O-linked Glycan Structures Research Articles

O-glycosylation contributes to mammalian glycoRNA biogenesis.

There is an increasing appreciation for the role of cell surface glycans in modulating interactions with extracellular ligands and participating in intercellular communication. We recently reported the existence of sialoglycoRNAs, where mammalian small RNAs are covalently linked to N-glycans through the modified base acp3U and trafficked to the cell surface. However, little is currently known about the role for O-glycosylation, another major class of carbohydrate polymer modifications. Here, we use parallel genetic, enzymatic, and mass spectrometry approaches to demonstrate that O-linked glycan biosynthesis is responsible for the majority of sialoglycoRNA levels. By examining the O-glycans associated with RNA from cell lines and colon organoids we find known and previously unreported O-linked glycan structures. Further, we find that O-linked glycans released from small RNA from organoids derived from ulcerative colitis patients exhibit higher levels of sialylation than glycans from healthy organoids. Together, our work provides flexible tools to interrogate O-linked glycoRNAs (O-glycoRNA) and suggests that they may be modulated in human disease.

C1GalT1 expression reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression

Although most cell membrane proteins are modified by glycosylation, our understanding of the role and actions of protein glycosylation is still very limited. β1,3galactosyltransferase (C1GalT1) is a key glycosyltransferase that controls the biosynthesis of the Core 1 structure of O-linked mucin type glycans and is overexpressed by many common types of epithelial cancers. This study reports that suppression of C1GalT1 expression in human colon cancer cells caused substantial changes of protein glycosylation of cell membrane proteins, many of which were ligands of the galactoside-binding galectin-3 and the macrophage galactose-type lectin (MGL). This led to significant reduction of cancer cell proliferation, adhesion, migration and the ability of tumour cells to form colonies. Crucially, C1GalT1 suppression significantly reduced galectin-3-mediated tumour cell-cell interaction and galectin-3-promoted tumour cell activities. In the meantime, C1GalT1 suppression substantially increased MGL-mediated macrophage-tumour cell interaction and macrophage-tumour cell phagocytosis and cytokine secretion. C1GalT1-expressing cancer cells implanted in chick embryos resulted in the formation of significantly bigger tumours than C1GalT1-suppressed cells and the presence of galectin-3 increased tumour growth of C1GalT1-expressing but not C1GalT1-suppressed cells. More MGL-expressing macrophages and dendritic cells were seen to be attracted to the tumour microenvironment in ME C1galt1-/-/Erb mice than in C1galt1f/f /Erb mice. These results indicate that expression of C1GalT1 by tumour cells reciprocally controls tumour cell-cell and tumour-macrophage interactions mediated by galectin-3 and MGL with double impact on cancer development and progression. C1GalT1 overexpression in epithelial cancers therefore may represent a fundamental mechanism in cancer promotion and in reduction of immune response/surveillance in cancer progression.

Structure and Role of O-Linked Glycans in Viral Envelope Proteins.

N- and O-glycans are both important constituents of viral envelope glycoproteins. O-linked glycosylation can be initiated by any of 20 different human polypeptide O-acetylgalactosaminyl transferases, resulting in an important functional O-glycan heterogeneity. O-glycans are organized as solitary glycans or in clusters of multiple glycans forming mucin-like domains. They are functional both in the viral life cycle and in viral colonization of their host. Negatively charged O-glycans are crucial for the interactions between glycosaminoglycan-binding viruses and their host. A novel mechanism, based on controlled electrostatic repulsion, explains how such viruses solve the conflict between optimized viral attachment to target cells and efficient egress of progeny virus. Conserved solitary O-glycans appear important for viral uptake in target cells by contributing to viral envelope fusion. Dual roles of viral O-glycans in the host B cell immune response, either epitope blocking or epitope promoting, may be exploitable for vaccine development. Finally, specific virus-induced O-glycans may be involved in viremic spread.

Suppression of O-Linked Glycosylation of the SARS-CoV-2 Spike by Quaternary Structural Restraints.

