An alpha2,3 sialyltransferase (ST3Gal I) is elevated in primary breast carcinomas

The MUC1 mucin is expressed on the luminal surface of most simple epithelial cells but in carcinomas, especially those of the breast and ovary, MUC1 is upregulated and aberrantly glycosylated. MUC1 contains a large amount of O-linked glycans which, in the mucin expressed by normal mammary epithelial cells, consist mainly of core 2 based structures carrying polylactosamine chains. However, the mucin expressed by breast carcinomas has shorter side-chains, often consisting of sialylated core 1 (Galbeta1-3GalNAc). in situ hybridization of primary breast tissue showed that a sialyltransferase (ST3Gal I), responsible for adding sialic acid to core 1 thereby terminating chain extension, is elevated in primary breast carcinomas when compared to normal or benign tissue. Furthermore, the level of mRNA expression encoding ST3Gal I is correlated to the intensity of staining seen with the antibody SM3, which specifically recognises underglycosylated, tumour associated MUC1. Thus, the aberrant glycosylation of MUC1 seen in breast carcinomas appears to be due, at least in part, to the elevation of ST3Gal I.     

小鼠肝癌与肝正常细胞系ST3Gal和ST6Gal家族mRNA表达差异研究

探讨肝癌细胞系Hepa1-6与肝正常细胞系BNL CL.2唾液酸糖基转移酶ST3Gal和ST6Gal家族mRNA表达的差异以及与细胞膜唾液酸含量的关系,采用RT-PCR方法检测ST3Gal唾液酸转移酶家族6个成员以及ST6Gal唾液酸转移酶家族2个成员mRNA表达差异,用凝集素芯片检测细胞膜表面唾液酸表达情况,结果显示:与正常细胞系BNL CL.2相比,hepa1-6细胞内唾液酸转移酶ST3GalⅠ、ST3GalⅣ、ST3GalⅥ呈现高表达,ST3GalⅤ低表达,ST3GalⅡ、ST3GalⅢ表达无显著性差异,两细胞系内均为检测出ST6GalⅠ表达,ST6GalⅡ表达无显著差异;hepa1-6细胞膜α2-3和α2-6连接唾液酸含量均显著增加;提示ST3GalⅠ、ST3GalⅣ、ST3GalⅤ、ST3GalⅥ可能与肝癌发生过程相关,ST3GalⅠ、ST3GalⅣ、ST3GalⅥ可能与肝癌细胞膜α2-3唾液酸含量增加相关,ST6Gal家族对细胞膜α2-6连接唾液酸含量增加无贡献.     

Purification and characterization of a soluble recombinant human ST6Gal I functionally expressed in Escherichia coli

A soluble and active form of recombinant human ST6Gal I was expressed in Escherichia coli. The gene encoding the soluble form of ST6Gal I lacking the membrane and cytosolic regions was introduced into a bacterial expression vector, pMAL-p2X, fused in frame with a maltose-binding protein (MBP) tag. Low-temperature cultivation at 13C during IPTG-induction significantly improved both solubility and MBP-tagging of the recombinant enzyme expressed in bacteria. The supernatant prepared by disruption of the cells demonstrated sialic acid transfer activity to both an oligosaccharide and a glycoprotein, asialofetuin, indicating that the enzyme expressed in bacteria is soluble and active. The MBP-tagged enzyme was efficiently purified by a combination of cation-exchange column and amylase-conjugated agarose column chromatography. The purified recombinant enzyme exerted enzymatic activity even in the absence of detergents in the reaction mixture. Acceptor substrate specificity of the enzyme was marginally different from that of rat liver ST6Gal I. These observations suggest that membrane and cytosolic regions of ST6Gal I may affect the properties of the enzyme. The purified recombinant enzyme was applied to convert desialylated fetuin to resialylated fetuin. Lectin blotting demonstrated that resialylated fetuin possesses a single Neu5Ac 2-6 residue. The resialylated fetuin efficiently blocked hemagglutination induced by influenza virus strain A/Memphis/1/71 (H3N2), indicating that resialylated carbohydrate chains on the protein are so active as to competitively inhibit virus-receptor interaction. In conclusion, soluble recombinant ST6Gal I obtained using our bacterial expression system is a valuable tool to investigate the molecular mechanisms of biological and pathological interactions mediated via carbohydrates. Published in 2005.The authors contributed equally to this work.     

