susceptibility to galectin-1, identifying a novel function for CD43 in controlling cell death. In addition, these studies demonstrate that different T cell glycoproteins on the same cell have distinct requirements for glycan modifications that allow recognition and cross-linking by galectin-1.     

Pathways of mucin O-glycosylation in normal and malignant rat colonic epithelial cells reveal a mechanism for cancer-associated Sialyl-Tn antigen expression

The Sialyl-Tn antigen (Sialyl alpha-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells. Its presence is associated with a poor prognosis in patients with colorectal and other cancers. We previously reported that Sialyl-Tn expression in LSC human colon cancer cells could be explained by a specific lack of the activity of core 1 beta3-Gal-transferase (Brockhausen et al., Glycoconjugate J. 15, 595-603, 1998) and an inability to synthesize the common O-glycan core structures. To support this mechanism, or find other mechanisms to explain Sialyl-Tn antigen expression, we investigated the O-glycosylation pathways in clonal rat colon cancer cell lines that were selected for positive or negative expression of Sialyl-Tn antigen, and compared these pathways to those in normal rat colonic mucosa. Normal rat colonic mucosa had very active glycosyltransferases synthesizing O-glycan core structures 1 to 4. Several sialyl-, sulfo- and fucosyltransferases were also active. An M type core 2 beta6-GlcNAc-transferase was found to be present in rat colon mucosa and all of the rat colon cancer cells. O-glycosylation pathways in rat colon cancer cells were significantly different from normal rat colonic mucosa; for example, rat colon cancer cells lost the ability to synthesize O-glycan core 3. All rat colon cancer cell lines, regardless of the Sialyl-Tn phenotype, expressed glycosyltransferases assembling complex O-glycans of core 1 and core 2 structures (unlike human LSC colon cancer cells which lack core 1 beta3-Gal-transferase activity). It was the activity of CMP-sialic acid:GalNAc-mucin alpha6-sialyltransferase that coincided with Sialyl-Tn expression. Sialyl-Tn negative cells had a several fold higher activity of core 2 beta6-GlcNAc-transferase which synthesizes complex O-glycans that may mask adjacent Sialyl-Tn epitopes. The results suggest a new mechanism controlling Sialyl-Tn expression in cancer cells.     

Cosmc和T-合酶在黏蛋白型O-聚糖合成中的重要作用及其与人类疾病的相关性

从核心1结构（Galβ1,3GalNAcα1-O-Ser/Thr, core 1 structure, T antigen）中衍生出来的黏蛋白型O-聚糖在很多生理过程中发挥重要的生物学功能。T-合酶 （core 1 β3-galactosyltransferase, T-synthase） 是合成核心1结构的唯一糖基转移酶,它主要的功能是将半乳糖（Galactose） 添加到GalNAcα1-Ser/Thr （Tn抗原） 糖链上。但是在人体和其他脊椎动物中有活性的T-合酶的形成需要一个重要的伴侣分子Cosmc;Cosmc功能丧失将直接导致T-合酶失活,其结果是机体细胞只能合成Tn抗原以及唾液酰化Tn （sialylTn, STn, Neu5Acα2,6GalNAcα1-O-Ser/Thr）。综述目前对T-合酶和Cosmc的研究以及他们在人类疾病（如异常O-聚糖表达相关的Tn综合征、IgA肾病和肿瘤）发生发展中的作用。     

Anti-pig antibody adsorption efficacy of {alpha}-Gal carrying recombinant P-selectin glycoprotein ligand-1/immunoglobulin chimeras increases with core 2 {beta}1, 6-N-acetylglucosaminyltransferase expression

We have previously described the construction of a P-selectin glycoprotein ligand-1-mouse immunoglobulin Fc fusion protein, which when transiently coexpressed with the porcine alpha1,3 galactosyltransferase in COS cells becomes a very efficient adsorber of xenoreactive, anti-pig antibodies. To relate the adsorption capacity with the glycan expression of individual fusion proteins produced in different cell lines, stable CHO-K1, COS, and 293T cells producing this fusion protein have been engineered. On alpha1,3 galactosyltransferase coexpression, high-affinity adsorbers were produced by both COS and 293T cells, whereas an adsorber of lower affinity was derived from CHO-K1 cells. Stable coexpression of a core 2 beta1,6 N-acetylglucosaminyltransferase in CHO-K1 cells led to increased alpha-Gal epitope density and improved anti-pig antibody adsorption efficacy. ESI-MS/MS of O-glycans released from PSGL-1/mIgG(2b) produced in an alpha1,3 galactosyl- and core 2 beta1,6 N-acetylglucosaminyltransferase expressing CHO-K1 cell clone revealed a number of structures with carbohydrate sequences consistent with terminal Gal-Gal. In contrast, no O-glycan structures with terminal Gal-Gal were identified on the fusion protein when expressed alone or in combination with the alpha1,3 galactosyltransferase in CHO-K1 cells. In conclusion, the density of alpha-Gal epitopes on PSGL-1/mIgG(2b) was dependent on the expression of O-linked glycans with core 2 structures and lactosamine extensions. The structural complexity of the terminal Gal-Gal expressing O-glycans with both neutral as well as sialic acid-containing structures is likely to contribute to the high adsorption efficacy.     

