Lipopolysaccharide O-antigen profiles of Helicobacter pylori strains from Southwest China.

BACKGROUND: Helicobacter pylori lipopolysaccharide (LPS) structures vary among strains of different geographic origin. The aim of this study was to characterize the LPS O-antigen profiles of H. pylori strains isolated from Southwest China, and to further analyze the association of Lewis antigen expression with clinical outcomes and antibiotic resistance. RESULTS: A total of 71 H. pylori isolates from Southwest China were included for LPS profiling by silver staining and Western blotting after SDS-PAGE electrophoresis. We demonstrated that all the clinical isolates had the conserved lipid A and core-oligosaccharide, whereas the O-antigen domains varied significantly among the isolates. Compared with the common presence of the glucan/heptan moiety in LPS O-antigen structure of European strains, the clinical isolates in this study appeared to lack the glucan/heptan moiety. The expression frequency of Lex, Ley, Lea, and Leb was 66.2% (47/71), 84.5% (60/71), 56.3% (40/71), and 31.0% (22/71), respectively. In total, the expression of type II Lex and/or Ley was observed in 69 (97.2%) isolates, while type I Lea and/or Leb were expressed in 49 (69.0%) isolates. No association of Lewis antigen expression with clinical outcomes or with antibiotic resistance was observed. CONCLUSIONS: H. pylori strains from Southwest China tend to produce heptan-deficient LPS and are more likely to express type I Lewis antigens as compared with Western strains. This may suggest that H. pylori evolves to change its LPS structure for adaptation to different hosts.

X-ray inhibits FUT4-mediated proliferation in A549 cells by downregulating SP1 expression.

Objective: To identify the mechanism of down-regulation of Lewis Y antigen caused by X-ray irradiation. METHODS: The present original research study was conducted at Zhejiang University City College, Hangzhou, Republic of China, from 2020 to 2022. Western blotting, Co-immunoprecipitation (CO-IP), electrophoretic mobility shift assay and Cell Counting Kit-8 (CCK8) were performed to confirm the effect of X-ray irradiation on A549 cell proliferation and its mechanism. Data was analysed using Statistical Package for Social Sciences (SPSS) 11.5. RESULTS: The expressions of fucosyltransferase IV and Lewis Y were decreased after X-ray irradiation, thus inhibiting the proliferation of A549 lung cancer cells. Deoxyribonucleic acid damage caused by the irradiation caused higher level of poly- adenosinediphosphate-ribosylated Specific Protein 1(SP1), and translocation of SP1 from the nucleus, decreasing the expression of fucosyltransferase IV and Lewis Y. Conclusion: There was a significant role of glycosylation in radiation therapy for lung cancer.

Intestinal Norovirus Binding Patterns in Nonsecretor Individuals.

Human norovirus (HuNoV) infection is associated with an active FUT2 gene, which characterizes the secretor phenotype. However, nonsecretor individuals are also affected by HuNoV infection although in a lesser proportion. Here, we studied GII.3, GII.4, and GII.17 HuNoV interactions in nonsecretor individuals using virus-like particles (VLPs). Only GII.4 HuNoV specifically interacted with nonsecretor saliva. Competition experiments using histo-blood group antigen (HBGA)-specific monoclonal antibodies (MAbs) demonstrate that GII.4 VLPs recognized the Lewis a (Lea) antigen. We also analyzed HuNoV VLP interactions on duodenum tissue blocks from healthy nonsecretor individuals. VLP binding was observed for the three HuNoV genotypes in 10 of the 13 individuals, and competition experiments demonstrated that VLP recognition was driven by an interaction with the Lea antigen. In 3 individuals, binding was restricted to either GII.4 alone or GII.3 and GII.17. Finally, we performed a VLP binding assay on proximal and distal colon tissue blocks from a nonsecretor patient with Crohn's disease. VLP binding to inflammatory tissues was genotype specific since GII.4 and GII.17 VLPs were able to interact with regenerative mucosa, whereas GII.3 VLP was not. The binding of GII.4 and GII.17 HuNoV VLPs was linked to Lea in regenerative mucosae from the proximal and distal colon. Overall, our data clearly showed that Lea has a pivotal role in the recognition of HuNoV in nonsecretors. We also showed that Lea is expressed in inflammatory/regenerative tissues and interacts with HuNoV in a nonsecretor individual. The physiological and immunological consequences of such interactions in nonsecretors have yet to be elucidated. IMPORTANCE Human norovirus (HuNoV) is the main etiological agent of viral gastroenteritis in all age classes. HuNoV infection affects mainly secretor individuals where ABO(H) and Lewis histo-blood group antigens (HBGAs) are present in the small intestine. Nonsecretor individuals, who only express Lewis (Le) antigens, are less susceptible to HuNoV infection. Here, we studied the interaction of common HuNoV genotypes (GII.3, GII.4, and GII.17) in nonsecretor individuals using synthetic viral particles. Saliva binding assays showed that only GII.4 interacted with nonsecretor saliva via the Lewis a (Lea) antigen Surprisingly, the three genotypes interacted with nonsecretor enterocytes via the Lea antigen on duodenal tissue blocks, which were more relevant for HuNoV/HBGA studies. The Lea antigen also played a pivotal role in the recognition of GII.4 and GII.17 particles by inflammatory colon tissue from a nonsecretor Crohn's disease patient. The implications of HuNoV binding in nonsecretors remain to be elucidated in physiological and pathological conditions encountered in other intestinal diseases.

