Glycan Core Research Articles

Novel structural features of the immunocompetent ceramide phospho-inositol glycan core from Trichomonas vaginalis

The ceramide phosphoinositol glycan core (CPI-GC) of the lipophosphoglycan of Trichomonas vaginalis is a major virulent factor of this common genitourinary parasite. While its carbohydrate composition has been reported before, its structure has remained largely unknown. We isolated the glycan portions of CPI-GC by nitrous acid deamination and hydrofluoric acid treatment and investigated their structures by methylation analysis and 1- and 2-D NMR. We found that the α-anomer of galactose is a major constituent of CPI-GC. The β-anomer was found exclusively at the non-reducing end of CPI-GC side chains. Furthermore the data showed that the rhamnan backbone is more complex than previously thought and that the inositol residue at the reducing end is linked to a 4-linked α-glucuronic acid (GlcA) residue. This appears to be the most striking and novel feature of this GPI-anchor type molecule.

Association of systemic lupus erythematosus with decreased immunosuppressive potential of the IgG glycome.

ObjectiveGlycans attached to the Fc portion of IgG are important modulators of IgG effector functions. Interindividual differences in IgG glycome composition are large and they associate strongly with different inflammatory and autoimmune diseases. IKZF1, HLA–DQ2A/B, and BACH2 genetic loci that affect IgG glycome composition show pleiotropy with systemic lupus erythematosus (SLE), indicating a potentially causative role of aberrant IgG glycosylation in SLE. We undertook this large multicenter case–control study to determine whether SLE is associated with altered IgG glycosylation.MethodsUsing ultra‐performance liquid chromatography analysis of released glycans, we analyzed the composition of the IgG glycome in 261 SLE patients and 247 matched controls of Latin American Mestizo origin (the discovery cohort) and in 2 independent replication cohorts of different ethnicity (108 SLE patients and 193 controls from Trinidad, and 106 SLE patients and 105 controls from China).ResultsMultiple statistically significant differences in IgG glycome composition were observed between patients and controls. The most significant changes included decreased galactosylation and sialylation of IgG (which regulate proinflammatory and antiinflammatory actions of IgG) as well as decreased core fucose and increased bisecting N‐acetylglucosamine (which affect antibody‐dependent cell‐mediated cytotoxicity).ConclusionThe IgG glycome in SLE patients is significantly altered in a way that decreases immunosuppressive action of circulating immunoglobulins. The magnitude of observed changes is associated with the intensity of the disease, indicating that aberrant IgG glycome composition or changes in IgG glycosylation may be an important molecular mechanism in SLE.

Preferred conformations ofN-glycan core pentasaccharide in solution and in glycoproteins

N-linked glycans are on protein surfaces and have direct and water/ion-mediated interactions with surrounding amino acids. Such contacts could restrict their conformational freedom compared to the same glycans free in solution. In this work, we have examined the conformational freedom of the N-glycan core pentasaccharide moiety in solution using standard molecular dynamics (MD) simulations as well as temperature replica-exchange MD simulations. Both simulations yield the comparable conformational variability of the pentasaccharide in solution, indicating the convergence of both simulations. The glycoprotein crystal structures are analyzed to compare the conformational freedom of the N-glycan on the protein surface with the simulation result. Surprisingly, the pentasaccharide free in solution shows more restricted conformational variability than the N-glycan on the protein surface. The interactions between the carbohydrate and the protein side chain appear to be responsible for the increased conformational diversity of the N-glycan on the protein surface. Finally, the transfer entropy analysis of the simulation trajectory also reveals an unexpected causality relationship between intramolecular hydrogen bonds and the conformational states in that the hydrogen bonds play a role in maintaining the conformational states rather than driving the change in glycosidic torsional states.

Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation

Recent progress in snake venomics has shed much light on the intra-species variation among the toxins from different geographical regions and has provided important information for better snakebite management. Most previous reports on snake venomics were based on venoms pooled from different snakes. In this study, we present the proteomic and glycomic profiles of venoms from individual Naja atra snakes. The results reveal wide dynamic range of three-finger toxins. Systematic classification based on cardiotoxin (CTX-) profiles of A2/A4 and A6, respectively, allowed the identification of two putative subspecies of Taiwan cobra from the eastern and western regions. We also identified four major N-glycan moieties on cobra snake venom metalloproteinase on the bi-antennary glycan core. ELISA showed that these glycoproteins (<3%) could elicit much higher antibody response in antiserum when compared to other high-abundance cobra venom toxins such as small molecular weight CTXs (~60%). By removing these high-molecular weight glycoproteins from the immunogen, we demonstrated better protection than that achieved with conventional crude venom immunization in mice challenged by crude venom. We conclude that both intra-species and inter-individual variations of proteomic and glycomic profiles of snake venomics should be considered to provide better antivenomic approach for snakebite management. Biological significanceBased on the proteomic and glycomic profiles of venoms obtained from individual snakes, we demonstrated a surprisingly wide dynamic range and geographical variation of three-finger toxins in cobra venomics. This provides a reasonable explanation for the variable neutralization effects of antivenom treatment on victims suffering from cobra snakebite and suggests a simple and economic method to produce potent antivenom with better efficacy. Since two major venomic profiles with distinct dynamic ranges were observed for Taiwan cobra venoms isolated from the eastern and western regions, the current venomic profile should be used as a quality control for future production of antivenom in clinical applications.

Glycomic Analysis of Life Stages of the Human Parasite Schistosoma mansoni Reveals Developmental Expression Profiles of Functional and Antigenic Glycan Motifs*[S

Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galβ1–4(Fucα1–3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcβ1–4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galβ1–3(Galβ1–6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated stretches enriched in mature eggs and miracidia. This global analysis of the developing schistosome's glycome provides new insights into how stage-specifically expressed glycans may contribute to different aspects of schistosome-host interactions.

Assignment of Core versus Antenna Fucosylation Types in Protein N-Glycosylation via Procainamide Labeling and Tandem Mass Spectrometry.

Fucosylation is an important feature of protein N-glycosylation as it has been reported to influence the efficacy of therapeutic proteins and as a potential disease biomarker. A common approach for characterizing protein N-glycans is to analyze the native glycans via tandem mass spectrometry (MS). However, tandem MS analysis of native N-glycans typically results in proton migration, which in turn leads to fucose residue migration from the glycan core to the antenna and vice versa. This phenomenon ultimately leads to ambiguous assignment of N-glycan fucosylation. Although the use of specific fucosidases has been successfully employed for assigning fucosylation, such strategies are often too cumbersome, expensive, and time-consuming for routine N-glycan analysis. As an alternative, we explore the influence of labeling N-glycans with procainamide hydrochloride to inhibit fucose migration during tandem MS analysis. The labeled N-glycan pool was separated and analyzed using ultraperformance liquid chromatography and a hydrophobic interaction liquid chromatography column coupled to a quadrupole time-of-flight mass spectrometer (UPLC-HILIC-QTOF-MS). The observation of the m/z 587.3 core fucose diagnostic peak corresponding to [GlcNAc + Fucose + Procainamide + H](+) in the tandem MS data of fucosylated N-glycans rapidly verifies core fucosylation while its absence signifies antennae fucosylation. This unique approach is here validated with human IgG (for core fucosylation) and human alpha-1-acid-glycoprotein (for antenna fucosylation). We further present a useful application toward the rapid verification of fucosylation types in a therapeutic protein (Rituximab).

Glycosylation and liver cancer.

Liver cancer is the fifth most common cancer, but the second leading cause of cancer death, in the world, with more than 700,000 fatalities annually. The major etiology of liver cancer is infection with an hepatotropic virus such as hepatitis B virus or hepatitis C virus infection. While chronic viral infection remains the main cause of liver disease and risk of hepatocellular carcinoma (HCC), rates of nonviral-associated HCC are occurring at an alarmingly increasing rate. Like many cancers, survival rates are closely associated with time of detection. If HCC is caught early, survival rates can be as high as 50%. Regrettably, most cases of HCC are caught late where survival rates can be as low as 2-7%. Thus, there has been great interest in discovering serum biomarkers that could be used to identify those with HCC. To this end, many groups have examined the N-linked glycans to identify changes that occur with HCC. As the liver secretes the vast majority of proteins into the serum, this has often been a starting point for study. In serum, alterations in core fucosylation, outer-arm fucosylation, increased sialylation, and glycan branching have been observed in patients with HCC. Similar findings have been found directly in HCC tissue suggesting that these glycan changes may play a role in tumor formation and development.

