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Small molecule monosaccharide analogs (e.g. 4F-GlcNAc, 4F-GalNAc) and acceptor decoys (e.g. ONAP, SNAP) are commonly used as metabolic glycoengineering tools to perturb molecular and cellular recognition processes. Azido-derivatized sugars (e.g. ManNAz, GlcNAz, GalNAz) are also used as bioorthogonal probes to assay the glycosylation status of cells and tissue. With the goal of obtaining a systems-level understanding of how these compounds work, we cultured cells with these molecules and systematically evaluated their impact on: (i) cellular nucleotide-sugar levels, and (ii) N-linked glycosylation. To this end, we developed a streamlined, simple workflow to quantify nucleotide-sugar levels using amide-based hydrophilic interaction liquid chromatography (HILIC) separation followed by negative-mode electrospray ionization mass spectrometry (ESI-MS/MS) using an Orbitrap detector. N-Glycans released from cells were also procainamide functionalized and quantified using positive-mode ESI-MS/MS. Results show that all tested compounds changed the baseline nucleotide-sugar levels, with the effect being most pronounced for the fluoro-HexNAc compounds. These molecules depressed UDP-HexNAc levels in cells by up to 80%, while concomitantly elevating UDP-4F-GalNAc and UDP-4F-GlcNAc. While the measured changes in nucleotide-sugar concentration were substantial in many cases, their impact on N-linked glycosylation was relatively small. This may be due to the high nucleotide-sugar concentrations in the Golgi, which far exceed the KM values of the glycosylating enzymes. Thus, the glycosylation system output exhibits 'robustness' even in the face of significant changes in cellular nucleotide-sugar concentrations.

Diabetes is a widely existing disease in patients at risk of pancreatic cancer. It originates from pancreatic endocrine gland whereas pancreatic cancer develops from exocrine glands. Leukocyte cell surface glycans are involved in leukocyte-endothelial cell adherence and retinal endothelial cell death in diabetic retinopathy. A stimulation of hexosamine biosynthesis pathway occurs in diabetes and in pancreatic cancer controlled by oncogene KRAS variant. The present study found a significant elevation of \(\alpha\)1-3 and \(\alpha\)1-4fucosyltransferase activities in pancreatic cancer serum. We examined 5 pancreatic non-tumor and 14 pancreatic tumor tissue specimens for quantitative changes in glycosyltransferse (GTs) activities in pancreatic tumorigenesis by following the incorporation of 14C or 3H monosaccharide (CPM) from 14C or 3H monosaccharide donors into specific acceptor catalyzed by 1 mg protein of TritonX-100 solubilized tissue extract. As compared to pancreatic non-tumor tissue specimens with a very low level of GTs activities, pancreatic tumor specimens on average contained 26.0, 42.9, 331.7, 121.0 and 62.8-fold of \(\alpha\)1-2, \(\alpha\)1-3, \(\alpha\)1-4, \(\alpha\)1-6 FTs and FT VI activities respectively. The major sialyltransferase \(\alpha\)2-3 (O)ST and sialomucin glycoproteins increased 95.4 and 4.0-fold; N-glycan \(\alpha\)Man: \(\beta\)1-2GlcNAc-T, chain elongating \(\beta\)Gal: \(\beta\)1-3 GlcNAc-T and N-glycan GalNAc capping \(\beta\)1-3/1-4 GalNAc-T were respectively 95.0, 2.7 and 14.8-fold and the mRNAs of FUT-4, \(\beta\)1-3 and \(\beta\)1-4 GalNAc-Ts were 8.3, 12.0 and 2.4-fold respectively. The increase in activity in neutrophils of retinopathy vs. normal was: \(\beta\)1-2- GlcNAc-T (9.0 fold), \(\beta\)1-3-GlcNAc-T (2.5), \(\alpha\)1-3- FT (3.5), \(\alpha\)1-6-FT (3.3), FTVII (1.9), \(\alpha\)GalNAc: \(\beta\)1-3-GalT (1.4), \(\beta\)GlcNAc: \(\beta\)1-4-Gal-T (2.1), \(\alpha\)2-3-(O)ST (2.1), \(\alpha\)2-3-(N)ST (4.5), \(\alpha\)2-6-(N)ST (8.1). GalNAc replacing Gal in LacNAc terminals results in changes of glycosyltransferase specificities and the modified GalNAc \(\beta\)1-4GlcNAc by FTs and STs bind to lectins such as WGA and SNA. In contrast to a low-level expression-difference of glycosyltransferases between tumor and non-tumor specimens from stomach, prostate and colon, a multifold increase in GTs in pancreatic tumor would indicate their significant role in invasion and intractability of pancreatic cancer. The high-level GT activities in diabetic neutrophils could contribute to the alterations in cell-cell interactions in diabetic retinopathy.

Skp1 is a subunit of the SCF ( S kp1/ C ullin 1/ F -box protein) class of E3 ubiquitin ligases that are important for eukaryotic protein degradation. Unlike its animal counterparts, Skp1 from Toxoplasma gondii is hydroxylated by an O2-dependent prolyl-4-hydroxylase (PhyA), and the resulting hydroxyproline can subsequently be modified by a five-sugar chain. A similar modification is found in the social amoeba Dictyostelium, where it regulates SCF assembly and O2-dependent development. Homologous glycosyltransferases assemble a similar core trisaccharide in both organisms, and a bifunctional α-galactosyltransferase from CAZy family GT77 mediates the addition of the final two sugars in Dictyostelium, generating Galα1, 3Galα1,3Fucα1,2Galβ1,3GlcNAcα1-. Here, we found that Toxoplasma utilizes a cytoplasmic glycosyltransferase from an ancient clade of CAZy family GT32 to catalyze transfer of the fourth sugar. Catalytically active Glt1 was required for the addition of the terminal disaccharide in cells, and cytosolic extracts catalyzed transfer of [3H]glucose from UDP-[3H]glucose to the trisaccharide form of Skp1 in a glt1-dependent fashion. Recombinant Glt1 catalyzed the same reaction, confirming that it directly mediates Skp1 glucosylation, and NMR demonstrated formation of a Glcα1,3Fuc linkage. Recombinant Glt1 strongly preferred the full core trisaccharide attached to Skp1 and labeled only Skp1 in glt1Δ extracts, suggesting specificity for Skp1. glt1-knock-out parasites exhibited a growth defect not rescued by catalytically inactive Glt1, indicating that the glycan acts in concert with the first enzyme in the pathway, PhyA, in cells. A genomic bioinformatics survey suggested that Glt1 belongs to the ancestral Skp1 glycosylation pathway in protists and evolved separately from related Golgi-resident GT32 glycosyltransferases.

Sialyltransferases (STs) catalyze the addition of sialic acids to the non-reducing ends of glycoproteins and glycolipids. In this work, we examined the acceptor specificity of five human α(2,3)sialyltransferases, namely ST3Gal -I, -II, -III, -IV and -VI. KM values for each of these enzymes is presented using radioactivity for acceptors containing Type-I (Galβ1,3GlcNAc), Type-II (Galβ1,4GlcNAc), Type-III (Galβ1,3GalNAc) and Core-2 (Galβ1,3(GlcNAcβ1,6)GalNAc) reactive groups. Several variants of acceptors inhibited ST3Gal activity emphasizing structural role of acceptor in enzyme-catalyzed reactions. In some cases, mass spectrometry was performed for structural verification. The results demonstrate human ST3Gal-I catalysis towards Type-III and Core-2 acceptors with KM = 5-50 μM and high VMax values. The KM for ST3Gal-I and ST3Gal-II was 100 and 30-fold lower, respectively, for Type-III compared to Type-I acceptors. Variants of Type-I and Type-II structures characterized ST3Gal-III, -IV and -VI for their catalytic specificity. This manuscript also estimates KM for human ST3Gal-VI using Type-I and Type-II substrates. Together, these findings built a platform for designing inhibitors of STs having therapeutic potential.