Design,synthesis and evaluation of carbamate-linked uridyl-based inhibitors of human ST6Gal I

Sialic acid at the terminus of cell surface glycoconjugates is a critical element in cell-cell recognition, receptor binding and immune responses. Sialyltransferases (ST), the enzymes responsible for the biosynthesis of sialylated glycans are highly upregulated in cancer and the resulting hypersialylation of the tumour cell surface correlates strongly with tumour growth, metastasis and drug resistance. Inhibitors of human STs, in particular human ST6Gal I, are thus expected to be valuable chemical tools for the discovery of novel anticancer drugs. Herein, we report on the computationally-guided design and development of uridine-based inhibitors that replace the charged phosphodiester linker of known ST inhibitors with a neutral carbamate to improve pharmacokinetic properties and synthetic accessibility. A series of 24 carbamate-linked uridyl-based compounds were synthesised by coupling aryl and hetaryl α-hydroxyphosphonates with a 5′-amino-5′-deoxyuridine fragment. The inhibitory activities of the newly synthesised compounds against recombinant human ST6Gal I were determined using a luminescent microplate assay, and five promising inhibitors with K_i’s ranging from 1 to 20 µM were identified. These results show that carbamate-linked uridyl-based compounds are a potential new class of readily accessible, non-cytotoxic ST inhibitors to be further explored.     

The relationship between ST6Gal I Golgi retention and its cleavage-secretion

The ST6Gal I is a sialyltransferase that modifies N-linked oligosaccharides of glycoproteins. Previous results suggested a role for luminal stem and active domain sequences in the efficiency of ST6Gal I Golgi retention. Characterization of a series of STtyr isoform deletion mutants demonstrated that the stem is sensitive to proteases and that preventing cleavage in this region leads to increased cell surface expression. A mutant lacking amino acids 32-104 (STDelta4) is not active or cleaved and secreted like the wild type STtyr, but does exhibit increased cell surface expression. It is probable that the STDelta4 mutant lacks the stem region and some amino acids of the active domain because the STDelta5 mutant lacking amino acids 86-104 is also not active but is cleaved and secreted. In contrast, deletion of stem amino acids between residues 32 and 86 in the STDelta1, STDelta2, and STDelta3 mutants does not inactive these enzyme forms, eliminate their cleavage and secretion, or increase their cell surface expression. Surprisingly, cleavage occurs even though the previously identified Asn63-Ser 64 cleavage site is missing. Further evaluation demonstrated that a cleavage site between Lys 40 and Glu 41 is used in COS cells. Mutagenesis of Lys 40 significantly decreased, but did not eliminate cleavage, suggesting that there are additional secondary sites of cleavage in the ST6Gal I stem.     

Conformational and electrostatic analysis of SN1 donor analogue glycomimetic inhibitors of ST3Gal-I mammalian sialyltransferase

Mammalian sialyltransferases play a role in the metastasis of various cancers in humans. Inhibitors of these enzymes will in principle be able to directly inhibit aberrant sialylation in cancer. Inhibitors of ST3Gal-I resembling the donor component of S_N1 Transition State structures were previously evaluated as part of a kinetics study. Here, using classical dynamics simulations and free energy perturbation calculations, we rationalize the performance of three of these donor analogue ST3Gal-I enzyme inhibitors. We find to inhibit the mammalian ST3Gal-I enzyme a donor analogue requires configurationally limited functionality. This is mediated by the binding of the inhibitor to the enzyme. The inhibitor’s ability to interact with Y194 and T272 through a charged group such as a carboxylate is especially important. Furthermore, a conformational rigid form approximating the donor substrate is central. Here this is achieved by an intramolecular hydrogen bond formed between the carboxylate group and one of the ribose hydroxyl groups of the cytidine monophosphate (CMP) leaving group. This intramolecular interaction results in the donor substrate conformer complimenting the form of the catalytic binding site. Finally the carboxylate charge is essential for electrostatic pairing with the binding site. Substituting this group for an alcohol or amide results in severe weakening of the ligand binding. The carboxylate thus proves an to be an irreplaceable functional group and an essential pharmacophore.     

