Remodeling of the major pig xenoantigen by N-acetylglucosaminyltransferase III in transgenic pig

We have been successful in generating several lines of transgenic mice and pigs that contain the human beta-d-mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) gene. The overexpression of the GnT-III gene in mice and pigs reduced their antigenicity to human natural antibodies, especially the Galalpha1-3Galbeta1-4GlcNAc-R, as evidenced by immunohistochemical analysis. Endothelial cell studies from the GnT-III transgenic pigs also revealed a significant down-regulation in antigenicity, including Hanganutziu-Deicher antigen, and dramatic reductions in both the complement- and natural killer cell-mediated pig cell lyses. Changes in the enzymatic activities of other glycosyltransferases, such as alpha1,3-galactosyltransferase, GnT-IV, and GnT-V, did not support cross-talk between GnT-III and these enzymes in the transgenic animals. In addition, we demonstrated the effect of GnT-III in down-regulating the xenoantigen of pig heart grafts, using a pig to cynomolgus monkey transplantation model, suggesting that this approach may be useful in clinical xenotransplantation in the future.     

Endothelial alpha 2,6-linked sialic acid inhibits VCAM-1-dependent adhesion under flow conditions.

We have previously shown that costimulation of endothelial cells with IL-1 + IL-4 markedly inhibits VCAM-1-dependent adhesion under flow conditions. We hypothesized that sialic acids on the costimulated cell surfaces may contribute to the inhibition. Northern blot analyses showed that Gal beta 1-4GlcNAc alpha 2, 6-sialyltransferase (ST6N) mRNA was up-regulated in cultured HUVEC by IL-1 or IL-4 alone, but that the expression was enhanced by costimulation, whereas the level of Gal beta 1-4GlcNAc/Gal beta 1-3GalNAc alpha2,3-sialyltransferase (ST3ON) mRNA was unchanged. Removing both alpha 2,6- and alpha 2,3-linked sialic acids from IL-1 + IL-4-costimulated HUVEC by sialidase significantly increased VCAM-1-dependent adhesion, whereas removing alpha 2,3-linked sialic acid alone had no effect; adenovirus-mediated overexpression of ST6N with costimulation almost abolished the adhesion, which was reversible by sialidase. The same treatments of IL-1-stimulated HUVEC had no effect. Lectin blotting showed that VCAM-1 is decorated with alpha 2,6- but not alpha 2,3-linked sialic acids. However, overexpression of alpha 2,6-sialyltransferase did not increase alpha 2,6-linked sialic acid on VCAM-1 but did increase alpha 2,6-linked sialic acids on other proteins that remain to be identified. These results suggest that alpha 2,6-linked sialic acids on a molecule(s) inducible by costimulation with IL-1 + IL-4 but not IL-1 alone down-regulates VCAM-1-dependent adhesion under flow conditions.     

alpha(1,2)-fucosylation prevents sialyl Lewis x expression and E-selectin-mediated adhesion of fucosyltransferase VII-transfected cells.

E-selectin is a cytokine-inducible, calcium-dependent endothelial cell adhesion molecule that plays a critical role in the leucocyte-endothelium interaction during inflammation and is thought to contribute to the metastatic dissemination of tumour cells. Like the other selectins, E-selectin binds to ligands carrying the tetrasaccharide sialyl-Lewis x (NeuAcalpha2,3Galbeta1,4[Fucalpha1, 3]GlcNAc)1 or its isomer sialyl-Lewis a (NeuAcalpha2, 3Galbeta1, 3[Fucalpha1,4]GlcNAc). We examined the effect of expressing the H-type alpha(1,2)-fucosyltransferase or the alpha(2, 6)-sialyltransferase on the synthesis of sialyl-Lewis x by alpha(1, 3)fucosyltransferase. We found that H-type alpha(1, 2)-fucosyltransferase but not alpha(2,6)-sialyltransferase, strongly inhibited sialyl-Lewis x expression and E-selectin adhesion. We assume that H-type alpha(1,2)-fucosyltransferase competes with the endogenous alpha(2,3)-sialyltransferase for the N-acetyllactosamine structures assigned to further serve as acceptors for alpha(1, 3)fucosyltransferase.     

Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells

Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required.We report here the partial cloning of the CMP-sialic, acid:Galbeta1,3GalNAcalpha2,3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galbeta1,3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express alpha1,3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field.     

Remodeling of the major mouse xenoantigen, Galalpha1-3Galbeta1-4GlcNAc-R, by N-acetylglucosaminyltransferase-III

beta-D-Mannoside beta-1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyses the attachment of an N-acetylglucosamine (GlcNAc) residue to mannose in the beta(1-4) configuration in N-glycans, and forms a bisecting GlcNAc. We have generated transgenic mice that contain the human GnT-III gene under the control of the mouse albumin enhancer/promoter [Lee et al., (2003)]. Overexpression of this gene in mice reduced the antigenicity of N-glycans to human natural antibodies, especially in the case of the alpha-Gal epitope, Galalpha1-3Galbeta1-4GlcNAc-R. Study of endothelial cells from the GnT-III transgenic mice revealed a significant reduction in antigenicity, and a dramatic decrease in both complement- and natural killer cell-mediated mouse cell lysis. Changes in the enzymatic activities of other glycosyltransferases, such as alpha1,3-galactosyltransferase, and alpha-6-D-mannoside beta-1,6 N-acetylglucosaminyltransferase V, did not point to any interaction between GnT-III and these enzymes in the transgenic mice, suggesting that this approach may be useful in clinical xenotransplantation.     

A novel viral alpha2,3-sialyltransferase (v-ST3Gal I): transfer of sialic acid to fucosylated acceptors

The substrate specificity of an alpha2,3-sialyltransferase (v-ST3Gal I) obtained from myxoma virus infected RK13 cells has been determined. Like mammalian sialyltransferase enzymes, the viral enzyme contains the characteristic L- and S-sialyl motif sequences in its catalytic domain. Analysis of the deduced amino acid sequences of cloned sialyltransferases suggests that v-ST3Gal I is closely related to mammalian ST3Gal IV. v-ST3Gal I catalyzes the transfer of sialic acid from CMP-NeuAc to Type I (Galbeta1-3GlcNAcbeta) II (Galbeta1-4GlcNAcbeta) and III (Galbeta1-3GalNAcbeta) acceptors. In addition, the viral enzyme also transfers sialic acid to the fucosylated acceptors Lewis(x) and Lewis(a). This substrate specificity is unlike any sialyltransferases described to date, though it is most comparable with those of mammalian ST3Gal IV enzymes. The products from reactions with fucosylated acceptors were characterized by capillary zone electrophoresis, (1)H-NMR spectroscopy and mass spectrometry. They were shown to be 2,3-sialylated Lewis(x) and 2,3-sialylated Lewis(a), respectively.     

Reversible sialylation: synthesis of cytidine 5'-monophospho-N-acetylneuraminic acid from cytidine 5'-monophosphate with alpha2,3-sialyl O-glycan-, glycolipid-, and macromolecule-based donors yields diverse sialylated products

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.     

N-acetylglucosaminyltransferase III antagonizes the effect of N-acetylglucosaminyltransferase V on alpha3beta1 integrin-mediated cell migration

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of beta1,6-GlcNAc branching of N-glycans, which contributes to metastasis. N-acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulting in the suppression of metastasis. It has long been hypothesized that the suppression of GnT-V product formation by the action of GnT-III would also exist in vivo, which will consequently lead to the inhibition of biological functions of GnT-V. To test this, we draw a comparison among MKN45 cells, which were transfected with GnT-III, GnT-V, or both, respectively. We found that alpha3beta1 integrin-mediated cell migration on laminin 5 was greatly enhanced in the case of GnT-V transfectant. This enhanced cell migration was significantly blocked after the introduction of GnT-III. Consistently, an increase in bisected GlcNAc but a decrease in beta1,6-GlcNAc-branched N-glycans on integrin alpha3 subunit was observed in the double transfectants of GnT-III and GnT-V. Conversely, GnT-III knockdown resulted in increased migration on laminin 5, concomitant with an increase in beta1,6-GlcNAc-branched N-glycans on the alpha3 subunit in CHP134 cells, a human neuroblastoma cell line. Therefore, in this study, the priority of GnT-III for the modification of the alpha3 subunit may be an explanation for why GnT-III inhibits GnT-V-induced cell migration. Taken together, our results demonstrate for the first time that GnT-III and GnT-V can competitively modify the same target glycoprotein and furthermore positively or negatively regulate its biological functions.     

