Blood group-related antigen Ley on human platelets and its involvement in platelet aggregation via a possible interaction with CD61.

BACKGROUND: The Ley antigen is a carbohydrate chain belonging to the ABH-Lewis blood group family. Ley has been reported to be present on red blood cells (RBCs) and granulocytes, but its distribution and function in platelets remain unknown. There are a variety of glycoproteins on platelets, which may carry the Ley antigen. This study aims to investigate the expression pattern and the function of Ley in human platelets. STUDY DESIGN AND METHODS: Flow cytometry, Western blot, and immunofluorescence assays were performed to determine Ley expression on human platelets. ADP (1.25-10 µM) and thrombin (0.05-1 IU/mL) were used to activate platelets in the presence or absence of prostaglandin E1 (PGE1) and the Ley expression was evaluated again by flow cytometry. Blockade was performed with an anti-Ley monoclonal antibody to verify the role of this epitope in platelet function. Finally, coimmunoprecipitation was performed to identify glycoproteins associated with Ley . RESULTS: Ley was expressed on human platelets independent of ABO blood type. Ley expression was decreased in a dose-dependent manner after activation with either ADP or thrombin, and this effect could be partially reversed by PGE1. Anti- Ley mAb treatment increased alpha-granule release and neutralized the inhibitory effect of the anti-CD61 antibody on platelet aggregation. In addition, Ley was proven to interact and colocalize with CD61. CONCLUSIONS: These results demonstrate nondifferential expression of Ley in platelets of different ABO blood types and suggest the involvement of Ley in platelet function, possibly via interaction with CD61.

High expressions of BCL6 and Lewis y antigen are correlated with high tumor burden and poor prognosis in epithelial ovarian cancer.

Aberrant regulation of BCL6 plays crucial oncogenic roles in various malignant tumors; howbeit, the function of BCL6 in tumorigenesis of ovarian cancer remains unclear. The aim of this study is to investigate the role of BCL6 in ovarian cancer. The methods of immunohistochemical staining, quantitative real-time polymerase chain reaction, immunocytochemical staining, and gene expression profile enrichment analysis were performed to identify the possible role of BCL6 in ovarian cancer. We observed that the expression of BCL6 was significantly higher in ovarian cancer tissues and correlated with higher tumor burden including advanced International Federation of Gynecology and Obstetrics stages, poor differentiation, Type II ovarian cancer, the presence of >1 cm residual tumor size, and appearance of recurrence or death (all p < 0.05). The expression patterns of Lewis y were similar to these of BCL6. Multivariate Cox analysis demonstrated that advanced International Federation of Gynecology and Obstetrics stage, lymph node metastasis, residual tumor size >1 cm, as well as high expressions of BCL6 and Lewis y antigen were independent factors of worse progression-free survival and overall survival (all p < 0.05). There was a positive correlation of the expressions of BCL6 and Lewis y antigen. The associated genes with BCL6 in response to Lewis y antigen were identified, including four upregulated genes ( SOCS3, STAT1, PPARG, and GADD45A) and three downregulated genes ( ACAN, E2F3, and ZBTB7B). In conclusion, the high expressions of BCL6 and Lewis y antigen are associated with development, high tumor burden, and worse prognosis of ovarian cancer and targeting BCL6 could be a novel therapeutic strategy for ovarian cancer treatment.

The expression of annexin II and Lewis y antigen in ovarian epithelial tumors and the correlation between them.