3D-QSAR analysis of sialyltransferase inhibitors

A predictive 3D-QSAR model that correlates the biological activities with the chemical structures of a series of sialyltransferase inhibitors, exemplified by the sugar:nucleotide derivatives, was developed by means of comparative molecular field analysis (CoMFA). The resulting cross-validated value (q(2)=0.629), non-cross-validated value (r(2)=0.965) and standard error of estimate (SEE=0.288) indicate that the obtained pharmacophore model indeed mimics the steric and electrostatic environment where inhibitors bind to the enzyme. The developed model also possesses promising predictive ability as discerned by the testing on the external test set, and should be useful to further understand the molecular nature of inhibitor-enzyme interactions and to aid in the design of more potent sialyltransferase inhibitors.     

Computational characterisation of the interactions between human ST6Gal I and transition‐state analogue inhibitors: insights for inhibitor design

Human β‐galactoside α‐2,6‐sialyltransferase I (hST6Gal I) catalyses the synthesis of sialylated glycoconjugates involved in cell–cell interactions. Overexpression of hST6Gal I is observed in many different types of cancers, where it promotes metastasis through altered cell surface sialylation. A wide range of sialyltransferase (ST) inhibitors have been developed based on the natural donor, cytidine 5′‐monophosphate N‐acetylneuraminic acid (CMP‐Neu5Ac). Of these, analogues that are structurally similar to the transition state exhibit the highest inhibitory activity. In order to design inhibitors that are readily accessible synthetically and with favourable pharmacokinetic properties, an investigation of the replacement of the charged phosphodiester‐linker, present in many ST inhibitors, with a potential neutral isostere such as a carbamate or a 1,2,3‐triazole has been undertaken. To investigate this, molecular docking and molecular dynamics simulations were performed. These simulations provided an insight into the binding mode of previously reported phosphodiester‐linked ST inhibitors and demonstrated that targeting the proposed sialyl acceptor site is a viable option for producing selective inhibitors. The potential for a carbamate‐ or triazole‐linker as an isosteric replacement for the phosphodiester in transition‐state analogue ST inhibitors was established using molecular docking. Molecular dynamics simulations of carbamate‐ and phosphodiester‐linked compounds revealed that both classes exhibit consistent interactions with hST6Gal I. Overall, the results obtained from this study provide a rationale for synthetic and biological evaluation of triazole‐ and carbamate‐linked transition‐state analogue ST inhibitors as potential new antimetastatic agents. Copyright © 2015 John Wiley & Sons, Ltd.     

Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli

Background  

The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity.     

Constitutively unmasked CD22 on B cells of ST6Gal I knockout mice: novel sialoside probe for murine CD22

The interaction of CD22 with glycoprotein ligands bearing the Siaalpha2,6Gal-R sequence is believed to modulate its function as a regulator of B cell signaling. Although a commercial sialoside-polyacrylamide (PAA) probe, NeuAc- alpha2,6Gal-PAA, has facilitated studies on ligand binding by human CD22, murine CD22 binds instead with high affinity to NeuGcalpha2,6Gal-R. A multivalent probe with this sequence was constructed to facilitate investigations of ligand binding in CD22 function using genetically defined murine models. The probe is based on the sialoside-PAA platform, which is then biotinylated for easy detection. A series of sialoside probes were constructed with two different length linker arms between the sialoside and the backbone and three different sialoside to PAA molar ratios. The NeuGcalpha2,6Gal-PAA probe is specific for CD22: it binds to sialidase-treated B cells of wild-type mice but not B cells of CD22-null mice. Additionally, because the probe only binds to sialidase-treated wild-type cells, it confirms that CD22 is constitutively "masked" on most B cells from wild-type mice by binding to ligands in cis. In contrast, the probe bound equally well to native or sialidase-treated B cells from the immunocompromised ligand-deficient ST6Gal I knockout mice, demonstrating that CD22 is constitutively "unmasked" in these cells.     