Engineering N-Glycosylation Pathway in Insect Cells: Suppression of β-N-Acetylglucosaminidase and Expression of β-1,4-Galactosyltransferase.

Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. It has been proposed that β-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation profile in several insect cells including Drosophila S2. Here, we describe GlcNAcase suppression strategy using RNA interference (RNAi) to avoid the formation of paucimannosidic glycans in insect S2 cells. In addition, we describe coexpression of β(1,4)-galactosyltransferase (GalT) as a strategy to improve N-glycosylation pattern and enable recombinant therapeutic proteins to be produced in S2 cells with more complex N-glycans.

Antigenic Potential of a Highly Conserved Neisseria meningitidis Lipopolysaccharide Inner Core Structure Defined by Chemical Synthesis

Neisseria meningitidis is a leading cause of bacterial meningitis worldwide. We studied the potential of synthetic lipopolysaccharide (LPS) inner core structures as broadly protective antigens against N. meningitidis. Based on the specific reactivity of human serum antibodies to synthetic LPS cores, we selected a highly conserved LPS core tetrasaccharide as a promising antigen. This LPS inner core tetrasaccharide induced a robust IgG response in mice when formulated as an immunogenic glycoconjugate. Binding of raised mouse serum to a broad collection of N. meningitidis strains demonstrated the accessibility of the LPS core on viable bacteria. The distal trisaccharide was identified as the crucial epitope, whereas the proximal Kdo moiety was immunodominant and induced mainly nonprotective antibodies that are responsible for lack of functional protection in polyclonal serum. Our results identified key antigenic determinants of LPS core glycan and, hence, may aid the design of a broadly protective immunization against N. meningitidis.

Altered glycosylation of complexed native IgG molecules is associated with disease activity of systemic lupus erythematosus.

In addition to the redundancy of the receptors for the Fc portion of immunoglobulins, glycans result in potential ligands for a plethora of lectin receptors found in immune effector cells. Here we analysed the exposure of glycans containing fucosyl residues and the fucosylated tri-mannose N-type core by complexed native IgG in longitudinal serum samples of well-characterized patients with systemic lupus erythematosus. Consecutive serum samples of a cohort of 15 patients with systemic lupus erythematosus during periods of increased disease activity and remission were analysed. All patients fulfilled the 1982 American College of Rheumatology classification criteria. Sera of 15 sex- and age-matched normal healthy blood donors served as controls. The levels and type of glycosylation of complexed random IgG was measured with lectin enzyme-immunosorbent assays. After specifically gathering IgG complexes from sera, biotinylated lectins Aleuria aurantia lectin and Lens culinaris agglutinin were employed to detect IgG-associated fucosyl residues and the fucosylated tri-mannose N-glycan core, respectively. In sandwich-ELISAs, IgG-associated IgM, IgA, C1q, C3c and C-reactive protein (CRP) were detected as candidates for IgG immune complex constituents. We studied associations of the glycan of complexed IgG and disease activity according to the physician's global assessment of disease activity and the systemic lupus erythematosus disease activity index 2000 documented at the moment of blood taking. Our results showed significantly higher levels of Aleuria aurantia lectin and Lens culinaris agglutinin binding sites exposed on IgG complexes of patients with systemic lupus erythematosus than on those of normal healthy blood donors. Disease activity in systemic lupus erythematosus correlated with higher exposure of Aleuria aurantia lectin-reactive fucosyl residues by immobilized IgG complexes. Top levels of Aleuria aurantia lectin-reactivity were found in samples taken during the highest activity of systemic lupus erythematosus. Our results show that native circulating IgG complexes from active systemic lupus erythematosus patients expose fucosyl residues and their glycan core is accessible to soluble lectins. Two putative mechanisms may contribute to the increased exposure of these glycans: (1) the canonical N-glycosylation site of the IgG-CH2 domain; (2) an IgG binding non-IgG molecule, like complement or C-reactive protein. In both cases the complexed IgG may be alternatively targeted to lectin receptors of effector cells, e.g. dendritic cells.