The main aim of this study was to explore the molecular structural relationship between annexin II (ANXA2) and Lewis y antigen by determining their expression patterns and clinical significance in ovarian epithelial carcinoma. The structural relationship between ANXA2 and Lewis y antigen was examined using immunoprecipitation and confocal laser scanning microscopy in two ovarian caner cell lines ES-2 and CaoV-3. We also constracted the stably transfected cell lines with low ANXA2 gene expression in order to detect the expression level between ANXA2 and Lewis y. ANXA2 and Lewis y were detected in tissues from malignant, borderline, benign, and normal ovarian tissues using immunohistochemical analysis. ANXA2 and Lewis y were present in both two ovarian cancer cells and ANXA2 contained Lewis y antigen. Moreover, expression of Lewis y antigen in ANXA2 from cell after transfection was higher than that before. Our immunohistochemistry data revealed significantly higher positive expression rates of ANXA2 in malignant ovarian tissues, compared to benign tumor and normal tissue, similar to Lewis y antigen levels in ovarian cancer. Notably, tissues displaying marked expression of ANXA2 simultaneously expressed high levels of Lewis y antigen. A linear correlation between the expression patterns of ANXA2 and Lewis y antigen was evident. Consistently, double-labeling immunofluorescence experiments illustrated co-localization of ANXA2 and Lewis y antigen within the same area. In conclusions, ANXA2 contains Lewis y antigen. Our results further demonstrate a close correlation between the expression levels of the two antigens, which are significantly high in ovarian cancer.

Screening of enzymatic synthesis and expression of Lewis determinants in human colorectal carcinoma.

BACKGROUND: Although colorectal carcinogenesis has been intensively studied, the published investigations do not provide a consistent description of how different carbohydrate determinants of colorectal epithelium are modified in colorectal cancer (CRC). OBJECTIVE: This study is an attempt to characterize the terminal fucosylation steps responsible for the synthesis of mono- Le(a)/Le(x)- and difucosylated -Le(b)/Le(y)- Lewis antigens in healthy and tumour CRC tissue. METHODS: An immunohistochemical study of Lewis antigens' expression was undertaken, along with screening of the fucosyltransferase (FT) activities involved in their synthesis, on healthy and tumour samples from 18 patients undergoing CRC. RESULTS: Analysis of alpha(1,2/3/4)FT activities involved in the sequential fucosylation of cores 1 and 2 showed significant increases in tumour tissue. Expressed as microU/mg and control vs. tumour activity (pfrom Wilcoxon's test), the FT activities for Le(a)/Le(b) synthesis were: lacto-N-biose alpha(1,2)/alpha(1,4)FT, 65.4 ± 19.0 vs. 186 ± 35.1 (p< 0.005); lacto-N-fucopentaose 1 alpha(1,4)FT, 64.9 ± 11.9 vs. 125.4 ± 20.7 (p< 0.005); Le(a) alpha(1,2)FT, 56.2 ± 7.2 vs. 130.5 ± 15.6 (p< 0.001). Similarly, for Le(x)/Le(y) synthesis were: N-acetyllactosamine alpha(1,2)-/alpha(1,3)FT, 53.4 ± 12.2 vs. 108.1 ± 18.9 (p< 0.001); 2'-Fucosyl-N-acetyllactosamine alpha(1,3)FT, 61.3 ± 10.7 vs. 126.4 ± 22.9 (p< 0.001); 2'-Fucosyllactose alpha(1,3)FT, 38.9 ± 10.9 vs. 143.6 ± 28.9 (p< 0.001); 2'-Methyllactose alpha(1,3)FT, 30.9 ± 4.8 vs. 66.1 ± 8.1 (p< 0.005); and Le(x) alpha(1,2)FT, 54.3 ± 11.9 vs. 88.2 ± 14.4 (p< 0.001). Immunohistochemical Le(y) expression was increased (p< 0.01 according to Wilcoxon's test) in tumour tissue, with 84.6% of specimens being positive: 7.7% weak, 15.4% moderate and 61.5% high intensity. CONCLUSIONS: Results suggest the activation of the biosynthesis pathways of mono- and difucosylated Lewis histo-blood antigens in tumour tissue from CRC patients, leading to the overexpression of Le(y), probably at the expense of Le(x).

Novel functions for glycosyltransferases Jhp0562 and GalT in Lewis antigen synthesis and variation in Helicobacter pylori.

