Carbohydrate carriers affect adhesion of H. pylori to immobilized Leb-oligosaccharide

The present study involved comparison of adhesion of Helicobacter pylori KH202 to immobilized Le(b)-oligosaccharide carried on different carriers, i.e. Leb-oligosaccharide conjugated with polyacrylamide, bovine serum albumin, and dipalmitoylphosphatidylethanolamine (Le(b)-PAA, Le(b)-BSA, and Le(b)-DPPE). All of the Le(b)-oligosaccharide-carrying neoglycoconjugates served as ligands for H. pylori. However, H. pylori required 10-fold and 100-fold quantities of Le(b)-antigen to adhere to Le(b)-PAA and to Le(b)-DPPE in comparison to the quantity of Le(b)-antigen needed to adhere to Le(b)-BSA, respectively. H. pylori adhesion to Le(b)-PAA and Le(b)-DPPE was clearly inhibited by Le(b)-oligosaccharide, but adhesion to Le(b)-BSA was hardly inhibited by the oligosaccharide. Therefore, the carbohydrate carrier affects the affinity of H. pylori KH202 toward Le(b)-antigen, although the bacteria recognize Le(b)-antigen regardless of the carbohydrate carrier.     

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.     

Amino acid sequence of an unique ribonuclease with a C-terminus rich in O-glycosylated serine and threonine from culture medium of Lentinus edodes

The mushroom Lentinus edodes produces three base-non-specific and acid ribonucleases, RNases Le2, Le37, and Le45. The latter two are excreted from mycelia into the medium. The primary structure of RNase Le37, which had a molecular mass of 37 kDa, was sequenced. It was a member of the RNase T2 family, as is RNase Le2. RNase Le37 was some 30 amino acid residues longer at the C-terminal end than RNase Le2. The C-terminal region of RNase LE37 was rich in O-glycosylated serine and threonine. In fungal glucoamylases and chitinases, which hydrolyze raw-starch and chitin, respectively, have structures resembling the structure of the C-terminal of RNase Le37.     

Characterization of Ribonucleases from Culture Medium of Lentinus edodes

Lentinus edodes (shiitake) cultivated in potato dextrose medium produced five RNases in the culture filtrate. The two major RNases (RNase Le37 and RNase Le45) were highly purified and their molecular masses, base specificities, N-terminal amino acid sequences, and amino acid compositions were analyzed and compared to RNase Le2 isolated from the fruit bodies of the same mushroom. RNase Le37 and RNase Le45 are base non-specific and adenylic acid preferential RNases like RNase Le2 and their N-terminal sequences are very similar to RNase Le2, but they are glycoproteins and their amino acid compositions are significantly different from that of RNase Le2. In addition to these enzymes, a guanylic acid-specific RNase with a molecular mass 13 kDa was partially purified. Since RNase Le2, which has very similar N-terminal sequence to RNase Le 37 and RNase Le 45, was not excreted from the mycelia, the analysis of the structures of these two excreted RNase may shade a light on the mechanism of excretion of RNases in this organism.     

Role of futC slipped strand mispairing in Helicobacter pylori Lewisy phase variation

The O antigen of the Helicobacter pylori lipopolysaccharide is composed of repeating units of fucosylated Lewis (Le) antigens. The alpha(1,2)-fucosyltransferase (futC) of H. pylori, which catalyzes the conversion of Le(x) to Le(y) by addition of fucose, is subject to slipped-strand mispairing involving a homonucleotide (poly-C) tract. To explore the distribution of Le phenotypes within H. pylori cells grown in vitro, 379 single colonies of strain J166 were examined for Le expression. Two major populations with reciprocal Le(x)/Le(y) phenotypes were identified. Phenotypes correlated with futC frame status, suggesting that strain J166 represents a mixed population with respect to futC poly-C tract length, which was confirmed by a translational reporter. After hundreds of generations in vitro, phenotypes did not change significantly, indicating that the observed J166 Le diversity reflects the founding population. Since slipped-strand mispairing in the futC poly-C tract was postulated to explain the Le(y) phenotypic change observed in J166 derivative strain 98-169 isolated 10 months after rhesus monkey challenge, in trans complementation with in-frame futC was performed. Le(y) synthesis was restored and Le(x) expression was reciprocally lowered. From these studies, we confirmed the principal role of futC slipped-strand mispairing in Le antigenic variation in vitro and in vivo.     

