Sialyltransferase Research Articles

A Sexual Dimorphism in Intrathymic Sialylation Survey Is Revealed by thetrans-Sialidase fromTrypanosoma cruzi

Sialylation is emerging as an important issue in developing thymocytes and is considered among the most significant cell surface modifications, although its physiologic relevance is far from being completely understood. It is regulated by the concerted expression of sialyl transferases along thymocyte development. After in vivo administration of trans-sialidase, a virulence factor from the American trypanosomatid Trypanosoma cruzi that directly transfers the sialyl residue among macromolecules, we found that the alteration of the sialylation pattern induces thymocyte apoptosis inside the "nurse cell complex." This suggests a glycosylation survey in the development of the T cell compartment. In this study, we report that this thymocyte apoptosis mechanism requires the presence of androgens. No increment in apoptosis was recorded after trans-sialidase administration in females or in antiandrogen-treated, gonadectomized, or androgen receptor mutant male mice. The androgen receptor presence was required only in the thymic epithelial cells as determined by bone marrow chimeric mouse approaches. The presence of the CD43 surface mucin, a molecule with a still undefined function in thymocytes, was another absolute requirement. The trans-sialidase-induced apoptosis proceeds through the TNF-alpha receptor 1 deathly signaling leading to the activation of the caspase 3. Accordingly, the production of the cytokine was increased in thymocytes. The ability of males to delete thymocytes altered in their sialylation pattern reveals a sexual dimorphism in the glycosylation survey during the development of the T cell compartment that might be related to the known differences in the immune response among sexes.

Role of sialyltransferases involved in the biosynthesis of Lewis antigens in human pancreatic tumour cells

The sialylated carbohydrate antigens, sialyl-Lewisx and sialyl-Lewisa, are expressed in pancreatic tumour cells and are related to their metastatic potential. While the action of the fucosyltransferases involved in the synthesis of these antigens has already been investigated, no studies have been carried out on the activity and expression of the alpha 2,3-sialyltransferases in pancreatic tumour cells. We describe the sialyltransferase (ST) activity, mRNA expression, and analysis of the cell carbohydrate structures in four human pancreatic adenocarcinoma cell lines of a wide range of neoplastic differentiation stages and in normal human pancreatic tissues. Total ST activity measured on asialofetuin, employing a CMP fluorescent sialic acid, varied among the pancreatic cell lines and could be correlated to the expression of their cell surface antigens. However, in some of the pancreatic cell lines, no relationship could be established with their ST3Gal III and IV mRNA expression. Human pancreatic tissues also showed ST expression and activity. However, it presented a much higher expression of neutral fucosylated structures than sialylated structures. In conclusion, ST activity levels in pancreatic cells could be correlated to their expression of sialylated epitopes, which indicates their involvement in the formation of the sialyl-Lewis antigens, in addition to fucosyltransferase activities.

Soyasaponin-I-modified invasive behavior of cancer by changing cell surface sialic acids

Objective Sialylation involving tumor formation and invasive behavior goes along with altered sialyltransferase (ST) activity. A potent ST inhibitor, soyasaponin I (SsaI), was discovered to selectively inhibit the cellular α2,3-sialyltranserase activity. In this study, we further test the effects of SsaI on modifying the metastatic and invasive behaviors of cancer cell lines. Methods Nonmetastatic breast cancer cell line, MCF-7, and highly metastastic breast cancer cell line, MDA-MB-231, were used to investigate the effects of SsaI on tumor cells. Results SsaI did not affect cell growth cycle and also failed to inhibit cell growth in this study (the concentration of SsaI ≤100 μM). SsaI was as predicted to successfully inhibit cellular α2,3-ST activity and depressed the dose-dependent tumor cell surface α2,3-sialic acid expression. In addition, different concentrations of SsaI did stimulate MCF-7 cell adhesion to collagen type I linearly and significantly enhanced cell adhesion to the Matrigel-matrix. Furthermore, SsaI significantly decreased MDA-MB-231 cell migration. Reverse transcriptase polymerase chain reaction for evaluating mRNA expression of ST3Gal I, III and IV showed that SsaI also down-regulated the expression of ST3Gal IV but did not affect the other two. Conclusions The results showed that SsaI was implicated in the invasive behavior of tumor cells, suggesting that altered α2,3-sialylation pathway played a crucial role in the adhesion and tumor metastases. SsaI is a good candidate for studying the biological roles of ST, and might provide a new preventive strategy in tumor metastasis.