Lewis (Le) antigens are fucosylated oligosaccharides present in the Helicobacter pylori lipopolysaccharide. Expression of these antigens is believed to be important for H. pylori colonization, since Le antigens also are expressed on the gastric epithelia in humans. A galactosyltransferase encoded by ß-(1,3)galT is essential for production of type 1 (Le(a) and Le(b)) antigens. The upstream gene jhp0562, which is present in many but not all H. pylori strains, is homologous to ß-(1,3)galT but is of unknown function. Because H. pylori demonstrates extensive intragenomic recombination, we hypothesized that these two genes could undergo DNA rearrangement. A PCR screen and subsequent sequence analyses revealed that the two genes can recombine at both the 5' and 3' ends. Chimeric ß-(1,3)galT-like alleles can restore function in a ß-(1,3)galT null mutant, but neither native nor recombinant jhp0562 can. Mutagenesis of jhp0562 revealed that it is essential for synthesis of both type 1 and type 2 Le antigens. Transcriptional analyses of both loci showed ß-(1,3)galT expression in all wild-type (WT) and mutant strains tested, whereas jhp0562 was not expressed in jhp0562 null mutants, as expected. Since jhp0562 unexpectedly displayed functions in both type 1 and type 2 Le synthesis, we asked whether galT, part of the type 2 synthesis pathway, had analogous functions in type 1 synthesis. Mutagenesis and complementation analysis confirmed that galT is essential for Le(b) production. In total, these results demonstrate that galT and jhp0562 have functions that cross the expected Le synthesis pathways and that jhp0562 provides a substrate for intragenomic recombination to generate diverse Le synthesis enzymes.

Differential glycosylation of MUC1 and CEACAM5 between normal mucosa and tumour tissue of colon cancer patients.

Altered glycosylation in epithelial cancers may play an important role in tumour progression, as it may affect tumour cell migration and antigen presentation by antigen presenting cells. We specifically characterise the glycosylation patterns of two tumour antigens that are highly expressed in cancer tissue and often detected in their secreted form in serum: the epithelial mucin MUC1 and carcinoembryonic antigen (CEA, also called CEACAM5). We analysed 48 colorectal cancer patients, comparing normal colon and tumour epithelium within each patient. Lectin binding was studied by a standardised CEA/MUC1 capture ELISA, using several plant lectins, and the human C-type lectins MGL and DC-SIGN, and Galectin-3. Peanut agglutinin (PNA) bound to MUC1 from tumour tissue in particular, suggests increased expression of the Thomsen-Friedenreich antigen (TF-antigen) (Core 1, Galß1-3GalNAc-Ser/Thr). Only small amounts of Tn-antigen (GalNAcα-Ser/Thr) expression was observed, but the human C-type lectin MGL showed increased binding to tumour-associated MUC1. Furthermore, sialylation was greatly enhanced. In sharp contrast, tumour-associated CEA (CEACAM5) contained high levels of the blood-group related carbohydrates, Lewis X and Lewis Y. This correlated strongly with the interaction of the human C-type lectin DC-SIGN to tumour-associated CEA, suggesting that CEA can be recognized and taken up by antigen presenting cells. In addition, increased mannose expression was observed and branched N-glycans were prominent, and this correlated well with human Galectin-3 binding. These data demonstrate that individual tumour antigens contain distinct glycan structures associated with cancer and, since glycans affect cellular interactions with its microenvironment, this may have consequences for progression of the disease.

Ganglioside biosynthesis in developing brains and apoptotic cancer cells: X. regulation of glyco-genes involved in GD3 and Sialyl-Lex/a syntheses.