Glycan structure alterations during cancer regulate disease progression and represent clinical biomarkers. The study determined the degree to which changes in glycosyltransferase activities during cancer can be related to aberrant cell-surface tumor associated carbohydrate structures (TACA). To this end, changes in sialyltransferase (sialylT), fucosyltransferase (fucT) and galactosyltransferase (galT) activity were measured in normal and tumor tissue using a miniaturized enzyme activity assay and synthetic glycoconjugates bearing terminal LacNAc Type-I (Galβ1-3GlcNAc), LacNAc Type-II (Galβ1-4GlcNAc), and mucin core-1/Type-III (Galβ1-3GalNAc) structures. These data were related to TACA using tissue microarrays containing 115 breast and 26 colon cancer specimen. The results show that primary human breast and colon tumors, but not adjacent normal tissue, express elevated β1,3GalT and α2,3SialylT activity that can form α2,3SialylatedType-IIIglycans (Siaα2-3Galβ1-3GalNAc). Prostate tumors did not exhibit such elevated enzymatic activities. α1,3/4FucT activity was higher in breast, but not in colon tissue. The enzymology based prediction of enhanced α2,3sialylated Type-III structures in breast tumors was verified using histochemical analysis of tissue sections and tissue microarrays. Here, the binding of two markers that recognize Galβ1-3GalNAc (peanut lectin and mAb A78-G/A7) was elevated in breast tumor, but not in normal control, only upon sialidase treatment. These antigens were also upregulated in colon tumors though to a lesser extent. α2,3sialylatedType-III expression correlated inversely with patient HER2 expression and breast metastatic potential. Overall, enzymology measurements of glycoT activity predict truncated O-glycan structures in tumors. High expression of the α2,3sialylated T-antigen O-glycans occur in breast tumors. A transformation from linear core-1 glycan to other epitopes may accompany metastasis.

Summary Metabolic decoys are synthetic analogs of naturally occurring biosynthetic acceptors. These compounds divert cellular biosynthetic pathways by acting as artificial substrates that usurp the activity of natural enzymes. While O-linked glycosides are common, they are only partially effective even at millimolar concentrations. In contrast, we report that N-acetylglucosamine (GlcNAc) incorporated into various thioglycosides robustly truncate cell surface N- and O-linked glycan biosynthesis at 10–100 μM concentrations. The >10-fold greater inhibition is in part due to the resistance of thioglycosides to hydrolysis by intracellular hexosaminidases. The thioglycosides reduce β-galactose incorporation into lactosamine chains, cell surface sialyl Lewis-X expression, and leukocyte rolling on selectin substrates including inflamed endothelial cells under fluid shear. Treatment of granulocytes with thioglycosides prior to infusion into mouse inhibited neutrophil homing to sites of acute inflammation and bone marrow by ∼80%–90%. Overall, thioglycosides represent an easy to synthesize class of efficient metabolic inhibitors or decoys. They reduce N-/O-linked glycan biosynthesis and inflammatory leukocyte accumulation.

Plant lectins through their multivalent quaternary structures bind intrinsically flexible oligosaccharides. They recognize fine structural differences in carbohydrates and interact with different sequences in mucin core 2 or complex-type N-glycan chain and also in healthy and malignant tissues. They are used in characterizing cellular and extracellular glycoconjugates modified in pathological processes. We study here, the complex carbohydrate-lectin interactions by determining the effects of substituents in mucin core 2 tetrasaccharide Galβ1-4GlcNAcβ1-6(Galβ1-3)GalNAcα-O-R and fetuin glycopeptides on their binding to agarose-immobilized lectins PNA, RCA-I, SNA-I and WGA. Briefly, in mucin core 2 tetrasaccharide (i) structures modified by α2-3/6-Sialyl LacNAc, LewisX and α1-3-Galactosyl LacNAc resulted in regular binding to PNA whereas compounds with 6-sulfo LacNAc displayed no-binding; (ii) strucures bearing α2-6-sialyl 6-sulfo LacNAc, or 6-sialyl LacdiNAc carbohydrates displayed strong binding to SNA-I; (iii) structures with α2-3/6-sialyl, α1-3Gal LacNAc or LewisX were non-binder to RCA-I and compounds with 6-sulfo LacNAc only displayed weak binding; (iv) structures containing LewisX, 6-Sulfo LewisX, α2-3/6-sialyl LacNAc, α2-3/6-sialyl 6-sulfo LacNAc and GalNAc Lewis-a were non-binding to WGA, those with α1-2Fucosyl, α1-3-Galactosyl LacNAc, α2-3-sialyl T-hapten plus 3'/6'sulfo LacNAc displayed weak binding, and compounds with α2-3-sialyl T-hapten, α2.6-Sialyl LacdiNAc, α2-3-sialyl D-Fucβ1-3 GalNAc and Fucα-1-2 D-Fucβ-1-3GalNAc displaying regular binding and GalNAc LewisX and LacdiNAc plus D-Fuc β-1-3 GalNAcα resulting in tight binding. RCA-I binds Fetuin triantennary asialoglycopeptide 100 % after α-2-3 and 25 % after α-2-6 sialylation, 30 % after α-1-2 and 100 % after α-1-3 fucosylation, and 50 % after α-1-3 galactosylation. WGA binds 3-but not 6-Fucosyl chitobiose core. Thus, information on the influence of complex carbohydrate chain constituents on lectin binding is apparently essential for the potential application of lectins in glycoconjugate research.

Our previous studies suggest that the α2,3sialylated T-antigen (NeuAcα2,3Galβ1,3GalNac-) and associated glycan structures are likely to be elevated during cancer. An easy and reliable strategy to label mucinous glycans that contain such carbohydrates can enable the identification of novel glycoproteins that are cancer associated. To this end, the present study demonstrates that the exchange sialylation property of mammalian ST3Gal-II can facilitate the labeling of mucin glycoproteins in cancer cells, tumor specimens, and glycoproteins in cancer sera. Results show that (i) the radiolabeled mucin glycoproteins of each of the cancer cell lines studied (T47D, MCF7, LS180, LNCaP, SKOV3, HL60, DU4475, and HepG2) is distinct either in terms of the specific glycans presented or their relative distribution. While some cell lines like T47D had only one single sialylated O-glycan, others like LS180 and DU4475 contained a complex mixture of mucinous carbohydrates. (ii) [14C]sialyl labeling of primary tumor cells identified a 25-35 kDa mucin glycoprotein unique to pancreatic tumor. Labeled glycoproteins for other cancers had higher molecular weight. (iii) Studies of [14C] sialylated human sera showed larger mucin glycopeptides and >2-fold larger mucin-type chains in human serum compared to [14C]sialyl labeled glycans of fetuin. Overall, the exchange sialylation property of ST3Gal-II provides an efficient avenue to identify mucinous proteins for applications in glycoproteomics and cancer research.

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLe(X)), and related lectin ligands on effector leukocytes. Based on anti-sLe(X) antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLe(X) formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLe(X) (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLe(X) structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLe(X) on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.

Predominant α-linked products can be generated in glycosylation involving galactosyl trichloroacetimidate donors with 2-naphthylmethyl (NAP) as the non-participating group at C-2 position. The above-mentioned donor was successfully utilized for the synthesis of α-galactosyl ceramide.

Syntheses of fluorinated mucin core 6 disaccharides and core 2 trisaccharides modified at the C-3 or C-4 position of the pertinent glucosamine residue required for mechanistic study of glycosyltransferases and sulfotransferases involved in the biosynthesis of O-glycans are reported. Novel fluorinated glucosaminyl donors were synthesized from 2-naphthylmethyl β- d -N-acetylglucosamine (β-O-NAP-GlcNAc) via double inversion of the C-3 or C-4 configuration. A one-step β-alkylation of GlcNAc was reported for the first time to afford β-O-NAP-GlcNAc in high yield, which constitutes the cornerstone of the synthetic strategy based on NAP-glycosides in oligosaccharides synthesis.

Stereoselective syntheses of two 3- O-Galsulfated trisaccharides GalNAcβ(1-4)[(3-SE)-Gal]β(1-3)GalNAcα- O-All and GalNAcβ(1-4)[(3-SE)-Gal]β(1-4)Glcβ- O-All were accomplished through the useof three novel glycosyl acceptors, namely, allyl 4,6- O-benzylidene-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-α- D-galactopyranoside, methyl 2,6-di- O-benzoyl-3- O-naphthylmethyl-α- D-galactopyranoside and allyl 6- O-acetyl-2- O-benzoyl-3- O-naphthylmethyl-β- D-galactopyranosyl-(1→4)-2,3,6-tri- O-benzoyl-β- D-glucopyranoside.These sulfated trisaccharides were expected to act as potentialreference compounds for human β4GalNAc transferase andcan be effectively used as antigens when linked to KLH.

