The enzymatic sulfation of glycoprotein carbohydrate units: blood group T-hapten specific and two other distinct Gal:3-O-sulfotransferases as evident from specificities and kinetics and the influence of sulfate and fucose residues occurring in the carbohydrate chain on C-3 sulfation of terminal Gal

Enzymatic 3-O-sulfation of terminal ß-Gal residueswas investigated by screening sulfotransferase activity presentin 37 human tissue specimens toward the following synthesizedacceptor moieties: Galß1,3GalNAc-O-Al, Galß1,4GlcNAcß-O-Al,Galß1,3GlcNAcß-O-Al, and mucin-type Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bnstructures containing a C-3 methyl substituent on either Gal.Two distinct types of Gal: 3-O-sulfotransferases were revealed.One (Group A) was specific for the Galß1, 3GalNAc-linkage and the other (Group B) was directed toward the Galß1,4GlcNAcbranch ß1,6 linked to the blood group T hapten. Enzymeactivities found in breast tissues were unique in showing astrict specificity for the T-hapten. Galß-O-allylor benzyl did not serve as acceptors for Group A but were veryactive with Group B. An exainination of activity present insix human sera revealed a specificity of the serum enzyme towardß1,3 linked Gal, particularly, the T-hapten withoutß1,6 branching. Group A was highly active toward T-haptenlacrylamidecopolymer, anti-freeze glycoprotein, and fetuin O-glycosidicasialo glycopeptide; less active toward fetuin triantennaryasialo glycopeptide; and least active toward bovine IgG diantennaryglycopeptide. Group B was moderately and highly active, respectively,with the latter two glycopeptides noted and least active withthe first two. Competition experiments performed with Galß1,3GaLNAc-O-Aland Galß1,4GlcNAcß1,6(Galß1,3)GalNAc-O-Bnhaving a C-3 substituent (methyl or sulfate) on either Gal reinforcedearlier findings on the specificity characteristics of GroupA and Group B. Group A displayed a wider range of optimal activity(pH 6.0–7.4), whereas Group B possessed a peak of activityat pH 7.2. Mg²⁺ stimulated Group A 55% and Group B 150%, whereasMn⁺² stimulated Group B 130% but inhibited Group A 75%. Ca²⁺stimulated Group B 100% but inhibited Group A 35%. Group A andGroup B enzymes appeared to be of the same molecular size (<100,000Da) as observed by Sephacryl S-100 HR column chromatography.The following effects upon Gal: 3-O- sulfotransferase activitiesby fucose, sulfate, and other substituents on the carbohydratechains were noted. (1) A methyl or GlcNAc substituent on C-6of GalNAc diminished the ability of Galß1,3GalNAc-O-Alto act as an acceptor for Group A. (2) An 1,3-fucosyl residueon the ß1,6 branch in the mucin core structure didnot affect the activity of Group A toward Gal linked ß1,3to GalNAc-. (3) Lewis x and Lewis a terminals did not serveas acceptors for either Group A or B enzymes. (4) Eliminationof Group B activity on Gal in the ß1,6 branch owingto the presence of a 3-fucosyl or 6-sulfo group on GlcNAc didnot hinder any action toward Gal linked ß1,3 to GalNAc.(5) Group A activity on Gal linked ß1,3 to GalNAcremained imaffected by 3'-sulfation of the ß1,6 branch.The reverse was true for Group B. (6) The acceptor activityof the T-hapten was increased somewhat upon C-6 sulfation ofGalNAc, whereas, C-6 slalylation resulted in an 85% loss ofactivity. (7) A novel finding was that Galß1,4GlcNAcß-O-Aland Galß1,3GlcNAcß-O-M, upon C-6 sulfationof the GlcNAc moiety, became 100% inactive and 5- to 7-foldactive, respectively, in their ability to serve as acceptorsfor Group B. human tissues glycoprotein galactose:sulfotransferase specificities kinetic properties     

Rodents infected with Schistosoma mansoni produce cytolytic IgG and IgM antibodies to the Lewis x antigen

