Structural convergence of antibody binding of carbohydrate determinants in Lewis Y tumor antigens

Antibodies targeting human epithelial carcinomas bearing Lewis Y (Le(y)) carbohydrate antigens provide a striking illustration of convergent immune recognition. We report a 1.9A resolution crystal structure of the Fab of a humanized antibody (hu3S193) in complex with the Le(y) tetrasaccharide, Fuc(alpha 1-->2)Gal(beta 1-->4)[Fuc(alpha 1-->3)]GlcNAc. Comparisons of the hu3S193 and BR96 antibodies bound to Le(y) tumor antigens revealed extremely similar mechanisms for recognition of the carbohydrate epitopes. Solvent plays a critical role in hu3S193 antibody binding to the Le(y) carbohydrate epitope. Specificity for Le(y) is maintained because a conserved pocket accepts an N-acetyl group of the core Gal(beta 1-->4)GlcNAc disaccharide. Closely related blood-group determinants (Le(a) and Le(b)) cannot enter the specificity pocket, making the Le(y) antibodies promising candidates for immunotherapy of epithelial cancer.     

Immunoreactivity of Lewis blood group and mucin peptide core antigens: correlations with grade of dysplasia and malignant transformation in the colorectal adenoma-carcinoma sequence

Previous studies on the immunoreactivity of various mucin peptide and carbohydrate antigens in neoplastic colorectal tissues led to at least in part contradictory results. Therefore, we investigated a series of 42 adenomas and 44 carcinomas applying monoclonal antibodies (mabs) directed against Lewis blood group antigens (sialyl-Le(a), Le(x), sialyl-Le(x), Le(y)) as well as mucin peptide cores (MUC1, MUC2 and MUC5AC) by immunohistochemistry. A statistically significant positive correlation between the development of high-grade dysplasia in colorectal adenomas and the immunoreactivity of Le(y) and MUC1 epitopes was observed, whereas MUC2 exhibited a significant negative correlation. The reactivity of the other epitopes did not show an association with the progression of malignant transformation. Colorectal carcinomas were subdivided according to their histopathological subtype. The immunohistochemical staining resulted in a significantly stronger MUC2 reactivity of mucinous vs. tubular adenocarcinomas. Immunoreactivity of the MUC1-specific mab, which does not react with the fully glycosylated peptide core, showed a statistically non-significant inverse tendency, whereas all carbohydrate antigens displayed a strong expression in both tumor subtypes. Furthermore, correlations between mucin peptide and carbohydrate epitope labelling were evaluated. Progression of the adenoma-carcinoma sequence was accompanied by an increase of Le(y) as well as MUC1 antigen and an increase of all Lewis antigens compared to MUC2 immunoreactivity. On the other hand, mucinous carcinomas exhibited an inverse pattern. In conclusion, these results demonstrate that Le(y) and MUC1 immunoreactivity correlate with malignant transformation in the colorectum, whereas MUC2 represents a marker for low-grade dysplasia and the subtype of mucinous carcinomas.     

Interaction of perch fucolectin with Lewis antigens

Interaction of fucolectin of perch Perca fluviatilis (PFL) with a set of Lewis antigens was studied by monitoring changes in its tryptophan fluorescence. PFL bound Le(c) (H type 1)-pentasaccharide (Ka = 6.6 x 10(3) M(-1)) and H type 6-trisaccharide (Ka = 2.5 x 10(3) M(-1)); essentially weaker, with Le(b)-hexasaccharide (Ka = = 4.0 x 10(2) M(-1)); and failed to interact with Le(a)-, Le(x)-, and Le(d)-containing oligosaccharides. PFL belongs to a new type of the fucolectins recognizing H-disaccharide Fuc alpha1-2Gal within various antigens, including H type 1/2 and Le(b).     

Phenotypic variation in molecular mimicry between Helicobacter pylori lipopolysaccharides and human gastric epithelial cell surface glycoforms. Acid-induced phase variation in Lewis(x) and Lewis(y) expression by H. Pylori lipopolysaccharides.