The application of systems biology methods in the emerging field of glycomics requires the collection and integration of glycosyltransferase data at the gene and enzyme level for the purpose of hypothesis generation. We systematically examined the relationship between gene expression, glycosyltransferase activity, glycan expression, and selectin-binding function in different systems, including human neutrophils, undifferentiated HL-60 (human promyelocytic cells), differentiated HL-60, and HL-60 synchronized in specific growth phases. Results demonstrate that 1) the sLeX (sialyl-Lewis-X) epitope is expressed in P-selectin glycoprotein ligand-1 (PSGL-1) from neutrophils at higher levels compared with HL-60. This variation may be due to differences in the relative activities of α1,3-fucosyltransferases and α2,3-sialyltransferases in these two cell types. 2) HL-60 cell differentiation along granulocyte lineage increased the activity of β1,4GalT and β1,3GlcNAcT by 1.6- to 3.2-fold. This may contribute to LacNAc chain extension as evidenced by the 1.7-fold increase in DSA-lectin (lectin recognizing LacNAc) binding to cells after differentiation. 3) The activity of enzymes contributing to sLeX formation in leukocytes likely varies as ST3[Galβ1,4GlcNAc] ≤ α1,3FT[sialyl-LacNAc] < β1,3GlcNAcT. 4) O-glycan specific glycosyltransferase activity does not undergo periodic variation with cell cycle phases. Overall, gene expression and enzyme activity data combined with knowledge of biochemistry can predict the resulting glycan structures and yield viable experimentally testable hypothesis.—Marathe, D. D., Chandrasekaran, E. V., Lau, J. T. Y., Matta, K. L., Neelamegham, S. Systems-level studies of glycosyltransferase gene expression and enzyme activity that are associated with the selectin binding function of human leukocytes.

Sialyltransferases transfer sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-NeuAc) to an acceptor molecule. Trans-sialidases of parasites transfer alpha2,3-linked sialic acid from one molecule to another without the involvement of CMP-NeuAc. Here we report another type of sialylation, termed reverse sialylation, catalyzed by mammalian sialyltransferase ST3Gal-II. This enzyme synthesizes CMP-NeuAc by transferring NeuAc from the NeuAcalpha2,3Galbeta1,3GalNAcalpha unit of O-glycans, 3-sialyl globo unit of glycolipids, and sialylated macromolecules to 5'-CMP. CMP-NeuAc produced in situ is utilized by the same enzyme to sialylate other O-glycans and by other sialyltransferases such as ST6Gal-I and ST6GalNAc-I, forming alpha2,6-sialylated compounds. ST3Gal-II also catalyzed the conversion of 5'-uridine monophosphate (UMP) to UMP-NeuAc, which was found to be an inactive sialyl donor. Reverse sialylation proceeded without the need for free sialic acid, divalent metal ions, or energy. Direct sialylation with CMP-NeuAc as well as the formation of CMP-NeuAc from 5'-CMP had a wide optimum range (pH 5.2-7.2 and 4.8-6.4, respectively), whereas the entire reaction comprising in situ production of CMP-NeuAc and sialylation of acceptor had a sharp optimum at pH 5.6 (activity level 50% at pH 5.2 and 6.8, 25% at pH 4.8 and 7.2). Several properties distinguish forward/conventional versus reverse sialylation: (i) sodium citrate inhibited forward sialylation but not reverse sialylation; (ii) 5'-CDP, a potent forward sialyltransferase inhibitor, did not inhibit the conversion of 5'-CMP to CMP-NeuAc; and (iii) the mucin core 2 compound 3-O-sulfoGalbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-benzyl, an efficient acceptor for ST3Gal-II, inhibited the conversion of 5'-CMP to CMP-NeuAc. A significant level of reverse sialylation activity is noted in human prostate cancer cell lines LNCaP and PC3. Overall, the study demonstrates that the sialyltransferase reaction is readily reversible in the case of ST3Gal-II and can be exploited for the enzymatic synthesis of diverse sialyl products.

The sialyl-T antigen sialylα2-3Galβ1-3GalNAc is a common O-glycan structure in human glycoproteins and is synthesized by sialyltransferase ST3Gal1. The enterohemorrhagic Escherichia coli serotype O104 has the rare ability to synthesize a sialyl-T antigen mimic. We showed here that the wbwA gene of the E. coli O104 antigen synthesis gene cluster encodes an α2,3-sialyltransferase WbwA that transfers sialic acid from CMP-sialic acid to Galβ1-3GalNAcα-diphosphate-lipid acceptor. Nuclear magnetic resonance (NMR) analysis of purified WbwA enzyme reaction product indicated that the sialyl-T antigen sialylα2-3Galβ1-3GalNAcα-diphosphate-lipid was synthesized. We showed that the conserved His-Pro (HP) motif and Glu/Asp residues of two EDG motifs in WbwA are important for the activity. The characterization studies showed that WbwA from E. coli O104 is a monofunctional α2,3-sialyltransferase and is distinct from human ST3Gal1 as well as all other known sialyltransferases due to its unique acceptor specificity. This work contributes to knowledge of the biosynthesis of bacterial virulence factors. IMPORTANCE This is the first characterization of a sialyltransferase involved in the synthesis of an O antigen in E. coli . The enzyme contributes to the mimicry of human sialyl-T antigen and has unique substrate specificity but very little sequence identity to other sialyltransferases. Thus, the bacterial sialyltransferase is related to the human counterpart only by the similarity of biochemical activity.

Syntheses of fluorinated mucin core 2 tri- and tetrasaccharides modified at the C-3 or C-4 position of the pertinent galactose residue are reported. These compounds were used for the study of sialyltransferases and 3-O-sulfotransferases involved in the biosynthesis of O-glycans. Our acceptor substrate specificity studies on three cloned sialyltransferases (Sia-Ts) revealed that a 3- or 4-fluoro substituent in beta1,4Gal resulted in poor acceptors for alpha2,6(N)Sia-T and alpha2,3(N)Sia-T, whereas 4-fluoro-Galbeta1,3GalNAcalpha was a good acceptor for alpha2,3(O)Sia-T. Uniquely, 4-F-Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-OBn was an inhibitor of alpha2,6(N)Sia-T activity but not alpha2,3(N)Sia-T activity. Further we found that the activities of only Gal 3-O-sulfotransferases and not sialyltransferases were adversely affected by a C-3 fluoro substituent at the other Gal terminal of mucin core 2. The strategy of building branched mucin core 2 structures by three glycosidation sequence coupling three classes of glycosyl donors with the reactivity-matching acceptors proved to be successful in syntheses of modified mucin-type core structures of O-glycan. The relative poor yields of the glycosylations using fluorinated galactosyl donors indicated that the fluorine modification dramatically decreased the donor reactivity due to electron-withdrawing effect.

Sialic acids are key determinants in many carbohydrates involved in biological recognition. We studied the acceptor specificities of three cloned sialyltransferases (STs) [alpha2,3(N)ST, alpha2,3(O)ST, and alpha2,6(N)ST] and another alpha2,3(O)ST present in prostate cancer cell LNCaP toward mucin core 2 tetrasaccharide [Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAcalpha-O-Bn] and Globo [Galbeta1,3GalNAcbeta1,3Galalpha-O-Me] structures containing sialyl, fucosyl, sulfo, methyl, or fluoro substituents by identifying the products by electrospray ionization tandem mass spectral analysis and other biochemical methods. The Globo precursor was an efficient acceptor for both alpha2,3(N)ST and alpha2,3(O)ST, whereas only alpha2,3(O)ST used its deoxy analogue (d-Fucbeta1,3GalNAcbeta1,3-Gal-alpha-O-Me); 2-O-MeGalbeta1,3GlcNAc and 4-OMeGalbeta1,4GlcNAc were specific acceptors for alpha2,3(N)ST. Other major findings of this study include: (i) alpha2,3 sialylation of beta1,3Gal in mucin core 2 can proceed even after alpha1,3 fucosylation of beta1,6-linked LacNAc. (ii) Sialylation of beta1,3Gal must precede the sialylation of beta1,4Gal for favorable biosynthesis of mucin core 2 compounds. (iii) alpha2,3 sialylation of the 6-O-sulfoLacNAc moiety in mucin core 2 (e.g., GlyCAM-1) is facilitated when beta1,3Gal has already been alpha2,3 sialylated. (iv) alpha2,6(N)ST was absolutely specific for the beta1,4Gal in mucin core 2. Either alpha1,3 fucosylation or 6-O-sulfation of the GlcNAc moiety reduced the activity. Sialylation of beta1,3Gal in addition to 6-O-sulfation of GlcNAc moiety abolished the activity. (v) Prior alpha2,3 sialylation or 3-O-sulfation of beta1,3Gal would not affect alpha2,6 sialylation of Galbeta1,4GlcNAc of mucin core 2. (vi) A 3- or 4-fluoro substituent in beta1,4Gal resulted in poor acceptors for the cloned alpha2,6(N)ST and alpha2,3(N)ST, whereas 4-fluoro- or 4-OMe-Galbeta1,3GalNAcalpha was a good acceptor for cloned alpha2,3(O)ST. (vii) 4-O-Methylation of beta1,4Gal abolished the acceptor ability toward alpha2,6(N)ST but increased the acceptor efficiency considerably toward alpha2,3(N)ST. (viii) Just like LNCaPalpha1,2-FT and Gal-3-O-sulfotransferase T2, the cloned alpha2,3(N)ST which modifies terminal Gal in Galbeta1,4GlcNAc also efficiently utilizes the terminal beta1,3Gal in the Globo backbone. Utilization of C-3 blocked compounds such as 3-O-sulfo-Galbeta1,3GalNAcbeta1,3Galalpha-OMe as acceptors by cloned alpha2,3(O)ST and analyses of the resulting products by lectin chromatography and mass spectrometry indicate that alpha2,3(O)ST is capable of attaching NeuAc to another position in C-3-substituted beta1,3Gal.