The synthesis of some mechanistic probes for sialic acid processing enzymes and the labeling of a sialidase fromTrypanosoma rangeli

Sialyl hydrolases, trans-sialidases, and sialyl transferases are biologically important enzymes that are responsible for the incorporation and removal of sialic acid residues, which decorate many cell surface glycocongugates. Two fluorinated sialic acid derivatives have been synthesized as mechanism-based inactivators, to probe the catalytic mechanisms through which sialidases and trans-sialidases operate. Both compounds are known to be covalent inactivators of a trans-sialidase from Trypanosoma cruzi. Here, 3-fluorosialosyl fluoride has been found to covalently label the catalytic nucleophile of a sialidase from T. rangeli, and the residue involved is shown to be Tyr346 within the sequence DENSGYSSVL. This is the first demonstration that sialidases operate through a covalent glycosyl-enzyme intermediate, strongly suggesting a common catalytic mechanism amongst all members of the sialidase superfamily. CMP-3-fluoro sialic acid is a competitive inhibitor of sialyl transferases and was synthesized via a two-step enzymatic process from commercially available N-acetyl mannosamine, 3-fluoropyruvic acid, and cytidine triphosphate in around 84% yield.Key words: sialidase, mechanism, labeling, nucleophile, inhibitor.

Changes in mucin‐type O‐glycosylation in breast cancer: implications for the host immune response

The major epithelial mucin, which is expressed by more than 90% of breast carcinomas and epithelial breast cancer cell lines, is MUC1. The MUC1 membrane mucin has a relatively simple structure, where a tandem repeat (TR) region carrying multiple O‐glycans forms the major part of the extracellular domain. The gene shows a size polymorphism, due to variation in the number of tandem repeats, each of which has five potential glycosylation sites. MUC1 has been found to be a good model for studying mucin‐type O‐glycosylation both in vitro and in vivo.Changes in the pattern of glycosylation of MUC1 have been observed in breast cancer (Burchell et al. 2001) and have led to an interest in MUC1 as a possible target antigen for immunotherapeutic intervention (Taylor‐Papadimitriou et al. 2000). The mucin expressed at high levels in the normal lactating mammary gland carries mainly core 2‐based O‐glycans (2–3 per tandem repeat). In breast cancers, the level of the ST3 Gal 1 enzyme is elevated (Burchell et al. 1999) and this is associated with increased numbers of Sialyl T O‐glycans being added, as predicted from in vitro studies, showing competition for the core 1 substrate between this sialyl transferase and the relevant core 2 enzyme (Dalziel et al. 2001). Moreover, the TR domain is more densely glycosylated. While the Sialyl T O‐glycan is commonly found on normal cells (e.g. resting T cells), it is cancer associated in the mammary gland. However, several lines of evidence indicate that this MUC1 glycoform, as expressed on breast cancers, can be immunosuppressive, maybe reflecting an inhibitory interaction with a lectin, possibly a siglec (Nath et al. 1999), on immune effector cells. In 25–30% of breast cancers, O‐glycosylation is terminated very early so that GalNAc (Tn) or NeuAc α2,6 GalNAc (Sialyl Tn) glycans appear. Sialyl Tn is highly tumour‐specific and is detected immunohistochemically only when the enzyme ST6GalNAcI is expressed. The Tn and Sialyl Tn will bind to different lectins, including MGL and could stimulate the immune response. Preliminary studies in mouse models suggest that an immunization protocol which includes MUC1‐carrying Sialyl Tn O‐glycans can protect against tumour growth, while clinical studies in breast cancer patients using immunogens based on the Sialyl Tn glycan are underway.