Understanding the glycosylation of the envelope spike (S) protein of SARS-CoV-2 is important in defining the antigenic surface of this key viral target. However, the underlying protein architecture may significantly influence glycan occupancy and processing. There is, therefore, potential for different recombinant fragments of S protein to display divergent glycosylation. Here, we show that the receptor binding domain (RBD), when expressed as a monomer, exhibits O-linked glycosylation, which is not recapitulated in the native-like soluble trimeric protein. We unambiguously assign O-linked glycosylation by homogenizing N-linked glycosylation using the enzymatic inhibitor, kifunensine, and then analyzing the resulting structures by electron-transfer higher-energy collision dissociation (EThcD) in an Orbitrap Eclipse Tribrid instrument. In the native-like trimer, we observe a single unambiguous O-linked glycan at T323, which displays very low occupancy. In contrast, several sites of O-linked glycosylation can be identified when RBD is expressed as a monomer, with T323 being almost completely occupied. We ascribe this effect to the relaxation of steric restraints arising from quaternary protein architecture. Our analytical approach has also highlighted that fragmentation ions arising from trace levels of truncated N-linked glycans can be misassigned as proximal putative O-linked glycan structures, particularly where a paucity of diagnostic fragments were obtained. Overall, we show that in matched expression systems the quaternary protein architecture limits O-linked glycosylation of the spike protein.

The Biological Significance of von Willebrand Factor O-Linked Glycosylation.

Glycosylation is a key posttranslational modification, known to occur on more than half of all secreted proteins in man. As such, the role of N- and O-linked glycan structures in modulating various aspects of protein biology is an area of much research. Given their prevalence, it is perhaps unsurprising that variations in glycan structures have been demonstrated to play critical roles in modulating protein function and have been implicated in the pathophysiology of human diseases. von Willebrand factor (VWF), a plasma glycoprotein that is essential for normal hemostasis, is heavily glycosylated, containing 13 N-linked and 10 O-linked glycans. Together, these carbohydrate chains account for 20% of VWF monomeric mass, and have been shown to modulate VWF structure, function, and half-life. In this review, we focus on the specific role played by O-linked glycans in modulating VWF biology. Specifically, VWF O-linked glycans have been shown to modulate tertiary protein structure, susceptibility to ADAMTS13 proteolysis, platelet tethering, and VWF circulatory half-life.

High expression of tissue O-linked glycans is associated with a malignant phenotype of cholangiocarcinoma.

ObjectiveThis study aimed to investigate the expression of O-linked glycoprotein glycans in tissue of patients with cholangiocarcinoma compared with adjacent normal tissue.MethodsSixty patients with cholangiocarcinoma were included in the study. Permethylated O-linked glycans from intrahepatic cholangiocarcinoma tissue and adjacent normal tissue were analyzed using nano-spray ionization-linear ion trap mass spectrometry. Histochemistry of peanut agglutinin lectin was used for detection and localization of galactose (Gal) 1, N-acetyl-galactosamine (GalNAc) 1.ResultsO-linked glycans from patients with cholangiocarcinoma were composed of di- to hexa-saccharides with a terminal galactose and sialic acids (N-acetylneuraminic acid [NeuAc]). A total of eight O-linked glycan structures were detected. Gal1GalNAc1 and Gal2 N-acetyl-glucosamine 1 GalNAc1 expression was significantly higher in tissue from patients with cholangiocarcinoma compared with adjacent normal tissue, while NeuAc1Gal1GalNAc1 expression was significantly lower. High Gal1GalNAc1 expression was significantly associated with the late stage of cholangiocarcinoma (stages II–IV), lymphatic invasion, and vascular invasion.ConclusionOur study shows expression of O-linked glycans in progression of cholangiocarcinoma and highlights the association of Gal1GalNAc1 with lymphatic and vascular invasion of cholangiocarcinoma.

Beta1-Adrenergic Receptor Regulation Revisited.

The actions and regulation of cardiomyocyte β1ARs differ in several respects from the properties described for the prototypical β2AR subtype; a mechanism to explain the unique properties of the β1AR subtype has never been obvious. This viewpoint summarizes recent studies that identify a novel signaling paradigm for the β1AR, implicating the N-terminus as a molecular determinant of β1AR responsiveness.

Tool for Rapid Analysis of Glycopeptide by Permethylation via One-Pot Site Mapping and Glycan Analysis.