Helicobacter pylori is an important gastroduodenal pathogen of humans whose survival in the gastric environment below pH 4 is dependent on bacterial production of urease, whereas above pH 4 urease-independent mechanisms are involved in survival, but that remain to be elucidated fully. Previous structural investigations on the lipopolysaccharides (LPSs) of H. pylori have shown that the majority of these surface glycolipids express partially fucosylated, glucosylated, or galactosylated N-acetyllactosamine (LacNAc) O-polysaccharide chains containing Lewis(x) (Le(x)) and/or Lewis(y) (Le(y)), although some strains also express type 1 determinants, Lewis(a), Lewis(b), and H-1 antigen. In this study, we investigated acid-induced changes in the structure and composition of LPS and cellular lipids of the genome-sequenced strain, H. pylori 26695. When grown in liquid medium at pH 7, the O-chain consisted of a type 2 LacNAc polysaccharide, which was glycosylated with alpha-1-fucose at O-3 of the majority of N-acetylglucosamine residues forming Le(x) units, including chain termination by a Le(x) unit. However, growth in liquid medium at pH 5 resulted in production of a more complex O-chain whose backbone of type 2 LacNAc units was partially glycosylated with alpha L-fucose, thus forming Le(x), whereas the majority of the nonfucosylated N-acetylglucosamine residues were substituted at O-6 by alpha-D-galactose residues, and the chain was terminated by a Le(y) unit. In contrast, detailed chemical analysis of the core and lipid A components of LPS and analysis of cellular lipids did not show significant differences between H. pylori 26695 grown at pH 5 and 7. Although putative molecular mechanisms affecting Le(x) and Le(y) expression have been investigated previously, this is the first report identifying an environmental trigger inducing phase variation of Le(x) and Le(y) in H. pylori that can aid adaptation of the bacterium to its ecological niche.     

Antigenic Mimicry Between Helicobacter pylori and Gastric Mucosa: Failure to Implicate Heat-Shock Protein Hsp60 Using Immunoelectron Microscopy

Background. Helicobacter pylori infection induces autoantibodies that cross-react with human gastric mucosa from infected individuals. Candidates for the antigens responsible for molecular mimicry causing autoreactivity include the heat-shock protein HspB (Hsp60, sometimes called Hsp54) or Lewis x and Lewis y carbohydrate antigens.
Objective. Our goal was to investigate the involvement of HspB (Hsp60) in autoreactivity between H. pylori and gastric biopsy tissue.
Materials and Methods. Immunoelectron microscopy was used to study cross-reactivity among biopsy tissues from a patient with gastritis, gastric ulcer, and duodenal ulcer and his own serum as well as reactivity with serum raised against HspB from H. pylori and monoclonal antibodies against Lewis antigens.
Results. The patient serum reacted with gastric mucosa, and the antibodies involved were predominantly IgG. Antibody raised to H. pylori HspB (Hsp60) reacted only with H. pylori cells but not with gastric mucosal tissue. In contrast, monoclonal antibodies specific for Lewis x and Lewis y antigens reacted with both H. pylori and human gastric epithelial tissue.
Conclusions. Hsp60 (Hsp54) is unlikely to be involved in autoreactivity seen in individuals infected with H. pylori. In contrast, we could not rule out the role of Lewis x and Lewis y carbohydrate antigens, expressed as a component of H. pylori lipopolysaccharides, in molecular mimicry and autoantibody production.     

Structural studies on lipopolysaccharides of serologically non-typable strains of Helicobacter pylori, AF1 and 007, expressing Lewis antigenic determinants.