Formation of insoluble oligomers correlates with ST6Gal I stable localization in the golgi

The ST6Gal I is a sialyltransferase that functions in the late Golgi to modify the N-linked oligosaccharides of glycoproteins. The ST6Gal I is expressed as two isoforms with a single amino acid difference in their catalytic domains. The STcys isoform is stably retained in the cell and is predominantly found in the Golgi, whereas the STtyr isoform is only transiently localized in the Golgi and is cleaved and secreted from a post-Golgi compartment. These two ST6Gal I isoforms were used to explore the role of the bilayer thickness mechanism and oligomerization in Golgi localization. Analysis of STcys and STtyr proteins with longer transmembrane regions suggested that the bilayer thickness mechanism is not the predominant mechanism used for ST6Gal I Golgi localization. In contrast, the formation and quantity of Triton X-100-insoluble oligomers was correlated with the stable or transient localization of the ST6Gal I isoforms in the Golgi. Nearly 100% of the STcys and only 13% of the STtyr were found as Triton-insoluble oligomers when Golgi membranes of COS-1 cells expressing these proteins were solubilized at pH 6.3, the pH of the late Golgi. In contrast, both proteins were found in the soluble fraction when these membranes were solubilized at pH 8.0. Analysis of other mutants suggested that a conformational change in the catalytic domain rather than increased disulfide bond-based cross-linking is the basis for the increased ability of STcys protein to form oligomers and the stable localization of STcys protein in the Golgi.     

The ST6Gal I sialyltransferase selectively modifies N-glycans on CD45 to negatively regulate galectin-1-induced CD45 clustering,phosphatase modulation,and T cell death

The addition of sialic acid to T cell surface glycoproteins influences essential T cell functions such as selection in the thymus and homing in the peripheral circulation. Sialylation of glycoproteins can be regulated by expression of specific sialyltransferases that transfer sialic acid in a specific linkage to defined saccharide acceptor substrates and by expression of particular glycoproteins bearing saccharide acceptors preferentially recognized by different sialyltransferases. Addition of alpha2,6-linked sialic acid to the Galbeta1,4GlcNAc sequence, the preferred ligand for galectin-1, inhibits recognition of this saccharide ligand by galectin-1. SAalpha2,6Gal sequences, created by the ST6Gal I enzyme, are present on medullary thymocytes resistant to galectin-1-induced death but not on galectin-1-susceptible cortical thymocytes. To determine whether addition of alpha2,6-linked sialic acid to lactosamine sequences on T cell glycoproteins inhibits galectin-1 death, we expressed the ST6Gal I enzyme in a galectin-1-sensitive murine T cell line. ST6Gal I expression reduced galectin-1 binding to the cells and reduced susceptibility of the cells to galectin-1-induced cell death. Because the ST6Gal I preferentially utilizes N-glycans as acceptor substrates, we determined that N-glycans are essential for galectin-1-induced T cell death. Expression of the ST6Gal I specifically resulted in increased sialylation of N-glycans on CD45, a receptor tyrosine phosphatase that is a T cell receptor for galectin-1. ST6Gal I expression abrogated the reduction in CD45 tyrosine phosphatase activity that results from galectin-1 binding. Sialylation of CD45 by the ST6Gal I also prevented galectin-1-induced clustering of CD45 on the T cell surface, an initial step in galectin-1 cell death. Thus, regulation of glycoprotein sialylation may control susceptibility to cell death at specific points during T cell development and peripheral activation.     