Gangliosides, the acidic glycosphingolipids (GSLs) containing N-acetylgalactosamine and sialic acid are ubiquitous in the central nervous system. At least six DSL-glycosyltransferase activities (GLTs Gangliosides, the acidic glycosphingolipids (GSLs) containing N-acetylgalactosamine and sialic acid (or NAc-Neuraminic acid) are ubiquitous in the central nervous system. At least six GSL-glycosyltransferase activities (GLTs) of Basu-Roseman pathway catalyzing the biosynthesis of these gangliosides have been characterized in developing chicken brains. Most of these glyco-genes are expressed in the early stages (7-17 days) of brain development and lowered in the adult stage, but the cause of reduction of enzymatic activities of these GLTs in the adult stages is not known. In order to study glyco-gene regulation we used four clonal metastatic cancer cells of colon and breast cancer tissue origin (Colo-205, SKBR-3, MDA-468, and MCF-3). The glyco-genes for synthesis of SA-LeX and SA-LeA (which contain N-acetylglucosamine, sialic acid and fucose) in these cells were modulated differently at different phases (between 2 and 48 h) of apoptotic inductions. L-PPMP, D-PDMP (inhibitor of glucosylceramide biosynthesis), Betulinic Acid (a triterpinoid isolated from bark of certain trees and used for cancer treatment in China), Tamoxifen a drug in use in the west for treatment of early stages of the disease in breast cancer patients), and cis-platin (an inhibitor of DNA biosynthesis used for testicular cancer patients) were used for induction of apoptosis in the above-mentioned cell lines. Within 2-6 h, transcriptional modulation of a number of glyco-genes was observed by DNA-micro-array (containing over 300 glyco genes attached to the glass cover slips) studies. Under long incubation time (24-48 h) almost all of the glyco-genes were downregulated. The cause of these glyco-gene regulations during apoptotic induction in metastatic carcinoma cells is unknown and needs future investigations for further explanations. These apoptotic agents could be employed as a new generation of anti-cancer drugs after properly delivered to the patients.

Differential expression of LeY and fucosyltransferase IV correlates with the receptivity of RL95-2 and HEC-1A human uterine epithelial cells.

Adhesion molecules expressed on the uterine endometrium are potential receptive markers in embryo implantation. RL95-2 and HEC-1A cell lines represent the high- and low-receptive endometrial epithelium respectively. LeY (Lewis Y) is a difucosylated oligosaccharide highly expressed in the endometrium of some species during implantation. α1, 3 fucosylation of LeY is catalysed by FUT4 (fucosyltransferase IV), the key synthesis enzyme for LeY. We investigated whether the difference in receptivity between the 2 cell lines was related to different expressions of LeY and FUT4. RL95-2 cells expressed a higher level of LeY and FUT4 than HEC-1A cells, as shown by immunofluorescent staining, RT-PCR (reverse transcription-PCR) or Western blotting. FUT4-siRNA (small interfering RNA) transfection down-regulated FUT4 and LeY in RL95-2 cells, and inhibited the adhesion of the embryonic cells (JAR) to RL95-2 cell monolayer. FUT4-cDNA, however, increased the expression of FUT4 and LeY in HEC-1A cells, and increased the adhesion of embryonic cells to HEC-1A cell monolayer. Alterations of LeY level by up- or down-regulation of FUT4 also mediated EGFR (epidermal growth factor receptor)/MAPK (mitogen-activated protein kinase) signalling pathway. To conclude, the expression of LeY and FUT4 correlates with endometrial receptivity, making them potential new markers for the evaluation of endometrial receptivity.

Elevated levels of Lewis y and integrin α5ß1 correlate with chemotherapeutic drug resistance in epithelial ovarian carcinoma.

OBJECTIVE: To measure Lewis y and integrin α(5)ß(1) expression in epithelial ovarian carcinoma and to correlate the levels of these molecules with ovarian carcinoma chemotherapy and prognosis. METHODS: The study population included 34 ovarian carcinoma patients with chemotherapeutic drug-resistance, six partially drug-sensitive cases, and 52 drug-sensitive cases (92 total). Immunochemistry was used to determine expression of Lewis y antigen and integrin α(5)ß(1) in ovarian carcinoma tissues, and correlation of these molecules with chemotherapy resistance was further investigated, Multi-factor logistic regression analysis was applied to investigate: age, surgical stage, grade, subtype of patient cases, metastasis of lymph nodes, residual tumor size, expression levels of Lewis y antigen and integrin α(5)ß(1) correlation with ovarian carcinoma chemotherapy resistance. RESULTS: The expression rates of Lewis y antigen and integrins α(5) and ß(1) were significantly greater in the drug-resistant group (91.17%, 85.29%, 88.24%) than the partially sensitive (50.00%, 33.33%, 50.00%) or sensitive groups (61.54%, 57.69%, 55.77%). Binary logistic regression analysis revealed that surgical stage, residual tumor size, and expression of integrin α(5) and Lewis y in ovarian carcinoma tissues were independent risk factors for chemotherapeutic drug resistance. CONCLUSIONS: Overexpression of Lewis y and integrin α(5) are strong risk factors for chemotherapeutic drug resistance in ovarian carcinoma patients.