To overcome the challenges in the analysis of protein glycosylation, we have developed a comprehensive and universal tool through permethylation of glycopeptides and their tandem mass spectrometric analysis. This method has the potential to simplify glycoprotein analysis by integrating glycan sequencing and glycopeptide analysis in a single experiment. Moreover, glycans with unique glycosidic linkages, particularly from prokaryotes, which are resistant to enzymatic or chemical release, could also be detected and analyzed by this methodology. Here we present a strategy for the permethylation of intact glycopeptides, obtained via controlled protease digest, and their characterization by using advanced mass spectrometry. We used bovine RNase B, human transferrin, and bovine fetuin as models to demonstrate the feasibility of the method. Remarkably, the glycan patterns, glycosylation site, and their occupancy by N-glycans are all detected and identified in a single experimental procedure. Acquisition on a high resolution tandem-MSn system with fragmentation methodologies such as high-energy collision dissociation (HCD) and collision induced dissociation (CID), provided the complete sequence of the glycan structures attached to the peptides. The behavior of 20 natural amino acids under the basic permethylation conditions was probed by permethylating a library of short synthetic peptides. Our studies indicate that the permethylation imparts simple, limited, and predictable chemical transformations on peptides and do not interfere with the interpretation of MS/MS data. In addition to this, permethylated O-glycans in unreduced form (released by β elimination) were also detected, allowing us to profile O-linked glycan structures simultaneously.

Proteomics Analysis of O-GalNAc Glycosylation in Human Serum by an Integrated Strategy.

The diversity of O-linked glycan structures has drawn increasing attention due to its vital biological roles. However, intact O-glycopeptides with different glycans are typically not well elucidated using the current methods. In this work, an integrated strategy was developed for comprehensive analysis of O-GalNAc glycosylation by combining hydrophilic interaction chromatography (HILIC) tip enrichment, beam-type collision induced decomposition (beam-CID) detection, and in silico deglycosylation method for spectra interpretation. In this strategy, the intact O-GalNAc glycopeptides were selectively enriched and the original spectra obtained by time-of-flight (TOF)-CID were preprocessed using an in silico deglycosylation method, enabling direct searching without setting multiple glycosylation modifications, which could significantly decrease the search space. This strategy was applied to analyze the O-GalNAc glycoproteome of human serum, leading to identification of 407 intact O-GalNAc glycopeptides from 93 glycoproteins. About 81% of the glycopeptides contained at least one sialic acid, which could reveal the microheterogeneity of O-GalNAc glycosylation. Up until now, this is the largest data set of intact O-GalNAc glycoforms from complex biological samples at the proteome level. Furthermore, this method is readily applicable to study O-glycoform heterogeneity in other complex biological systems.

Recombinant therapeutic proteins produced in plants: towards engineering of human-type O-and N-glycosylation

Background and Purpose : Recombinant DNA technology has allowed expression of different heterologous proteins in many host systems, ranging from prokaryotic to eukaryotic organisms. Therapeutic properties of recombinant proteins are very often affected by the composition and heterogeneity of their glycans. Conventional expression systems for recombinant pharmaceuticals typically do not address this problem and result with products that contain a mixture of glycoforms that are neither identical to human glycans nor optimized for enhanced biological activity. Over the last decade plants have been developed as production platforms for recombinant proteins with pharmaceutical or industrial applications. Namely, plant expression systems contain very small differences in the post-translational modifications, mainly glycosylation, which can partly be overpowered by glycoengineering, whose goal is production of recombinant proteins with highly homogenous glycosylation that closely resembles the native system. This review attempts to present current accomplishments in the production of plant-derived glycoconjugates with humanized N- and O-glycans. Materials and Methods: Main goal of N-glycoengineering is to reduce or eliminate plant-specific N-glycans, and at the same time to introduce mammalian-specific N-glycans through the several approaches. The easiest way is to change intracellular targeting of plant-made recombinant proteins and to ensure their retention in the ER; next approach is to eliminate the addition of plant-specific glycans; while the final step is engineering the plant glycosylation pathway to introduce mammalian glycotransferases into plants with generation of biantennary and multi-antennary structures on complex N-glycans. Due to significant differences in O-glycosylation between humans and plants, different approaches to engineering of O-glycosylation have been taken. Besides having their typical O-glycoslyation on Hyp-residues, plants in general miss the machinery for production of mammalian-type O-glycosylation. Attempts have been made to mimic mammalian O-glycosylation in plants, specifically the mucin-type addition of GalNAc residues. Result: Efficient generation of bisected tetraantennary complex N-glycans without typical plant glycoepitopes on human erythropoietin (hEPO) and human transferrin (hTF) was obtained in Nicotiana benthamiana plants, thus demonstrating generation of recombinant proteins with human-type N-glycosylation at great uniformity. As for the O-glycosylation, attempts to produce mucin-type O-GalNAc and disialylated core 1 O-linked glycan structures on hEPO in N. benthamiana transgenic plants proved to be successful. Moreover, although small amounts of Hyp residues were found on recombinant EPO, no plant-specific O-glycans were detected, which demonstrates that plants are eligible candidates for production of recombinant therapeutics with fully humanized O- and N-glycans. Conclusion : Plants and methods of plant molecular farming offer a powerful expression platform for the production of a variety of recombinant proteins, which show similar, or even higher, biological activity then protein or native homologs in cultured mammalian cells currently used for large-scale production.