Schistosoma mansoni is a blood fluke that produces glyco-conjugatescontaining the Lewis x antigen (Le^x) Galß1     

Selection of carbohydrate-binding cell phenotypes using oligosaccharide-coated magnetic particles

Neoglycoconjugate coated magnetic beads were assessed for theirability to selectively isolate human cells with known anti-carbohydratereactivity. Four lung cancer cell lines, NCI-H146, NCI-N417D,SKMES-1, EKVX; two acute lymphoblastic leukemia lines, MOLT-4and CCRF-CEM; and the anti- Le^c (isolactosamine) hybridoma,LU-BCRU-G7, were tested. The neoglycoconjugates (biotinylatedpseudopolysaccharides) bound uniformly to streptavidin coatedmagnetic beads as demonstrated by FTTC labeled lectin. Streptavidinbeads alone did not bind to any of the cell types. The anti-Le^c hybridoma cell line, LU BCRU-G7, demonstrated binding onlyto Le^c pseudopolysaccharide coated magnetic beads. Subsequentincubation in the presence of unlabeled pseudopolysaccharideresulted in the release of the beads from the cell surface.Although there was some heterogeneity within the individuallung and leukemic cell lines, positive cells showed strong rosetteformation with the coated beads. The A_dl disaccharide coatedbeads showed binding in all four lung cancer cell lines, withthe Le^c and the H (type1) pseudopolysaccharide-bead conjugatesonly reactive in the N417 and H146 SCLC lines. The range ofL-selectin ligand-coated beads were all successful in bindingto the acute lymphoblastic leukemia cell lines MOLT4 and CCRF-CEM.This approach provides a versatile model for the study of cell-surfacecarbohydrate interactions that should find application in manyareas of cell biology. carbohydrate-coated magnetic beads cell selec-tion lectins neoglycoconjugates pseudopolysaccharides     

Alterations of O-glycan biosynthesis in human colon cancer tissues

Human colon cancer is associated with antigenic and structuralchanges in mucin-type carbohydrate chains (O-glycans). To elucidatethe control of the biosynthesis of these O-glycans in coloncancer, we have studied glycosyltransferase and sulphotransferaseactivities involved in the assembly of elongated O-glycan structures.We analysed homogenates prepared from cancer tissue, adjacentnormal and distal normal tissue from 20 patients. Several transferaseactivities showed pronounced changes in cancer tissue. The changescorrelate with previous findings of a loss of O-glycans in cancermucins, but did not always correlate with levels of Tn, sialyl-Tn,T and Le^x antigens in homogenates or with the differentiationstatus and Duke's stages of the cancer tissue or the patient'sblood type, sex and age. UDP-GlcNAc: Gal NAc-R ß3-N-acetylglucosaminyltransferase(where GlcNAc is N-acetyl-D-glucosamine and GalNAc is N-acetyl-D-galactosamine)synthesizing O-glycan core 3, GlcNAcß1-3GalNAc-, CMP-sialicacid: GalNAc-peptide     

Human melanoma and Chinese hamster ovary cells galactosylate n-alkyl-{beta}-glucosides using UDP gal:GlcNAc {beta}1,4 galactosyltransferase