Amino acid sequence and characterization of a nuclease (nuclease Le3) from Lentinus edodes

The fruit body of shiitake (Lentinus edodes) produces two acid nucleases, nuclease Le1 and nuclease Le3, both of which are thought to be candidates for the enzyme that produces a flavorful substance, 5'-GMP, and the primary structure of one of the nucleases, nuclease Le1, has been analyzed by both protein chemistry and gene cloning [Biosci. Biotechnol. Biochem. 64, 948-957 (2000)]. In this study the amino acid sequence of nuclease Le3 was analyzed by protein chemistry and gene cloning. Nuclease Le3 is a glycoprotein that contains 280 amino acid residues, and the molecular mass of the protein moiety of nuclease Le3 is 31,045. The nucleotide sequence of the cDNA and genomic DNA encoding nuclease Le3 revealed the presence of an 18-residue putative signal peptide. Nuclease Le3 contains 170, 108, and 98 amino acid residues that are identical to residues of nuclease Le1, nuclease P1, and nuclease S, respectively. The amino acid residues involved in coordination with Zn2+ atoms in nuclease P1 are all conserved in nuclease Le3. Nuclease Le3 contains 9 half-cystine residues, and 7 of them are located in the same positions as in nuclease Le1.     

着床期小鼠子宫内膜Le~y糖蛋白的动态变化

为了解子宫内膜Ｌｅ~ｙ糖蛋白在胚泡着床期间的变化,以及与胚泡表面阶段特异性Ｌｅ~ｙ抗原出现的关系,应用对Ｌｅ~ｙ寡糖特异的ＡＨ－６单抗为探针,通过ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔ免疫酶标染色,观察了小鼠子宫内膜Ｌｅ~ｙ糖蛋白在着床期的动态变化和分布特点。结果表明：（１）未孕及着床前后的小鼠子宫内膜均含Ｌｅ~ｙ糖蛋白Ｍｒ５０～２００ｋＤ）,未孕内膜的含量明显高于着床期间的样品;（２）在着床日（Ｄ４）样品可见微量１６ｋＤ的Ｌｅ~ｙ糖蛋白条带;（３）未孕至Ｄ４的子宫内膜均有Ｌｅ~ｙ糖蛋白分泌至宫腔,但呈渐减趋势,未孕样品Ｌｅ~ｙ糖蛋白主要为分泌型,而Ｄ４样品Ｌｅ~ｙ糖蛋白主要存在于子宫内膜,宫腔液含量很少;结果提示子宫内膜Ｌｅ~ｙ糖蛋白在着床期间具有含量、组成和分布上的动态变化,这些变化可能与胚泡着床以及胚泡在宫腔内其表面Ｌｅ~ｙ糖抗原转为阳性密切有关。     

Catalytic Reaction of Basidiomycete Lentinula edodes Cytochrome P450, Le.CYP1 Enzyme Produced in Yeast

The basidiomycete Lentinula edodes (Le.) cytochrome P450, Le.CYP1 was functionally expressed in Saccharomyces cerevisiae. The microsomal fraction containing Le.CYP1 was prepared from the recombinant yeast and the Le.CYP1 was analyzed. The 7-ethoxycoumarin and benzo(a)pyrene were found to be the substrates of Le.CYP1 enzyme. Le.CYP1 converted 7-ethoxycoumarin to 7-hydroxycoumarin.     

超滤分离和鉴定三种香菇多糖

用热水从香菇子实体中浸提出香菇多糖,采用两种超滤陶瓷膜将粗多糖分级成三部分Le1,Le2和Le3。所有的这三种多糖都由两组分所组成,采用凝胶过滤色谱测定了多糖分子量,13CNMR和IR光谱测定显示多糖Le1为含α糖甙键的多糖,多糖Le3为含β糖甙键的多糖。采用气相色谱法测定了三种多糖的单糖组成,结果显示三种多糖都由葡糖糖,阿拉伯糖,木糖,甘露糖和半乳糖组成,Le1,Le2和Le3中阿拉伯糖、木糖、甘露糖、半乳糖、葡萄糖的摩尔比分别为0.15∶0.52∶1.00∶1.20∶7.20、0.21∶0.68∶1.00∶1.02∶11.56、0.29∶0.42∶1.00∶0.85∶16.20。三种多糖Le1,Le2和Le3的平均分子量分别为4.02×104、2.16×105和8.93×105。     

Characterization of anti-Leb antibody purified from serum of immunized rabbits.