Unraveling the Novel Structure and Biosynthetic Pathway of O-Linked Glycans in the Golgi Apparatus of the Human Pathogenic Yeast Cryptococcus neoformans

Cryptococcus neoformans is an encapsulated basidiomycete causing cryptococcosis in immunocompromised humans. The cell surface mannoproteins of C. neoformans were reported to stimulate the host T-cell response and to be involved in fungal pathogenicity; however, their O-glycan structure is uncharacterized. In this study, we performed a detailed structural analysis of the O-glycans attached to cryptococcal mannoproteins using HPLC combined with exoglycosidase treatment and showed that the major C. neoformans O-glycans were short manno-oligosaccharides that were connected mostly by α1,2-linkages but connected by an α1,6-linkage at the third mannose residue. Comparison of the O-glycan profiles from wild-type and uxs1Δ mutant strains strongly supports the presence of minor O-glycans carrying a xylose residue. Further analyses of C. neoformans mutant strains identified three mannosyltransferase genes involved in O-glycan extensions in the Golgi. C. neoformans KTR3, the only homolog of the Saccharomyces cerevisiae KRE2/MNT1 family genes, was shown to encode an α1,2-mannosyltransferase responsible for the addition of the second mannose residue via an α1,2-linkage to the major O-glycans. C. neoformans HOC1 and HOC3, homologs of the Saccharomyces cerevisiae OCH1 family genes, were shown to encode α1,6-mannosyltransferases that can transfer the third mannose residue, via an α1,6-linkage, to minor O-glycans containing xylose and to major O-glycans without xylose, respectively. Moreover, the C. neoformans ktr3Δ mutant strain, which displayed increased sensitivity to SDS, high salt, and high temperature, showed attenuated virulence in a mouse model of cryptococcosis, suggesting that the extended structure of O-glycans is required for cell integrity and full pathogenicity of C. neoformans.

In vitro enzymatic treatment to remove O-linked mannose from intact glycoproteins

The methylotrophic yeast Pichia pastoris is an attractive expression system for heterologous protein production due to its ability to perform posttranslational modifications, such as glycosylation, and secrete large amounts of recombinant protein. However, the structures of N- and O-linked oligosaccharide chains in yeast differ significantly from those of mammalian cells. The most common O-linked glycan structures added by P. pastoris are typically polymers of between one and four α-linked mannose residues, with a subset of glycans being capped by a β-1,2-mannose disaccharide or phosphomannose residue. Such mannosylation of recombinant proteins is considered a key factor in immunomodulation, with mannose-specific receptors binding and promoting enhanced immune responses. As a result of engineering the N-linked glycosylation pathway of P. pastoris, the recombinant proteins expressed in this system are devoid of phospho- and β-mannose on O-linked glycans, leaving only α-mannose polymers. Here we screen a library of α-mannosidases for their ability to decrease the extent of O-mannosylation on glycoproteins secreted from this expression system. In doing so, we demonstrate the utility of the α-1,2/3/6-mannosidase from Jack bean in not only reducing extended O-linked mannose chains but also in specifically hydrolyzing the Man-α-O-Ser/Thr glycosidic bond on intact glycoproteins. As such, this presents for the first time a strategy to remove O-linked glycosylation from intact glycoproteins expressed in P. pastoris. We additionally show that this strategy can be used to significantly decrease the extent of O-mannosylation on commercial products produced in other similar expression systems.

Determination of glycan motifs using serial lectin affinity chromatography.

