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Stereoselective syntheses of 6-OSO3Na-Gal-β-(1→3)-GalNAc-α-OR (10), 6-OSO3Na-Gal-β-(1→3)-6-OSO3Na-GalNAc-α-OR (11), 3-OSO3Na-Gal-β-(1→3)GalNAc-α-OR (13), 6-OSO3Na-Gal-β-(1→3) [NeuAc-α-(2→6)]-GalNAc-α-OR (15), Gal-β-(1→3) [NeuAc-α-(2→6)]-GlcNAc-β-OR (18) and 6-OSO3Na-Gal-β-(1→3) [NeuAc-α-(2→6)]-GlcNAc-β-OR (19) were accomplished through utilization of two key glycosyl donors, namely, 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-α-d-galactopyranosyl bromide (3) and 2,4,6-tri-O-acetyl-3-O-chloroacetyl-α-d-galactopyranosyl bromide (4). The synthesized compounds were evaluated as ligands for CD22 adhesion protein. Synthesis of 6-O-SO3NaGal-β-(1→3)-GalNAc-α-OR (10), 6-O-SO3NaGal-β-(1→3)-6-O-SO3NaGalNAc-α-OR (11), 3-O-SO3NaGal-β-(1→3)GalNAc-α-OR (13), 6-O-SO3NaGal-β-(1→3) NeuAc-α-(2→6) -GalNAc-α-OR (15), Gal-β-(1→3) NeuAc-α- (2→6) -GlcNAc-β-OR (18) and 6-O-SO3NaGal-β-(1→3) NeuAc-α-(2→6) -GlcNAc-β-OR (19) were accomplished

Blood group H type 1 [Fuc alpha (1,2)Gal beta (1,3)GlcNAc beta-->] is known as the precursor structure of the blood group determinant, Lewis b [Fuc alpha (1,2)Gal beta (1,3)(Fuc alpha (1,4))GlcNAc beta-->]. Recently, a new biosynthetic route for Lewis b from Lewis a [Gal beta (1,3)(Fuc alpha (1,4))GlcNAc-->] was identified in human gastric carcinoma cells, colon carcinoma Colo 205, and ovarian tumor. The present study demonstrates the association of this new type of alpha (1,2)-L-fucosyltransferase (FT) activity with the Lewis-type alpha (1,3/4)-L-FT as follows: (i) the alpha (1,4)- and novel alpha (1,2)-FT activities of Colo 205 were much less inhibited than the alpha (1,3)-FT activity by N-ethylmaleimide [Ki(microM) = 714.0, 119.0, and 6.5 respectively]. (ii) The alpha (1,4)- and novel alpha (1,2)-FT activities emerged from a Sephacryl S-200 column in identical positions. (iii) A specific inhibitor (copolymer from 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-allyl and acrylamide) of alpha(1,4)-FT activity inhibited both alpha(1,4)- and alpha(1,2)-FT activities in Sephacryl S-200 column effluent to almost the same extent (approximately 80%); (iv) separation of the Lewis-type alpha(1,3/4)-FT from the plasma-type alpha(1,3)-FT by specific elution of the affinity column (bovine IgG glycopep-Sepharose) with lactose and further purification on a Sephacryl S-100 HR column showed that (a) the alpha(1,3)-FT activity was the inherent capacity of the Lewis-type FT (Colo 205 fraction L) since approximately 90% of both the alpha(1,4)- and alpha(1,3)-FT activities is inhibited by the copolymer, (b) the unique ability of catalyzing the alpha(1,2)-L-fucosylation of Gal in Lewis a structure and also the alpha(1,3)-L-fucosylation of Glc in lactose-based structure belonged to the Lewis type enzyme (Colo 205 fraction L), (c) a measurement of the [14C]fucosyl products arising from the two acceptors Galbeta(1,3)(4,6-di-O-Me)GlcNAcbeta-O-Bn and 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-A1 (specific for alpha(1,2) and alpha(1,4), respectively) taken in the same incubation mixture showed mutual inhibition by the acceptors ([Km for the alpha(1,4)-specific acceptor, 3-sulfo-Galbeta(1,3)GlcNAcbeta-O-A], increased from 32 to 50 microM in the presence of 7.5 mM Galbeta(1,3)(4,6-di-O-Me)GlcNAcbeta-O-Bn, whereas Ki for the mutual inhibition of alpha(1,2)-FT activity by the former was 102 microM], and (d) the Lewis-type FT, in contrast to the plasma type FT, was highly effective in fucosylating complex glycopeptides. (iv) A cloned FT (FT III:Lewis type) and the Colo 205 Lewis-type FT (fraction L) showed similar activities toward various acceptors; the enzymatic product resulting from the action of cloned FT on Galbeta(1,3)(Fucalpha(1,4))GlcNAc-beta-O-Bn was identified by FAB mass spectrometry as the difucosyl compound. (v) An examination of six human cell lines indicated that the novel alpha(1,2)-FT activity associates with the alpha(1,4)-FT activity.