Clinical usefulness of alterations in sialic acid, sialyl transferase and sialoproteins in breast cancer

Sialic acid, the end moieties of the carbohydrate chains are biologically important and essential for functions of glycoconjugates and are reported to be altered in cancer patients. Two hundred and twenty five breast cancer (BC) patients, 100 patients with benign breast disease (BBD) and 100 healthy females (controls) were enrolled for the study. Eight hundred and twenty four follow-up samples of 225 breast carcinoma patients were also evaluated. The association of sialic acid forms, sialyltransferase and α-2-6 sialoproteins levels with presence and extent as well as prognosis of breast carcinoma was studied. Serum sialic acid forms and sialyltransferase revealed significantly elevated levels among untreated breast cancer patients as compared to the controls, patients with BBD as well as cancer patients in remission. Non-responders showed comparable levels of the markers with those found in breast cancer patients at the time of diagnosis. Higher levels of sialic acid forms at diagnosis were associated with poor prognosis. A positive correlation between serum levels of different forms of sialic acids and extent of malignant disease was observed. The changes in serum proteins with terminal α-2-6 sialic acid correlated well with alterations in the levels of sialic acid forms and sialyltransferase. Malignant tissues showed elevated levels of sialic acid and sialyltransferase as compared to surrounding normal tissues.The results suggested potential utility of these markers in evaluation of clinical outcome.

Should we use carbohydrate deficient transferrin as a marker for alcohol abusers?

Carbohydrate deficient transferrin (CDT) is one of the conventional markers for chronic alcohol consumption, is used by researchers and clinicians. A number of enzymes are affected by ethanol intake. The induction or inhibition of sialyl transferase and plasma sialidase may be involved in the CDT level elevation. An alteration of protein transport during post-translational modification could be a primary mechanism in the impairment of protein metabolism associated with chronic alcohol abuse. Transferrin being a steroid responsive protein, sex-based hormonal variations might contribute to the lower sensitivity of CDT. Varying hormonal statuses such as pregnancy, use of contraceptives, menopause/ menstrual cycle can alter iron homeostasis in women. CDT levels are markedly affected by iron homeostasis. Several CDT assay methods appeared promising, but it is not readily apparent which technique is the most accurate. Moreover, false-positive results of CDT have been reported in non-alcohol related hepatic failure and in rare conditions. Therefore clinical interpretation of CDT needs careful assessment in patients with alcohol-related or non-alcohol-related health disorders.

Genetic analysis of Canadian isolates of C:2a:P1.2,5 and B:2a:P1.2,5 Neisseria meningitidis strains belonging to the hypervirulent clone of ET-15.

Isolates of the hypervirulent Neisseria meningitidis clone ET-15 found to express the serogroup B antigen were investigated and compared with representative members of serogroup B and C isolates. Clonal-clustering methods clearly grouped the B:ET15 isolates with C:ET15 isolates, indicating the only major difference between the two groups was in the capsule expressed. The organization of the cps operon from the B:ET15 isolates was found to be consistent with typical serogroup B isolates and differed from serogroup C isolates only in the sialyl transferase gene present. This suggests that these strains arose via recombination of the sialyl transferase gene. Specific points of recombination could not be identified, however, the majority (64%) of the B:ET15 isolates contained a copy of pseudo-IS1106 downstream of the cps operon indicating the potential for a common ancestral origin. The combination of pulsed-field gel electrophoresis (PFGE) and sequence analysis of targeted regions of the cps operon permitted the differentiation of most B:ET15 isolates indicating that they likely arose from separate genetic events and do not represent the emergence and spread of a new clone. However, two isolates that appeared identical by all methods employed were temporally and geographically related although no epidemiological evidence is available indicating a link between these strains.

Gene expression analysis reveals evidence for inactivation of the TGF-beta signaling cascade in autonomously functioning thyroid nodules.