We previously showed that human melanoma, CHO and other cellscan convert ß-xylosides into structural analogs ofganglioside G_M3. We have investigated several potential acceptorsincluding a series of n-alkyl-ß-D-glucosides (n =6–9). All were labeled with ³H-galactose when incubatedwith human melanoma cells. Octyl-ß-D-glucoside (GlcßOctyl)was the best acceptor, whereas neither octyl--D-glucoside norN-octanoyl-methylglucamine (MEGA 8) were labeled. Analysis ofthe products by a combination of chromatographic methods andspecific enzyme digestions showed that the acceptors first receiveda single Galß1,4 residue followed by an 2,3 linkedsialic acid. Synthesis of these products did not affect cellviability, adherence, protein biosynthesis, or incorporationof radio-labeled precursors into glycoprotein, glycolipid orproteoglycans. To determine which ß1,4 galactosyltransferase synthesized Galß1,4GlcßOctyl,we analyzed similar incubations using CHO cells and a mutantCHO line (CHO 761) which lacks GAG-core specific ß1,4galactosyltransferase. The mutant cells showed the same levelof incorporation as the control, eliminating this enzyme asa candidate. Thermal inactivation kinetics using melanoma cellmicrosomes and rat liver Golgi to galactosylate GlcßOctylshowed the same half-life as UDP-Gal:GlcNAc ß1,4 galactosyltransferase,whereas LacCer synthase was inactivated at a much faster rate.We show that GlcßOctyl is a substrate for purifiedbovine milk UDP-Gal:GlcNAc ß1,4 galactosyltransferaseFurthermore, the galactosylation of GlcßOctyl by CHOcell microsomes can be competitively inhibited by GlcNAc orGlcNAcßMU . These results indicate that UDP-Gal:GlcNAcß1,4 galactosyltransferase is the enzyme used forthe synthesis of the alkyl lactosides when cells or rat liverGolgi are incubated with alkyl ß glucosides. alkylglucosides galactosyltransferase glycolipid artificial acceptors     

Characterization of serum {beta}-glucuronyltransferase involved in chondroitin sulfate biosynthesis

We studied a glucuronyltransferase involved in chondroitin sulfate(CS) biosynthesis in a preparation obtained from fetal bovineserum by heparin-Sepharose affinity chromatography. This enzymetransferred GlcA from UDP-GlcA to the nonreducing GalNAc residuesof polymeric chondroitin. It required Mn²⁺ for maximal activityand showed a sharp pH optimum between pH 5.5 and 6.0. The apparentKm value of the glucuronyltransferase for UDP-GlcA was 51 µM.The specificity was investigated using structurally definedacceptor substrates, which consisted of chemically synthesizedtri-, penta-, and heptasaccharide-serines and various odd-numberedoligosaccharides with a GalNAc residue at the nonreducing terminus,prepared from chondroitin and CS by chondroitinase ABC digestionfollowed by mercuric acetate treatment. The enzyme utilizeda heptasaccharide-serine GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3Galß1-3Galß1-4Xylß1-O-Serand a pentasaccharide-serine GalNAcß 4GlcAß1-3Galß1-3Galß1-4Xylß1-O-Seras acceptors. In contrast, neither a trisaccharide-serine Galß1-3Galß1-4Xylß1-O-Sernor an     

Human Lewis {alpha}1->3/4fucosyltransferase: specificity of fucose transfer to GlcNAc{beta}1->3Gal{beta}1->4Glc{beta}1->1Cer (LcOse3Cer)

Biosynthesis of the Le^x series of carbohydrate antigens proceedsby fucose transfer in 13-linkage to the penultimate GlcNAc residueof a neolacto-series oligosaccharide acceptor, a reaction catalysedby multiple enzymes expressed in human tissues. Particularlybroad acceptor specificity, including the ability to catalysefucose transfer to both lacto- and neolacto-series acceptorsas well as the precursor Lc₃ structure (where Lc₃ lactotriaosylceramide,is GlcNAcß13Galß14Glcß1Cer), existsfor one human fucosyltransferase form, the Lewis 13/4fucosyltransferase(FucT-III). To determine if fucose transfer to Lc₃may representan alternate early step in Le^xor Le^a antigen biosynthesis withthis enzyme, the chemical structure of the fucosylated Lc₃ reactionproduct formed by the Lewis 13/4fucosyltransferase from Colo205 cells has been defined. Transfer of [¹⁴C]fucose to Lc₃ yieldeda labelled product migrating as a tetrasaccharide on thin layerchromatography plates. This product remained an acceptor forboth ß13- and ß14-galactosyl transfer onthe terminal GlcNAc residue. The product was degraded to a fucosylatedtrisaccharide derivative by bovine kidney ß-N-acetylglucosaminidase.Fast atom bombardment mass spectrometry and methylation analysisconfirmed that the product was composed exclusively of the followingstructure containing a fucose linked to the 3-position of theinternal Glc residue: GlcNAcß13Galß14Glcß11Cer Such a structure does not represent an intermdiate in Le^xorLe^a antigen biosynthesis. Thus, the evidence suggests that Le^xor Le^a antigen synthesis results exclusively from fucosylationof complete core chains. fucosyltransferase lacto-series LcOse₃Cer Lewis antigen transfer specificity     