The sequence in the assembly of the functional unit of selectin ligands containing sulfate, sialic acid, and fucose and also tumor-associated O-glycan structures was studied by examining the specificities of alpha 2,3-sialyltransferases (ST). The first enzyme, porcine liver ST, was 57, 37, and 79% active (Km: 0.105, 0.420, and 0.200 mM), respectively, toward 6-sulfo, 6-sialyl, or 6-O-methyl derivatives of the Gal beta 1,3GalNAc alpha- unit; C-3 or C-6 substitution on Gal abolished sialylation. An acrylamide copolymer (MW approximately 40,000) containing approximately 40 T-haptens and asialo Cowper's gland mucin (MW approximately 200,000) containing approximately 48 T-haptens was 5-fold more active as an acceptor as compared to Gal beta 1, 3GalNAc alpha-O-Al on a molecular weight basis. The second enzyme, a cloned alpha-2,3-ST specific for lactose-based structure, was 70, 102, and 108% active (Km: 0.500, 0.210, and 0.330 mM), respectively, toward 6-sialyl, 6-sulfo, or 6-O-methyl derivatives of the Gal beta 1,3GlcNAc beta- unit; C-3 and C-6 substitution on Gal abolished sialylation. Gal beta 1,4GlcNAc beta- and its 6-sulfo derivative were approximately 20% active; the Lewis a structure, Gal beta 1,3- (Fuc alpha 1,4)GlcNAc beta-, was not an acceptor. The acrylamide copolymers containing approximately 40 units of Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, or fetuin triantennary asialo or bovine IgG diantennary glycopeptides were respectively 5.9-, 5.4-, 0.7-, and 0.1-fold as active. A transfer of 7-9 mol of NeuAc per mole of the above copolymers was catalyzed by this ST, the sialyl linkage being susceptible to alpha 2,3-specific sialidase. A partially purified Colo 205 Lewis type (alpha 1, 3/4) fucosyltransferase catalyzed the formation of 3'-sialyl-6-sulfo Lewis a from [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta-O-Allyl and copolymer containing [9-3H]NeuAc alpha 2, 3Gal beta 1, 3(6-sulfo)GlcNAc beta- units, using GDP[14C]Fuc as fucosyl donor. The third enzyme, HL-60 ST, was 103% active with Gal beta 3(6-sulfo)GalNAc alpha- but was only 8% active with 6-sialo compound; it showed 11.6-fold greater activity with the copolymer of T-hapten. Further, we observed the alpha 2,3 sialylation of Gal beta 1,4GlcNAc beta- but not Gal beta 1,3GlcNAc beta- by HL60-ST, consistent with the occurrence of 3'-sialyl LacNAc and 3'-sialyl Lewis x units in leukosialin of HL60.(ABSTRACT TRUNCATED AT 400 WORDS)

CD22, a B cell-specific receptor of the immunoglobulin superfamily, has been demonstrated to bind to oligosaccharides containing α2-6-linked sialic acid (Sia) residues. Previously, we demonstrated that the minimal structure recognized by this lectin is the trisaccharide Siaα2-6Galβ1-4GlcNAc, as found on N-linked, O-linked, or glycolipid structures (Powell, L., and Varki, A.(1994) J. Biol. Chem. 269, 10628-10636). Here we utilize a soluble immunoglobulin fusion construct (CD22Rg) to determine directly by equilibrium dialysis the stoichiometry (2:1) and dissociation constant (32 μM) for Neu5Acα2-6Galβ1-4Glc binding. Inhibition assays performed with over 30 different natural and synthetic sialylated and/or sulfated compounds are utilized to define in greater detail specific structural features involved in oligosaccharide-protein binding. Specifically, the critical features required for binding include the exocyclic hydroxylated side chain of the Sia residue and the α2-6 linkage position to the underlying Gal unit. Surprisingly, alterations of the 2-, 3-, and 4-positions of the latter residue have limited effect on the binding. The nature of the residue to which the Gal is attached may affect binding. Bi(α2-6)-sialylated biantennary oligosaccharides are capable of simultaneously interacting with both lectin sites present on the dimeric CD22Rg fusion construct, giving a marked improvement in binding over monosialylated compounds. Furthermore, data are presented indicating that full-length native CD22, expressed on the surface of Chinese hamster ovary cells, is structurally and functionally a multimeric protein, demonstrating a higher apparent affinity for multiply sialylated compounds over monosialylated compounds. These observations provide a mechanism for strong CD22-dependent cell adhesion despite the relatively low Kd for protein-sugar binding.

Sialylated or sulfated derivatives and acrylamide copolymers of blood group T-(Galβ1,3GalNAcα-) and Tn-(GalNAcα) haptens were studied for their interaction with the lectins of peanut (PNA),Agaricus bisporus-(ABA),Helix pomatia-(HPA) andVicia villosa B4-(VVA), using asialo Cowper's gland mucin (ACGM), which contains both T and Tn epitopes, as the coating substrate in enzyme linked lectin assay. Both T and Tn copolymers (∼40 haptens) showed high affinity and strict specificity; although the T-copolymer at 0.05–0.07 µm concentration caused 50% inhibition of interaction of either PNA or ABA with ACGM, there was little inhibition of the HPA and VVA interactions at over 100 times that concentration. The Tn-copolymer at 0.02–0.05 µm inhibited HPA or VVA interaction with ACGM by 50% but gave virtually no inhibition of PNA and ABA binding. Sialyl, sulfate or methyl group substitution on C-6 of GalNAc of the T-haptene did not prevent interaction with PNA but almost abolished interaction with ABA. In contrast, sialyl or sulfate group on C-6 and sulfate on C-3 of Gal in Galβ1,3GalNAcα- inhibited almost completely the interaction of PNA with ACGM but had only a slight effect on the interaction of ABA; C-6 substitution with either sialic acid or sulfate on GalNAcα- almost abolished the interaction of both HPA and VVA with ACGM. Preliminary studies revealed a significant depression in the serum level of anti-T (two to three-fold decrease) and anti-Tn (∼ two-fold decrease) antibodies in breast cancer compared with normal control subjects when the acrylamide T- and Tn-copolymers were used as coating substrates in enzyme linked immunoassays.