Partners in crime: The Lewis Y antigen and fucosyltransferase IV in Helicobacter pylori-induced gastric cancer.

Helicobacter pylori (H. pylori) is a major causative agent of chronic gastritis, gastric ulcer and gastric carcinoma. H. pylori cytotoxin associated antigen A (CagA) plays a crucial role in the development of gastric cancer. Gastric cancer is associated with glycosylation alterations in glycoproteins and glycolipids on the cell surface. H. pylori cytotoxin associated antigen A (CagA) plays a significant role in the progression of gastric cancer through post-translation modification of fucosylation to develop gastric cancer. The involvement of a variety of sugar antigens in the progression and development of gastric cancer has been investigated, including type II blood group antigens. Lewis Y (LeY) is overexpressed on the tumor cell surface either as a glycoprotein or glycolipid. LeY is a difucosylated oligosaccharide, which is catalyzed by fucosyltransferases such as FUT4 (α1,3). FUT4/LeY overexpression may serve as potential correlative biomarkers for the prognosis of gastric cancer. We discuss the various aspects of H. pylori in relation to fucosyltransferases (FUT1-FUT9) and its fucosylated Lewis antigens (LeY, LeX, LeA, and LeB) and gastric cancer. In this review, we summarize the carcinogenic effect of H. pylori CagA in association with LeY and its synthesis enzyme FUT4 in the development of gastric cancer as well as discuss its importance in the prognosis and its inhibition by combination therapy of anti-LeY antibody and celecoxib through MAPK signaling pathway preventing gastric carcinogenesis.

Lewis y Expressed in Oral Squamous Cell Carcinoma Attenuates Malignant Properties via Down-regulation of EGF Signaling.

BACKGROUND/AIM: Lewis y is expressed in oral squamous cell carcinoma (OSCC) cells and tumors. Previously, we reported that Lewis y was not expressed in invasion areas, and attenuation of proliferation and invasion in OSCC cells was caused by over-expression of Lewis y. However, the roles of Lewis y in the attenuation of malignant properties have not been clarified. In this study, we investigated the roles of Lewis y in OSCC. MATERIALS AND METHODS: The levels of Lewis y on EGFR and the phosphorylation levels of EGFR in OSCC cells were analyzed by immunoprecipitation and western blot. EGFR cross-linking and binding kinetics of EGF were performed. RESULTS: Upon EGF stimulation, phosphorylation and dimer formation of EGFR were more prominent in Lewis y- cells. EGF binding kinetics showed reduced binding sites in Lewis y+ cells. CONCLUSION: Lewis y reduced EGF binding to EGFR, leading to suppression of malignant properties through suppression of EGF signaling.

Synthesis of Fucose Derivatives with Thiol Motifs towards Suicide Inhibition of Helicobacter pylori.

The syntheses of six thiol-exhibiting monosaccharides towards suicide inhibition of Helicobacter pylori are reported. Blood group Antigen Binding Adhesin (BabA), a bacterial membrane-bound lectin, binds to human ABO and Lewis b blood group structures displayed on the surface of host epithelial cells. Crystal structures of the carbohydrate-recognition domain revealed a conserved disulfide bonded loop that anchors a critical fucose residue in these blood group structures. Disruption of this loop by N-acetylcysteine results in reduced BabA-mediated adherence to human gastric tissue sections and attenuated virulence in Lewis b-expressing transgenic mice. With a view of creating specific inhibitors of the lectin, we designed and successfully synthesised six fucose-derived compounds with thiol motifs to engage in a thiol-disulfide exchange with this disulfide bond of BabA and form a glycan-lectin disulfide linkage. Branching and extending the fucose backbone with 2- and 3-carbon thiol motifs delivered a range of candidates to be tested for biological activity against BabA.