Structure-function relationships in 3alpha-hydroxysteroid dehydrogenases: a comparison of the rat and human isoforms

3alpha-Hydroxysteroid dehydrogenases (3alpha-HSDs) inactivate steroid hormones in the liver, regulate 5alpha-dihydrotestosterone (5alpha-DHT) levels in the prostate, and form the neurosteroid, allopregnanolone in the CNS. Four human 3alpha-HSD isoforms exist and correspond to AKR1C1-AKR1C4 of the aldo-keto reductase (AKR) superfamily. Unlike the related rat 3alpha-HSD (AKR1C9) which is positional and stereospecific, the human enzymes display varying ratios of 3-, 17-, and 20-ketosteroid reductase activity as well as 3alpha-, 17beta-, and 20alpha-hydroxysteroid oxidase activity. Their k(cat) values are 50-100-fold lower than that observed for AKR1C9. Based on their product profiles and discrete tissue localization, the human enzymes may regulate the levels of active androgens, estrogens, and progestins in target tissues. The X-ray crystal structures of AKR1C9 and AKR1C2 (human type 3 3alpha-HSD, bile acid binding protein and peripheral 3alpha-HSD) reveal that the AKR1C2 structure can bind steroids backwards (D-ring in the A-ring position) and upside down (beta-face inverted) relative to the position of a 3-ketosteroid in AKR1C9 and this may account for its functional plasticity. Stopped-flow studies on both enzymes indicate that the conformational changes associated with binding cofactor (the first ligand) are slow; they are similar in both enzymes but are not rate-determining. Instead the low k(cat) seen in AKR1C2 (50-fold less than AKR1C9) may be due to substrate "wobble" at the plastic active site.     

Structure-function studies of the G-domain from human gem, a novel small G-protein

Gem, a member of the Rad,Gem/Kir subfamily of small G-proteins, has unique sequence features. We report here the crystallographic structure determination of the Gem G-domain in complex with nucleotide to 2.4 A resolution. Although the basic Ras protein fold is maintained, the Gem switch regions emphatically differ from the Ras paradigm. Our ensuing biochemical characterization indicates that Gem G-domain markedly prefers GDP over GTP. Two known functions of Gem are distinctly affected by spatially separated clusters of mutations.     

Structure-function relationships in 3-phosphoglycerate kinase: role of the carboxy-terminal peptide

Yeast 3-phosphoglycerate kinase (PGK) is a monomeric enzyme (Mr approximately 45,000) composed of two globular domains. Each domain corresponds approximately to the amino- and carboxy-terminal halves of the polypeptide chain. The carboxy-terminal end extends over the interdomain "hinge" region and packs against the amino-terminal domain. It has been proposed that domain movement, resulting in closure of the active site cleft, is essential for the catalytic function of PGK. Large-scale conformational changes have also been postulated to explain activation of the enzyme by sulfate ions. Using site-specific mutagenesis, we have removed a 15-amino-acid carboxy-terminal fragment, in order to probe its role in the substrate- and sulfate-induced conformational changes. The truncated enzyme exhibited approximately 1% of the activity of native PGK and lost the ability to undergo sulfate-induced activation. The Km for ATP was essentially unchanged (Km = 0.23 mM) in comparison to the native enzyme (Km = 0.30 mM), whereas the Km value for 3-phosphoglycerate was increased about eightfold (Km = 3.85 mM and 0.50 mM, respectively). These results suggest that the carboxy-terminal segment is important for the mechanism of the substrate- and sulfate-induced conformational transitions. CD spectra and sedimentation velocity measurements indicate that the carboxy-terminal peptide is essential for structural integrity of PGK. The increased susceptibility of the truncated enzyme to thermal inactivation implies that the carboxy-terminal peptide also contributes to the stability of PGK.