Epitope Recognition of Antibodies against a Yersinia pestis Lipopolysaccharide Trisaccharide Component

Today, the process of selecting carbohydrate antigens as a basis for active vaccination and the generation of antibodies for therapeutic and diagnostic purposes is based on intuition combined with trial and error experiments. In efforts to establish a rational process for glycan epitope selection, we employed glycan array screening, surface plasmon resonance, and saturation transfer difference (STD)-NMR to elucidate the interactions between antibodies and glycans representing the Yersinia pestis lipopolysaccharide (LPS). A trisaccharide epitope of the LPS inner core glycan and different LPS-derived oligosaccharides from various Gram-negative bacteria were analyzed using this combination of techniques. The antibody-glycan interaction with a heptose substructure was determined at atomic-level detail. Antibodies specifically recognize the Y. pestis trisaccharide and some substructures with high affinity and specificity. No significant binding to LPS glycans from other bacteria was observed, which suggests that the epitopes for just one particular bacterial species can be identified. On the basis of these results we are beginning to understand the rules for structure-based design and selection of carbohydrate antigens.

Liquid–liquid diffusion crystallization improves the X-ray diffraction of EndoS, an endo-β-N-acetylglucosaminidase fromStreptococcus pyogeneswith activity on human IgG

Endoglycosidase S (EndoS) is an enzyme secreted by Streptococcus pyogenes that specifically hydrolyzes the β-1,4-di-N-acetylchitobiose core glycan on immunoglobulin G (IgG) antibodies. One of the most common human pathogens and the cause of group A streptococcal infections, S. pyogenes secretes EndoS in order to evade the host immune system by rendering IgG effector mechanisms dysfunctional. On account of its specificity for IgG, EndoS has also been used extensively for chemoenzymatic synthesis of homogeneous IgG glycoprotein preparations and is being developed as a novel therapeutic for a wide range of autoimmune diseases. The structural basis of its enzymatic activity and substrate specificity, however, remains unknown. Here, the purification and crystallization of EndoS are reported. Using traditional hanging-drop and sitting-drop vapor-diffusion crystallization, crystals of EndoS were grown that diffracted to a maximum of 3.5 Å resolution but suffered from severe anisotropy, the data from which could only be reasonably processed to 7.5 Å resolution. When EndoS was crystallized by liquid-liquid diffusion, it was possible to grow crystals with a different space group to those obtained by vapor diffusion. Crystals of wild-type endoglycosidase and glycosynthase constructs of EndoS grown by liquid-liquid diffusion diffracted to 2.6 and 1.9 Å resolution, respectively, with a greatly diminished anisotropy. Despite extensive efforts, the failure to reproduce these liquid-liquid diffusion-grown crystals by vapor diffusion suggests that these crystallization methods each sample a distinct crystallization space.

A New Approach for the Synthesis of Hyperbranched N-Glycan Core Structures from Locust Bean Gum

A novel protocol for the synthesis of general N-glycan core structures was established by means of Manβ(1→4)Man peracetate derived from a naturally abundant locust bean gum as a key starting material. Phenyl (2-O-benzyl-4,6-O-benzylidine-β-D-mannopyranosyl)-(1→4)-3,6-di-O-benzyl-2-azido-2-deoxy-1-thio-β-D-glucopyranoside facilitated the synthesis of key intermediates leading to hyperbranched N-glycan core structures.

Knockout of an endogenous mannosyltransferase increases the homogeneity of glycoproteins produced in Pichia pastoris

The yeast Pichia pastoris is a common host for the recombinant production of biopharmaceuticals, capable of performing posttranslational modifications like glycosylation of secreted proteins. However, the activity of the OCH1 encoded α-1,6-mannosyltransferase triggers hypermannosylation of secreted proteins at great heterogeneity, considerably hampering downstream processing and reproducibility. Horseradish peroxidases are versatile enzymes with applications in diagnostics, bioremediation and cancer treatment. Despite the importance of these enzymes, they are still isolated from plant at low yields with different biochemical properties. Here we show the production of homogeneous glycoprotein species of recombinant horseradish peroxidase by using a P. pastoris platform strain in which OCH1 was deleted. This och1 knockout strain showed a growth impaired phenotype and considerable rearrangements of cell wall components, but nevertheless secreted more homogeneously glycosylated protein carrying mainly Man8 instead of Man10 N-glycans as a dominant core glycan structure at a volumetric productivity of 70% of the wildtype strain.