Genotypic and phenotypic variation of Lewis antigen expression in geographically diverse Helicobacter pylori isolates.

BACKGROUND: Helicobacter pylori are a persistent colonizer of the human gastric mucosa, which can lead to the development of peptic ulcer disease and gastric adenocarcinomas. However, H. pylori can asymptomatically colonize a host for years. One factor that has been hypothesized to contribute to such persistence is the production of Lewis (Le) antigens in the lipopolysaccharide layer of the bacterial outer membrane as a form of molecular mimicry, because humans also express these antigens on their gastric mucosa. Humans and H. pylori both are polymorphic for Le expression, which is driven in H. pylori by variation at the Le synthesis loci. In this report, we sought to characterize Le genotypic and phenotypic variation in geographically diverse H. pylori isolates. MATERIALS AND METHODS: From patients undergoing endoscopy in 29 countries, we determined Le phenotypes of 78 H. pylori strains and performed genotyping of the galT and ß-(1,3)galT loci in 113 H. pylori strains. RESULTS: Le antigen phenotyping revealed a significant (p < .0001) association between type 1 (Le(a) and Le(b) ) expression and strains of East Asian origin. Genotyping revealed a significant correlation between strain origin and the size of the promoter region upstream of the Le synthesis gene, galT (p < .0001). CONCLUSION: These results indicate that the heterogeneity of human Le phenotypes is reflected in their H. pylori colonizing strains and suggest new loci that can be studied to assess the variation of Le expression.

Incomplete synthesis of the Sda/Cad blood group carbohydrate in gastrointestinal cancer.

Cancer-associated changes in cell surface carbohydrates, including incomplete synthesis of normal carbohydrate epitopes, strongly affect malignant and metastatic potential. Here, we report that compensating for the cancer-associated loss of a single glycosyltransferase, beta1,4N-acetylgalactosaminyltransferase T2, dramatically decreased cell surface expression of both E-selectin ligands (sialyl Lewis(x) and sialyl Lewis(a)). This modification was associated with elevated expression of the Sd(a) carbohydrate determinant, which is expressed in normal gastrointestinal mucosa and is strikingly downregulated in cancer tissues. Loss of E-selectin ligands resulted in decreased adhesion of cancer cells to activated human endothelial cells in vitro and eventually suppressed metastatic potential in vivo.

Induction of sialyl-Lex expression by herpes simplex virus type 1 is dependent on viral immediate early RNA-activated transcription of host fucosyltransferase genes.

We have previously shown that varicella-zoster virus (VZV) and cytomegalovirus (CMV) infection of diploid human fibroblasts (HEL) results in neo-expression of Lewis antigens sialyl Lewis x (sLe(x)) and Lewis y (Le(y)), respectively, after transcriptional activation of different combinations of dormant human fucosyltransferase genes (FUT1, FUT3, FUT5, and FUT6), whose gene products are responsible for the synthesis of Le antigens. Here, we show that herpes simplex virus type 1 (HSV-1) also induces sLe(x) expression dependent on induction of FUT3, FUT5, and FUT6 transcription in infected cells. HSV-1 induction of FUT5 was subsequently used as a model system for analyzing the mechanism of viral activation of dormant fucosyltransferase genes. We show that this is a rapid process, which gives rise to elevated FUT5 RNA levels already at 90 min postinfection. Augmented FUT5 transcription was found to be dependent on transcription of viral genes, but not dependent on the immediate early proteins ICP0 and ICP4, as demonstrated by experiments with HSV-1 mutants defective in expression of these genes. Augmented FUT5 transcription takes place in cycloheximide-treated HSV-1-infected cells, suggesting a more direct role for IE viral RNA during activation of cellular FUT5.