Purification and characterization of avian {beta}1,4 galactosyltransferase: comparison with the mammalian enzyme

Avian ß1,4 galactosyltransferase (GalTase) was purifiedfrom chicken serum, partially characterized, and compared tomammalian GalTase using antibody cross-reactivity, North-ernblot hybridization and amino acid sequence analysis. The enzymewas purified to apparent homogeneity by lactalbumin(LA)-agaroseaffinity chromatography followed by preparative SDS-polyacrylamidegel electrophoresis, and identified as two proteins of apparentmolecular masses of 39 and 46 kD. Chicken serum GalTase hada K_m for UDPGal of 42 µM, for GlcNAc of 10 mM and hadoptimal activity in the presence of 10–20 mM MnCl₂ Substrateand linkage specificity analyses indicated that the purifiedenzyme behaves as a traditional Gal ß1,4 GlcNAc:GalTase,since: (i) the avian ß1,4 GalTase bound to -LA; (ii)terminal GlcNAc residues served as good acceptors for chickenserum GalTase; (iii) the enzyme was inhibited by high concentrationsof GlcNAc; (iv) the galactosylated product was sensitive toß1,4-specific ß-galactosidase. Finally,the disaccharide reaction product comigrated with authenticß1,4 N-acetyllactosamine standard. No other GalTaseactivities were detectable using a battery of defined glycosidesubstrates. Polyclonal antibodies raised against the two gel-purifiedGalTase proteins showed reactivity with avian GalTase by ELISAand immunoprecipitation assays. The antibodies also inhibitedGalTase activity toward both high mol. wt and monosaccharideacceptor substrates. Despite similar kinetics and substratespecificity, the avian and mammalian GalTases showed littleoverall structural similarity, since polyclonal anti-avian GalTaseIgG failed to react with mammalian GalTase purified from bovinemilk, and conversely anti-bovine milk GalTase IgG did not reactwith the avian enzyme. Furthermore, in Northern blot analysis,no hybridization was detected when chicken embryo liver poly(A)⁺RNA was probed with a mouse GalTase cDNA, even under conditionsof reduced stringency. Amino acid sequence analysis identifiedthree of five tryptic peptides that are homologous to the mammaliansequence within a putative substrate binding domain and thecarboxy terminal domain of the enzyme. Their overall structuraldisparity leads us to believe that regions of homology betweenthe avian and mammalian GalTases may represent active sitesof the enzyme. avian ß1,4 galactosyltransferase homology mammalian purification     

Production of sialyltrisaccharides using β-galactosidase andtrans-sialidase in one pot

Sialyltrisaccharides based on β-galactosyldisaccharides were synthesized using β-galactosidase andtrans-sialidase in one pot. Using β-galactosidase fromBacillus circulans andtrans-sialidase fromTrypanosoma cruzi simultaneously, 6 mM sialyltrisaccharides composed of about 95% NeuAcα(2,3)Galβ(1,4)GlcNAc and 5% NeuAcα(2,3)Galβ(1,6)GlcNAc were produced from a reaction mixture containing 25 mM 0-nitrophenyl-β-D-galactopyranoside, 100 mM N-acety lglucosamine and 10 mM p-nitrophenyl-α-D-N-acetylneuraminic acid. One beauty of this reaction was that a secondary hydrolysis of the disaccharide intermediate occurring between the activated galactopyranoside and N-acetylglucosamine was prevented. Using β-galactosidase fromEscherichia coli and the sametrans-sialidase, 15 mM sialyltrisaccharides composed of about 90% NeuAcα(2,3)Galβ(1,6)GlcNAc and 10% NeuAcα(2,3)Galβ (1,4)GlcNAc were produced from a reaction mixture containing 400 mM galactose, 800 mM N-acetylglucosamine and 20 mMp-nitrophenyl-α-D-N-acetylneuraminic acid. In this study, the reverse-galactosylation reaction between galactose and N-acetylglucosamine was dominant since the disaccharide intermediate mainly resulted in the sialylated product.     