Identification of Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a Regulator of N-Acetylglucosaminyltransferase GnT-IX (GnT-Vb)

Our previous studies on a β1,6-N-acetylglucosaminyltransferase, GnT-IX (GnT-Vb), a homolog of GnT-V, indicated that the enzyme has a broad GlcNAc transfer activity toward N-linked and O-mannosyl glycan core structures and that its brain-specific gene expression is regulated by epigenetic histone modifications. In this study, we demonstrate the existence of an endogenous inhibitory factor for GnT-IX that functions as a key regulator for GnT-IX enzymatic activity in Neuro2a (N2a) cells. We purified this factor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), as evidenced by mass spectrometry and by the knockdown and overexpression of ENPP3 in cultured cells. Kinetic analyses revealed that the mechanism responsible for the inhibition of GnT-IX caused by ENPP3 is the ENPP3-mediated hydrolysis of the nucleotide sugar donor substrate, UDP-GlcNAc, with the resulting generation of UMP, a potent and competitive inhibitor of GnT-IX. Indeed, ENPP3 knockdown cells had significantly increased levels of intracellular nucleotide sugars and displayed changes in the total cellular glycosylation profile. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3-mediated hydrolysis of nucleotide sugars would have widespread and significant impacts on glycosyltransferase activities and would be responsible for altering the total cellular glycosylation profile and modulating cellular functions.

Array-assisted Characterization of a Fucosyltransferase Required for the Biosynthesis of Complex Core Modifications of Nematode N-Glycans

Fucose is a common monosaccharide component of cell surfaces and is involved in many biological recognition events. Therefore, definition and exploitation of the specificity of the enzymes (fucosyltransferases) involved in fucosylation is a recurrent theme in modern glycosciences. Despite various studies, the specificities of many fucosyltransferases are still unknown, so new approaches are required to study these. The model nematode Caenorhabditis elegans expresses a wide range of fucosylated glycans, including N-linked oligosaccharides with unusual complex core modifications. Up to three fucose residues can be present on the standard N,N'-diacetylchitobiose unit of these N-glycans, but only the fucosyltransferases responsible for transfer of two of these (the core α1,3-fucosyltransferase FUT-1 and the core α1,6-fucosyltransferase FUT-8) were previously characterized. By use of a glycan library in both array and solution formats, we were able to reveal that FUT-6, another C. elegans α1,3-fucosyltransferase, modifies nematode glycan cores, specifically the distal N-acetylglucosamine residue; this result is in accordance with glycomic analysis of fut-6 mutant worms. This core-modifying activity of FUT-6 in vitro and in vivo is in addition to its previously determined ability to synthesize Lewis X epitopes in vitro. A larger scale synthesis of a nematode N-glycan core in vitro using all three fucosyltransferases was performed, and the nature of the glycosidic linkages was determined by NMR. FUT-6 is probably the first eukaryotic glycosyltransferase whose specificity has been redefined with the aid of glycan microarrays and so is a paradigm for the study of other unusual glycosidic linkages in model and parasitic organisms.

The carbohydrate switch between pathogenic and immunosuppressive antigen‐specific antibodies

IgG antibodies have one conserved N-glycosylation site at Asn 297 in each of their constant heavy chain regions. These Fc glycans influence the overall structure and pro- or anti-inflammatory effector functions of IgG antibodies. The biantennary core glycan structure, consisting of four N-acetyl-glucosamine (GlcNAc) and three mannose residues, can be further decorated with fucose, a bisecting GlcNAc and terminal galactose or galactose plus sialic acid. Non-galactosylated (agalactosylated; G0) IgG antibodies have long been associated with pro-inflammatory effector functions in autoimmune patients with rheumatoid arthritis (RA). In contrast, it has been shown that sialylated IgGs are responsible for anti-inflammatory effects of intravenous immunoglobulin (IVIG; purified IgG from pooled human plasma), which is administered at high doses (2g/kg) for the systemic treatment of autoimmune patients. It has become increasingly evident that pro-inflammatory immune responses, such as autoimmune reactions, primarily induce antigen-specific G0 IgGs, whereas tolerance induces immunosuppressive galactosylated and sialylated IgGs. Under physiological conditions, differentially glycosylated IgGs mediate their pro- or anti-inflammatory effector functions obviously as immune complexes (IC) in an antigen-specific manner. Therefore, antigen-specific galactosylated and sialylated IgGs may be a promising therapeutic tool for re-establishing tolerance against defined (self-) antigens in autoimmune or allergic patients. Here, we summarize these findings and outline our viewpoint on the development and function of differentially glycosylated antigen-specific IgG antibodies.