In contrast to other Helicobacter pylori strains, which have serologically detectable Lewis(x)+ (Le(x)) and Lewis(y)++ (++Le(y)) antigenic determinants in the O-specific polysaccharide chains of the lipopolysaccharides, H. pylori AF1 and 007 were non-typable with anti-Le(x) and anti-Le(y) antibodies. The carbohydrate portions of the lipopolysaccharides were liberated by mild acid hydrolysis and subsequently studied by sugar and methylation analyses, 1H-NMR spectroscopy and electrospray ionization-mass spectrometry. Compared with each other, and with lipopolysaccharides of strains studied previously, the lipopolysaccharides of both AF1 and 007 showed similarities, but also differences, in the structures of the core region and O-specific polysaccharide chains. The O-specific polysaccharide chains of both strains consisted of a short or long polyfucosylated poly-N-acetyl-beta-lactosamine chains, which were distinguished from those of other strains by a high degree of fucosylation producing a polymeric Le(x)chain terminating with Le(x) or Le(y) units:[sequence: see text] where n = 0 or 1 in strain AF1 and 0 in strain 007, m = 0-2, 6-7 in strain AF1 and m = 0-2, 6-7 or approximately 40 in strain 007, the medium-size species being predominant. Therefore, compared with other strains, the lack of reactivity of lipopolysaccharide of H. pylori AF1 and 007 with anti-Le(x) and anti-Le(y) may reflect the presence of a polymeric Le(x) chain and has important implications for serological and pathogenesis studies. As the substitution pattern of a D-glycero-D-manno-heptose residue in the outer core varied in the two strains, and an extended DD-heptan chain was present in some lipopolysaccharide species but not in others, this region was less conservative than the inner core region. The inner core L-glycero-D-manno-heptose region of both strains carried a 2-aminoethyl phosphate group, rather than a phosphate group, as reported previously for other H. pylori strains.     

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.     

Expression of histo-blood group antigens by lipopolysaccharides of Helicobacter pylori strains from asian hosts: the propensity to express type 1 blood-group antigens

Past studies have shown that the cell surface lipopolysaccharides (LPSs) of the ubiquitous human gastric pathogen Helicobacter pylori (a type 1 carcinogen) isolated from people residing in Europe and North America express predominantly type 2 Lewis x (Le(x)) and Le(y) epitopes and, infrequently, type 1 Le(a), Le(b), and Le(d) antigens. This production of Lewis blood-group structures by H. pylori LPSs, similar to those found in the surfaces of human gastric cells, allows the bacterium to mimic its human niche. In this study, LPSs of H.pylori strains extracted from patients living in China, Japan, and Singapore were chemically and serologically analyzed. When compared with Western H.pylori LPSs, these Asian strains showed a stronger tendency to produce type 1 blood groups. Of particular interest, and novel observations in H.pylori, the O-chain regions of strains F-58C and R-58A carried type 1 Le(a) without the presence of type 2 Le(x), strains R-7A and H607 were shown to have the capability of producing the type 1 blood group A antigen, and strains CA2, H507, and H428 expressed simultaneously the difucosyl isomeric antigens, type 1 Le(b) and type 2 Le(y). The apparent proclivity for the production of type 1 histo-blood group antigens in Asian H.pylori LPSs, as compared with Western strains, may be an adaptive evolutionary effect in that differences in the gastric cell surfaces of the respective hosts might be significantly dissimilar to select for the formation of different LPS structures on the resident H.pylori strain.     

IgG antibodies to Lewis type 2 antigens in serum of H. pylori-infected and noninfected blood donors of different Lewis(a,b) blood-group phenotype

Individuals of the Le(b+)/secretor phenotype revealed a stronger natural immune response to Le(x) and Le(y) epitopes irrespective of Helicobacter pylori serologic status. In contrast, H. pylori-infected Le(b-) type individuals showed a significantly higher proportion of strong responders to Le(x) antigen compared with the H. pylori-uninfected subgroup. The data suggest that the immune response to Lewis type 2 determinants is related to both the H. pylori serologic status and the Le(a,b) phenotype of the host.     