Knockdown of α2,3-Sialyltransferases Impairs Pancreatic Cancer Cell Migration, Invasion and E-selectin-Dependent Adhesion.

Aberrant sialylation is frequently found in pancreatic ductal adenocarcinoma (PDA). α2,3-Sialyltransferases (α2,3-STs) ST3GAL3 and ST3GAL4 are overexpressed in PDA tissues and are responsible for increased biosynthesis of sialyl-Lewis (sLe) antigens, which play an important role in metastasis. This study addresses the effect of α2,3-STs knockdown on the migratory and invasive phenotype of PDA cells, and on E-selectin-dependent adhesion. Characterization of the cell sialome, the α2,3-STs and fucosyltransferases involved in the biosynthesis of sLe antigens, using a panel of human PDA cells showed differences in the levels of sialylated determinants and α2,3-STs expression, reflecting their phenotypic heterogeneity. Knockdown of ST3GAL3 and ST3GAL4 in BxPC-3 and Capan-1 cells, which expressed moderate to high levels of sLe antigens and α2,3-STs, led to a significant reduction in sLex and in most cases in sLea, with slight increases in the α2,6-sialic acid content. Moreover, ST3GAL3 and ST3GAL4 downregulation resulted in a significant decrease in cell migration and invasion. Binding and rolling to E-selectin, which represent key steps in metastasis, were also markedly impaired in the α2,3-STs knockdown cells. Our results indicate that inhibition of ST3GAL3 and ST3GAL4 may be a novel strategy to block PDA metastasis, which is one of the reasons for its dismal prognosis.

Identification by mass spectrometry and immunoblotting of xenogeneic antigens in the N- and O-glycomes of porcine, bovine and equine heart tissues.

Animal bioprosthetic heart valves (BHV) are used to replace defective valves in patients with valvular heart disease. Especially young BHV recipients may experience a structural valve deterioration caused by an immune reaction in which α-Gal and Neu5Gc are potential target antigens. The expression of these and other carbohydrate antigens in animal tissues used for production of BHV was explored. Protein lysates of porcine aortic and pulmonary valves, and porcine, bovine and equine pericardia were analyzed by Western blotting using anti-carbohydrate antibodies and lectins. N-glycans were released by PNGase F digestion and O-glycans by ß-elimination. Released oligosaccharides were analyzed by liquid chromatography - tandem mass spectrometry. In total, 102 N-glycans and 40 O-glycans were identified in animal heart tissue lysates. The N- and O-glycan patterns were different between species. α-Gal and Neu5Gc were identified on both N- and O-linked glycans, N,N´-diacetyllactosamine (LacdiNAc) on N-glycans only and sulfated O-glycans. The relative amounts of α-Gal-containing N-glycans were higher in bovine compared to equine and porcine pericardia. In contrast to the restricted number of proteins carrying α-Gal and LacdiNAc, the distribution of proteins carrying Neu5Gc-determinants varied between species and between different tissues of the same species. Porcine pericardium carried the highest level of Neu5Gc-sialylated O-glycans, and bovine pericardium the highest level of Neu5Gc-sialylated N-glycans. The identified N- and O-linked glycans, some of which may be immunogenic and remain in BHVs manufactured for clinical use, could direct future genetic engineering to prevent glycan expression rendering the donor tissues less immunogenic in humans.

Molecular basis of P[II] major human rotavirus VP8* domain recognition of histo-blood group antigens.