Serial lectin affinity chromatography is a convenient technique for characterizing glycan motifs (terminal glycan structures) of glycoproteins or released glycans. When these glycoconjugates are applied serially or in parallel to lectin-immobilized columns, information regarding the glycan motifs can be obtained. We demonstrate lectin affinity chromatographic methods for determining O-linked glycan structures of MUC1 purified from a breast cancer cell line, YMB-S, N-linked glycan structures of serum prostate-specific antigen from prostate cancer, and serum alkaline phosphatases from choriocarcinoma. These lectin-fractionated samples are analyzed quantitatively by measuring radioactivity, antigen contents are analyzed using enzyme-linked immunosorbent assay, and enzymatic activities are assessed.

Engineering of Sialylated Mucin-type O-Glycosylation in Plants

Proper N- and O-glycosylation of recombinant proteins is important for their biological function. Although the N-glycan processing pathway of different expression hosts has been successfully modified in the past, comparatively little attention has been paid to the generation of customized O-linked glycans. Plants are attractive hosts for engineering of O-glycosylation steps, as they contain no endogenous glycosyltransferases that perform mammalian-type Ser/Thr glycosylation and could interfere with the production of defined O-glycans. Here, we produced mucin-type O-GalNAc and core 1 O-linked glycan structures on recombinant human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) by transient expression in Nicotiana benthamiana plants. Furthermore, for the generation of sialylated core 1 structures constructs encoding human polypeptide:N-acetylgalactosaminyltransferase 2, Drosophila melanogaster core 1 β1,3-galactosyltransferase, human α2,3-sialyltransferase, and Mus musculus α2,6-sialyltransferase were transiently co-expressed in N. benthamiana together with EPO-Fc and the machinery for sialylation of N-glycans. The formation of significant amounts of mono- and disialylated O-linked glycans was confirmed by liquid chromatography-electrospray ionization-mass spectrometry. Analysis of the three EPO glycopeptides carrying N-glycans revealed the presence of biantennary structures with terminal sialic acid residues. Our data demonstrate that N. benthamiana plants are amenable to engineering of the O-glycosylation pathway and can produce well defined human-type O- and N-linked glycans on recombinant therapeutics.

Binding of DC-SIGN to glycoproteins expressed in glycoengineered Pichia pastoris

Previous studies have shown that glycoproteins expressed in wild-type Pichia pastoris bind to Dendritic cell-SIGN (DC-Specific Intercellular adhesion molecule-3 Grabbing Nonintegrin), a mannose-binding receptor found on dendritic cells in peripheral tissues which is involved in antigen presentation and the initiation of an immune response. However, the binding of DC-SIGN to glycoproteins purified from P. pastoris strains engineered to express humanized N- and O-linked glycans has not been tested to date. In this study, the binding of glycoproteins with specific high-mannose or human N- and O-linked glycan structures to DC-SIGN was tested. Proteins with humanized N-glycans including Man5 structures and O-glycans (up to as many as 24) with single mannose chain length showed DC-SIGN binding that was comparable to that measured for a CHO-produced IgG1 which lacks O-linked mannose. Glycoproteins with wild-type N-glycans and mannotriose and higher O-glycans bound to DC-SIGN in a manner that was strongly inhibited by either the use of enzymatic N-deglycosylation or sodium meta-periodate oxidation. Mannan purified from humanized P. pastoris also showed lower ability to inhibit DC-SIGN binding to glycoproteins with wild type fungal glycosylation than mannan purified from wild type strains. This study shows that humanized P. pastoris can produce glycoproteins that do not bind to DC-SIGN.

O-linked glycosylation of von Willebrand factor modulates the interaction with platelet receptor glycoprotein Ib under static and shear stress conditions

AbstractWe have examined the effect of the O-linked glycan (OLG) structures of VWF on its interaction with the platelet receptor glycoprotein Ibα. The 10 OLGs were mutated individually and as clusters (Clus) on either and both sides of the A1 domain: Clus1 (N-terminal side), Clus2 (C-terminal side), and double cluster (DC), in both full-length-VWF and in a VWF construct spanning D′ to A3 domains. Mutations did not alter VWF secretion by HEK293T cells, multimeric structure, or static collagen binding. The T1255A, Clus1, and DC variants caused increased ristocetin-mediated GPIbα binding to VWF. Platelet translocation rate on OLG mutants was increased because of reduced numbers of GPIbα binding sites but without effect on bond lifetime. In contrast, OLG mutants mediated increased platelet capture on collagen under high shear stress that was associated with increased adhesion of these variants to the collagen under flow. These findings suggest that removal of OLGs increases the flexibility of the hinge linker region between the D3 and A1 domain, facilitating VWF unfolding by shear stress, thereby enhancing its ability to bind collagen and capture platelets. These data demonstrate an important functional role of VWF OLGs under shear stress conditions.