Oligosaccharide specificity of the fucolectin from the bark of laburnum Laburnum anagyroides

A comparative study of thin carbohydrate specificity of the lectin from the bark of laburnum Laburnum anagyroides (LABA) and fucolectin from asparagus pea Tetragonolobus purpureus (TPA) was performed using inhibition of agglutination of the complex formed by H-active neoglycoprotein and nanoparticles of colloidal gold. Both lectins bound most strongly the H type 2 oligosaccharides comprising O-glycanes; however, TPA was almost unable to discriminate between them. LABA bound more weakly the H type 6 trisaccharide (Fuc alpha 1-2Gal beta 1-4Glc) and difucosyllactose (Fuc alpha 1-2Gal beta 1-4[Fuc alpha 1-3]Glc), a glucoanalogue of the Le(y) antigen, and, even more weakly, the Le(a) pentasaccharide lacto-N-fucopentaose II (Gal beta 1-3[Fuc alpha 1-4]GlcNAc beta 1-3Gal beta 1-4Glc). However, LABA did not bind the antigens Le(b), Le(c), and Le(d), very poorly interacted with the terminal Le(x), and somewhat more strongly bound the internal Le(x). The lectin also had a hydrophobic binding site. Both lectins exhibited a cluster effect with polymeric ligands (neoglycoproteins).     

Glycosyltransferases involved in type 1 chain and Lewis antigen biosynthesis exhibit glycan and core chain specificity

Sialyl Lewis A (SLe(a)), Lewis A (Le(a)), and Lewis B (Le(b)) have been studied in many different biological contexts, for example in microbial adhesion and cancer. Their biosynthesis is complex and involves beta1,3-galactosyltransferases (beta3Gal-Ts) and a combined action of alpha2- and/or alpha4-fucosyltransferases (Fuc-Ts). Further, O-glycans with different core structures have been identified, and the ability of beta3Gal-Ts and Fuc-Ts to use these as substrates has not been resolved. Therefore, to examine the in vivo specificity of enzymes involved in SLe(a), Le(a), and Le(b) synthesis, we have transiently transfected CHO-K1 cells with relevant human glycosyltransferases and, on secreted reporter proteins, detected the resulting Lewis antigens on N- and O-linked glycans using western blotting and Le-specific antibodies. beta3Gal-T1, -T2, and -T5 could synthesize type 1 chains on N-linked glycans, but only beta3Gal-T5 worked on O-linked glycans. The latter enzyme could use both core 2 and core 3 precursor structures. Furthermore, the specificity of FUT5 and FUT3 in Le(a) and Le(b) synthesis was different, with FUT5 fucosylating H type 1 only on core 2, but FUT3 fucosylating H type 1 much more efficient on core 3 than on core 2. Finally, FUT1 and FUT2 were both found to direct alpha2-fucosylation on type 1 chains on both N- and O-linked structures. This knowledge enables us to engineer recombinant glycoproteins with glycan- and core chain-specific Lewis antigen substitution. Such tools will be important for investigations on the fine carbohydrate specificity of Le(b)-binding lectins, such as Helicobacter pylori adhesins and DC-SIGN, and may also prove useful as therapeutics.     

Binding specificity of Salmonella plasmid-encoded fimbriae assessed by glycomics

The Salmonella enterica serotype Typhimurium (S. Typhimurium) genome encodes 12 intestinal colonization factors of the chaperone/usher fimbrial assembly class; however, the binding specificity is known for only one of these adhesins, known as type 1 fimbriae. Here we explored the utility of glycomics to determine the carbohydrate binding specificity of plasmid-encoded fimbriae from S. Typhimurium. A cosmid carrying the pef operon was introduced into Escherichia coli and expression of fimbrial filaments composed of PefA confirmed by flow cytometry and immune-electron microscopy. Plasmid-encoded fimbriae were purified from the surface of E. coli, and the resulting preparation was shown to contain PefA as the sole major protein component. The binding of purified plasmid-encoded fimbriae to a glycanarray suggested that this adhesin specifically binds the trisaccharide Galbeta1-4(Fucalpha1-3)GlcNAc, also known as the Lewis X (Le(x)) blood group antigen. Results from the glycanarray were validated by enzyme-linked immunosorbent assay (ELISA) in which plasmid-encoded fimbriae bound Le(x)-coated wells in a concentration-dependent manner. The binding of plasmid-encoded fimbriae to Le(x)-coated wells could be inhibited by co-incubation with soluble Le(x) antigen. Our results establish glycomic analysis as a promising new approach for determining the carbohydrate binding specificity of bacterial adhesins.     