OP0189 Anti-Citrullinated Protein Antibody Specific FC Glycosylation Patterns in Patients with Arthralgia

BackgroundIn rheumatoid arthritis (RA), anti-citrullinated protein antibodies (ACPA) of the IgG isotype exhibit a specific, pro-inflammatory Fc glycosylation profile characterized by a low content of galactose and sialic acid residues...

Clinicopathological correlation of endocan expression and survival in epithelial ovarian cancer

Endothelial-cell-specific molecule-1 or endocan is a proteoglycan with tumorigenic activity through both its glycan and protein cores. Endocan mRNA is identified as one of the most significant molecular signatures defining a poor prognosis in lung, breast, kidney, and hepatocellular cancer. To assess the clinical value of endocan expression in ovarian cancer tissues in association with other prognostic factors and its impact on overall survival. Oncology unit of Zagazig University Hospitals, Egypt. Prospective observational cohort. One hundred primary ovarian cancer patients were recruited as study group, another 100 patients undergoing hysterectomy and oophorectomy due to uterine fibroid were the control group. Angiogenesis was determined by immunohistochemical staining, using anti-endocan, and anti vascular endothelial growth factor (VEGF) monoclonal antibodies. Endocan was expressed in endothelium of ovarian cancer tissue specimens in all patients with no expression in endothelium of normal ovarian tissue in the control group. VEGF was also expressed in endothelium of all specimens of ovarian cancer tissue, compared with 70% expression in normal ovarian tissue specimens in the control group. A significant association was found between endocan-microvessel density (MVD) and tumor histology, tumor size, staging, and grading. No significant association was found between VEGF expression and any of the clinicopathological variables. Overall survival of patients was inversely associated with endocan-MVD (P < 0.01). Multivariate analysis showed that endocan-MVD was an independent prognostic marker for overall survival of epithelial ovarian cancer (P < 0.01). Endocan could be a reliable marker to predict the survival in epithelial ovarian cancer patients.

The VP8* Domain of Neonatal Rotavirus Strain G10P[11] Binds to Type II Precursor Glycans

Naturally occurring bovine-human reassortant rotaviruses with a P[11] VP4 genotype exhibit a tropism for neonates. Interaction of the VP8* domain of the spike protein VP4 with sialic acid was thought to be the key mediator for rotavirus infectivity. However, recent studies have indicated a role for nonsialylated glycoconjugates, including histo-blood group antigens (HBGAs), in the infectivity of human rotaviruses. We sought to determine if the bovine rotavirus-derived VP8* of a reassortant neonatal G10P[11] virus interacts with hitherto uncharacterized glycans. In an array screen of >600 glycans, VP8* P[11] showed specific binding to glycans with the Galβ1-4GlcNAc motif, which forms the core structure of type II glycans and is the precursor of H type II HBGA. The specificity of glycan binding was confirmed through hemagglutination assays; GST-VP8* P[11] hemagglutinates type O, A, and B red blood cells as well as pooled umbilical cord blood erythrocytes. Further, G10P[11] infectivity was significantly enhanced by the expression of H type II HBGA in CHO cells. The bovine-origin VP4 was confirmed to be essential for this increased infectivity, using laboratory-derived reassortant viruses generated from sialic acid binding rotavirus SA11-4F and a bovine G10P[11] rotavirus, B223. The binding to a core glycan unit has not been reported for any rotavirus VP4. Core glycan synthesis is constitutive in most cell types, and modification of these glycans is thought to be developmentally regulated. These studies provide the first molecular basis for understanding neonatal rotavirus infections, indicating that glycan modification during neonatal development may mediate the age-restricted infectivity of neonatal viruses.