Tritium labelling of amino sugars at C-2 by alkaline epimerization in tritiated water

N-Acetyl-D-[2-³H]glucosamine was synthesized from N-acetyl-D-mannosamineby alkaline 2-epimerization in pyridine containing ³H₂O andnickelous acetate. The reaction involves reversible formationof an enol intermediate and therefore also resulted in incorporationof tritium into N-acetylmannosamine. After completed reaction,the two N-acetylhexosamines were separated from other radioactiveproducts and Morgan-Elson chromogens by chromatography on acolumn of Sephadex G-10, which was eluted with 10% ethanol,and were then separated from each other by chromatography onSephadex G-15 in 0·27 M sodium borate (pH 7·8).The location of the incorporated tritium was established bytreatment of the N-acetylhexosamines with borate under the conditionsof the Morgan-Elson reaction, which converts the sugars to Kuhn'schromogen I with concomitant loss of the C-2 hydrogen. As expected,this treatment resulted in the formation of ³H₂O, indicatingthat the tritium was located at C-2. [2-³H]Glucosamine was preparedby acid hydrolysis of the labelled N-acetylglucosamine and wasconverted to [2-³H]glucosamine 6-phosphate by incubation withhexokinase and ATP. The sugar phosphate was used as a substratefor glucosamine 6-phosphate deaminase (isomerase, EC 5.3.1.10 [EC] )in a simple ³H₂O release assay. N-acetyl[2-³H]glucosamine N-acetyl[2-³H]mannosamine [2-³H]glucosamine glucosamine 6-phosphate deaminase [2-³H]mannosamine     

Schistosoma mansoni cercarial glycolipids are dominated by Lewis X and pseudo-Lewis Y structures

The oligosaccharide structures of glycolipids from cercariaeof the human blood fluke, Schistosoma mansoni, were analyzedin the form of their corresponding, pyridylaminated oligosaccharidesby methylation analysis, partial hydrolysis, exoglycosidasetreatment, on-target exoglyco­sidase cleavage and matrix-assistedlaser desorption/ionization time-of-flight mass spectrometry.The six, dominant chemical structures present have been determinedas: GalNAc(ß1–4)Glc1-ceramide; GlcNAc(ß1–3)Gal­NAc(ß1–4)Glc1-ceramide;Gal(ß1–4)GlcNAc(ß1–3)Gal­NAc(ß1–4)Glc1-ceramide;Gal(ß1–4)[Fuc(     

Isolation, characterization and inactivation of the mouse Mgat3 gene: the bisecting N-acetylglucosamine in asparagine-linked oligosaccharides appears dispensable for viability and reproduction

The biosynthesis of complex asparagine (N)-linked oligosaccharidesin vertebrates proceeds with the linkage of N-acetylglucosamine(GlcNAc) to the core mannose residues. UDP-N-acetylglucosamine:ß-D-mannosideß1–4 N-acetylglucosaminyltransferase III (GlcNAc-TIII,EC2.4.1.144) catalyzes the addition of GlcNAc to the mannosethat is itself ß1–4 linked to underlying N-acetylglucosamine.GlcNAc-TIII thereby produces what is known as a ‘bisecting’GlcNAc linkage which is found on various hybrid and complexN-glycans. GlcNAc-TIII can also play a regulatory role in N-glycanbiosynthesis as addition of the bisecting GlcNAc eliminatesthe potential for     

Binding of galectin-1 (gal-1) to the Thomsen–Friedenreich (TF) antigen on trophoblast cells and inhibition of proliferation of trophoblast tumor cells in vitro by gal-1 or an anti-TF antibody