Molecular events that lead to the development of autonomously functioning thyroid nodules (AFTNs) are somatic mutations of the thyrotropin receptor (TSHR) in approximately 60% of the nodules and less frequently, somatic mutations in the Gsalpha protein. However, AFTNs without known mutations indicate that other causes remain to be identified. Moreover, the impact of constitutively activating TSHR mutations on the signal transduction network of the thyroid epithelial cell is unknown. We therefore investigated gene expression in 15 AFTNs and their surrounding tissue using Affymetrix GeneChips. Most prominently, data analysis revealed a changed pattern of gene expression in the TGF-beta signaling cascade and 25 differentially regulated genes in AFTNs, including thyroid peroxidase, type I iodothyronine deiodinase and sialyltransferase (SIAT) 1. Strikingly coexpression of SIAT 1 and TSHR in COS-7 cells increased TSH binding and cell surface expression of the TSHR. Moreover, differences in gene expression patterns for AFTNs with and without TSHR mutations indicate specific alterations of signal transduction in AFTNs without TSHR mutations. These results suggest that AFTNs with TSHR mutations harbor further mechanisms of forward stimulation. Furthermore, they give important leads to elucidate the molecular etiology of AFTNs without TSHR mutations.

Sensitivity to human serum of gammaretroviruses produced from pig endothelial cells transduced with glycosyltransferase genes.

Reduction of pig cell-surface alpha-galactosyl (Gal) epitope, Galalpha1, 3Galbeta1, 4GlcNAc-R, by the introduction of glycosyltransferase genes is effective in suppressing hyperacute rejection (HAR) in pig-to-human xenotransplantation. The transmission of porcine endogenous retroviruses (PERVs) has been recognized as a potential risk factor associated with xenotransplantation. In this study, effects of the introduction of glycosyltransferase genes to pig cells on the sensitivity of gammaretroviruses to human serum were investigated. Pig endothelial cells (PEC), PEC transduced with alpha1,2 fucosyltransferase (FT), alpha2,3 sialyltransferase (ST), or N-acetylglucosaminyltransferase III (GnT-III), and human embryonic kidney (HEK) 293 cells were transduced with the LacZ gene with the packaging signal of murine leukemia virus (MuLV) under the control of the long terminal repeat of MuLV by a pseudotype infection. Then, the cells were further infected with PERV subtype B (PERV-B) or feline leukemia virus subgroup B (FeLV-B). Culture supernatants of the infected cells were mixed with human serum (HS) and then inoculated to HEK293 cells. The inoculated cells were histochemically stained and lacZ-positive blue foci were counted. Glycosyltransferase activity, xenoantigenicity, and alpha-Gal epitope density in the cells were measured at the time of the infection experiments. PERV-B or FeLV-B particles from the parental PEC were efficiently neutralized by HS, while those from PEC transduced with alpha1,2FT, alpha2,3ST or GnT-III were less sensitive to HS. The transduced PEC exhibited high levels of activity of the introduced glycotransferases, and expressed fewer xenoantigens and cell-surface alpha-Gal epitopes. Our results suggest that gammaretroviruses including PERVs produced by transgenic pigs, that are generally modified to reduce the cell-surface alpha-Gal epitope to overcome the HAR in xenotransplantation, are less sensitive to HS.

The role of protein phosphorylation in [formula omitted]2,6( N)-sialyltransferase activity

Sialoglycoproteins play a key role in both brain development and neuronal plasticity with their sialylation state being controlled by the sialyltransferase (STN) family of enzymes. In this study, we have determined the role of specific kinase enzymes in the expression and catalytic activity of the α2,6 STN (ST6N) isozyme. The catalytic activity was moderately decreased following the inhibition of GSK3β with LiCl. However, there was a significant increase in catalytic activity following activation of protein kinase C (PKC) by phorbol ester. There was no change in the expression levels of the enzyme protein following any of the treatments. The changes in enzyme catalytic activity were also mirrored by the expression of both protein-bound sialic acid and the polysialic acid oligosaccharide group attached to the neural cell adhesion molecule, NCAM. These results provide further evidence for the role of second messenger-associated kinase enzymes in the modulation of the cell glycosylation potential.

The mitochondria-associated endoplasmic-reticulum subcompartment (MAM fraction) of rat liver contains highly active sphingolipid-specific glycosyltransferases.