Synthesis of two trisaccharides and a tetrasaccharide, namely, alpha-Gal-(1--3)-beta-Gal-(1--3)-GlcNAc-beta-OBn (6), alpha-Gal-(1--3)-beta-Gal-(1--4)-GlcNAc-beta-OBn (9) and alpha-Gal-(1--3)-beta-Gal-(1--4)-GlcNAc-beta-(1--6)-GalNAc- alpha-OBn (19) was accomplished through development and utilization of a key alpha-galactosyl donor, methyl 2,3,4,6-tetra-O-(4- methoxybenzyl)-1-thio-beta-D-galactopyranoside (1).

We report the first large scale heterologous expression of a recombinant yeast a-l,2-mannosyltransferase in E. coli. The enzyme was isolated from 10-L and 50-L fermentations, purified and used for mannosylation reactions. The specificity of the recombinant enzyme was extensively studied by using mannose derivatives, oligosaccharides, and analogs as acceptors, and the results show that the enzyme exhibits high activities toward ManOMe and disaccharides connected by an a-1,2-mannosidic linkage. The recombinant E. coli cells were also used as a catalyst for glycosylation reaction, and mannosylation of saccharides and glycopeptides proceeded in moderate to good yields.

4-Nitrophenyl 2,3-O-isopropylidine-α-D-mannopyranoside 2 was condensed with O-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl bromide 1 and 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl bromide 11 in the presence of mercuric cyanide. Products were deprotected to yield, respectively, 4-nitrophenyl O-α-D-mannopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→6)-α-D-mannopyranoside 6 and 4-nitrophenyl O-α-D-mannopyranosyl-(1→6)-α-D-mannopyranoside 14. The 4-nitrophenyl group of 6 was reduced to give title trisaccharide. Bromide 1 was also condensed with methyl 2,3,4-tri-O-benzyl-α-D-manopyranoside 3 in the presence of silver trifluoromethanesulfonate and tetramethylurea to give protected trisaccharide derivative which was deprotected to furnish, methyl O-α-D-mannopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→6)-α-D-mannopyranoside 10. The identities of all protected and deprotected compounds were supported by 1H and 13C NMR spectral data.

Adhesion of A-121 human ovarian carcinoma cells to extracellular matrix is partly mediated via interaction between galaptin, an endogenous beta-galactoside-binding lectin present in extracellular matrix, and specific cell surface carbohydrate receptors identified as lysosomal associated membrane proteins, lamp-1 and lamp-2. In this study, we report that adhesion of human ovarian carcinoma cells to polystyrene plates coated with polymerized human splenic galaptin can be inhibited by polyclonal antibodies raised against lamp-1 and lamp-2 molecules and by pretreatment of A-121 human ovarian carcinoma cells with glucosamine analogs: 2-acetamido-1,4,6-tri-O-acetyl-3- deoxy-3-fluoro-alpha-D-glucopyranose (3-F-GlcNAc) and 2-acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-alpha-D-glucopyranose (4-F-GlcNAc). A 48-h exposure of A-121 cells to individual sugar analogs, or to a combination of the two, resulted in a concentration-dependent inhibition of cellular attachment to polymerized galaptin. Both drugs inhibited glycoprotein biosynthesis as measured by cellular incorporation of labeled [3H]glucosamine and [3H]fucose with negligible effects on [3H]thymidine and [3H]leucine incorporation and cell growth. As a result of drug action on glycoprotein biosynthesis, an alteration in the structure of the galaptin receptor was noted by indirect immunofluorescence and Western blot analysis. Moreover, probing gels of cell extracts with anti-lamp antibodies or Datura stramonium lectin demonstrated significant changes in the reactivity and pattern of glycoprotein staining, suggesting an effect of sugar analogs on the glycosylation of various cellular receptor molecules. The greatest change was observed when tumor cells were exposed to a combination of the two sugar analogs. These studies suggest that specific endogenous lectins and their surface receptors play a role in tumor cell adhesion and perhaps metastasis and may serve as suitable targets for therapeutic exploitation.

A human colonic adenoma cell line PC/AA derived from a familial polyposis coli patient was passaged in culture to form an intermediate premalignant clonogenic variant AA/C1 and, upon treatment with differentiating and carcinogenic agents, a cell line AA/C1/SB10 which is tumourigenic in nude mice. These three mucin-secreting cell lines have been used as a model to study the changes in O-glycan biosynthesis during the progression to cancer. Several glycosyltransferases involved in the synthesis, elongation and termination of the common O-glycan core structures were found to decrease in the progression sequence towards adenocarcinoma. Higher activity of a number of enzymes was seen in the intermediate cell line. O-glycan biosynthesis in the original PC/AA cell line was closest to the normal human colonic phenotype, since all four common mucin O-glycan cores and their extended structures could be synthesized; core 3 beta 3-GlcNAc-transferase and alpha 6-sialytransferase acting on GalNAc-mucin were still detectable and core 2 beta 6-GlcNAc-transferase activity was accompanied by core 4 and I beta 6-GlcNAc-transferase activities. During progression towards adenocarcinoma, the expression of alpha 6-sialyltransferase, core 3 beta 3-GlcNAc-transferase, core 4 and I beta 6-GlcNAc-transferases were turned off. Using monoclonal antibodies, Tn antigen, sialyl-Tn antigen, O-acetyl-sialomucin and sialyl-Lea determinants were not detected in secreted or cellular mucin isolated from any of the cell lines. The exposure of MUC1 epitopes was seen in the malignant line, whereas sialyl-Lex determinants were found only in the premalignant PC/AA line. Sulfotransferase activities using core 1 substrate, Gal beta 1-3GalNAc alpha-benzyl, were high in PC/AA cells and progressively decreased upon development to adenocarcinoma, and this decrease correlated with mucin sulfation. In summary, the synthesis of less abundant, sialylated, fucosylated and extended, unbranched core 1 structures should be facilitated in the malignant cells. This is the first report of glycosyltransferase changes in human premalignant cells developing to tumourigenic cells. The data demonstrate that these cell lines are an excellent model to study the changes and regulation of mucin oligosaccharide biosynthesis during progression to cancer.