Skp1 is an adaptor-like protein in E3(SCF)-ubiquitin ligases and other multiprotein complexes of the cytoplasm and nucleus. In Dictyostelium, Skp1 is modified by an unusual pentasaccharide containing a Galalpha1-Fuc linkage, whose formation is examined here. A cytosolic extract from Dictyostelium was found to yield, after 2400-fold purification, an activity that could transfer Gal from UDP-Gal to both a Fuc-terminated glycoform of Skp1 and synthetic Fuc conjugates in the presence of Mn(2+) and dithiothreitol. The microsomal fraction was devoid of activity. The linkage formed was Galalpha1,3Fuc based on co-chromatography with only this synthetic isomer conjugate, and sensitivity to alpha1,3/6-galactosidase. Skp1 exhibited an almost 1000-fold lower K(m) and 35-fold higher V(max) compared with a simple alpha-fucoside, but this advantage was abolished by denaturation or alkylation of Cys residues. A comparison of a complete series of synthetic glycosides representing the non-reducing terminal mono-, di-, and trisaccharides of Skp1 revealed, surprisingly, that the disaccharide is most active owing primarily to a V(max) advantage, but still much less active than Skp1 itself because of a K(m) difference. These findings indicate that alpha-GalT1 is a cytoplasmic enzyme whose modification of Skp1 requires proper presentation of the terminal acceptor disaccharide by a folded Skp1 polypeptide, which correlates with previous evidence that the Galalpha1,3Fuc linkage is deficient in expressed mutant Skp1 proteins.

Sulfated glycoconjugates regulate biological processes such as cell adhesion and cancer metastasis. We examined the acceptor specificities and kinetic properties of three cloned Gal:3-O-sulfotransferases (Gal3STs) ST-2, ST-3, and ST-4 along with a purified Gal3ST from colon carcinoma LS180 cells. Gal3ST-2 was the dominant Gal3ST in LS180. While the mucin core-2 structure Galβ1,4GlcNAcβ1,6(3-O-MeGalβ1,3)GalNAcα-O-Bn (where Bn is benzyl) and the disaccharide Galβ1,4GlcNAc served as high affinity acceptors for Gal3ST-2 and Gal3ST-3, 3-O-MeGalβ1,4GlcNAcβ1,-6(Galβ1,3)GalNAcα-O-Bn and Galβ1,3GalNAcα-O-Al (where Al is allyl) were efficient acceptors for Gal3ST-4. The activities of Gal3ST-2 and Gal3ST-3 could be distinguished with the Globo H precursor (Galβ1,3GalNAcβ1,3Galα-O-Me) and fetuin triantennary asialoglycopeptide. Gal3ST-2 acted efficiently on the former, while Gal3ST-3 showed preference for the latter. Gal3ST-4 also acted on the Globo H precursor but not the glycopeptide. In support of the specificity, Gal3ST-2 activity toward the Galβ1,4GlcNAcβ unit on mucin core-2 as well as the Globo H precursor could be inhibited competitively by Galβ1,4GlcNAcβ1,6(3-O-sulfoGalβ1,3)GalNAcα-O-Bn but not 3-O-sulfoGalβ1,-4GlcNAcβ1,6(Galβ1,3)GalNAcα-O-Bn. Remarkably these sulfotransferases were uniquely specific for sulfated substrates: Gal3ST-3 utilized Galβ1,4(6-O-sulfo)-GlcNAcβ-O-Al as acceptor, Gal3ST-2 acted efficiently on Galβ1,3(6-O-sulfo)GlcNAcβ-O-Al, and Gal3ST-4 acted efficiently on Galβ1,3(6-O-sulfo)GalNAcα-O-Al. Mg2+, Mn2+, and Ca2+ stimulated the activities of Gal3ST-2, whereas only Mg2+ augmented Gal3ST-3 activity. Divalent cations did not stimulate Gal3ST-4, although inhibition was noted at high Mn2+ concentrations. The fine substrate specificities of Gal3STs indicate a distinct physiological role for each enzyme.

The synthesis of the common and well-documented Siaalpha 2,6 to Galbeta 1,4GlcNAc structure (Sia6LacNAc) is principally mediated by the sialyltransferase ST6Gal I, which is particularly highly expressed in liver, lactating mammary gland, intestinal epithelia of newborn animals, and B cells. Multiple independent promoters govern the expression of Siat1, the ST6Gal I gene. In liver, elevation of hepatic and serum ST6Gal is part of the acute phase reaction, the hepatic response to systemic trauma, and is governed by the inducible, liver-specific promoter-regulatory region, P1. A constitutive and nontissue-specific promoter, P3, mediates low-level, basal hepatic Siat1 transcription. We generated a mouse specifically unable to use the transcriptional initiation site uniquely used in P1-mediated ST6Gal I expression. These animals, Siat1deltaP1, are viable and display reduced ST6Gal I mRNA in liver with concomitantly reduced sialyltransferase activities in liver and in serum. Siat1deltaP1 animals are unable to elevate hepatic Siat1 mRNA as part of the inflammatory response induced by turpentine. Surprisingly, serum glycoprotein components exhibit normal extent of sialylation, with no noticeable difference in binding to SNA, the alpha2,6-sialyl-specific lectin. Siat1deltaP1 animals also exhibit an outwardly normal B cell response. On intraperitoneal challenge with the pathogen Salmonella typhimurium, a significantly greater accumulation of neutrophils within the peritoneal space was observed in Siat1deltaP1 animals compared to wild-type mice. Siat1deltaP1 mice also exhibit a greater bacterial burden in liver and spleen, accompanied by more pronounced spleno-/hepatomegaly and greater leukocyte infiltration into affected organs than their wild-type counterparts.

A convergent pathway for the syntheses of core 2 oligosaccharide analogues 1 and 2, and a natural form sialylated and sulfated hexasaccharide 3 was developed. Construction of pentasaccharides 24, 27 and hexasaccharide 28 was achieved by complete regioselective glycosylation of the 6-OH in the acceptors 5, 7 and 8, respectively, owing to the much higher reactivity of the primary hydroxyl group over the secondary axial hydroxyl group in these structures. Stereoselective sialylation was accomplished using donor 10 with defined configuration established through X-ray crystallographic analysis. Target oligosaccharides 1-3 were then obtained by the systematic deprotection of intermediates 24, 27 and 29. With these target oligosaccharides 1-3 obtained, biological evaluations of these molecules as enzyme substrates was undertaken and selectin binding studies are planned.