Introduction of bisecting GlcNAc into integrin alpha5beta1 reduces ligand binding and down-regulates cell adhesion and cell migration

The enzyme beta1,4-N-acetylglucosaminyltransferase III (GnT-III) catalyzes the addition of a bisecting GlcNAc residue to glycoproteins, resulting in a modulation in biological function. Our previous studies showed that the transfection of the GnT-III gene into B16 melanoma cells results in a suppression of invasive ability and lung colonization. The suppression has been postulated to be due to an increased level of E-cadherin expression on the cell surface, which in turn leads to the up-regulation of cell-cell adhesion. In this study, we report on the effects of overexpression of GnT-III on cell-matrix adhesion. The overexpression of GnT-III, but not that of an enzymatic inactive GnT-III (D323A), inhibits cell spreading and migration on fibronectin, a specific ligand for integrin alpha(5)beta(1), and the focal adhesion kinase phosphorylation. E(4)-PHA lectin blot analyses showed that the levels of bisecting GlcNAc structures on the integrin alpha(5) subunit as well as alpha(2) and alpha(3) subunits immunoprecipitated from GnT-III transfectants were substantially increased. In addition, the affinity of the binding of integrin alpha(5)beta(1) to fibronectin was significantly reduced by the introduction of the bisecting GlcNAc, to the alpha(5) subunit. These findings suggest that the modification of N-glycan of integrin by GnT-III inhibits its ligand binding ability, subsequently leading to the down-regulation of integrin-mediated signaling.     

Enhanced activity of recombinant beta-secretase from Drosophila melanogaster S2 cells transformed with cDNAs encoding human beta1,4-galactosyltransferase and Galbeta1,4-GlcNAc alpha2,6-sialyltransferase

beta-Secretase (betaSEC) was expressed in Drososphila melanogaster Schneider 2 (S2) cells transformed with cDNAs encoding beta1,4-galactosyltransferase (GalT) and Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (ST). The apparent molecular weight of recombinant beta-secretase was increased from 56kDa to 61kDa. A lectin blot analysis indicated that recombinant beta-secretase from S2betaSEC/GalT-ST cells (S2 cells co-transformed with cDNAs encoding beta-secretase, glycosyltransferases, GalT, and ST) contained the glycan residues of beta1,4-linked galactose and alpha2,6-linked sialic acid. Two dimensional electrophoresis revealed that recombinant beta-secretase from S2betaSEC/GalT-ST cells had a lower isoelectric point compared to beta-secretase from control S2betaSEC cells (S2 cells transformed only with beta-secretase cDNA). Recombinant beta-secretase from transformed S2 cells was also present as heterogeneous forms. The enzyme activity of recombinant beta-secretase from S2betaSEC/GalT-ST cells was enhanced up to 260% compared to control S2betaSEC cells. We have shown that an exogeneous human glycosyltransferases cDNA can be introduced into S2 cells to extend the N-glycan processing capabilities of the insect cell line, and that the extended glycosylation improves the activity of recombinant beta-secretase.     

Engineering of coordinated up- and down-regulation of two glycosyltransferases of the O-glycosylation pathway in Chinese hamster ovary (CHO) cells