A stereoselective synthesis of Gal-β-(1→4)-6-O-SO 3 Na-GlcNac-β-(1→6)-Man-α-OR (as a part of gp 120) containing an anomeric p-nitrophenyl group was accomplished through the use of ethyl 3,4,6-tri-O-chloroacetyl-2-deoxy-2-phthalimido-1-thio-β-D-glucopyranoside.

The present study reports a control on the biosynthesis of fucosylated structures, serving as ligands for selectins by demonstrating the potential of 3-sialyl or 3-sulfo Galβ1,3/4GlcNAcβ-containing glycoconjugates as high affinity substrates for α1,3/4-L-fucosyltransferases and as substrate inhibitors at higher concentrations. The synthetic sulfated saccharides and the triantennary sialoglycopeptide from fetuin were potent competitive inhibitors of the transfer of fucose to non-anionic saccharide acceptors and the corresponding triantennary asialoglycopeptide respectively catalyzed by a partially purified α1,3/4-L-fucosyltransferase preparation from Colo 205 (specific activity:transfer of 113.1 nmol Fuc to 2′-FucosylLacNAc per h per mg protein); Ki for the inhibitions by triantennary sialoglycopeptide, 3-SulfoGalβ1,3GlcNAcβ-O-Allyl and a copolymer from 3-SulfoGalβ1,3GlcNAcβ-O-Allyl and acrylamide were 51.9 μM, 500 μM and 67.0 μM, respectively. Further, the α1,3-specific anionic acceptor, 3′-SulfoLacNAc, also inhibited the α1,4- activity; Km for the α1,4-specific acceptor, 2-methylGalβ1,3GlcNAcβ-O-Bn increased from 0.40 mM to 1.35 mM in presence of 3.0 mM 3′-sulfoLacNAc, whereas Ki for the mutual inhibition of α1,3-activity by the former was found to be high (3.64 mM). Furthermore, the phenomenon of substrate inhibition, serving as acceptors at lower concentrations and as inhibitors at higher concentrations, was exhibited by the anionic acceptors; the Hill plots gave the Ki values 342.7 μM, 13.03 mM and 13.36 mM respectively for fetuin triantennary sialo glycopeptide, 3′-sulfoLacNAc and 3-sulfoGalβ1,3GlcNAcβ-O-Allyl.

Human colon cancer is associated with antigenic and structural changes in mucin-type carbohydrate chains (O-glycans). To elucidate the control of the biosynthesis of these O-glycans is colon cancer, we have studied glycosyltransferase and sulphotransferase activities involved in the assembly of elongated O-glycan structures. We analysed homogenates prepared from cancer tissue, adjacent normal and distal normal tissue from 20 patients. Several transferase activities showed pronounced changes in cancer tissue. The changes correlate with previous findings of a loss of O-glycans in cancer mucins, but did not always correlate with levels of Tn, sialyl-Tn, T and Lex antigens in homogenates or with the differentiation status and Duke's stages of the cancer tissue or the patient's blood type, sex and age. UDP-GlcNAc: Gal NAc-R beta 3-N-acetylglucosaminyltransferase (where GlcNAc is N-acetyl-D-glucosamine and GalNAc is N-acetyl-D-galactosamine) synthesizing O-glycan core 3, GlcNAc beta 1-3GalNAc-, CMP-sialic acid: GalNAc-peptide alpha 6-sialyltransferase synthesizing the sialyl-Tn antigen and sulphotransferase activities towards O-glycan core 1, Gal beta 1-3GalNAc-, were found to be decreased in cancer. UDP-GlcNAc: Gal beta 1-3GalNAc beta 6-N-acetylglucosaminyltransferase was also decreased in cancer concomitant with a loss of the ability to synthesize the I antigen and core 4, GlcNAc beta 1-6(GlcNAc beta 1-3) GalNAc-, CMP-sialic acid: Gal beta 1-3GalNAc-R alpha 3-sialyltransferase and GDP-fucose: Gal beta-R alpha 2-fucosyltransferase, synthesizing the blood group H determinant, were found to be 4- and 3- to 8-fold increased, respectively, in cancer compared to normal tissue. The data suggest that the biosynthesis of antigens and mucin-bound O-glycan structures in colon cancer is subject to complex control mechanisms.