Galectin-1 (gal-1), a member of the mammalian β-galactoside-binding proteins, recognizes preferentially Galβ1-4GlcNAc sequences of several cell surface oligosaccharides. We demonstrate histochemically that the lectin recognizes appropriate glycotopes on the syncytiotrophoblast and extravillous trophoblast layer from second trimester human placenta and on BeWo chorion carcinoma cells. Gal-1 binding to BeWo cells was diminished by the Thomsen–Friedreich (TF)-disaccharide (Galβ1-3GalNAc-) conjugated to polyacrylamide (TF–PAA). Gal-1 also inhibited BeWo cell proliferation in a concentration-dependent manner. Similar antiproliferative effects were also observed with an anti-TF monoclonal antibody (mAb, A78-G/A7). Therefore, we conclude that ligation of Galβ1-4GlcNAc and Galβ1-3GalNAc epitopes on BeWo cells may have regulatory effects on cell proliferation.     

Further studies of the binding specificity of the leukocyte adhesion molecule, L-selectin, towards sulphated oligosaccharides--suggestion of a link between the selectin- and the integrin-mediated lymphocyte adhesion systems

This communication is concerned with the binding specficityof the leukocyte-adhesion molecule L-selectin (leukocyte homingreceptor) towards structurally defined sulphated oligosaccharidesof the blood group Le^a and Le^x series, and of the glycolsaminoglycanseries heparin, chondroitin sulphate and keratan sulphate. Therecombinant soluble form of the rat L-selectin (L-selectin-IgGFc chimera) investigated here was shown previously to bind tolipid-linked oligosaccharides 3-O, 4-O and 6-O sulphated atgalactose, such as sulphatides and a mixture of 3-sulphatedLe^a/Le^x type tetrasaccharides isolated from ovarian cystadenoma,as well as to the HNK-1 glycolipid with 3-O sulphated glucuronicacid. In the present study, the L-selectin investigated in bothchromatogram binding and plastic microwell binding experimentsusing neoglycolipids was found to bind to the individual 3-sulphatedLe^a and Le^x sequences (penta-, tetra- and trisaccharides), andwith somewhat lower intensities to their non-fucosylated analogues.Glycosaminoglycan disaccharides of keratan sulphate, heparinand chondroitin sulphate types were also bound by L-selectinin one or both assay systems, leading to the conclusion thatclustered glycosaminoglycan oligosaccharides with 6-O sulphationof N-acetylgalactctosamine, N-acetylglucosamine or glucosamine,4-O sulphation of N-acetylgalactosamine, 2-O sulphation of uronicacid, N-sulphation of glucosamine and, to a lesser extent, thenon-sulphated uronic acid-contahing disaccharides, can supportL-selectin adhesion. As inflammatory chemokines (short-rangestimulators of lymphocyte migration which trigger integrin activation)are known to bind to endothelial glycosaminoglycans, we proposethat the binding of the lymphocyte membrane L-selectin to endothelialglycosaminoglycans may provide a link between the selectin-mediatedand integrin-mediated adhesion systems in leukocyte extravasationcascades. The posibility is also raised that lymphocyte L-selectininteractions with glycosaminoglycans may contribute to pathologiesof glycosaminoglycan-rich tissues, e.g. cartilage loss in rheumatoidarthritis and inflammatory lesions of the cornea. glycosaminoglycans leukocyte adhesion cascades neoglycolipids oligosaccharide presentation sulphated oligosaccharides     

{beta}1,4-Galactosyltransferase activity in B cells detected using a simple ELISA-based assay