Production of O-linked oligosaccharides that interact with selectins to mediate cell-cell adhesion occurs in one segment of a branched glycan biosynthesis network. Prior efforts to direct the branched pathway towards selectin-binding oligosaccharides by amplifying enzymes in this branch of the network have had limited success, suggesting that metabolic engineering to simultaneously inhibit the competing pathway may also be required. We report here the partial cloning of the CMP-sialic acid:Galbeta1,3GalNAcalpha2, 3-sialyltransferase (ST3Gal I) gene from Chinese hamster ovary (CHO) cells and the simultaneous inhibition of expression of CHO cell ST3Gal I gene and overexpression of the human UDP-GlcNAc:Galbeta1, 3GalNAc-R beta1,6-N-acetylglucosaminyltransferase (C2GnT) gene. A tetracycline-regulated system adjoined to tricistronic expression technology allowed "one-step" transient manipulation of multiple enzyme activities in the O-glycosylation pathway of a previously established CHO cell line already engineered to express alpha1, 3-fucosyltransferase VI (alpha1,3-Fuc-TVI). Tetracycline-regulated co-expression of a ST3Gal I fragment, cloned in the antisense orientation, and of C2GnT cDNA resulted in inhibition of the ST3Gal I enzymatic activity and increase in C2GnT activity which varied depending on the extent of tetracycline reduction in the cell culture medium. This simultaneous regulated inhibition and activation of the two key enzyme activities in the O-glycosylation pathway of mammalian cells is an important addition to the metabolic engineering field.     

The Hexapeptide inhibitor of Galbeta 1,3GalNAc-specific alpha 2,3-sialyltransferase as a generic inhibitor of sialyltransferases

The mammalian Galbeta1,3GalNAc-specific alpha2,3-sialyltransferase (ST3Gal I) was expressed as a secreted glycoprotein in High Five (Trichoplusia ni) cells. Using this recombinant ST3Gal I, we screened the synthetic hexapeptide combinatorial library to explore a sialyltransferase inhibitor. We found that the hexapeptide, NH(2)-GNWWWW, exhibited the most strong inhibition of ST3Gal I among five different hexapeptides that were finally selected. The kinetic analysis of ST3Gal I inhibition demonstrated that this hexapeptide could act as a competitive inhibitor (K(i) = 1.1 microm) on CMP-NeuAc binding to the enzyme. Moreover, the hexapeptide was shown to strongly inhibit both N-glycan-specific alpha2,3- and alpha2,6-sialyltranferase in vitro, suggesting that this peptide may inhibit the broad range of sialyltransferases regardless of their linkage specificity. The inhibitory activity in vivo was investigated by RCA-I lectin blot analyses and by metabolic d-[6-(3)H]GlcNH(2) radiolabeling analyses of N- and O-linked oligosaccharides in Chines hamster ovary cells. Our results demonstrate that the hexapeptide can act as a generic inhibitor of the N- and O-glycan-specific sialyltransferases in mammalian cells, which results in the significantly reduced NeuAc expression on cellular glycoproteins in vivo.     

Reduction of the major xenoantigen on glycosphingolipids of swine endothelial cells by various glycosyltransferases

The effect of the various glycosyltransferases on glycosphingolipids was examined, using transfected swine endothelial cell (SEC) lines. The reactivity of parental SEC to normal human serum (NHS) and Griffonia simplicifolia IB(4) (GSIB4) lectin, which binds to the Gal alpha1-3 Gal beta 1-4 GlcNAc-R (alpha-galactosyl epitope), was reduced by approximately 20% by the treatment with D-PDMP (D-threo-1-phenyl-2-decan- oylamino-3-morpholino-1-propanol), suggesting that glycosphingolipids contained by SEC have a considerable amount of the alpha-galactosyl epitope. The overexpression of two different types of glycosyltransferase, N-acetylglucosaminyl transferase III (GnT-III), as well as alpha2, 6-sialyltransferase (ST6Gal I), alpha2,3-sialyltransferase (ST3Gal III), and alpha1,2-fucosyltransferase (alpha1,2FT), suppresses the total antigenicity of SEC significantly. However, the reduction in reactivities toward NHS and GSIB4 lectin in the case of GnT-III transfectants was milder than those in other transfectants. Western blot analysis indicated that the glycoproteins in all transfectants had diminished reactivity to NHS and GSIB4 lectin to approximately the same extent. Therefore, the neutral glycosphingolipids of these transfectants were separated by thin layer chromatography, followed by immunostaining with NHS and GSIB4 lectin. The levels of the alpha-galactosyl epitope in glycosphingolipids were not decreased in the GnT-III transfectants but were in the ST6Gal I, ST3Gal III, and alpha1,2FT transfectants. These data indicate that ST6Gal I, ST3Gal III, and alpha1,2FT reduced the alpha-galactosyl epitope in both glycoproteins and glycosphingolipids, while GnT-III reduced them only in glycoproteins.     