Objective: Approximately 50% of patients with ovarian cancer who have normal CA 125 levels at the completion of therapy have persistent disease. In an effort to improve the ability to detect small volume disease, we have evaluated the usefulness of N -acetylglucosamine:β1,4-galactosyltransferase as a potential biomarker for the detection of subclinical disease after the completion of primary therapy for ovarian cancer. Study Design: The sera of 33 patients with stage IIIC epithelial ovarian cancer in complete clinical remission after chemotherapy (CA 125 N -acetylglucosamine:β1,4-galactosyltransferase activity. The values were determined from sera that had been obtained before primary cytoreductive operation and before second-look surgery after the completion of platinum-based chemotherapy. Determinations of the levels of CA 125 were performed with the Bayer Immuno ITM CA-125 II assay. N -acetylglucosamine:β1,4-galactosyltransferase activity was determined by measuring the transfer of galactose from uridine diphosphate– carbon 14–labeled galactose to the terminal N -acetylglucosamine residue of a very well-defined synthetic acceptor, N -acetylglucosamine:β1,6GalNAcα-o-benzyl, which is a portion of the core structure of mucin glycoproteins. The cutoff value of N -acetylglucosamine:β1,4-galactosyltransferase was determined to be 22,000 counts/min, based on the analysis of 25 healthy control subjects. Correlation between serum CA 125 and N -acetylglucosamine:β1,4-galactosyltransferase levels was determined with the use of the Pearson correlation coefficient. The ability of galactosyltransferase to identify small volume disease correctly was also evaluated. Results: There was a significant correlation between serum CA 125 and N -acetylglucosamine:β1,4-galactosyltransferase levels before the operation ( r = 0.57; P =.03) but not before second-look surgery ( r = 0.10; P =.57). Thirteen patients (39.4%) had residual disease at second-look surgery. Elevated N -acetylglucosamine:β-1,4galactosyltransferase activity >22,000 cpm correctly identified 10 of these patients (76.9%). The sensitivity, specificity, and positive and negative predictive values of N -acetylglucosamine:β1,4-galactosyltransferase activity (>22,000 counts/min) for the prediction of residual disease at second-look surgery were 77%, 45%, 48%, and 77%, respectively. Conclusion: Our comparative study of serum CA 125 and N -acetylglucosamine:β1,4-galactosyltransferase levels showed a significant correlation between the two tumor markers before the beginning of ovarian cancer therapy. This correlation disappeared before second-look surgery because 60% of patients with normal serum CA 125 and N -acetylglucosamine:β1,4-galactosyltransferase levels. CA 125 antigen appears to be inferior to N -acetylglucosamine:β1,4-galactosyltransferase in the detection of small-volume residual disease. N -acetylglucosamine:β1,4-galactosyltransferase may be useful as a biomarker in the monitoring of patients with ovarian cancer when the serum CA 125 level is normal. These findings require confirmation in larger studies. (Am J Obstet Gynecol 2002;187:575-80.)

Galectin-4 is a member of galectin family and has two carbohydrate recognition domains. Although galectin-4 has been thought to function in cell adhesion, its precise carbohydrate binding specificity has not yet been clarified. We studied the carbohydrate binding specificity of galectin-4 comparatively with that of galectin-3, using surface plasmon resonance, galectin-3- or -4-Sepharose column chromatography and the inhibition assay of their binding to immobilized asialofetuin. Galectin-3 broadly recognized lactose, type 1, type 2, and core 1. The substitution at the C-2 and C-3 position of beta-galactose in these oligosaccharides with alpha-fucose, alpha-GalNAc, alpha-Neu5Ac, or sulfate increased the binding ability for galectin-3, whereas the substitution at the C-4 or C-6 position diminished the affinity. In contrast, galectin-4 had quite weak affinity to lactose, type 1, and type 2 (K(d) congruent with 8 x 10(-4) M). Galectin-4 showed weak binding ability to core 1 and C-2' or -3'-substituted lactose, type 1, and type 2 with alpha-fucose, alpha-GalNAc, or sulfate (K(d) : 5 x 10(-5) approximately 3 x 10(-4) M). Interestingly, the K(d) value, 3.4 x 10(-6) M, of SO(3)(-)-->3Galbeta1-->3GalNAc-O-Bn to galectin-4 at 25 degrees C was two orders of magnitude lower than that of core 1-O-Bn. 3'-Sialylated core 1 had very weak affinity to galectin-4, suggesting that 3'-O-sulfation of core 1 is critical for the recognition. These results suggest that galectin-4 has a unique carbohydrate binding specificity and interacts with O-linked sulfoglycans.

Searchable mass spectral libraries for glycans may be enhanced using a B2 ion library. Using a quadrupole ion-trap mass spectrometer, successive fragmentations of sodiated oligosaccharides were carried out in the positive ion mode. In B,Y-type fragmentation, disaccharide B2 ions are generated which correspond to specific glycosidic linkages using progressive MS stages. Fragmentation of “B2 ions” corresponding to glycosidic linkages such as Hex–Fuc, Hex–Hex, Hex–HexNAc, HexNAc–Hex and HexNAc–HexNAc, were systematically studied in low energy CID and collected to form a “B2 library”. Linkages produce characteristic fragmentation patterns in the absence of cross-ring fragmentation. Patterns of “B2 ions” rely on relative stability of glycosidic bonds and carbohydrate–metal complexes in the gas phase. MSn studies of linear, branched trisaccharides and tetrasaccharides show that isomers for which B2 ion information is not available are rarely a problem in practice by their absence in an isomeric sequence or by their scarcity in nature. This MS strategy for linkage determination of carbohydrates aided by a “B2 library” was developed with a scope for expansion, providing an improved tool for glycomics. We validated this method examining levels of expressed activities of two glycosyl transferases in cancer cell lines: β3(B3GALNT2) and β4GalNAcT(B4GALNT3&4) that generate GalNAcβ3GlcNAcβ and GalNAcβ4GlcNAcβ. Biological significance Glycosylation is an important class of the “postranslationome”, which includes manifold aspects of post-translational protein modification, affecting protein conformation, providing ligands for protein receptors [1] , [2] , [3] , [4] , [5] , and encoding unique haptenic [6] , [7] or antigenic markers for oncology [8] , [9] , [10] , [11] and other applications. Identification of individual monomeric units, linkages, ring size, branching and anomerity has posed significant challenges to mass spectrometrists. MSn is a growing key instrumental method to differentiate among isomers [12] . While the potential isomers in oligosaccharides are impossibly large [12] , likely possibilities can be limited by the biological system, including the expressed glycosyl transferases [13] , [14] , [15] , [16] , [17] , [18] , [19] , [20] . Mass spectra from sequential stages of collision activation (MSn) can supply structural details for precise characterization of linkage, monomer ID, substitutions, anomerity and branching [21] , [22] , [23] , [24] , [25] . There is a fundamental need for high throughput tools in glycomics to complement proteome studies. In that regard, nothing could be more important than searchable spectral library files for structural confirmation. The National Academy of Science (NAS) report ( http://glyco.nas.edu ) recommends the need of more than 10,000 synthetic structures of carbohydrates to advance the field of glycomics. This study demonstrates that the general reproducibility of ion trap spectra, and energy independence from modes of ionization and collisional activation, make compiling an MSn library for carbohydrate identification an achievable research target [26] . We intend to use the new B2 library for carbohydrate differences found on cancers, where we profile the glycosyltransferases to predict classes of potential structures, and use the library for MS identification of the expected cohort of altered structures.

Affinity Gel-UDP was utilized to purify GlcNAc:beta1,4Gal/GalNAc transferases (Ts) from human lymphoma spleen, ovarian tumor, and ovarian cancer sera. Mn(2+) was found to be an absolute requirement for activity. Two molecular species containing both beta1,4Gal/GalNAc-T activities were discernible when the purified ovarian tumor microsomal enzyme was subjected to Sephacryl S-100 HR column chromatography as well as native polyacylamide gel-electrophoresis. Acceptor specificity studies of the affinity-purified lymphoma spleen and ovarian tumor microsomal enzymes and the conventionally purified, as well as the cloned, bovine milk GlcNAc:beta1,4Gal-Ts using a number of synthetic acceptors showed that the beta(1,6)-linked GlcNAc moiety to alpha-GalNAc was the most efficient acceptor. As compared to the purified milk enzyme, the recombinant form exhibited sixfold GlcNAc:beta1,4 GalNAc-T activity and up to eightfold GlcNAc6SO3beta-:beta1,4Gal-T activity. Further, the recombinant enzyme catalyzed the transfer of GalNAc to the terminal beta-linked GlcNAc6SO3 moiety. Alpha-lactalbumin (alpha-LA) inhibited up to 85%, the transfer of Gal to the GlcNAc moiety linked either to Man or GlcNAc. On the contrary, alpha-LA had no significant influence on the transfer of GalNAc to the above acceptors. alpha-LA had no appreciable effect on the recombinant enzyme, except for the transfer of Gal or GalNAc to Glc. Both alpha- and beta-glucosides, as well as alpha-N-acetylglucosaminide, did not serve as acceptors.