Although most glycosphingolipids (GSLs) are thought to be located in the outer leaflet of the plasma membrane, recent evidence indicates that GSLs and their precursor, ceramide, are also associated with intracellular organelles and, particularly, mitochondria. GSL biosynthesis starts with the formation of ceramide in the endoplasmic reticulum (ER), which is transported by controversial mechanisms to the Golgi apparatus, where stepwise addition of monosaccharides on to ceramides takes place. We now report the presence of GSL-biosynthetic enzymes in a subcompartment of the ER previously characterized and termed 'mitochondria-associated membrane' (MAM). MAM is a membrane bridge between the ER and mitochondria that is involved in the biosynthesis and trafficking of phospholipids between the two organelles. Using exogenous acceptors coated on silica gel, we demonstrate the presence of ceramide glucosyltransferase (Cer-Glc-T), glucosylceramide galactosyltransferase and sialyltransferase (SAT) activities in the MAM. Estimation of the marker-enzyme activities showed that glycosyltransferase activities could not be ascribed to cross-contamination of MAM by Golgi membranes. Cer-Glc-T was found to have a marked preference for ceramide bearing phytosphingosine as sphingoid base. SAT activities in MAM led to the synthesis of G(M3) ganglioside and small amounts of G(D3). G(M1) was also synthesized along with G(M3) upon incubation of the fraction with exogenous unlabelled G(M3), underlying the presence of other sphingolipid-specific glycosyltransferases in MAM. On the basis of our results, we propose MAM as a privileged compartment in providing GSLs for mitochondria.

Recognition of cell surface acceptors by two human α-2,6-sialyltransferases produced in CHO cells

The action of sialyltransferases (STs) on cell surface glycoconjugates is a key process in shaping cell phenotype in a variety of cells mostly involved in migratory and adhesive pathways. The factors determining cell-specific pattern of glycosylation are so far poorly understood. Most STs are resident proteins of the Golgi apparatus, where acceptors are sialylated while they are in transit to the cell surface. To identify putative structural features that may account for their acceptor preference, we analyzed 53 cloned animal and human STs. We could identify conserved regions and peptide motifs representative of ST subfamilies, located at the C-terminal end of the hypervariable region upstream from the L-sialyl motif. Residues 93-100 in human ST6Gal I (hST6Gal I) were shown to be crucial for enzymatic activity when deleted and expressed in CHO cells. The Δ100 hST6Gal I mutant protein was fully recognized by polyclonal anti-hST6Gal I antibodies and followed the intracellular secretory pathway. This indicated that the conserved QVWxKDS sequence is essential for the whole catalytic domain to acquire a biologically active conformation. When full-length epitope-tagged hST6Gal I and hST6GalNAc I constructs were transfected in CHO cells, the α-2,6 sialylated glycotope was found to be largely restricted to intracellular resident acceptors and enzymatic activity based on fluorescent lectin staining. In contrast, both enzymes deprived of their membrane anchor and part of the hypervariable region but still possessing the conserved domains exhibited a very efficient transfer of sialic acid to cell surface glycoconjugates. Colocalization of the ST6Gal I mutant proteins with early and late Golgi markers such as giantin or rab6 proteins confirmed that soluble STs migrate forward in these subcompartments where they can act upon newly synthesized acceptors and follow the secretory pathway. It is thus concluded that downstream from the transmembrane domain, native STs possess peptide sequences that allow them to sialylate glycoprotein acceptors selectively along their transit within Golgi stacks.

ADP-ribosylation factor 1 of Arabidopsis plays a critical role in intracellular trafficking and maintenance of endoplasmic reticulum morphology in Arabidopsis.