Diagnosis of epithelial mesothelioma using tree-based regression analysis and a minimal panel of antibodies.

AIMS: Immunohistochemistry with panels of antibodies is a standard procedure to distinguish between malignant mesothelioma and metastatic adenocarcinoma. Most studies assess only the sensitivity and specificity for single antibodies, even when the paper concludes by recommending an antibody panel. It was the aim of this study to use a novel statistical approach to identify a minimal panel of antibodies, which would make this distinction in the majority of cases. METHODS: Two hundred consecutive cases of pleural malignancy (173 pleural mesotheliomas of epithelial type and 27 cases of secondary adenocarcinoma) were investigated using a standard panel of 12 antibodies (CAM5.2, CK5/6, calretinin, HBME-1, thrombomodulin, WT-1, EMA, CEA, CD15, B72.3, BG8, and TTF-1). Regression and classification tree-based methods were applied to select the best combination of markers. The modelling procedures used employ successive, hierarchical predictions computed for individual cases to sort them into homogeneous classes. RESULTS: Labelling for calretinin and lack of labelling for BG8 were sufficient for definite correlation with a diagnosis of malignant mesothelioma. CD15 provided further differentiating information in some cases. CONCLUSION: A panel of three antibodies was sufficient in most cases to diagnose, or to exclude, epithelial mesothelioma. Calretinin exhibits the strongest correlative power of the antibodies tested.

Studies of Lewis antigens and H. pylori adhesion in CHO cell lines engineered to express Lewis b determinants.

Many microbes bind and adhere via adhesins to host cell carbohydrates as an initial step for infection. Therefore, cell lines expressing Lewis b (Le(b)) determinants were generated as a potential model system for Helicobacter pylori colonization and infection, and their expression of blood group Lewis determinants was characterized. CHO-K1 cells were stably transfected with selected glycosyltransferase cDNAs, and two Le(b) positive clones, 1C5 and 2C2, were identified. Expression of Lewis (Le(a), Le(b), Le(x), and Le(y)) determinants was analyzed by flow cytometry of intact cells, SDS-PAGE/Western blot of solubilized glycoproteins, and thin layer chromatography immunostaining of isolated glycolipids (GL). Binding of H. pylori to cells was examined by microscopy and quantified. Flow cytometry showed that 1C5 and 2C2 were Le(a) and Le(b) positive. 1C5 expressed Le(b) on O-linked, but not N-linked, glycans and only weakly on GLs. In contrast, 2C2 expressed Le(b) on N-, O-glycans, and GLs. Furthermore, both clones expressed Le(a) on N- and O-glycans but not on GLs. 2C2, but not 1C5, stained positively for Le(y) on N-linked glycans and GLs. Both clones, as well as the parental CHO-K1 cells, expressed Le(x) on GLs. A Le(b)-binding H. pylori strain bound to the 1C5 and 2C2 cells. In summary, two glycosyltransferase transfected CHO-K1 cell clones differed regarding Lewis antigen expression on N- and O-linked glycans as well as on GLs. Both clones examined supported adhesion of a Le(b)-binding H. pylori strain and may thus be a useful in vitro model system for H. pylori colonization/infection studies.

Lewis antigen expression by Helicobacter pylori strains colonizing different regions of the stomach of individual patients.

The diversity in the expression of Lewis antigens (Le) of 226 single colonies of Helicobacter pylori isolated from four regions of the stomach of eight adults is shown. Le(y) was expressed more in strains colonizing antrum than in strains colonizing fundus, whereas Le(x) was more common in fundus strains. cagA(+) strains were more associated with Le-negative strains.

A phase I biodistribution and pharmacokinetic trial of humanized monoclonal antibody Hu3s193 in patients with advanced epithelial cancers that express the Lewis-Y antigen.