Total syntheses of the GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1) oligosaccharide structures: [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-(6-O-SO3Na)-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (1) and [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (2) through a novel sialyl LewisX tetrasaccharide donor are described. Employing sequential glycosylation strategy, the starting trisaccharide was regio- and stereoselectively constructed through coupling of a disaccharide imidate with the monosaccharide acceptor phenyl-6-O-naphthylmethyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside with TMSOTf as a catalyst without affecting the SPh group. The novel sialyl Lewisx tetrasaccharide donor 3 was then obtained by alpha-L-fucosylation of trisaccharide acceptor with the 2,3,4-tri-O-benzyl-1-thio-beta-L-fucoside donor. The structure of the novel sialyl Lewisx tetrasaccharide was established by a combination of 2D DQF-COSY and 2D ROESY experiments. Target oligosaccharides 1 and 2 were eventually constructed through heptasaccharide which was obtained by regioselective assembly of advanced sialyl Lewisx tetrasaccharide donor 3 and a sialylated trisaccharide acceptor in a predictable and controlled manner. Finally, target heptasaccharides 1 and 2 were fully characterized by 2D DQF-COSY, 2D ROESY, HSQC, HMBC experiments and FAB mass spectroscopy.

The syntheses of two sulfated pentasaccharides: β- d -Gal6SO 3 Na-(1→3)-[β- d -Gal-(1→4)-α- l -Fuc-(1→3)-β- d -GlcNAc-(1→6)]-α- d -GalNAc→OMe ( 1 ) and β- d -Gal6SO 3 Na-(1→3)-[β- d -Gal-(1→4)-α- l -Fuc-(1→3)-β- d -GlcNAc6SO 3 Na-(1→6)]-α- d -GalNAc→OMe ( 2 ) by using Lewis X trisaccharides 12 and 16 as glycosyl donors are described. Sulfated oligosaccharides 1 – 2 and intermediate compounds are fully characterized by 2D 1 H– 1 H DQF-COSY and 2D ROESY experiments.

The total syntheses of several complex oligosaccharide moieties that occur in the core structure of sulfated mucins are reported. A trisaccharide acceptor was obtained through regio- and stereoselective sialylation of methyl (6-O-pivaloyl-beta-D-galactopyanosyl)(1-->3)-4,6-O-benzylidene-2-a cetamido-2-deoxy-alpha-D-galactopyranoside with a novel sialyl donor. A tetrasaccharide, pentasaccharide, and hexasaccharide were constructed in predictable and controlled manner with high regio- and stereoselectivity after the successful preparation and employment of a disaccharide donor, trisaccharide donor, disaccharide acceptor, and trisaccharide acceptor building blocks. Finally, a mild oxidative cleaving method was adopted for the selective removal of 2-naphthylmethyl (NAP) in the presence of benzyl groups.

The syntheses of three trisaccharides: α-Neu5Ac-(2→3)-β- d -Gal-(1→4)-β- d -GlcNAc→OMe, α-Neu5Ac-(2→3)-β- d -Gal6SO 3 Na-(1→4)-β- d -GlcNAc→OMe, and α-Neu5Ac-(2→3)-β- d -Gal-(1→3)-α- d -GalNAc→OBn were accomplished by using either methyl (phenyl 5-acetamido-4,7,8,9-tetra- O -acetyl-3,5-dideoxy-2-thio-β- d - glycero - d - galacto -2-nonulopyranoside)onate or methyl (phenyl N -acetyl-5-acetamido-4,7,8,9-tetra- O -acetyl-3,5-dideoxy-2-thio-β- d - glycero - d - galacto -2-nonulopyranoside)onate as the sialyl donor. The N , N -diacetylamino sialyl donor appears to be more reactive than its parent acetamido sugar when allowed to react with an disaccharide acceptor under the same glycosylation conditions. The trisaccharides, as well as the intermediate products, were fully characterized by 2D DQF 1 H– 1 H COSY and 2D ROESY spectroscopy.

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 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 .

A new ex vivo method for assaying adhesion of cancer cells to the greater omentum has been developed using mouse greater omentum and [3H]labelled human gastric and mouse colorectal cancer cells. Since the adhesion rates were found to increase up to 18 h and labelled cells seemed to be stable during the period, the present method could be useful for investigating adhesion of cancer cells to the greater omentum, which must occur at the first step of the peritoneal dissemination. The adhesion of cancer cells to the greater omentum was inhibited by a series of chemically synthesized oligosaccharides and Galβ1,3[3OMeGalβ1,4GlcNAcβ1,6]αBn was found to be the best inhibitor. The anti-tumor effect of this novel tetrasaccharide in vivo was shown in preliminary experiments using Balb/c mice and colon26 cells.

The first chemical synthesis of F-4-GlcAβ(1→3)GlcNAc-UDP is described here. This compound can serve as a novel substrate to study the catalytic mechanism of hyaluronic acid synthases (HASs) and has potential to be donor for these enzymes that extend HA chain at the reducing end. Moreover, it may also behave as inhibitor for the enzymes that act on non-reducing end in the assembly of HA chain.

Sialoglycans on the cell surface of human colon cancer (HCC) cells have been implicated in cellular adhesion and metastasis. To clarify the role of N-acetylneuraminic acid (NeuAc) linked alpha2,3 to galactose (Gal) on the surface of HCC cells, we studied the intercellular adhesion of HCC cell lines expressing increasing NeuAcalpha2,3Gal-R. Our model system consisted of the HCC SW48 cell line, which inherently possesses low levels of cell surface alpha2,3 and alpha2,6 sialoglycans. To generate SW48 clonal variants with elevated cell surface NeuAcalpha2,3Gal-R linkages, we transfected the expression vector, pcDNA3, containing either rat liver cDNA encoding Galbeta1,3(4)GlcNAc alpha2,3 sialyltransferase (ST3Gal III) or human placental cDNA encoding Galbeta1,3GalNAc/Galbeta1,4GlcNAc alpha2,3 sialyltransferase (ST3Gal IV) into SW48 cells. Selection of neomycin-resistant clones (600 microgram G418/ml) having a higher percentage of cells expressing NeuAcalpha2,3Gal-R (up to 85% positive Maackia amurenis agglutinin staining compared with 30% for wild type cells) was performed. These ST3Gal III and ST3Gal IV clonal variants demonstrated increased adherence to IL-1beta-activated human umbilical vein endothelial cells (HUVEC) (up to 90% adherent cells compared with 63% for wild type cells). Interestingly, ST3Gal III and ST3Gal IV clonal variants also bound non-activated HUVEC up to 4-fold more effectively than wild type cells. Cell surface NeuAcalpha2,3Gal-R expression within the various SW48 clonal variants correlated directly with increased adhesion to HUVEC (r=0.84). Using HCC HT-29 cells, which express high levels of surface NeuAcalpha2,3Gal-R, addition of synthetic sialyl, sulfo or GalNAc Lewis X structures were found to specifically inhibit intercellular adhesion. At 1.0mM, NeuAcalpha2,3Galbeta1,3(Fucalpha1, 4)GlcNAc-OH and Galbeta1,4(Fucalpha1,3)GlcNAcbeta1,6(SE-6Galbeta1++ +, 3)GalNAcalpha1-O-methyl inhibited HT-29 cell adhesion to IL-1beta-stimulated HUVEC by 100% and 68%, respectively. GalNAcbeta1, 4(Fucalpha1,3)GlcNAcbeta1-O-methyl and GalNAcbeta1,4(Fucalpha1, 3)GlcNAcbeta1,6Manalpha1,6Manbeta1-0-C30H61, however, did not possess inhibitory activity. In conclusion, these studies demonstrated that cell surface NeuAcalpha2,3Gal-R expression is involved in HCC cellular adhesion to HUVEC. These specific carbohydrate-mediated intercellular adhesive events may play an important role in tumor angiogenesis, metastasis and growth control.