Structural Basis for Distinct Binding Properties of the Human Galectins to Thomsen-Friedenreich Antigen

The Thomsen-Friedenreich (TF or T) antigen, Galβ1-3GalNAcα1-O-Ser/Thr, is the core 1 structure of O-linked mucin type glycans appearing in tumor-associated glycosylation. The TF antigen occurs in about 90% of human cancer cells and is a potential ligand for the human endogenous galectins. It has been reported that human galectin-1 (Gal-1) and galectin-3 (Gal-3) can perform their cancer-related functions via specifically recognizing TF antigen. However, the detailed binding properties have not been clarified and structurally characterized. In this work, first we identified the distinct TF-binding abilities of Gal-1 and Gal-3. The affinity to TF antigen for Gal-3 is two orders of magnitude higher than that for Gal-1. The structures of Gal-3 carbohydrate recognition domain (CRD) complexed with TF antigen and derivatives, TFN and GM1, were then determined. These structures show a unique Glu-water-Arg-water motif-based mode as previously observed in the mushroom galectin AAL. The observation demonstrates that this recognition mode is commonly adopted by TF-binding galectins, either as endogenous or exogenous ones. The detailed structural comparisons between Gal-1 and Gal-3 CRD and mutagenesis experiments reveal that a pentad residue motif (51AHGDA55) at the loop (g1-L4) connecting β-strands 4 and 5 of Gal-1 produces a serious steric hindrance for TF binding. This motif is the main structural basis for Gal-1 with the low affinity to TF antigen. These findings provide the intrinsic structural elements for regulating the TF-binding activity of Gal-1 in some special conditions and also show certain target and approach for mediating some tumor-related bioactivities of human galectins.

Novel O-linked glycans containing 6'-sulfo-Gal/GalNAc of MUC1 secreted from human breast cancer YMB-S cells: Possible carbohydrate epitopes of KL-6(MUC1) monoclonal antibody

Human serum Krebs von den Lugen-6 (KL-6) antigen is a MUC1 glycoprotein (KL-6/MUC1) recognized by anti-KL-6 monoclonal antibody (KL-6/mAb) and has been utilized as a diagnostic marker for interstitial pneumonia. KL-6/mAb is thought to recognize the specific glycopeptides sequence of MUC1, but the precise glycan structure of the epitope is unclear. In this study, we determined the carbohydrate structures of KL-6/MUC1 to search the carbohydrate epitopes for KL-6/mAb. KL-6/MUC1 was purified from the culture medium of human breast cancer YMB-S cells by KL-6/mAb-affinity chromatography; the O-linked glycan structures were determined in combination with paper electrophoresis, several lectin column chromatographies, sialidase digestion and methanolysis. KL-6/MUC1 contained core 1 and extended core 1 glycans modified with one or two sialic acid/sulfate residues. Based on these structures, several synthetic glycans binding to anti-KL-6/mAb were compared with one another by surface plasmon resonance. Sequentially, related radiolabeled oligosaccharides were enzymatically synthesized and analyzed for binding to a KL-6/mAb-conjugated affinity column. 3'-sialylated, 6'-sulfated LNnT [Neu5Acα2-3(SO(3)(-)-6)Galβ1-4GlcNAcβ1-3Galβ1-4Glc], 3'-sialylated, 6-sulfated core 1 [Neu5Acα2-3Galβ1-3(SO(3)(-)-6)GalNAc] and disulfated core 1 SO(3)(-)-3Galβ1-3(SO(3)(-)-6)GalNAc exhibited substantial affinity for KL-6/mAb, and 3'-sulfated core 1 derivatives [SO(3)(-)-3Galβ1-3(±Neu5Acα2-6)GalNAc] and 3'-sialylated core 1 weakly interacted with KL-6/mAb. These results indicated that the possible carbohydrate epitopes of KL-6/mAb involve not only 3'-sialylated core 1 but also novel core 1 and extended core 1 with sulfate and sialic acid residues. Epitope expressing changes with suppression or over-expression of the Gal6ST (Gal 6-O-sulfotransferase) gene, suggesting that Gal6ST is involved in the biosynthesis of the unique epitopes of KL-6/mAb.