Initial cell attachment of rotavirus (RV) to specific cell surface glycan receptors, which is the essential first step in RV infection, is mediated by the VP8* domain of the spike protein VP4. Recently, human histo-blood group antigens (HBGAs) have been identified as receptors or attachment factors for human RV strains. RV strains in the P[4] and P[8] genotypes of the P[II] genogroup share common recognition of the Lewis b (Leb) and H type 1 antigens, however, the molecular basis of receptor recognition by the major human P[8] RVs remains unknown due to lack of experimental structural information. Here, we used nuclear magnetic resonance (NMR) spectroscopy-based titration experiments and NMR-derived high ambiguity driven docking (HADDOCK) methods to elucidate the molecular basis for P[8] VP8* recognition of the Leb (LNDFH I) and type 1 HBGAs. We also used X-ray crystallography to determine the molecular details underlying P[6] recognition of H type 1 HBGAs. Unlike P[6]/P[19] VP8*s that recognize H type 1 HBGAs in a binding surface composed of an α-helix and a ß-sheet, referred as the "ßα binding site", the P[8] and P[4] VP8*s bind Leb HBGAs in a previously undescribed pocket formed by the edges of two ß-sheets, referred to as the "ßß binding site". Importantly, the P[8] and P[4] VP8*s retain binding capability to non-Leb type 1 HBGAs using the ßα binding site. The presence of two distinct binding sites for Leb and non-Leb HBGA glycans in the P[8] and P[4] VP8* domains suggests host-pathogen co-evolution under structural and functional adaptation of RV pathogens to host glycan polymorphisms. Assessment and understanding of the precise impact of this co-evolutionary process in determining RV host ranges and cross-species RV transmission should facilitate improved RV vaccine development and prediction of future RV strain emergence and epidemics.

Cholesteryl α-D-glucoside 6-acyltransferase enhances the adhesion of Helicobacter pylori to gastric epithelium.

Helicobacter pylori, the most common etiologic agent of gastric diseases including gastric cancer, is auxotrophic for cholesterol and has to hijack it from gastric epithelia. Upon uptake, the bacteria convert cholesterol to cholesteryl 6'-O-acyl-α-D-glucopyranoside (CAG) to promote lipid raft clustering in the host cell membranes. However, how CAG appears in the host to exert the pathogenesis still remains ambiguous. Herein we identified hp0499 to be the gene of cholesteryl α-D-glucopyranoside acyltransferase (CGAT). Together with cholesteryl glucosyltransferase (catalyzing the prior step), CGAT is secreted via outer membrane vesicles to the host cells for direct synthesis of CAG. This significantly enhances lipid rafts clustering, gathers adhesion molecules (including Lewis antigens and integrins α5, ß1), and promotes more bacterial adhesion. Furthermore, the clinically used drug amiodarone was shown as a potent inhibitor of CGAT to effectively reduce the bacterial adhesion, indicating that CGAT is a potential target of therapeutic intervention.

Expression of Lewis (b) blood group antigen interferes with oral dienogest therapy among women with adenomyosis.

Adenomyosis is frequently observed in premenopausal women, and oral dienogest is the recommended treatment to target the underlying pathology and improve the symptoms. This retrospective study investigated the association of Lewis (b) antigen expression with outcomes of dienogest therapy among women with adenomyosis. Records from a total of 342 adenomyosis patients were analysed, who were prescribed with oral dienogest for a maximum of 16 weeks. Expression levels of Lewis (b) antigen were measured to categorize all patients into either Le (b)- and Le(b)+ groups. Treatment outcomes, in terms of uterine volume, menstrual flow, pain symptoms and quality of life, were compared between the two groups. While oral dienogest therapy showed considerable clinical efficacy in both groups of patients, the extent of improvements in treatment outcomes was significantly more pronounced in Le (b)- group than Le (b)+ group, with respect to treatment time, uterine symptoms, menstrual flow, pain symptoms and quality of life. No difference in adverse effects was observed between the two groups. Expression of Lewis (b) blood group antigen interferes with oral dialogist therapy among women with adenomyosis.

Identification of carbohydrate peripheral epitopes important for recognition by positive-ion MALDI multistage mass spectrometry.

Carbohydrate sequences are important for various biological processes. It has recently been estimated to have 100,000-500,000 carbohydrate structures in mammalian glycome. However, the peripheral carbohydrate determinants on N- and O-glycoproteins, glycolipids, polysaccharides and secreted free sugars are limited in numbers. Among these blood-group-related antigens the ABO(H)- and Lewis-types are particularly important. Negative-ion MS/MS has been successfully used in assignment of these epitopes on free reducing sugars but cannot be applied to reduced sugars, e.g. O-glycans typically released from mucins as alditols, or in positive-ion detection of either reducing or reduced oligosaccharides. In the present study, we investigate the fragmentation features of permethylated reducing and reduced sugars under positive-ion conditions of multi-stage MALDI-MS, and propose the concept of epitope ion and epitope spectrum for determination of peripheral blood-group related epitopes on secreted human milk oligosaccharides and N-glycans as reducing sugars and O-glycans as reduced alditols in conjunction with MALDI-MS glycan profiling.