We found earlier in human breast and colon tumors, an augmented level of Gal : 3-O-sulfotransferase activities showing, respectively, an acceptor preference to blood group T-hapten (Group A enzymes) or Galbeta1,4GlcNAc (Group B enzymes) on the mucin Core 2 structure [Chandrasekaran EV, Jain RK, Vig R, and Matta KL (1997) Glycobiology 7: 753-68]. The present study reports these enzyme activities in human tumor cell lines and additional tumor specimens. The human colon tumor epithelial cell lines, akin to their parent tumors, express Group B enzyme activity. The acceptor specificity and kinetic properties, such as divalent metal ion activation and pH dependent activity profile, of the colon cancer line LS180 enzyme activity are identical to those of colon tissue specimens. Consistent with breast tumor specimens, the Group A enzyme activity is present in human breast tumor epithelial cell lines, with some exceptions. The Gal : 3-O-sulfotransferases show specific binding to Aleuria aurantia lectin, suggesting the presence of asparagine linked carbohydrate chains containing an inner core alpha1,6-fucosyl residue on these enzymes. Calf lymph nodes contain GlcNAc : 6-O-sulfotransferase as well as Group A Gal : 3-O-sulfotransferase activities, which differ in pH dependent profiles, pH optima (7.6 and 7.0, respectively) and the influence of Mn2+.

The chemical synthesis of beta-D-Gal-(1-->3)-6-O-SO3Na-beta-D-GlcNAc-(1-->6)-alpha-D-Man-O-+ ++C6H4NO2 (1) and beta-D-Gal-(1-->3)-6-O-SO3Na-beta-D-GlcNAc-(1-->2)-alpha-D-Man-OMe (2) is reported using a key glycosyl donor, phenyl O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->3)-4,6-di-O- chloroacetyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside (3).

The carbohydrate-binding site of galectin 1, a vertebrate β-galactoside-binding lectin, has a pronounced specificity for the βGal(1→3)- and βGal(1→4)GlcNAc sequences. The binding inhibition study reported herein was carried out to determine whether sulfation of saccharides would influence their binding by galectin 1. The presence of 6′-OSO3- on LacNAc greatly reduces the inhibitory potency relative to LacNAc. 3′-OSO3-LacNAc, 3′-OSO3-Galβ(1→3)GlcNAcβ1-OBzl and 3-OSO3-Galβ1-OMe are more potent inhibitors than the non-sulfated parent compounds. Surprisingly, 2′-OSO3-LacNAc showed over 40 fold less inhibitory potency relative to LacNAc. Ovarian carcinoma A121 cells were shown to synthesize sulfated macromolecules that bind to galectin 1. Modulation in vivo of saccharide sulfation may lead to modulation of galectin 1 interaction with glycoconjugates; hence, sulfation could play a role in modulating lectin functions.

We showed previously that HL-60 and F9 mouse embryonal carcinoma cells will take up and deblock peracetylated Galbeta1-4GlcNAcbeta-O-naphthalenemethanol (Galbeta1-4GlcNAc-NM) and use the disaccharide as a primer of oligosaccharide chains (Sarkar, A. K., Fritz, T. A., Taylor, W. H., and Esko, J. D. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 3323-3327). We now report that another disaccharide, acetylated GlcNAcbeta1-3Gal-naphthalenemethanol (GlcNAcbeta1-3Gal-NM), has even greater potency and that both compounds will inhibit sialyl LewisX (sLex)-dependent cell adhesion. When fed to U937 cells, acetylated forms of Galbeta1-4GlcNAc-NM and GlcNAcbeta1-3Gal-NM primed oligosaccharides in a dose-dependent manner. Analysis of compounds assembled on Galbeta1-4GlcNAc-NM showed only one product, namely Galbeta1-4(Fucalpha1-3)GlcNAc-NM. In contrast, GlcNAcbeta1-3Gal-NM generated Galbeta1-4GlcNAcbeta1-3Gal-NM, Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Gal-NM, NeuAcalpha2-3Galbeta1-4GlcNAcbeta1-3Gal-NM, and NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1- 3Gal-NM. Both compounds decreased the incorporation of [3H]fucose into cellular glycoconjugates, without affecting the incorporation of [3H]mannosamine, a precursor of sialic acid residues. Moreover, the overall extent of sialylation was not affected based on the reactivity of cells to fluorescein isothiocyanate-conjugated Maackia amurensis lectin. Priming inhibited expression of sLex on cell surface glycoconjugates, which reduced E-selectin-dependent cell adhesion to tumor necrosis factor-alpha-activated human umbilical vein endothelial cells. GlcNAcbeta1-3Gal-NM and Galbeta1-4GlcNAc-NM represent starting points for making enzyme-specific, site-directed inhibitors of glycosyltransferases that could act in living cells.

Stereoselective syntheses of 3-O-sulfo and 3-O-sialyl LewisX analogs 1 and 2 were accomplished. Compound 1 showed greater inhibitory activity than the natural ligand sialyl LewisX against P and L-selectin.

Selectins interact with glycoconjugate ligands in important normal and pathological situations. While high affinity recognition of natural ligands is associated with alpha 1-3(4)fucosylated, alpha 2-3sialylated (and/or sulfated) lactosamine sequences, small oligosaccharides that potently inhibit the selectins have not been found. One possibility suggested by other investigators is that high affinity may require unusual sequences not yet tested, for example, the "major capping group" (6'-sulfo-sialyl Le(x)) of the L-selectin ligand GlyCAM-1. To explore this possibility, we synthesized a spectrum of novel synthetic and semisynthetic oligosaccharides related to those on natural ligands. In studying these molecules, we noted that binding of recombinant soluble selectins to immobilized sialyl Le(a) or 3'-sulfo-Le(x) is markedly inhibited by concentrations of chloride above the physiological range. This indicates the ionic nature of the interactions, and shows that buffers typically used in screening assays for inhibitors are not optimal. Using parameters that more closely approximate physiological conditions, we confirmed that alpha 2-3-linked sialic acids, and alpha 1-3(4)fucosylation are important for recognition. Similar results obtained with both types of immobilized targets for the three selectins indicated that the binding sites for sialic acid and sulfate are very close, or identical. While O-sulfate esters mostly improved L- and P-selectin recognition, effects depended upon their position and number. Furthermore, sulfation can also impart some "negative" specificity: the major capping group does not interact with E-selectin. The branched Core 2 sequence seemed to enhance L- and P-selectin binding, however, the best inhibitors still appeared to be sialyl Le(a) and 3'-sulfo-Le(x), with the aglycone group of the latter affecting binding. Of particular note, the "major capping group" of GlyCAM-1 was not an unusually potent nor highly selective inhibitor of L-selectin, even when studying the interaction of L-selectin with native GlyCAM-1 itself.

Enzymatic 3-O-sulfation of terminal beta-Gal residues was investigated by screening sulfotransferase activity present in 37 human tissue specimens toward the following synthesized acceptor moieties: Galbeta1,3GalNAc alpha-O-Al, Galbeta1,4GlcNAcbeta-O-Al, Galbeta1,3GlcNAcbeta-O-Al, and mucin-type Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn structures 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 Galbeta1, 3GalNAc alpha- linkage and the other (Group B) was directed toward the Galbeta1,4GlcNAc branch beta1,6 linked to the blood group T hapten. Enzyme activities found in breast tissues were unique in showing a strict specificity for the T-hapten. Galbeta-O-allyl or benzyl did not serve as acceptors for Group A but were very active with Group B. An examination of activity present in six human sera revealed a specificity of the serum enzyme toward beta1,3 linked Gal, particularly, the T-hapten without beta1,6 branching. Group A was highly active toward T-hapten/acrylamide copolymer, anti-freeze glycoprotein, and fetuin O-glycosidic asialo glycopeptide; less active toward fetuin triantennary asialo glycopeptide; and least active toward bovine IgG diantennary glycopeptide. Group B was moderately and highly active, respectively, with the latter two glycopeptides noted and least active with the first two. Competition experiments performed with Galbeta1,3GalNAc alpha-O-Al and Galbeta1,4GlcNAcbeta1,6(Galbeta1,3)GalNAc alpha-O-Bn having a C-3 substituent (methyl or sulfate) on either Gal reinforced earlier findings on the specificity characteristics of Group A and Group B. Group A displayed a wider range of optimal activity (pH 6.0-7.4), whereas Group B possessed a peak of activity at pH 7.2. Mg2+ stimulated Group A 55% and Group B 150%, whereas Mn+2 stimulated Group B 130% but inhibited Group A 75%. Ca2+ stimulated Group B 100% but inhibited Group A 35%. Group A and Group B enzymes appeared to be of the same molecular size (