An anti-Le(b) antibody was produced in sera of rabbits by immunization with human saliva from blood group O Le(a-b+) secretor and purified by sequential use of silica beads immobilized with H type 1, Le(a) and Le(b). The purified antibody agglutinated only Le(a-b+) red cells irrespective of their ABO blood type. Hemagglutination reaction with the antibody of blood group O Le(a-b+) red cells was inhibited not only by saliva samples from blood group Le(a-b+) secretors and Synsorb beads immobilized with Le(b) hapten, but also weakly by Synsorb immobilized with Y and H type 2 haptens.     

An investigation of the interactions of E-selectin with fuco-oligosaccharides of the blood group family

This investigation is concerned with assignments of Lewis(a) (Le(a)) and Le(x) analogs on linear and branched di- to hexasaccharide backbones as components of the recognition motifs for E-selectin. The influence of the location of fucose residue(s) was investigated using 14 structurally defined and variously fucosylated oligosaccharides in biotinylated form or as neoglycolipids in static binding assays, in microwells, and on thin-layer chromatograms. Results of the two assay systems were in agreement overall and showed that the recognition motifs for E-selectin include 4-fucosyl-lacto (Le(a)) and 3-fucosyl-neo-lacto (Le(x)) sequences strictly at capping positions and not Le(x) at an internal position as a part of VIM-2 antigen sequence. There is greater potency of the Le(a) over the Le(x) series. Additional fucose residues alpha1-2-linked to neighboring galactoses or alpha1-3-linked to inner N-acetyglucosamines or to reducing-terminal glucose residues of the tetrasaccharide backbone had little or no effect on the selectin binding. E-selectin binding to the Le(a) or Le(x )capping motif on a 3-linked branch was equivalent to the binding on the corresponding linear backbone. A lack of E-selectin binding to the Le(x) motif capping a 6-linked branch and to the Le(x) trisaccharide linked to biotin via a nine-carbon spacer indicates that the -GlcNAcbeta1-3Gal- sequence on the oligosaccharide backbone adjoining the Le(x) is a part of recognition motif for E-selectin. These findings contribute to understanding the molecular basis of E-selectin recognition and could influence future designs of selectin antagonists as possible therapeutic substances.     

Expression of fucosyltransferases in skin, conjunctiva, and cornea during human development

During human development, type-1-precursor, sialyl-Le a, and Le x antigens were present in the periderm of skin and eye at week 6. The Le x antigen disappeared from cornea at 10 weeks and then from skin at 20 weeks. H-type-1, Le a, Le b, sialyl-Le a, H-type-2, sialyl-Le x, and Le y were found in cornea, conjunctiva, and periderm between 10 and 20 weeks. They disappear from the skin (at week 20) and progressively reappear in skin derivatives, especially in the epithelium of sweat glands. The secretory part of the sweat gland is type-1-precursor and H-type-1 positive while its excretory part is Le a, Le b, sialyl-Le a, and Le y positive. On the eye surface the disappearance of Le x at 10 weeks and of the H-type-1, sialyl-Le x, and Le y at week 35 starts in the central cornea in front of the lens. The corneal epithelium and the conjunctiva have similar antigens to those of excretory and secretory parts of the sweat gland, respectively. Invaginations and folding of the epidermis might preserve the embryonic staining. We propose that fucosylation patterns are associated with the embryonic origin and differentiation stage of tissue. The early and transient presence of Le x is associated with FUT4 or FUT9 activities, while the late appearance of Lewis antigens is related to other alpha3-fucosyltransferases.     