Lymphocytic ß1,4-galactosyltransferase (ß1,4-GalTase,EC 2.4.1.38 [EC] ) activity was measured in B cells using a neoglycoprotein,N-acetylglucosamine-phenylisothlocyanate-bovine serum albumin(GlcNAc-pITC-BSA), as an acceptor substrate in a novel enzyme-linkedimmunosorbent assay (ELISA)-based method. This assay provedto be much simpler to use than the lengthy and expensive radiochemicalassays commonly used, and has the additional advantage thatit specifically detects the enzyme mediating transfer via theGalß1,4GlcNAc linkage. A F(ab')₂ antibody againstGalTase was able to specifically inhibit the reaction. Greatersensitivity for ß1,4-GalTase activity was obtainedusing GlcNAc-pITC-BSA as an acceptor substrate rather than ovalbumin.Low levels of ß-galactosidase activity were detectablein lymphocyte cell lysates at acidic pH, although such activitywas not detectable at the neutral pH used in the ß1,4-GalTaseactivity assay. Using this assay with the GlcNAc-pITC-BSA acceptor,similar ß1,4-GalTase activities were observed in CD19⁺B cells from patients with rheumatoid arthritis (RA) to thoseseen in normal control individuals. ELISA ß1,4-galactosyltransferase lymphocyte neoglycoprotein radiochemical     

The unusual tetrasaccharide sequence GlcA{beta}-3GalNAc(4-sulfate)({beta}1-4GlcA(2-sulfate){beta}1-3GalNAc(6-sulfate) found in the hexasaccharides prepared by testicular hyaluronidase digestion of shark cartilage chondroitin sulfate D

Eight hexasaccharide fractions were isolated from commercialshark cartilage chondroitin sulfate D by means of gel nitrationchromatography and HPLC on an amine-bound silica column afterexhaustive digestion with sheep testicular hyaluronidase. Capillaryelectrophoresis of the enzymatic digests as well as one- andtwo-dimensional 500 MHz ¹H-NMR spectroscopy demonstrated thatthese hexasaccharides share the common core saccharide structureGlcAß1-3GalNAcß1-4GlcAß1-3GalNAcß1-4GlcAß1-3GalNAcwith three, four, or five sulfate groups in different combinations.Six structures had the same sulfation profiles as those of theunsaturated hexasaccharides isolated from the same source afterdigestion with chondroitinase ABC (Sugahara et al., Eur. J.Biochem., 293, 871–880, 1996) and the other two have notbeen reported so far. In the new components, a D disaccharideunit, GlcA(2-sulfate)ß1-3GalNAc(6-sulfate), characteristicof chondroitin sulfate D was arranged on the reducing side ofan A disaccharide unit, GlcAß1-3GalNAc(4-sulfate),forming an unusual A-D tetrasaccharide sequence, GlcAß1-3GalNAc(4-sulfate)-4GlcA(2-sulfate)ß1-3GaINAc(6-sulfate)which is known to be recognized by the monoclonal antibody MO225.These findings support the notion that the tetrasaccharide sequence,GlcAß1-3GalNAc(4-sulfate)ß1-4GlcAß1-3GalNAc(6-sulfate)is included in the acceptor site of a hitherto unreported 2-O-sulfotransferaseresponsible for its synthesis. The sulfated hexasaccharidesisolated in this study will be useful as authentic oligosaccharideprobes and enzyme substrates in studies of sulfated glycosaminogly-cans. sulfated hexasaccharides chondroitin sulfate D hyaluronidase ¹ H-NMR     

Terminal {beta}-linked tyvelose creates unique epitopes in Trichinella spiralis glycan antigens

Indirect evidence that the immunodominant N-glycans of the parasite,Trichinella spiralis are capped by novel ß-linked3,6-dideoxy-D-arabinohexopyranosyl residues (tyvelase, Tyv)was obtained from immunochemical assays employing monoclonalantibodies and synthetic oligosaccharides. Three of four previouslycharacterized monoclonal antibodies generated from the lymphocytesof T.spiralis infected rats bind BSA glycoconjugates bearingthe synthetic epitope ß-D-Tyvp(1     

Immunogenicity of synthetic conjugates of Lewisy oligosaccharide with proteins in mice: towards the design of anticancer vaccines