ADP-ribosylation factors (Arf), a family of small GTP-binding proteins, play important roles in intracellular trafficking in animal and yeast cells. Here, we investigated the roles of two Arf homologs, Arf1 and Arf3 of Arabidopsis, in intracellular trafficking in plant cells. We generated dominant negative mutant forms of Arf 1 and Arf3 and examined their effect on trafficking of reporter proteins in protoplasts. Arf1[T31N] inhibited trafficking of H(+)-ATPase:green fluorescent protein (GFP) and sialyltransferase (ST):GFP to the plasma membrane and the Golgi apparatus. In addition, Arf1[T31N] caused relocalization of the Golgi reporter protein ST:GFP to the endoplasmic reticulum (ER). In protoplasts expressing Arf1[T31N], ST:red fluorescent protein remained in the ER, whereas H(+)-ATPase:GFP was mistargeted to another organelle. Also, expression of Arf1[T31N] in protoplasts resulted in profound changes in the morphology of the ER. The treatment of protoplasts with brefeldin A had exactly the same effect as Arf1[T31N] on various intracellular trafficking pathways. In contrast, Arf3[T31N] did not affect trafficking of any of these reporter proteins. Inhibition experiments using mutants with various domains swapped between Arf1 and Arf3 revealed that the N-terminal domain is interchangeable for trafficking inhibition. However, in addition to the T31N mutation, motifs in domains II, III, and IV of Arf1 were necessary for inhibition of trafficking of H(+)-ATPase:GFP. Together, these results strongly suggest that Arf1 plays a role in the intracellular trafficking of cargo proteins in Arabidopsis, and that Arf1 functions through a brefeldin A-sensitive factor.

Selective and signal-dependent recruitment of membrane proteins to secretory granules formed by heterologously expressed von Willebrand factor.

von Willebrand factor (vWF) is a large, multimeric protein secreted by endothelial cells and involved in hemostasis. When expressed in AtT-20 cells, vWF leads to the de novo formation of cigar-shaped organelles similar in appearance to the Weibel-Palade bodies of endothelial cells in which vWF is normally stored before regulated secretion. The membranes of this vWF-induced organelle, termed the pseudogranule, are uncharacterized. We have examined the ability of these pseudogranules, which we show are secretagogue responsive, to recruit membrane proteins. Coexpression experiments show that the Weibel-Palade body proteins P-selectin and CD63, as well as the secretory organelle membrane proteins vesicle-associated membrane protein-2 and synaptotagmin I are diverted away from the endogenous adrenocorticotropic hormone-containing secretory granules to the vWF-containing pseudogranules. However, transferrin receptor, lysosomal-associated membrane protein 1, and sialyl transferase are not recruited. The recruitment of P-selectin is dependent on a tyrosine-based motif within its cytoplasmic domain. Our data show that vWF pseudogranules specifically recruit a subset of membrane proteins, and that in a process explicitly driven by the pseudogranule content (i.e., vWF), the active recruitment of at least one component of the pseudogranule membrane (i.e., P-selectin) is dependent on residues of P-selectin that are cytosolic and therefore unable to directly interact with vWF.

Redistribution of membrane proteins between the Golgi apparatus and endoplasmic reticulum in plants is reversible and not dependent on cytoskeletal networks.

We have fused the signal anchor sequences of a rat sialyl transferase and a human galactosyl transferase along with the Arabidopsis homologue of the yeast HDEL receptor (AtERD2) to the jellyfish green fluorescent protein (GFP) and transiently expressed the chimeric genes in tobacco leaves. All constructs targeted the Golgi apparatus and co-expression with DsRed fusions along with immunolabelling of stably transformed BY2 cells indicated that the fusion proteins located all Golgi stacks. Exposure of tissue to brefeldin A (BFA) resulted in the reversible redistribution of ST-GFP into the endoplasmic reticulum. This effect occurred in the presence of a protein synthesis inhibitor and also in the absence of microtubules or actin filaments. Likewise, reformation of Golgi stacks on removal of BFA was not dependent on either protein synthesis or the cytoskeleton. These data suggest that ER to Golgi transport in the cell types observed does not require cytoskeletal-based mechanochemical motor systems. However, expression of an inhibitory mutant of Arabidopsis Rab 1b (AtRab1b(N121I) significantly slowed down the recovery of Golgi fluorescence in BFA treated cells indicating a role for Rab1 in regulating ER to Golgi anterograde transport.

Proton leak and CFTR in regulation of Golgi pH in respiratory epithelial cells.