Glycosyltransferases (glycoTs) catalyze the transfer of monosaccharides from nucleotide-sugars to carbohydrate-, lipid-, and protein-based acceptors. We examined strategies to scale down and increase the throughput of glycoT enzymatic assays because traditional methods require large reaction volumes and complex chromatography. Approaches tested used (i) microarray pin printing, an appropriate method when glycoT activity was high; (ii) microwells and microcentrifuge tubes, a suitable method for studies with cell lysates when enzyme activity was moderate; and (iii) C(18) pipette tips and solvent extraction, a method that enriched reaction product when the extent of reaction was low. In all cases, reverse-phase thin layer chromatography (RP-TLC) coupled with phosphorimaging quantified the reaction rate. Studies with mouse embryonic stem cells (mESCs) demonstrated an increase in overall β(1,3)galactosyltransferase and α(2,3)sialyltransferase activity and a decrease in α(1,3)fucosyltransferases when these cells differentiate toward cardiomyocytes. Enzymatic and lectin binding data suggest a transition from Lewis(x)-type structures in mESCs to sialylated Galβ1,3GalNAc-type glycans on differentiation, with more prominent changes in enzyme activity occurring at later stages when embryoid bodies differentiated toward cardiomyocytes. Overall, simple, rapid, quantitative, and scalable glycoT activity analysis methods are presented. These use a range of natural and synthetic acceptors for the analysis of complex biological specimens that have limited availability.

Galectin-1 (Gal-1) has been shown to play a major role in tumor immune escape by inducing apoptosis of effector leukocytes and correlating with tumor aggressiveness and disease progression. Thus, targeting the Gal-1/Gal-1 ligand axis represents a promising cancer therapeutic approach. Here, to test the Gal-1-mediated tumor immune evasion hypothesis and demonstrate the importance of Gal-1-binding N-acetyllactosamines in controlling the fate and function of antitumor immune cells, we treated melanoma- or lymphoma-bearing mice with peracetylated 4-fluoro-glucosamine (4-F-GlcNAc), a metabolic inhibitor of N-acetyllactosamine biosynthesis, and analyzed tumor growth and immune profiles. We found that 4-F-GlcNAc spared Gal-1-mediated apoptosis of T cells and natural killer (NK) cells by decreasing their expression of Gal-1-binding determinants. 4-F-GlcNAc enhanced tumor lymphocytic infiltration and promoted elevations in tumor-specific cytotoxic T cells and IFN-γ levels, while lowering IL-10 production. Collectively, our data suggest that metabolic lowering of Gal-1-binding N-acetyllactosamines may attenuate tumor growth by boosting antitumor immune cell levels, representing a promising approach for cancer immunotherapy.

Glycoproteins modulate biological function such as signaling, immune response and tissue development by interaction through terminal sugars (1). These sugars or glycans are commonly either N- linked to asparagine, or O- linked to Ser/Thr residues on protein scaffolds. Among these, mucins are O-glycan rich glycoproteins that often contain tandem repeats of peptide sequences characterized by a high content of Ser, Thr and Pro residues (2–4). Each tissue exhibits a unique pattern of mucinous proteins that can be modified under pathological conditions. Such regulation of mucin expression in cancer epithelial cells influences cell adhesion and tumor invasiveness. Moreover, cancer associated mucins that contain incomplete O-glycan chains are highly immunogenic and these are potential targets for immunotherapy. Whereas the study of N-glycans has progressed well, in part, due to the availability of highly-specific endoglycosidases that can release intact N-glycans, similar universal glycosidases for mucin-type O-glycans are not available. New tools for the study of O-glycosylation are thus desirable. The current study addresses this need by presenting a novel strategy to enzymatically radiolabel O-glycans, and also glycolipids. O-glycosylation is initiated by the attachment of GalNAc via α-linkages to hydroxyl groups of Ser/Thr that are exposed on the protein surface such as at coils, turns or linker regions. This step is catalyzed by a family of specific UDP GalNAc: polypeptide N-acetylgalacto-saminyltransferases (5,6). Further extension of these glycans is regulated, in large measure, by the distribution of glycosyltransferases and sulfotransferases that are primarily localized in the cellular Golgi (7,8). Sialic acid residues are typically found at the terminal non-reducing ends of O-glycans expressed both on cell-surface and secreted glycoproteins. The attachment of these residues is mediated by enzymes belonging to the sialyltransferase family. While these enzymes are typically thought to uni-directionally catalyze the transfer of sialic acid from a sugar nucleotide donor (CMP-NeuAc) to an acceptor substrate, we recently showed that this reaction can be reversible at least for the case of one mammalian/rat sialyltransferase ST3Gal-II (9). Here, using this process called `reverse sialylation', we demonstrated that ST3Gal-II can synthesize CMP-NeuAc from 5′-CMP and NeuAcα2,3Galβ1,3GalNAc- units of O-glycans, and glycolipids. In addition to this, we now report that this enzyme can also catalyze the direct exchange of NeuAc between CMP-NeuAc and the NeuAcα2,3Galβ1,3GalNAc- units of O-glycans and glycolipids, in the absence of exogenous 5'-CMP. While the precise mechanism for this exchange process is yet to be established, this may be partially attributed to the formation of 5'-CMP in the reaction mixture due to the break-down of CMP-NeuAc. These unique catalytic properties of ST3Gal-II could be utilized for the facile radiolabeling in vitro of sialyl residues in mucin-type structures.