Norwalk virus-like particle hemagglutination by binding to h histo-blood group antigens

Noroviruses are a major cause of epidemic acute nonbacterial gastroenteritis worldwide. Here we report our discovery that recombinant Norwalk virus virus-like particles (rNV VLPs) agglutinate red blood cells (RBCs). Since histo-blood group antigens are expressed on gut mucosa as well as RBCs, we used rNV VLP hemagglutination (HA) as a model system for studying NV attachment to cells in order to help identify a potential NV receptor(s). rNV VLP HA is dependent on low temperature (4 degrees C) and acidic pH. Of the 13 species of RBCs tested, rNV VLPs hemagglutinated only chimpanzee and human RBCs. The rNV VLPs hemagglutinated all human type O (11 of 11), A (9 of 9), and AB (4 of 4) RBCs; however, few human type B RBC samples (4 of 14) were hemagglutinated. HA with periodate- and neuraminidase-treated RBCs indicated that rNV VLP binding was carbohydrate dependent and did not require sialic acid. The rNV VLPs did not hemagglutinate Bombay RBCs (zero of seven) that lack H type 2 antigen, and an anti-H type 2 antibody inhibited rNV VLP HA of human type O RBCs. These data indicated that the H type 2 antigen functions as the rNV VLP HA receptor on human type O RBCs. The rNV VLP HA was also inhibited by rNV VLP-specific monoclonal antibody 8812, an antibody that inhibits VLP binding to Caco-2 cells. Convalescent-phase sera from NV-infected individuals showed increased rNV VLP HA inhibition titers compared to prechallenge sera. In carbohydrate binding assays, the rNV VLPs bound to synthetic Lewis d (Le(d)), Le(b), H type 2, and Le(y) antigens, and these antigens also inhibited rNV VLP HA of human type O RBCs. Overall, our results indicate that carbohydrate antigens in the gut are a previously unrecognized factor in NV pathogenesis.     

Conformational studies of Lewis X and Lewis A trisaccharides using NMR residual dipolar couplings.

The conformations of the histo-blood group carbohydrate antigens Lewis X (Le(x)) and Lewis A (Le(a)) were studied by NMR measurements of one-bond C-H residual dipolar couplings in partially oriented liquid crystal solutions. A strategy for rapid calculation of the difference between theoretical and experimental dipolar couplings of a large number of model structures generated by computer simulations was developed, resulting in an accurate model structure for the compounds. Monte Carlo simulations were used to generate models for the trisaccharides, and orientations of each model were sought that could reproduce the experimental residual dipolar coupling values. For both, Le(a) and Le(x), single low energy models giving excellent agreement with experiment were found, implying a compact rigidly folded conformation for both trisaccharides. The new approach was also applied to the pentasaccharides lacto-N-fucopentaose 2 (LNF-2) and lacto-N-fucopentaose 3 (LNF-3) proving its consistency and robustness. For describing the conformation of tightly folded oligosaccharides, a definition for characterization of ring planes in pyranoside chairs is proposed and applied to the analysis of the relation between the fucose and galactose residues in the epitopes, revealing the structural similarity between them.     

Glycan array analysis of the antigen repertoire targeted by tumor-binding antibodies

Immunization with whole cells has been used extensively to generate monoclonal antibodies, produce protective immune responses, and discover new disease antigens. While glycans are abundant on cell surfaces, anti-glycan immune responses have not been well-characterized. We used glycan microarrays to profile 49 tumor-binding monoclonal antibodies generated by immunizing mice with whole cancer cells. A substantial proportion (41%) of the tumor binding antibodies bound carbohydrate antigens. The antibodies primarily recognize a group of 5 glycan antigens: Sialyl Lewis A (SLeA), Lewis A (LeA), Lewis X (LeX), blood group A (BG-A), and blood group H on a type 2 chain (BG-H2). The results have important implications for monoclonal antibody production and cancer vaccine development.     