Molecular cloning of a novel alpha2,3-sialyltransferase (ST3Gal VI) that sialylates type II lactosamine structures on glycoproteins and glycolipids

A novel member of the human CMP-NeuAc:beta-galactoside alpha2, 3-sialyltransferase (ST) subfamily, designated ST3Gal VI, was identified based on BLAST analysis of expressed sequence tags, and a cDNA clone was isolated from a human melanoma line library. The sequence of ST3Gal VI encoded a type II membrane protein with 2 amino acids of cytoplasmic domain, 32 amino acids of transmembrane region, and a large catalytic domain with 297 amino acids; and showed homology to previously cloned ST3Gal III, ST3Gal IV, and ST3Gal V at 34, 38, and 33%, respectively. Extracts from L cells transfected with ST3Gal VI cDNA in a expression vector and a fusion protein with protein A showed an enzyme activity of alpha2, 3-sialyltransferase toward Galbeta1,4GlcNAc structure on glycoproteins and glycolipids. In contrast to ST3Gal III and ST3Gal IV, this enzyme exhibited restricted substrate specificity, i.e. it utilized Galbeta1,4GlcNAc on glycoproteins, and neolactotetraosylceramide and neolactohexaosylceramide, but not lactotetraosylceramide, lactosylceramide, or asialo-GM1. Consequently, these data indicated that this enzyme is involved in the synthesis of sialyl-paragloboside, a precursor of sialyl-Lewis X determinant.     

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.     

IL-6 and IL-8 increase the expression of glycosyltransferases and sulfotransferases involved in the biosynthesis of sialylated and/or sulfated Lewisx epitopes in the human bronchial mucosa

Bronchial mucins from patients suffering from CF (cystic fibrosis) exhibit glycosylation alterations, especially increased amounts of the sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3]GlcNAc-R) and 6-sulfo-sialyl-Lewis(x) (NeuAcalpha2-3Galbeta1-4[Fucalpha1-3][SO(3)H-6]GlcNAc-R) terminal structures. These epitopes are preferential receptors for Pseudomonas aeruginosa, the bacteria responsible for the chronicity of airway infection and involved in the morbidity and early death of CF patients. However, these glycosylation changes cannot be directly linked to defects in CFTR (CF transmembrane conductance regulator) gene expression since cells that secrete airway mucins express no or very low amounts of the protein. Several studies have shown that inflammation may affect glycosylation and sulfation of various glycoproteins, including mucins. In the present study, we show that incubation of macroscopically healthy fragments of human bronchial mucosa with IL-6 (interleukin-6) or IL-8 results in a significant increase in the expression of alpha1,3/4-fucosyltransferases [FUT11 (fucosyltransferase 11 gene) and FUT3], alpha2-6- and alpha2,3-sialyltransferases [ST3GAL6 (alpha2,3-sialyltransferase 6 gene) and ST6GAL2 (alpha2,6-sialyltransferase 2 gene)] and GlcNAc-6-O-sulfotransferases [CHST4 (carbohydrate sulfotransferase 4 gene) and CHST6] mRNA. In parallel, the amounts of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes at the periphery of high-molecular-mass proteins, including MUC4, were also increased. In conclusion, our results indicate that IL-6 and -8 may contribute to the increased levels of sialyl-Lewis(x) and 6-sulfo-sialyl-Lewis(x) epitopes on human airway mucins from patients with CF.     