 Many human carcinomas overexpress the Lewis^y (Le^y) blood-group epitope [Fucα1→2Galβ1→4 (Fucα1→3)GlcNAcβ1→3Gal-]. With a view to developing Le^y based vaccines we have examined the immunogenicity of Le^y-protein conjugates in mice. Le^y pentasaccharide was synthesized as its allyl glycoside and coupled to keyhole limpet hemocyanin (KLH) by reductive amination or by a novel method utilizing a maleido-derivitized alkyl carboxyhydrazide as a bridging group to 2-iminothiolane-derivitized KLH. Le^y oligosaccharide was also coupled to bovine serum albumin by reductive amination. Immunization of groups of mice with the three conjugates, together with the immunological adjuvant QS21, showed that Le^y oligosaccharide directly coupled to KLH was the most efficient conjugate for eliciting IgG and IgM antibody responses to naturally occurring forms of Le^y epitopes carried on mucins and glycolipids. These antibodies were also reactive with and cytotoxic to a human breast cancer cell line expressing Le^y (MCF-7). These experiments suggest that Le^y-KLH antigen and QS21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma patients. Received: 28 March 1997 / Accepted: 2 September 1997     

Effects of a Plant-Growth Regulator, Prohexadione, on the Biosynthesis of Gibberellins in Cell-Free Systems Derived from Immature Seeds

Inhibition of the biosynthesis of gibberellins by prohexadione,3,5-dioxo-4-propionylcyclo-hexanecarboxylic acid, was studiedwith cell-free systems derived from immature seeds of Cucur-bitamaxima, Phaseolus vulgaris and Pisum sativum. Prohexadione,at a concentration of 10–⁴ M, inhibited C-7 oxidationof GA₁₂-aldehyde, C-20 oxidation of GA₁₅, conversion of C₂₀-gib-berellinsto C₁₉-gibberellins, 3ß-hydroxylation, 2,3-dehydrogenationof GA²⁰, 2,3-epoxidation of GA₅ and 2ß-hydroxylationof GA₉ and GA₂₀. The 3ß-hydroxylase activity appearedto be more sensitive to prohexadione than were the C-20 oxygenaseand the 2ß-hydroxylase activities. The conversionof mevalonic acid to GA₁₂-aldehyde and the 13-hydroxylationof GA₁₂ were not affected by prohexadione at a concentrationof 3 ? 10^–4 M. All of the steps inhibited by prohexadioneare oxidation steps catalyzed by soluble enzymes that require2-oxoglutarate, Fe²⁺ and oxygen, and all of them occur distalto the synthesis of GA₁₂-aldehyde in the biosynthesis of gibberellins. (Received April 4, 1990; Accepted September 14, 1990)     

KSGal6ST Is Essential for the 6-Sulfation of Galactose within Keratan Sulfate in Early Postnatal Brain

Keratan sulfate (KS) comprises repeating disaccharides of galactose (Gal) and N-acetylglucosamine (GlcNAc). Residues of Gal and GlcNAc in KS are potentially modified with sulfate at their C-6 positions. The 5D4 monoclonal antibody recognizes KS structures containing Gal and GlcNAc, both 6-sulfated, and has been used most extensively to evaluate KS expression in mammalian brains. We previously showed that GlcNAc6ST1 is an enzyme responsible for the synthesis of the 5D4 epitope in developing brain and in the adult brain, where it is induced after injury. It has been unclear which sulfotransferase is responsible for Gal-6-sulfation within the 5D4 KS epitope in developing brains. We produced mice deficient in KSGal6ST, a Gal-6-sulfotransferase. Western blotting and immunoprecipitation revealed that all 5D4-immunoreactivity to proteins, including phosphacan, were abolished in KSGal6ST-deficient postnatal brains. Likewise, the 5D4 epitope, expressed primarily in the cortical marginal zone and subplate and dorsal thalamus, was eliminated in KSGal6ST-deficient mice. Disaccharide analysis showed the loss of Gal-6-sulfate in KS of the KSGal6ST-deficient brains. Transfection studies revealed that GlcNAc6ST1 and KSGal6ST cooperated in the expression of the 5D4 KS epitope in HeLa cells. These results indicate that KSGal6ST is essential for C-6 sulfation of Gal within KS in early postnatal brains.