PURPOSE: We report a first-in-man trial of a humanized antibody (hu3S193) against the Le(y) antigen. EXPERIMENTAL DESIGN: Patients with advanced Le(y)-positive cancers received four infusions of hu3S193 at weekly intervals, with four dose levels (5, 10, 20, and 40 mg/m(2)). The first infusion of hu3S193 was trace labeled with Indium-111, and biodistribution, pharmacokinetics, tumor uptake, and immune response were evaluated in all patients. RESULTS: A total of 15 patients (7 male/8 female; age range, 42-76 years; 6 breast, 8 colorectal cancer, and 1 non-small-cell lung cancer) were entered into the study. Transient grade 1 to 2 nausea and vomiting was observed following infusion of hu3S193 at the 40 mg/m(2) dose level only. There was one episode of dose-limiting toxicity with self-limiting Common Toxicity Criteria grade 3 elevated alkaline phosphatase observed in one patient with extensive liver metastases. The biodistribution of (111)In-hu3S193 showed no evidence of any consistent normal tissue uptake, and (111)In-hu3S193 uptake was observed in cutaneous, lymph node, and hepatic metastases. Hu3S193 displayed a long serum half-life (T(1/2)beta = 189.63 +/- 62.17 h). Clinical responses consisted of 4 patients with stable disease and 11 patients with progressive disease, although one patient experienced a 89% decrease in a lymph node mass, and one patient experienced inflammatory symptoms in cutaneous metastases, suggestive of a biological effect of hu3S193. No immune responses (human anti-human antibody) to hu3S193 were observed. CONCLUSION: Hu3S193 is well tolerated and selectively targets tumors, and the long half-life and biological function in vivo of this antibody makes it an attractive potential therapy for patients with Le(y)-expressing cancers.

Relevance of fucosylation and Lewis antigen expression in the bacterial gastroduodenal pathogen Helicobacter pylori.

Helicobacter pylori is a prevalent bacterial, gastroduodenal pathogen of humans that can express Lewis (Le) and related antigens in the O-chains of its surface lipopolysaccharide. The O-chains of H. pylori are commonly composed of internal Le(x) units with terminal Le(x) or Le(y) units or, in some strains, with additional units of Le(a), Le(b), Le(c), sialyl-Le(x) and H-1 antigens, as well as blood groups A and B, thereby producing a mosaicism of antigenic units expressed. The genetic determination of the Le antigen biosynthetic pathways in H. pylori has been studied, and despite striking functional similarity, low sequence homology occurs between the bacterial and mammalian alpha(1,3/4)- and alpha(1,2)-fucosyltransferases. Factors affecting Le antigen expression in H. pylori, that can influence the biological impact of this molecular mimicry, include regulation of fucosyltransferase genes through slipped-strand mispairing, the activity and expression levels of the functional enzymes, the preferences of the expressed enzyme for distinctive acceptor molecules and the availability of activated sugar intermediates. Le mimicry was initially implicated in immune evasion and gastric adaptation by the bacterium, but more recent studies show a role in gastric colonization and bacterial adhesion with galectin-3 identified as the gastric receptor for polymeric Le(x) on the bacterium. From the host defence aspect, innate immune recognition of H. pylori by surfactant protein D is influenced by the extent of LPS fucosylation. Furthermore, Le antigen expression affects both the inflammatory response and T-cell polarization that develops after infection. Although controversial, evidence suggests that long-term H. pylori infection can induce autoreactive anti-Le antibodies cross-reacting with the gastric mucosa, in part leading to the development of gastric atrophy. Thus, Le antigen expression and fucosylation in H. pylori have multiple biological effects on pathogenesis and disease outcome.

Towards abolition of immunogenic structures in insect cells: characterization of a honey-bee (Apis mellifera) multi-gene family reveals both an allergy-related core alpha1,3-fucosyltransferase and the first insect Lewis-histo-blood-group-related antigen-synthesizing enzyme.