Mucins are large, highly O-glycosylated proteins that are present at the surface of most epithelial cells and are involved in the protection and lubrication of the epithelium (Gendler and Spicer 1995). The expression of mucin genes is organ-and cell-type specific. The increased expression and altered glycosylation of mucins influence cellular growth, differentiation, transformation, adhesion and immune surveillance (Hollingworth and swanson 2004) and are associated with the development of cancer. Mucins have been implicated in the biologic behavior and progression of several cancers. During carcinogenesis, aberrant glycosylation leads to the development of tumor subpopulations with different adhesion properties (Taniguchi et al. 1996; Hiraiwa et al. 1996). Alterations of mucins during the pathogenesis of cancer have been well documented (Hakomori et al. 2002). The under glycosylation of mucins results in the creation of tumor associated cryptic carbohydrate core structures Tn, sialyl Tn and T (Taylor-Papadimitriou et al. 1999). The Gram-negative bacterium Helicobacter pyroli infects over 50% of the world’s population and causes various gastric diseases such as chronic gastritis, peptic ulcer and gastric cancer. Peripheral lymph node addressin (PNAd) containing 6-sulfo sialyl Lewis Xcapped O-glycans on high endothelial venule (HEV)-like vessels are closely associated with pathogenesis of H pyroli-related diseases. The ┙1,4 GlcNAc-capped O-glycans expressed by gastric gland mucous cell-derived mucin prevents colonization of H pyroli. Thus, the differential expression of distinct O-glycans in stomach provides therapeutic potentialities based on specific carbohydrate modulation (Nakayama et al. 1999; Reis et al. 2000 and Kobayashi et al. 2009). Werther et al. (1996) examined the frequency of sialyl Tn expression and its prognostic value in gastric cancer by immunohistochemical analysis of 340 gastric tumors and found that sialyl Tn expression is a marker of gastric cancer progression suggesting that cancer associated mucins play a role in the malignant behavior of the tumor. Santos-Silva et al. (2005) have reported that polymorphism in the MUC1 tandem repeat exerts influence on the expression of cryptic carbohydrate core structures in gastric cancer cells and the aggressive gastric tumors tend to express T antigen. Immunohistochemical study of Amado et al. (1998) for the expression of dimeric sialyl Lewisx in 97 gastric carcinomas revealed a correlation

To elucidate control mechanisms ofO-glycan biosynthesis in leukemia and to develop biosynthetic inhibitors we have characterized core 2 UDP-GlcNAc:Galβ1-3GalNAc-R(GlcNAc to GalNAc) β6-N-acetylglucosaminyl-transferase (EC 2.4.1.102; core 2 β6-GlcNAc-T) and CMP-sialic acid: Galβ1-3GalNAc-R α3-sialyltransferase (EC 2.4.99.4; α3-SA-T), two enzymes that are significantly increased in patients with chronic myelogenous leukemia (CML) and acute myeloid leukemia (AML). We observed distinct tissue-specific kinetic differences for the core 2 β6-GlcNAc-T activity; core 2 β6-GlcNAc-T from mucin secreting tissue (named core 2 β6-GlcNAc-T M) is accompanied by activities that synthesize core 4 [GlcNAcβ1-6(GlcNAcβ1-3)GalNAc-R] and blood group I [GlcNAcβ1-6(GlcNAcβ1-3)Galβ-R] branches; core 2 β6-GlcNAc-T in leukemic cells (named core 2 β-GlcNAc-T L) is not accompanied by these two activities and has a more restricted specificity. Core 2 β6-GlcNAc-T M and L both have an absolute requirement for the 4- and 6-hydroxyls ofN-acetylgalactosamine and the 6-hydroxyl of galactose of the Galβ1-3GalNAcα-benzyl substrate but the recognition of other substituents of the sugar rings varies, depending on the tissue. α3-sialytransferase from human placenta and from AML cells also showed distinct specificity differences, although the enzymes from both tissues have an absolute requirement for the 3-hydroxyl of the galactose residue of Galβ1-3GalNAcα-Bn. Galβ1-3(6-deoxy)GalNAcα-Bn and 3-deoxy-Galβ1-3GalNAcα-Bn competitively inhibited core 2 β6-GlcNAc-T and α3-sialyltransferase activities, respectively.

A stereoselective synthesis of Galβ1→3 (Fucα1→4) GlcNAcβ-Or (Lewis a) structures containing an anomeric p-nitrophenyl or benzyl group was accomplished through the use of methyl 3,4-O-isopropylidene-2-O-(4-methoxybenzyl)-1-thio-β-L-fucopyranoside. The compounds were used as acceptors to show in human ovarian tumors and a colon carcinoma cell line (Colo 205), the presence of a new type of α1,2-L-fucosyltransferase which converts the blood group determinant Lewis a to Lewis b

The reaction of phenyl 3,4,6-tri-O-acetyl-2-deoxy-2-phthaIimido-l-thio-β-D-glucopyranoside with methyl 3,4,6-tri-O-benzyl-α-D-mannopyranoside catalysed by iodonium ion (TfOH-NIS) followed by deacylation-acetylarion afforded disaccharide 11. which was readily converted (in four steps) to bromide 12. A similar glycosylarion with phenyl 2,3,4,6-tetra-O-acetyl-l-thio-D-glucopyranoside of benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-α-D-glucopyranoside 16 followed by O-deacetylation of the resulting intermediate gave disaccharide 18. The 4,6-O-benzylidene derivative of 18 was acetylated then deacetaled to give diol 21. This diol acceptor was condensed with bromide 12 (promoted by mercuric cyanide) to give the partially protected tetrasaccharide derivative 22 which was O-deacetylated and then subjected to catalytic hydrogenation to furnish the title tetrasaccharide 6. The structure assigned to 6 was supported by 1H and 13C NMR spectral data and FAB mass spectroscopy.