BabA and LPS inhibitors against Helicobacter pylori: pectins and pectin-like rhamnogalacturonans as adhesion blockers.

The first step in the development of Helicobacter pylori pathogenicity is receptor-mediated adhesion to gastric epithelium. Adhesins of H. pylori not only enable colonisation of the epithelium, with BabA interacting with Lewisb, but also interaction of lipopolysaccharide (LPS) with galectin-3 contributes to attachment of H. pylori to the host cells. Anti-adhesive compounds against H. pylori have been described, but specific analytical assays for pinpointing the interaction with BabA are limited. LPS-galectin-3 inhibitors have not been described until now. A sandwich ELISA with recombinant BabA547-6K was developed to investigate the interaction of BabA with Lewisb-HSA. Isothermal titration calorimetry gave thermodynamic information on the interaction between BabA, Lewisb-HSA and anti-adhesive compounds. A highly esterified rhamnogalacturonan from Abelmoschus esculentus inhibited the adhesion of H. pylori to adherent gastric adenocarcinoma (AGS) cells (IC50 550 µg/mL) and interacted with BabA (IC50 17 µg/mL). Pectins with similar rhamnogalacturonan structure showed weak anti-adhesive activity. Highly branched rhamnogalacturonans with low uronic acid content and high degree of esterification are potent BabA inhibitors. BabA represents a promising target for the development of anti-adhesive drugs against H. pylori. The rhamnogalacturonan influenced also the binding affinity of H. pylori to recombinant galectin-3 in a concentration-dependent manner with an IC50 of 222 µg/mL. Similar effects were obtained with pectin from apple fruits, while pectins from other sources were inactive.

Sulfated Lewis A trisaccharide on oviduct membrane glycoproteins binds bovine sperm and lengthens sperm lifespan.

A fraction of sperm deposited at mating or insemination reaches the oviduct isthmus, where sperm are retained and thereby form a reservoir. This reservoir delays capacitation, prevents polyspermy, selects a fertile population of sperm, and, foremost, increases sperm lifespan. The molecular interactions underlying the formation of a sperm reservoir are becoming clearer in mammals. Sperm lectins bind to oviductal glycans to form the reservoir. Herein, we found that the highest percentage of bovine sperm bound to the 3'-O-sulfated form of Lewis A (suLeA) trisaccharide and sialylated Lewis A and that fluoresceinated versions of each localized to receptors on the anterior head of the sperm. Following capacitation, binding to suLeA decreased significantly, a potential explanation for sperm release from the reservoir. MS and immunohistochemistry analyses indicated that suLeA motifs were present predominantly on O-linked glycans initiated by GalNAc residues, but no sialylated Lewis A was detected. To determine whether sperm binding to isolated suLeAin vitro could mimic in vivo sperm binding to oviduct cells and increase sperm longevity, we immobilized suLeA and incubated it with sperm. Using free-swimming sperm and sperm bound to immobilized laminin as controls, we observed that over 96 h, the viability of free-swimming sperm decreased to 10%, and that of sperm bound to immobilized laminin decreased to about 50%, whereas viability of sperm bound to immobilized suLeA was highest throughout the incubation and 60% at 96 h. These results indicate that bovine sperm binding to oviduct suLeA retains sperm for reservoir formation and extends sperm lifespan.

Maternal and Infant Factors Associated with Human Milk Oligosaccharides Concentrations According to Secretor and Lewis Phenotypes.

Human milk oligosaccharides (HMOs) are multifunctional carbohydrates naturally present in human milk that act as prebiotics, prevent pathogen binding and infections, modulate the immune system and may support brain development in infants. HMOs composition is very individualized and differences in HMOs concentrations may affect the infant's health. HMOs variability can be partially explained by the activity of Secretor (Se) and Lewis (Le) genes in the mother, but non-genetic maternal factors may also be involved. In this cross-sectional, observational study, 78 single human milk samples ranging from 17 to 76 days postpartum (median: 32 days, IQR: 25-46 days) were collected from breastfeeding Brazilian women, analyzed for 16 representative HMOs by liquid chromatography coupled to mass spectrometry and associations between maternal and infant factors with HMOs concentrations were investigated. HMOs concentrations presented a high variability even in women with the same SeLe phenotype and associations with maternal allergic disease, time postpartum and with infant's weight, weight gain and sex. Overall, we present unprecedented data on HMOs concentrations from breastfeeding Brazilian women and novel associations of maternal allergic disease and infant's sex with HMOs concentrations. Differences in HMOs composition attributed to maternal SeLe phenotype do not impact infant growth, but higher concentrations of specific HMOs may protect against excessive weight gain.