Glycoproteins from honey-bee (Apis mellifera), such as phospholipase A2 and hyaluronidase, are well-known major bee-venom allergens. They carry N-linked oligosaccharide structures with two types of alpha1,3-fucosylation: the modification by alpha1,3-fucose of the innermost core GlcNAc, which constitutes an epitope recognized by IgE from some bee-venom-allergic patients, and an antennal Lewis-like GalNAcbeta1,4(Fucalpha1,3)GlcNAc moiety. We now report the cloning and expression of two cDNAs encoding the relevant active alpha1,3-FucTs (alpha1,3-fucosyltransferases). The first sequence, closest to that of fruitfly (Drosophila melanogaster) FucTA, was found to be a core alpha1,3-FucT (EC 2.4.1.214), as judged by several enzyme and biochemical assays. The second cDNA encoded an enzyme, most related to Drosophila FucTC, that was shown to be capable of generating the Le(x) [Galbeta1-4(Fucalpha1-3)GlcNAc] epitope in vitro and is the first Lewis-type alpha1,3-FucT (EC 2.4.1.152) to be described in insects. The transcription levels of these two genes in various tissues were examined: FucTA was found to be predominantly expressed in the brain tissue and venom glands, whereas FucTC transcripts were detected at highest levels in venom and hypopharyngeal glands. Very low expression of a third homologue of unknown function, FucTB, was also observed in various tissues. The characterization of these honey-bee gene products not only accounts for the observed alpha1,3-fucosylation of bee-venom glycoproteins, but is expected to aid the identification and subsequent down-regulation of the FucTs in insect cell lines of biotechnological importance.

Overexpression of fucosyltransferase IV in A431 cell line increases cell proliferation.

Fucosyltransferase IV is an essential enzyme that catalyzes the synthesis of fucosylated oligosaccharides by transferring GDP-fucose to the terminal N-acetylglucosamine with the alpha1,3-linkage. Lewis Y oligosaccharide has a terminal alpha1,3-linked fucose residue and elevation of Lewis Y level is seen in many epithelial cancers. The mechanism of Lewis Y elevation in neoplastic cells is still largely unknown. To study the impact of fucosyltransferase IV on Lewis Y expression and its role on neoplastic cell proliferation, a pEGFP-N1-FUT4 recombinant plasmid was developed and stably transfected into A431 cells. We found that fucosyltransferase IV overexpression promoted cell proliferation and increased the expression of proliferating cell nuclear antigen that correlated with Lewis Y augmentation. Cell cycle analysis demonstrated that fucosyltransferase IV overexpression facilitated cell cycle progression. In conclusion, fucosyltransferase IV overexpression augments Lewis Y expression to trigger neoplastic cell proliferation. These studies suggest that fucosyltransferase IV may serve as a potential therapeutic target for the treatment of Lewis Y-positive epithelial cancers.

Characteristic expression of Lewis-antigenic glycolipids in human ovarian carcinoma-derived cells with anticancer drug-resistance.

By comparing ovarian carcinoma-derived KF28 cells with the corresponding anticancer drug-resistant cells, the taxol- and cisplatin-resistant properties were found to be closely related with MDR1 and BSEP, and MRP2 transporters, respectively. In addition to the transporters expression, the amounts of glycolipids, particularly their longer carbohydrate structures, in the resistant cells increased to 3-4-fold of those in the sensitive cells due to enhanced transcription of the respective glycosyltransferases. The major glycolipids in the sensitive and resistant cells were GlcCer and Gb(3)Cer, respectively, and extension of the carbohydrate structure into Lewis antigen characteristically occurred in the resistant cells. Le(b), which was not detected in the cisplatin-resistant cells, was present in the taxol-resistant cells, while Le(x) was present in the cisplatin-resistant cells at a higher concentration than in the taxol-resistant cells. 2-Hydroxy fatty acids were significantly abundant in glycolipids of the resistant cells, but they were not detected in free ceramides or sphingomyelin, indicating that the enhanced synthesis of glycolipids in the resistant cells was not linked with the removal pathway for virulent ceramides derived from sphingomyelin. The resistant cells with abundant glycolipids exhibited lower membrane fluidity than the KF28 cells, and this property might be involved in the anticancer drug-resistance.