Transfer of synthetic sialic acid analogues to N- and O-linked glycoprotein glycans using four different mammalian sialyltransferases

This paper presents kinetic properties of the transfer of several synthetic 9-substituted sialic acid analogues onto N- or O-linked glycoprotein glycans by four purified mammalian sialyltransferases: Gal beta 1,4GlcNac alpha 2,6sialyltransferase, Gal beta-1,4(3)GlcNAc alpha 2,3-sialyltransferase, Gal beta 1,3GalNAc alpha 2,3sialyltransferase, and GalNAc alpha 2,6sialyltransferase. The substituents at C-9 of the sialic acid analogues introduce special biochemical characteristics: 9-Amino-NeuAc represents, up to the present, the first derivative that is resistant toward bacterial, viral, and mammalian sialidases but is transferred by a sialyltransferase. 9-Acetamido-NeuAc, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc differ in size and hydrophobic character from each other and from parent NeuAc. 9-Azido-NeuAc may be used to introduce a photoreactive label. The kinetic properties of the four sialyltransferases with regard to the donor CMP-glycosides differed distinctly depending on the structure of the substituent at C-9. CMP-9-amino-NeuAc was only accepted as donor substrate by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase (rat liver), but the Km value was 14-fold higher than that of parent CMP-NeuAc. In contrast, 9-azido-NeuAc was readily transferred by each of these four enzymes. 9-Acetamido-NeuAc, which is a receptor analogue for influenza C virus, 9-benzamido-NeuAc, and 9-hexanoylamido-NeuAc were also accepted by each sialyltransferase, but incorporation values differed significantly depending on the enzyme used. For the first time, the resialylation of asialo-alpha 1-acid glycoprotein with 9-substituted sialic acid analogues by Gal beta 1,4GlcNAc alpha 2,6sialyltransferase is demonstrated.     

Photobacterium sp. JT-ISH-224 produces two sialyltransferases, alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase

A novel bacterium, Photobacterium sp. JT-ISH-224, that produces alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase, was isolated from the gut of a Japanese barracuda. The genes that encode the enzymes were cloned from the genomic library of the bacterium using the genes encoding alpha-/beta-galactoside alpha2,3-sialyltransferase from P. phosphoreum and beta-galactoside alpha2,6-sialyltransferase from P. damselae as probes. The nucleotide sequences were determined, and open reading frames of 1,230 and 1,545 bp for encoding an alpha2,3-sialyltransferase and an alpha2,6-sialyltransferase of 409- and 514-amino acid residues, respectively, were identified. The alpha2,3-sialyltransferase had 92% amino acid sequence identity with the P. phosphoreum alpha2,3-sialyltransferase, whereas the alpha2,6-sialyltransferase had 54% amino acid sequence identity with the P. damselae alpha2,6-sialyltransferase. For both enzymes, the DNA fragments that encoded the full-length protein and its truncated form lacking the putative signal peptide sequence were amplified by a polymerase chain reaction and cloned into an expression vector. Each gene was expressed in Escherichia coli, and the lysate from each strain had enzymatic activity. The alpha2,3-sialyltransferase catalysed the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to lactose, alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside with low apparent K(m) and the alpha2,6-sialyltransferase catalysed the transfer of NeuAc from CMP-NeuAc to lactose with low apparent K(m).     

High-level expression of human glycosyltransferases in insect cells as biochemically active form

cDNAs, encoding human beta1,4-galactosyltransferase (hGalT I, EC 2.4.1.22), human Galbeta1,3(4)-GlcNAc alpha2,3-sialyltransferase (hST3GalIII, EC 2.4.99), and human Galbeta1,4-GlcNAc alpha2,6-sialyltransferase (hST6Gal I, EC 2.4.99.1), were cloned from human cell lines. In order to express these glycosyltransferases as secreted form in insect cells, cDNAs were inserted into a novel baculovirus transfer vector equipped with the mouse IgM signal peptide and IgG binding domain of the Staphylococcus aureus protein A as an N-terminal fusion partner. About 14 mg hGalT I, 8 mg hST3GalIII, and 6.4 mg hST6Gal I were purified from 1 liter of recombinant baculovirus infected insect cell culture media. The specific activities of recombinant hGalT I and hST6Gal I were determined as 0.65 and 1.6 U/mg protein, respectively. These results indicated that the recombinant hGalT I and hST6Gal I retained enzyme activities at similar level to those of the authentic one although they were fused with the IgG binding domain at the N-terminus. Taken together, the mouse IgM signal peptide and IgG binding domain of the protein A could be efficiently used as an N-terminus fusion partner for the over-expression of heterologous proteins in insect cells.