Work addressing whether cystic fibrosis transmembrane conductance regulator (CFTR) plays a role in regulating organelle pH has remained inconclusive. We engineered a pH-sensitive excitation ratiometric green fluorescent protein (pHERP) and targeted it to the Golgi with sialyltransferase (ST). As determined by ratiometric imaging of cells expressing ST-pHERP, Golgi pH (pH(G)) of HeLa cells was 6.4, while pH(G) of mutant (DeltaF508) and wild-type CFTR-expressing (WT-CFTR) respiratory epithelia were 6.7-7.0. Comparison of genetically matched DeltaF508 and WT-CFTR cells showed that the absence of CFTR statistically increased Golgi acidity by 0.2 pH units, though this small difference was unlikely to be physiologically important. Golgi pH was maintained by a H(+) vacuolar (V)-ATPase countered by a H(+) leak, which was unaffected by CFTR. To estimate Golgi proton permeability (P(H(+))), we modeled transient changes in pH(G) induced by inhibiting the V-ATPase and by acidifying the cytosol. This analysis required knowing Golgi buffer capacity, which was pH dependent. Our in vivo estimate is that Golgi P(H(+)) = 7.5 x 10(-4) cm/s when pH(G) = 6.5, and surprisingly, P(H(+)) decreased as pH(G) decreased.

Soyasaponin I, a Potent and Specific Sialyltransferase Inhibitor

A growing number of reports demonstrate that hypersialylation, which is observed in certain pathological processes, such as oncogenic transformation, tumor metastasis, and invasion, is associated with enhanced sialyltransferase (ST) activity. There is therefore a need for the development of ST inhibitors to modulate ST activity and thus alleviate the disease processes caused by STs. In the present study, soyasaponin I had been discovered to be a potent and specific ST inhibitor by screening strategy from 7500 samples including micribial extracts and natural products. Kinetic analysis shows that it is a CMP-Neu5Ac competitive inhibitor with for ST3Gal I with an inhibition constant (Ki) of 2.1 μM. In addition, it is only active against ST, but not against the other tested glycosyltransferases and glycosidases. Our study is the first report to discover ST inhibitor by screening method and also to provide the new chemical structure information that should be useful in the development of other novel ST inhibitors.

Sialylation of lipooligosaccharide cores affects immunogenicity and serum resistance of Campylobacter jejuni.

Three genes involved in biosynthesis of the lipooligosaccharide (LOS) core of Campylobacter jejuni MSC57360, the type strain of the HS:1 serotype, whose structure mimics GM(2) ganglioside, have been cloned and characterized. Mutation of genes encoding proteins with homology to a sialyl transferase (cstII) and a putative N-acetylmannosamine synthetase (neuC1), part of the biosynthetic pathway of N-acetylneuraminic acid (NeuNAc), have identical phenotypes. The LOS cores of these mutants display identical changes in electrophoretic mobility, loss of reactivity with cholera toxin (CT), and enhanced immunoreactivity with a hyperimmune polyclonal antiserum generated against whole cells of C. jejuni MSC57360. Loss of sialic acid in the core of the neuC1 mutant was confirmed by fast atom bombardment mass spectrometry. Mutation of a gene encoding a putative beta-1,4-N-acetylgalactosaminyltransferase (Cgt) resulted in LOS cores intermediate in electrophoretic mobility between that of wild type and the mutants lacking NeuNAc, loss of reactivity with CT, and a reduced immunoreactivity with hyperimmune antiserum. Chemical analyses confirmed the loss of N-acetylgalactosamine (GalNAc) and the presence of NeuNAc in the cgt mutant. These data suggest that the Cgt enzyme is capable of transferring GalNAc to an acceptor with or without NeuNAc and that the Cst enzyme is capable of transferring NeuNAc to an acceptor with or without GalNAc. A mutant with a nonsialylated LOS core is more sensitive to the bactericidal effects of human sera than the wild type or the mutant lacking GalNAc.

Chemo-enzymatic synthesis of fluorinated sugar nucleotide: useful mechanistic Probes for glycosyltransferases

An effective procedure for the synthesis of 2-deoxy-2-fluoro-sugar nucleotides via Selectfluor-mediated electrophilic fluorination of glycals with concurrent nucleophilic addition or chemo-enzymatic transformation has been developed, and the fluorinated sugar nucleotides have been used as probes for glycosyltransferases, including fucosyltransferase III, V, VI, and VII, and sialyl transferases. In general, these fluorinated sugar nucleotides act as competitive inhibitors versus sugar nucleotide substrates and form a tight complex with the glycosyltransferase.