Acquisition of P-selectin binding activity by en bloc transfer of sulfo Le(x) trisaccharide to the cell surface: comparison to a sialyl Le(x) tetrasaccharide transferred on the cell surface

Sialyl Le(x), NeuNAcalpha2 --> 3Galbeta1 --> 4(Fucalpha1 --> 3)GlcNAcbeta --> R, is known to be a ligand for E-selectin in various assays. The sulfated counterpart of sialyl Le(x), sulfo Le(x), (Sulfo --> 3) Galbeta1 --> 4 (Fucalpha1 --> 3) GlcNAcbeta --> R, was also shown to be a ligand for E-selectin in solid-phase assays employing immobilized oligosaccharides. In order to determine whether sulfo Le(x) structure on the cell surface also works as E-selectin or P-selectin ligand, a novel approach for in vitro transfer of oligosaccharides (S. Tsuboi, Y. Isogai, N. Hada, J. K. King, O. Hindsgaul, and M. Fukuda (1996) J. Biol. Chem. 271, 27213-27216) was utilized. A synthetic GDP-fucose harboring sialyl Le(x) or sulfo Le(x) oligosaccharide was enzymatically transferred to Chinese hamster ovary (CHO) cells with a milk fucosyltransferase. The resultant cells, CHO-sialyl Le(x) and CHO-sulfo Le(x) were tested for adhesion to E-selectin. IgG or P-selectin. IgG chimeric protein coated on plates. The results indicate that CHO-sialyl Le(x) adhered efficiently to E-selectin, while adhesion of CHO-sulfo Le(x) was very poor despite the fact that near equal number of the ligands had been attached to the cell surface. In contrast, CHO-sulfo Le(x) adhered efficiently to P-selectin, while CHO-sialyl Le(x) adhered modestly to P-selectin. These results demonstrate that sialyl Le(x) and sulfo Le(x) structures on the cell surface differ substantially in their ability to adhere to E- and P-selectin.     

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.     

Internode length in Pisum. Biochemical expression of the le gene in darkness

Ross, J. J. and Reid, J. B. 1989. Internode length in Pisum. Biochemical expression of the Le gene in darkness.
The Le gene appears to be biochemically expressed in dark-grown pea ( Pisum sativum L.) plants since the previously reported difference in metabolism of [³H]-GA₃₀ between light-grown Le and Le plants was also observed in darkness. Furthermore, both light- and dark-grown Le plants contained more endogenous GA¹, -like substance than did comparable Le plants. Darkness did not appear to significantly increase the accumulation of GA¹, in either Le or Le plants, although confirmation of GA¹ levels by gas chromatography-selected ion monitoring is still required. The results support previous findings that the overall metabolism of [³H]-GA₂₀, is accelerated by darkness. The evidence presented here supports previous suggestions that darkness acts on internode length by increasing some aspect of GA sensitivity.     

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.     

The discovery, biology, and drug development of sialyl Lea and sialyl Lex

The discoveries of sialylated, fucosylated lacto-, and neolacto-type carbohydrate structures were accomplished with the aid of analytical methods and monoclonal antibodies such as the immunostaining of thin layer chromatograms. Based on the use of such antibodies, these structures, notably sialyl Le(a) and sialyl Le(x), were demonstrated to be highly expressed in many malignant cancers. A diagnostic assay using one of these antibodies (CA19-9) is now established as one of the more commonly used assays for pancreatic and gastrointestinal cancers worldwide. Upon further study, several laboratories have demonstrated that the level of expression of these carbohydrate tumor markers is also positively correlated with patient survival and is a prognostic indicator of metastatic disease. Concurrent with this finding, both sialyl Le(a) and sialyl Le(x) were shown to bind to a family of carbohydrate-binding proteins involved in the extravasation of cells from the bloodstream, called the selectins. Thus, sialyl Le(a) and sialyl Le(x) expressed on cell surfaces play functional roles in medical conditions that require extravasation of cells from the bloodstream which include a wide range of inflammatory diseases and cancer metastasis. Many studies have confirmed the function of sialyl Le(a) and sialyl Le(x) in animal models of these diseases and the inhibition of binding of sialyl Le(a) and sialyl Le(x) to the selectins is a validated drug target in the pharmaceutical industry. Thus, a new class of drugs, arising from the field of glycobiology, is based on the rational design of small molecule drugs that mimic the structures sialyl Le(a) and sialyl Le(x) and can potently inhibit their functional binding to the selectins.