O -(2-Acetamido-3,4,6-tri- O -acetyl-2-deoxy-β- d -glucopyranosyl)-(1 → 2)-3,4,-di- O -acetyl-6- O -methyl-α- d -mannopyranosyl bromide and O -(2-acetamido-3,4,6-tri- O -acetyl-2-deoxy-β- d -glucopyranosyl)-(1 → 2)-3- O -acetyl-4,6-di- O -methyl-α- d -mannopyranosyl bromide were each condensed with 4-nitrophenyl 2,3-di- O -acetyl-β- d -glucopyranoside, and the products were deprotected to yield, respectively, β- d -Glc p NAc-(1 → 2)-6- O -Me-α- d -Man p -(1 → 6)-β- d -Glc p and β- d -Glc p NAc-(1 → 2)-4,6-di- O -Me-α- d -Man p -(1 → 6)-β- d -Glc p , as their 4-nitrophenyl glycosides. These trisaccharides are expected to function as inhibitors for N -acetylglucosaminyltransferase V.

The synthesis of two tetrasaccharides and one trisaccharide containing lacto-N-biose I (β-d-Galp-(1 → 3)-β-d-GlcpNAc) as their terminal unit was accomplished through development and utilization of a key glycosyl donor, namely, phenyl O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-(1 → 3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-1-thio-β-d-glucopyranoside.

A stereoselective synthesis of α-D-glucopyranosyl linked oligosaccharides containing a anomeric 4-nitrophenyl or benzyl group was accomplished through the use of methyl 2,3,4,6-tetra-O-(4-methoxybenzyl-1-thio-β-D-glucopyranoside ( 4 ) as a key glycosyl donor.

The 2-naphthylmethyl (NAP) group is a versatile group for protection of hydroxyl functions. It is stable to 4% TFA in CHCl 3 , hot 80% HOAc–H 2 O, SnCl 2 –AgOTf and HCl–EtOH, but it can readily be removed with DDQ in CH 2 Cl 2 .

An efficient synthesis of the mucin core-2 analogue 1a Download : Download high-res image (83KB) Download : Download full-size image Figure 1 . This C-2 position is blocked for commonly occurring α(1,2)- l -fucosyltransferase. was accomplished using N-p-nitrobenzyloxycarbonyl(PNZ)-protected trichloroacetimidate 4 Download : Download high-res image (134KB) Download : Download full-size image Scheme 2 . Reagents and conditions: (i) 4 (1.5 equiv), TESOTf (0.24 equiv), CH2Cl2, 4 A molecular sieves, rt, 15 min, 89%; (ii) Na2S2O4 (8 equiv), MeCN:EtOH:H2O (v/v/v 1:1:1), 10 min; (iii) Ac2O–MeOH; (iv) 75% acetic acid, 45 °C, 2 h, 90%; (v) 80% acetic acid, 40 °C, 2 h, 92%; (vi) 4 (1.2 equiv), TESOTf, CH2Cl2, −65 °C, 1 h, 78%; (vii) pyridine–Ac2O, DMAP; (viii) 4 (1.2 equiv), BF3·Et2O (0.5 equiv), CH2Cl2, −60 °C, 30 min, 82%. as a novel glycosyl donor.

The total synthesis of a sialylated and sulfated oligosaccharide 1 representative of a structure occurring in respiratory mucins has been accomplished. Our strategy depends upon the employment of 2-naphthymethyl (NAP) protection for hydroxyl functions. Choice of the well-defined sialyl donor 15 was made because of its enhanced reactivity over the parent compound 14 for glycosylation.

The stereo- and regioselective total synthesis of branched mucin core 2-structure 2, which contains the NeuAcα- 2,3(SO3Na-6)Galβ1,3GalNAcα sequence, is accomplished through the use of the key glycosyl donor 19.

Concentrations of blood group A-specified alpha(1-->3)-N-acetylgalactosaminyltransferase (A enzyme) were measured in human plasma of blood groups A1, A2, and A3 by using chemically synthesized H disaccharides and H type 1 and type 2 trisaccharides attached to hydrophobic aglycones as acceptors. When the trisaccharides were used as acceptors, enzyme activities were reduced in samples from A2 and A3. However, the H disaccharides were shown to be good acceptors even for enzymes from A2 and A3, and no significant difference in enzyme concentration was detected in any of the plasma tested.

A stereoselective synthesis of α-D-glucopyranosyl linked oligosaccharides containing a anomeric 4-nitrophenyl or benzyl group was accomplished through the use of methyl 2,3,4,6-tetra-O-(4-methoxybenzyl-1-thio-β-D-glucopyranoside ( 4 ) as a key glycosyl donor.

Several N-acetyllactosamine (LacNAc) derivatives were tested as acceptors for alpha 1,3-L-fucosyltransferase present in human ovarian cancer sera and ovarian tumor. The enzyme of the soluble fraction of tumor was purified to apparent homogeneity by chromatography on bovine IgG glycopeptide-Sepharose followed by Sephacryl S-200 (M(r) 80% activity was found as alpha 1,2-fucosyltransferase and the rest as alpha 1,3-fucosyltransferase. A screening of 21 ovarian cancer and 3 normal sera (3'-sulfo LacNAc as acceptor) showed 17-572% increase (average increase, 188%) of alpha 1,3-fucosyltransferase activity in cancer.(ABSTRACT TRUNCATED AT 400 WORDS)