Modifications of proteins by O-glycosylation determine many of the properties and functions of proteins. We wish to understand the mechanisms of O-glycosylation and develop inhibitors that could affect glycoprotein functions and alter cellular behavior.We expressed recombinant soluble human Gal- and GlcNAc-transferases that synthesize the O-glycan cores 1 to 4 and are critical for the overall structures of O-glycans. We determined the properties and substrate specificities of these enzymes using synthetic acceptor substrate analogs. Compounds that were inactive as substrates were tested as inhibitors.Enzymes significantly differed in their recognition of the sugar moieties and aglycone groups of substrates. Core 1 synthase was active with glycopeptide substrates but GlcNAc-transferases preferred substrates with hydrophobic aglycone groups. Chemical modifications of the acceptors shed light on enzyme-substrate interactions. Core 1 synthase was weakly inhibited by its substrate analog benzyl 2-butanamido-2-deoxy-α-d-galactoside while two of the three GlcNAc-transferases were selectively and potently inhibited by bis-imidazolium salts which are not substrate analogs.This work delineates the distinct specificities and properties of the enzymes that synthesize the common O-glycan core structures 1 to 4. New inhibitors were found that could selectively inhibit the synthesis of cores 1, 2 and 3 but not core 4.These studies help our understanding of the mechanisms of action of enzymes critical for O-glycosylation. The results may be useful for the re-engineering of O-glycosylation to determine the roles of O-glycans and the enzymes critical for O-glycosylation, and for biotechnology with potential therapeutic applications.

Rejection of pig-to-human or pig-to-primate xenografts is mediated by the natural anti-Gal antibody, which interacts with alpha-galactosyl epitopes (i.e., Gal alpha1-3Gal beta1-4GlcNAc-R) abundantly expressed on porcine cells. The objective of this study was to determine the ability of various synthetic oligosaccharides to inhibit the binding of anti-Gal IgG molecules to porcine endothelial cells in vitro. Such inhibition ultimately may help to reduce or to prevent the in vivo antibody-dependent cell cytotoxicity (ADCC) reaction. In the absence of complement-mediated hyperacute rejection, the ADCC induced by anti-Gal IgG molecules is likely to cause the chronic rejection of xenografts. The synthetic free alpha-galactosyl epitope (Gal alpha1-3Gal beta1-4GlcNAc) was found to be 300-fold more effective than melibiose or alpha-methyl galactoside in inhibiting anti-Gal binding to porcine endothelial cells, and to prevent >90% of the antibody binding at a concentration of 1 mM. The disaccharide Gal alpha1-3Gal was ten-fold less effective than the free alpha-galactosyl epitope. Accordingly, the affinity of the disaccharide to anti-Gal, as measured by equilibrium dialysis, was seven-fold lower than that of the trisaccharide. The effective concentration of oligosaccharides inhibiting anti-Gal is independent of the antibody affinity, but is dependent on the concentration of the antibody. Based on the small difference in affinity between Gal alpha1-3Gal beta1-4GlcNAc and Gal alpha1-3Gal beta1-4GIc, and the large difference in the price of N-acetyllactosamine vs. lactose, it is suggested that lactose may be considered as an appropriate starting material for synthesizing large amounts of a trisaccharide that effectively neutralizes anti-Gal.

The assembly of complex structures bearing the H determinant was examined by characterizing the specificities of a cloned blood group H gene-specified alpha 1,2-L-fucosyltransferase (FT) toward a variety of sulfated, sialylated, or fucosylated Gal beta 1,3/4GlcNAc beta- or Gal beta 1,3GalNAc alpha-based acceptor structures. (a) As compared to the basic type 2, Gal beta 1,4GlcNAc beta-(K(m) = 1.67 mM), the basic type 1 was 137% active (K(m) = 0.83 mM). (b) On C-6 sulfation of Gal, type 1 became 142.1% active and type 2 became 223.0% active (K(m) = 0.45 mM). (c) On C-6 sulfation of GlcNAc, type 2 showed 33.7% activity. (d) On C-3 or C-4 fucosylation of GlcNAc, both types 1 and 2 lost activity. (e) Type 1 showed 70.8% and 5.8% activity, respectively, on C-6 and C-4 O-methylation of GlcNAc. (f) Type 1 retained 18.8% activity on alpha 2,6-sialylation of GlcNAc. (g) Terminal type 1 or 2 of extended chain had lower activity. (h) With Gal in place of GlcNAc in type 1, the activity became 43.2%. (i) Compounds with terminal alpha 1,3-linked Gal were inactive. (j) Gal beta 1,3GalNAc alpha- (the T-hapten) was approximately 0.4-fold as active as Gal beta 1,4GlcNAc beta-. (k) C-6 sulfation of Gal on the T-hapten did not affect the acceptor activity. (l) C-6 sulfation of GalNAc decreased the activity to 70%, whereas on C-6 sulfation of both Gal and GalNAc the T-hapten lost the acceptor ability. (m) C-6 sialylation of GalNAc also led to inactivity. (n) beta 1,6 branching from GalNAc of the T-hapten by a GlcNAc residue or by units such as Gal beta 1, 4GlcNAc-, Gal beta 1,4(Fuc alpha 1,3)GlcNAc-, or 3-sulfoGal beta 1,4GlcNAc- resulted in 111.9%, 282.8%, 48.3%, and 75.3% activities, respectively. (o) The enhancement of enzyme affinity by a sulfo group on C-6 of Gal was demonstrated by an increase (approximately 5-fold) in the K(m) for Gal beta 1,4GlcNAc beta 1,6(Gal beta 1,3)GalNAc alpha-O-Bn in presence of 6-sulfoGal beta 1,- 4GlcNAc beta-O-Me (3.0 mM). (p) Among the two sites in Gal beta 1, 4GlcNAc beta 1,6(Gal beta 1,3) GalNAc alpha-O-Bn, the enzyme had a higher affinity ( > 3-fold) for the Gal linked to GlcNAc. (q) With respect to Gal beta 1,- 3GlcNAc beta-O-Bn (3.0 mM), fetuin triantennary asialo glycopeptide (2.4 mM), bovine IgG diantennary glycopeptide (2.8 mM), asialo Cowper's gland mucin (0.06 mM), and the acrylamide copolymers (0.125 mM each) containing Gal beta 1,3GlcNAc beta-, Gal beta 1,3(6-sulfo)GlcNAc beta-, Gal beta 1,3GalNAc alpha-, Gal beta 1,3Gal beta-, or Gal alpha 1,3Gal beta- units were 153.6%, 43.0%, 6.2%, 52.5%, 94.9%, 14.7%, 23.6%, and 15.6% active, respectively. (r) Fucosylation by alpha 1,2-L-FT of the galactosyl residue which occurs on the antennary structure of the bovine IgG glycopeptide was adversely affected by the presence of an alpha 1,6-L-fucosyl residue located on the distant glucosaminyl residue that is directly attached to the asparagine of the protein backbone. This became evident from the 4-fold activity of alpha 1,2-L-FT toward bovine IgG glycopeptide after approximately 5% removal of alpha 1,6-linked Fuo.

Allyl 2- acetamido -4,6-O-(4- methoxybenzylidene )-2- deoxy -α- d -galactopyranoside (1) was condensed with either 2,3,4,6- tetra -O- acetyl -α- d -galactopyranosyl bromide (2) or 2,3,4- tri -O- benzoyl 6-O- bromoacetyl -α- d -galactopyranosyl bromide (14) in the presence of mercuric cyanide. Selective substitution with methyl, sulfo or both at desired positions, followed by the removal of protecting groups, afforded allyl O-(β d -galactopyranosyl )-(1 → 3)-2- acetamido-2-deoxy -6-O- methyl -α- d -galactopyranoside (5), allyl O-(6-O- sulfo -α- d -galactopyranosyl sodium salt)-(1 → 3)-2-acetamido-2-deoxy -6-O- methyl -α- d -galactopyranoside (10), allyl O-(β- d -galactopyranosyl)-(1 → 3)-2-acetamido-2-deoxy -6-O- sulfo -α- d -galactopyranoside sodium salt (13), allyl O-(6-O- sulfo -β- d -galactopyranosyl sodium salt)-(1 → 3)-2-acetamido-2-deoxy -α- d -galactopyranoside (17) and allyl O-(3-O- sulfo -β- d -galactopyranosyl sodium salt)-(1 → 3)-2-acetamido-2-deoxy -α- d -galactopyranoside (22). The structures of compounds 5, 10, 13, 17 and 22 were established by 13C NMR and FAB mass spectroscopy.