Glycomic Analyses of Mouse Models of Congenital Muscular Dystrophy

Dystroglycanopathies are a subset of congenital muscular dystrophies wherein α-dystroglycan (α-DG) is hypoglycosylated. α-DG is an extensively O-glycosylated extracellular matrix-binding protein and a key component of the dystrophin-glycoprotein complex. Previous studies have shown α-DG to be post-translationally modified by both O-GalNAc- and O-mannose-initiated glycan structures. Mutations in defined or putative glycosyltransferase genes involved in O-mannosylation are associated with a loss of ligand-binding activity of α-DG and are causal for various forms of congenital muscular dystrophy. In this study, we sought to perform glycomic analysis on brain O-linked glycan structures released from proteins of three different knock-out mouse models associated with O-mannosylation (POMGnT1, LARGE (Myd), and DAG1(-/-)). Using mass spectrometry approaches, we were able to identify nine O-mannose-initiated and 25 O-GalNAc-initiated glycan structures in wild-type littermate control mouse brains. Through our analysis, we were able to confirm that POMGnT1 is essential for the extension of all observed O-mannose glycan structures with β1,2-linked GlcNAc. Loss of LARGE expression in the Myd mouse had no observable effect on the O-mannose-initiated glycan structures characterized here. Interestingly, we also determined that similar amounts of O-mannose-initiated glycan structures are present on brain proteins from α-DG-lacking mice (DAG1) compared with wild-type mice, indicating that there must be additional proteins that are O-mannosylated in the mammalian brain. Our findings illustrate that classical β1,2-elongation and β1,6-GlcNAc branching of O-mannose glycan structures are dependent upon the POMGnT1 enzyme and that O-mannosylation is not limited solely to α-DG in the brain.

Autoantibodies to aberrantly glycosylated MUC1 in early stage breast cancer are associated with a better prognosis

IntroductionDetection of serum biomarkers for early diagnosis of breast cancer remains an important goal. Changes in the structure of O-linked glycans occur in all breast cancers resulting in the expression of glycoproteins that are antigenically distinct. Indeed, the serum assay widely used for monitoring disease progression in breast cancer (CA15.3), detects a glycoprotein (MUC1), but elevated levels of the antigen cannot be detected in early stage patients. However, since the immune system acts to amplify the antigenic signal, antibodies can be detected in sera long before the antigen. We have exploited the change in O-glycosylation to measure autoantibody responses to cancer-associated glycoforms of MUC1 in sera from early stage breast cancer patients.MethodsWe used a microarray platform of 60mer MUC1 glycopeptides, to confirm the presence of autoantibodies to cancer associated glycoforms of MUC1 in a proportion of early breast cancer patients (54/198). Five positive sera were selected for detailed definition of the reactive epitopes using on chip glycosylation technology and a panel of glycopeptides based on a single MUC1 tandem repeat carrying specific glycans at specific sites. Based on these results, larger amounts of an extended repertoire of defined MUC1 glycopeptides were synthesised, printed on microarrays, and screened with sera from a large cohort of breast cancer patients (n = 395), patients with benign breast disease (n = 108) and healthy controls (n = 99). All sera were collected in the 1970s and 1980s and complete clinical follow-up of breast cancer patients is available.ResultsThe presence and level of autoantibodies was significantly higher in the sera from cancer patients compared with the controls, and a highly significant correlation with age was observed. High levels of a subset of autoantibodies to the core3MUC1 (GlcNAcβ1-3GalNAc-MUC1) and STnMUC1 (NeuAcα2,6GalNAc-MUC1) glycoforms were significantly associated with reduced incidence and increased time to metastasis.ConclusionsAutoantibodies to specific cancer associated glycoforms of MUC1 are found more frequently and at higher levels in early stage breast cancer patients than in women with benign breast disease or healthy women. Association of strong antibody response with reduced rate and delay in metastases suggests that autoantibodies can affect disease progression.