Molecular Mechanism of the Flexible Glycan Receptor Recognition by Mumps Virus.

Mumps virus (MuV) is an important aerosol-transmitted human pathogen causing epidemic parotitis, meningitis, encephalitis, and deafness. MuV preferentially uses a trisaccharide containing α2,3-linked sialic acid as a receptor. However, given the MuV tropism toward glandular tissues and the central nervous system, an additional glycan motif(s) may also serve as a receptor. Here, we performed a large-scale glycan array screen with MuV hemagglutinin-neuraminidase (MuV-HN) attachment proteins by using 600 types of glycans from The Consortium for Functional Glycomics Protein-Glycan Interaction Core in an effort to find new glycan receptor motif(s). According to the results of the glycan array, we successfully determined the crystal structures of MuV-HN proteins bound to newly identified glycan motifs, sialyl LewisX (SLeX) and the oligosaccharide portion of the GM2 ganglioside (GM2-glycan). Interestingly, the complex structures showed that SLeX and GM2-glycan share the same configuration with the reported trisaccharide motif, 3'-sialyllactose (3'-SL), at the binding site of MuV-HN, while SLeX and GM2-glycan have several unique interactions compared with those of 3'-SL. Thus, MuV-HN protein can allow an additional spatial modification in GM2-glycan and SLeX at the second and third carbohydrates from the nonreducing terminus of the core trisaccharide structure, respectively. Importantly, MuV entry was efficiently inhibited in the presence of 3'-SL, SLeX, or GM2-glycan derivatives, which indicates that these motifs can serve as MuV receptors. The α2,3-sialylated oligosaccharides, such as SLeX and 3'-sialyllactosamine, are broadly expressed in various tissues, and GM2 exists mainly in neural tissues and the adrenal gland. The distribution of these glycan motifs in human tissues/organs may have bearing on MuV tropism.IMPORTANCE Mumps virus (MuV) infection is characterized by parotid gland swelling and can cause pancreatitis, orchitis, meningitis, and encephalitis. MuV-related hearing loss is also a serious complication because it is usually irreversible. MuV outbreaks have been reported in many countries, even in high-vaccine-coverage areas. MuV has tropism toward glandular tissues and the central nervous system. To understand the unique MuV tropism, revealing the mechanism of receptor recognition by MuV is very important. Here, using a large-scale glycan array and X-ray crystallography, we show that MuV recognizes sialyl LewisX and GM2 ganglioside as receptors, in addition to a previously reported MuV receptor, a trisaccharide containing an α2,3-linked sialic acid. The flexible recognition of these glycan receptors by MuV may explain the unique tropism and pathogenesis of MuV. Structures will also provide a template for the development of effective entry inhibitors targeting the receptor-binding site of MuV.

Human adenovirus type 5 increases host cell fucosylation and modifies Ley antigen expression.

Certain viral infections are known to modify the glycosylation profile of infected cells through the overexpression of specific host cell fucosyltransferases (FUTs). Infection with CMV (cytomegalovirus), HCV (hepatitis C virus), HSV-1 (herpes simplex virus type-1) and VZV (varicella-zoster virus) increase the expression of fucosylated epitopes, including antigens sLex (Siaα2-3 Galß1-4(Fucα1-3)GlcNAcß1-R) and Ley (Fucα1-2 Galß1-4(Fucα1-3)GlcNAcß1-R). The reorganization of the glycocalyx induced by viral infection may favor the spread of viral progeny, and alter diverse biological functions mediated by glycans, including recognition by the adaptive immune system. In this work, we aimed to establish whether infection with human adenovirus type 5 (HAd5), a well-known viral vector and infectious agent, causes changes in the glycosylation profile of A549 cells, used as a model of lung epithelium, a natural target of HAd5. We demonstrate for the first time that HAd5 infection causes a significant increase in the cell surface de novo fucosylation, as assessed by metabolic labeling, and that such modification is dependent on the expression of viral genes. The main type of increased fucosylation was determined to be in α1-2 linkage, as assessed by UEA-I lectin binding and supported by the overexpression of FUT1 and FUT2. Also, HAd5-infected cells showed a heterogeneous change in the expression profile of the bi-fucosylated Ley antigen, an antigen associated with enhanced cell proliferation and inhibition of apoptosis.