Synthetic glycosides containing the core, -Glc-NAc beta 1,6GalNAc alpha-, acted as acceptors for beta-galactosyltransferase of human ovarian tumor. A significant amount of Gal was transferred from UDP-Gal (100 nmol) to the alpha-benzylglycoside of LacNAc beta 1,6GalNAc (LGBn) (25.1 nmol of Gal) and the alpha-ortho-nitrophenylglycosides of LacNAc beta 1,6GalNAc (22.0 nmol of Gal), GlcNAc beta 1,6GalNAc (15.5 nmol of Gal), and Fuc alpha 1,3GlcNAc beta 1,6GalNAc (25.9 nmol of Gal); LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn (where Bn is benzyl) was almost inactive (only 1.2 nmol of Gal), indicating the Gal transfer to the alpha-GalNAc moiety. The product from LGBn was isolated in microgram quantities and identified by fast atom bombardment mass spectrometry as LacNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn. The alpha GalNAc:beta 1,3Gal transferase was present in high concentration in ovarian tumor tissue (ovarian cancer serum----1.4; ascitic fluid----0.9; tumor----17.4). Asialo Cowper's gland mucin (ACGM) at 5 mg/ml reaction mixture inhibited the transfer of Gal to LGBn (25.2 and 53.4% respectively for 2 and 18 h incubation at 37 degrees C); inhibition by LGBn was 13.4 and 24.5%, respectively. In contrast to the inhibition by ACGM (25.2-31.6%), there was substantial increase (13.4-35.7%) in the inhibition by LGBn, when the incubation for 2 h at 37 degrees C was continued for 40 h at 4 degrees C, indicating the high affinity of LGBn for the enzyme at lower temp. Km for LGBn in presence of ACGM was 7.6 mM and in absence, 2.7 mM; Km for ACGM (M(r) 200,000) in presence of LGBn was 16.1 microM and Ki for ACGM (as the inhibitor) was 41.7 microM. In comparison with two normal ovarian tissues, the enzyme was found to be low (55-67%) in three ovarian tumors and high (146-260%) in two ovarian and one uterus tumors, as measured with ACGM; the synthetic acceptors showed similar activities. The enzyme had nearly the same extent of activity in the pH range 6-8. Fuc alpha 1,3GlcNAc beta 1,6GalNAc alpha-O-ONP had the highest affinity for the enzyme. The present study demonstrates the feasibility of beta 1,3Gal attachment on alpha GalNAc, which has already been substituted by beta 1,6GlcNAc, then elongated by beta 1,4Gal and also terminated by alpha 1,3Fuc.

Michael addition and acrylamide copolymerization of single N-acryloylated carbohydrate precursors of the Thomsen Friedenreich antigen (T-antigen) and an ABH type 2 human blood group trisaccharide determinant afforded both neoglycoprotein and glycopolymer conjugates suitable for immunochemical studies.

The “armed” methyl 2,3,4-tri- O -benzyl-1-thio-β- l -fucopyranoside was reacted with “disarmed” phenyl O -(tetra- O -acetyl-β- d -galactopyranosyl)-(1→4)-6- O -benzyl-2-deoxy-2-phthalimido-1-thio-β- d -glucopyranoside in the presence of CuBr 2 -Bu 4 NBr complex to give phenyl O -(2,3,4,6-tetra- O -acetyl-β- d -galactopyranosyl)-(1→4)- O -[(2,3,4-tri - O -benzyl-α- l -fucopyranosyl)-(1→3])-6- O -benzyl-2-deoxy-2-phthalimido-1-thio-β- d -glucopyranoside ( 6 ) as a novel glycosyl donor. The glycosylating capability of 6 was further examined using N -iodosuccinimide-triflic acid as a reagent. This led to the synthesis of a tetrasaccharide and a pentasaccharide incorporating the X-antigenic structure represented by 6 .

The specificity of glycosyltransferases is a major control factor in the biosynthesis of O-glycans. The enzyme that synthesizes O-glycan core 1, i.e., UDP-galactose:N-acetylgalactosamine-α-R β3-galactosyltransferase (β3-Gal-T; EC 2.4.1.122), was partially purified from rat liver. The enzyme preparation, free of pyrophosphatases, β4-galactosyltransferase, β-galactosidase, and N-acetylglucosaminyltransferase I, was used to study the specificity and inhibition of the β3-Gal-T. β3-Gal-T activity is sensitive to changes in the R-group of the GalNAcα-R acceptor substrate and is stimulated when the R-group is a peptide or an aromatic group. Derivatives of GalNAcα-benzyl were synthesized and tested as potential substrates and inhibitors. Removal or substitution of the 3-hydroxyl or removal of the 4-hydroxyl of GalNAc abolished β3-Gal-T activity. Compounds with modifications of the 3- or 4-hydroxyl of GalNAcα-benzyl did not show significant inhibition. Removal or substitution of the 6-hydroxyl of GalNAc reduced activity slightly and these derivatives acted as competitive substrates. Derivatives with epoxide groups attached to the 6-position of GalNAc acted as substrates and not as inhibitors, with the exception of the photosensitive 6-O-(4,4-azo)pentyl-GalNAcα-benzyl, which inhibited Gal incorporation into GalNAcα-benzyl. The results indicate that the enzyme does not require the 6-hydroxyl of GalNAc, but needs the 3- and the axial 4-hydroxyl as essential requirements for binding and activity. In the usual biochemical O-glycan pathway, core 2 (GlcNAcβ6[Galβ3]GalNAcα-) is formed from core 1 (Galβ3GalNAc-R). We have now demonstrated an alternate pathway that may be of importance in human tissues.Key words: β3-Gal-transferase, mucin synthesis, O-glycan core 1, enzyme specificity, enzyme inhibition.