Expression of the Lewis group carbohydrate antigens during Xenopus development

We have examined the pattern of expression of the Lewis group carbohydrate antigens during the development of African toad Xenopus laevis. One of these antigens, Lewis x (Le(x), also known as SSEA-1), was previously shown to be involved in cell-cell adhesion in early mouse embryos and teratocarcinoma stem cells. Recently another member of these antigens, sialyl-Le(x), was found to be one of the major ligands for the selectin family of cell-cell adhesion molecules. In order to study the role of carbohydrate-mediated cell adhesion during Xenopus development, we first studied the expression pattern of the Le(x). We found that Le(x)was not expressed in early embryos, started to be expressed at the tail bud stage in anterior regions of the body such as the cement gland or head skin, and was gradually showed more posterial expression at later stages. At tadpole stage, it was also expressed on specific cell bodies in brain, and in axon region in brain and neural retina. Antibodies against Le(x)blocked neurite outgrowth in the explant culture of tadpole brain. One of the candidates for Le(x)carrier protein in the tadpole brain is a 200 kDa glycoprotein detected by Western blotting. In adult tissues, it was expressed in brain, testis, and gut, but not in kidney, lung, spleen, ovary, or muscle. We also examined the expression patterns of other Lewis group antigens. Among them, sialyl-Le(x)was expressed on endothelial cells and on leukocytes, suggesting the possibility that it functions as a ligand for selectin in Xenopus.     

Expression of cell surface Lewis X and Y antigens and FUT4 mRNA is increased in Jurkat cells undergoing apoptosis

Cell surface molecules undergo specific changes during apoptosis, including the expression of phosphatidylserine (PS) and some proteins and alterations in sugar chains. Among the various sugar chains on the cell surface, Lewis X (Le(X)) and Lewis Y (Le(Y)) antigens are key determinants for a variety of biological processes. We studied the changes in Le(X) and Le(Y) expression in Jurkat cells, a human T cell line, during apoptosis. Flow cytometry showed that Le(X) and Le(Y) antigen expression was enhanced on the cell surface during apoptosis induced by anti-Fas antibody. To clarify the mechanism of enhanced Le(X) and Le(Y) expression, we assessed the expression levels of fucosyltransferase (FUT1, 2, 3-5-6, 4, and 9) mRNAs that are predominantly expressed in Jurkat cells and which are considered to form Le(X) and Le(Y). The expression of FUT4 mRNA was up-regulated after exposing cells to anti-Fas antibody. Moreover, the increase in Le(X) and Le(Y) antigen levels was significantly suppressed by caspase 3 or 8 inhibitors. These results indicated that the induction of FUT (mainly FUT4), the gene expression of which is mediated by signals downstream of caspase 3, increases Le(X) and Le(Y) expression in apoptotic cells.     

Are Autoantibodies against Lewis Antigens Involved in the Pathogenesis of Helicobacter pylori-lnduced Peptic Ulcers?

We examined whether anti-Lewis x (Le(x)) and y (Le(y)) autoantibodies affect the pathogenesis of Helicobacter pylori-induced peptic ulcers. Of 11 patients with peptic ulcers, 10 patients had both anti-Le(x) and -Le(y) immunoglobulin G (IgG) antibodies, and 1 patient had only anti-Le(x) antibody. After successful eradication, we measured the serum titer of anti-Le(x) and -Le(y) antibodies. Six patients had a reduction of the titers of anti-Le(x) and/or -Le(y) antibodies, whereas no notable changes were detected in 5 patients in the follow-up. This result suggests that anti-Le(x) and -Le(y) autoantibodies had no critical role in the development of H. pylori-induced peptic ulcer.