Abnormal glycosylation with hypersialylated O-glycans in patients with Sialuria

Sialuria is an inborn error of metabolism characterized by coarse face, hepatomegaly and recurrent respiratory tract infections. The genetic defect in this disorder results in a loss of feedback control of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine-kinase by CMP-N-acetylneuraminic acid (CMP-NeuAc) resulting in a substantial overproduction of cytoplasmic free sialic acid. This study addresses fibroblast CMP-NeuAc levels and N- and O-glycan sialylation of serum proteins from Sialuria patients. CMP-NeuAc levels were measured with HPLC in fibroblasts. Isoelectric focusing (IEF) of serum transferrin and of apolipoprotein C-III (apoC-III) was performed on serum of three Sialuria patients. Isoforms of these proteins can be used as specific markers for the biosynthesis of N- and core 1 O-glycans. Furthermore, total N- and O-linked glycans from serum proteins were analyzed by HPLC. HPLC showed a clear overproduction of CMP-NeuAc in fibroblasts of a Sialuria patient. Minor changes were found for serum N-glycans and hypersialylation was found for core 1 O-glycans on serum apoC-III and on total serum O-glycans in Sialuria patients. HPLC showed an increased ratio of disialylated over monosialylated core 1 O-glycans. The hypersialylation of core 1 O-glycans is due to the increase of NeuAcalpha2,6-containing structures (mainly NeuAcalpha2-3Galbeta1-3[NeuAcalpha2-6]GalNAc). This may relate to KM differences between GalNAc-alpha2,6-sialyltransferase and alpha2,3-sialyltransferases. This is the first study demonstrating that the genetic defect in Sialuria results in a CMP-NeuAc overproduction. Subsequently, increased amounts of alpha2,6-linked NeuAc were found on serum core 1 O-glycans from Sialuria patients. N-glycosylation of serum proteins seems largely unaffected. Sialuria is the first metabolic disorder presenting with hypersialylated O-glycans.     

Location, location, location: new insights into O-GalNAc protein glycosylation

O-GalNAc glycosylation of proteins confers essential structural, protective and signaling roles in eumetazoans. Addition of O-glycans onto proteins is an extremely complex process that regulates both sites of attachment and the types of oligosaccharides added. Twenty distinct polypeptide GalNAc-transferases (GalNAc-Ts) initiate O-glycosylation and fine-tuning their expression provides a mechanism for regulating this action. Recently, a new mode of regulation has emerged where activation of Src kinase selectively redistributes Golgi-localized GalNAc-Ts to the ER. This relocalization results in a strong increase in the density of O-glycan decoration. In this review, we discuss how different mechanisms can regulate the number and the types of O-glycans decorating proteins. In addition, we speculate how Src-dependent relocation of GalNAc-Ts could play an important role in cancerous cellular transformation.     

A novel strategy for evasion of NK cell immunity by tumours expressing core2 O-glycans

The O-glycan branching enzyme, core2 β-1,6-N-acetylglucosaminyltransferase (C2GnT), forms O-glycans containing an N-acetylglucosamine branch connected to N-acetylgalactosamine (core2 O-glycans) on cell-surface glycoproteins. Here, we report that upregulation of C2GnT is closely correlated with progression of bladder tumours and that C2GnT-expressing bladder tumours use a novel strategy to increase their metastatic potential. Our results showed that C2GnT-expressing bladder tumour cells are highly metastatic due to their high ability to evade NK cell immunity and revealed the molecular mechanism of the immune evasion by C2GnT expression. Engagement of an NK-activating receptor, NKG2D, by its tumour-associated ligand, Major histocompatibility complex class I-related chain A (MICA), is critical to tumour rejection by NK cells. In C2GnT-expressing bladder tumour cells, poly-N-acetyllactosamine was present on core2 O-glycans on MICA, and galectin-3 bound the NKG2D-binding site of MICA through this poly-N-acetyllactosamine. Galectin-3 reduced the affinity of MICA for NKG2D, thereby severely impairing NK cell activation and silencing the NK cells. This new mode of NK cell silencing promotes immune evasion of C2GnT-expressing bladder tumour cells, resulting in tumour metastasis.