Co­expression of Lewis y antigen and CD147 in epithelial ovarian cancer is correlated with malignant progression and poor prognosis.

CD147 is a highly glycosylated transmembrane protein expressed on the surface of tumor cells. In the present study, the expression and clinical significance of the Lewis y antigen and CD147 in epithelial ovarian cancer (EOC) were analyzed, and the function and correlation in between the expression of Lewis y and CD147 were evaluated using immunohistochemical staining, reverse transcription­quantitative polymerase chain reaction analysis, immunocytochemical staining, immunoprecipitation and western blotting. The results showed that the expression of CD147 was higher in EOC tissues and correlated with a higher tumor burden. Lewis y and CD147 exhibited similar expression patterns and their expression was positively correlated. The results of the immunofluorescence and immunoprecipitation experiments showed that Lewis y and CD147 colocalized in the cell membrane and cytoplasm. Lewis y antigen, but not Lewis x or sialyl Lewis x, was predominantly expressed in the highly glycosylated form of CD147. These changes occurred at the post­transcriptional level. As an important component of CD147, Lewis y promoted CD147­mediated cell adhesion and the expression of matrix metalloproteinase 2. In conclusion, Lewis y antigen and CD147 were significantly upregulated in ovarian tumors, and the altered expression of Lewis y may cause changes in CD147. The two molecules are associated with carcinogenesis and the development of ovarian cancer, and Lewis y antigen is a component of the CD147 structure.

N-Glycomic and Transcriptomic Changes Associated with CDX1 mRNA Expression in Colorectal Cancer Cell Lines.

The caudal-related homeobox protein 1 (CDX1) is a transcription factor, which is important in the development, differentiation, and homeostasis of the gut. Although the involvement of CDX genes in the regulation of the expression levels of a few glycosyltransferases has been shown, associations between glycosylation phenotypes and CDX1 mRNA expression have hitherto not been well studied. Triggered by our previous study, we here characterized the N-glycomic phenotype of 16 colon cancer cell lines, selected for their differential CDX1 mRNA expression levels. We found that high CDX1 mRNA expression associated with a higher degree of multi-fucosylation on N-glycans, which is in line with our previous results and was supported by up-regulated gene expression of fucosyltransferases involved in antenna fucosylation. Interestingly, hepatocyte nuclear factors (HNF)4A and HNF1A were, among others, positively associated with high CDX1 mRNA expression and have been previously proven to regulate antenna fucosylation. Besides fucosylation, we found that high CDX1 mRNA expression in cancer cell lines also associated with low levels of sialylation and galactosylation and high levels of bisection on N-glycans. Altogether, our data highlight a possible role of CDX1 in altering the N-glycosylation of colorectal cancer cells, which is a hallmark of tumor development.

Relationship Between CA 19.9 and the Lewis Phenotype: Options to Improve Diagnostic Efficiency.

BACKGROUND/AIM: Approximately 10% of patients are unable to synthesize CA 19.9 (Lewis-negative), and these results are erroneously considered false-negatives. The aim of this study was to confirm that CA 19.9 cannot be detected by immunoassays in Lewis-negative patients. MATERIALS AND METHODS: CA 19.9 levels were measured by immunological assays and Lewis phenotype was determined by the haemagglutination reaction. RESULTS: Patients with Lewis phenotype (a+b-) or (a-b+) had significantly higher CA 19.9 levels than Lewis-negative patients with active cancer (p<0.001), no-evidence of disease (NED) patients (p<0.001) or patients with benign disease (p<0.001). Ninenty-four percent of patients (33/35) with undetectable CA 19.9 had a Lewis-negative phenotype. Additionally, 94.7% (34/36) of patients with Lewis-negative phenotypes had undetectable CA 19.9 serum levels. CONCLUSION: Patients with undetectable CA 19.9 serum levels tend to be Lewis-negative, and CA 19.9 is not useful in diagnosis or follow-up.