The novel role of circular RNA ST3GAL6 on blocking gastric cancer malignant behaviours through autophagy regulated by the FOXP2/MET/mTOR axis.

Gastric cancer (GC) ranks third in mortality among all cancers worldwide. Circular RNAs (circRNAs) play an important role in the occurrence and development of gastric cancer. Forkhead box P2 (FOXP2), as a transcription factor, is closely associated with the development of many types of tumours. However, the regulatory network between FOXP2 and circRNAs remains to be explored. In our study, circST3GAL6 was significantly downregulated in GC and was associated with poor prognosis in GC patients. Overexpression of circST3GAL6 inhibited the malignant behaviours of GC cells, which was mediated by inducing apoptosis and autophagy. In addition, we demonstrated that circST3GAL6 regulated FOXP2 through the mir-300 sponge. We further found that FOXP2 inhibited MET Proto-Oncogene (MET), which was the initiating factor that regulated the classic AKT/mTOR pathway of autophagy. In conclusion, our results suggested that circST3GAL6 played a tumour suppressive role in gastric cancer through miR-300/FOXP2 axis and regulated apoptosis and autophagy through FOXP2-mediated transcriptional inhibition of the MET axis, which may become a potential target for GC therapy.

Therapeutic CMP-Nonulosonates against Multidrug-Resistant Neisseria gonorrhoeae.

Neisseria gonorrhoeae deploys a unique immune evasion strategy wherein the lacto-N-neotetraose termini of lipooligosaccharide (LOS) are "capped" by a surface LOS sialyltransferase (Lst), using extracellular host-derived CMP-sialic acid (CMP-Neu5Ac in humans). LOS sialylation enhances complement resistance by recruiting factor H (FH; alternative complement pathway inhibitor) and also by limiting classical pathway activation. Sialylated LOS also engages inhibitory Siglecs on host leukocytes, dampening innate immunity. Previously, we showed that analogues of CMP-sialic acids (CMP-nonulosonates [CMP-NulOs]), such as CMP-Leg5,7Ac2 and CMP-Neu5Ac9N3, are also substrates for Lst. Incorporation of Leg5,7Ac2 and Neu5Ac9N3 into LOS results in N. gonorrhoeae being fully serum sensitive. Importantly, intravaginal administration of CMP-Leg5,7Ac2 attenuated N. gonorrhoeae colonization of mouse vaginas. In this study, we characterize and develop additional candidate therapeutic CMP-NulOs. CMP-ketodeoxynonulosonate (CMP-Kdn) and CMP-Kdn7N3, but not CMP-Neu4,5Ac2, were substrates for Lst, further elucidating gonococcal Lst specificity. Lacto-N-neotetraose LOS capped with Kdn and Kdn7N3 bound FH to levels ∼60% of that seen with Neu5Ac and enabled gonococci to resist low (3.3%) but not higher (10%) concentrations of human complement. CMP-Kdn, CMP-Neu5Ac9N3, and CMP-Leg5,7Ac2 administered intravaginally (10 µg/d) to N. gonorrhoeae-colonized mice were equally efficacious. Of the three CMP-NulOs above, CMP-Leg5,7Ac2 was the most pH and temperature stable. In addition, Leg5,7Ac2-fed human cells did not display this NulO on their surface. Moreover, CMP-Leg5,7Ac2 was efficacious against several multidrug-resistant gonococci in mice with a humanized sialome (Cmah-/- mice) or humanized complement system (FH/C4b-binding protein transgenic mice). CMP-Leg5,7Ac2 and CMP-Kdn remain viable leads as topical preventive/therapeutic agents against the global threat of multidrug-resistant N. gonorrhoeae.

Sialylation of MUC4ß N-glycans by ST6GAL1 orchestrates human airway epithelial cell differentiation associated with type-2 inflammation.

Although type-2-induced (T2-induced) epithelial dysfunction is likely to profoundly alter epithelial differentiation and repair in asthma, the mechanisms for these effects are poorly understood. A role for specific mucins, heavily N-glycosylated epithelial glycoproteins, in orchestrating epithelial cell fate in response to T2 stimuli has not previously been investigated. Levels of a sialylated MUC4ß isoform were found to be increased in airway specimens from asthmatic patients in association with T2 inflammation. We hypothesized that IL-13 would increase sialylation of MUC4ß, thereby altering its function and that the ß-galactoside α-2,6-sialyltransferase 1 (ST6GAL1) would regulate the sialylation. Using human biologic specimens and cultured primary human airway epithelial cells (HAECs),we demonstrated that IL-13 increases ST6GAL1-mediated sialylation of MUC4ß and that both were increased in asthma, particularly in sputum supernatant and/or fresh isolated HAECs with elevated T2 biomarkers. ST6GAL1-induced sialylation of MUC4ß altered its lectin binding and secretion. Both ST6GAL1 and MUC4ß inhibited epithelial cell proliferation while promoting goblet cell differentiation. These in vivo and in vitro data provide strong evidence for a critical role for ST6GAL1-induced sialylation of MUC4ß in epithelial dysfunction associated with T2-high asthma, thereby identifying specific sialylation pathways as potential targets in asthma.

ST8SIA2 promotes oligodendrocyte differentiation and the integrity of myelin and axons.

ST8SIA2 is a polysialyltransferase that attaches polysialic acid to the glycoproteins NCAM1 and CADM1. Polysialylation is involved in brain development and plasticity. ST8SIA2 is a schizophrenia candidate gene, and St8sia2-/- mice exhibit schizophrenia-like behavior. We sought to identify new pathological consequences of ST8SIA2 deficiency. Our proteomic analysis suggested myelin impairment in St8sia2-/- mice. Histological and immune staining together with Western blot revealed that the onset of myelination was not delayed in St8sia2-/- mice, but the content of myelin was lower. Ultrastructure analysis of the corpus callosum showed thinner myelin sheaths, smaller and irregularly shaped axons, and white matter lesions in adult St8sia2-/- mice. Then we evaluated oligodendrocyte differentiation in vivo and in vitro. Fewer OLIG2+ cells in the cortex and corpus callosum, together with the higher percentage of undifferentiated oligodenroglia in St8sia2-/- mice suggested an impairment in oligodendrocyte generation. Experiment on primary cultures of oligodendrocyte precursor cells (OPCs) confirmed a cell-autonomous effect of ST8SIA2 in oligodendroglia, and demonstrated that OPC to oligodendrocyte transition is inhibited in St8sia2-/- mice. Concluding, ST8SIA2-mediated polysialylation influences on oligodendrocyte differentiation, and oligodendrocyte deficits in St8sia2 mice are a possible cause of the demyelination and degeneration of axons, resembling nerve fiber alterations in schizophrenia. GLIA 2016;65:34-49.

Intrinsically de-sialylated CD103(+) CD8 T cells mediate beneficial anti-glioma immune responses.

BACKGROUND: Cancer vaccines reproducibly cure laboratory animals and reveal encouraging trends in brain tumor (glioma) patients. Identifying parameters governing beneficial vaccine-induced responses may lead to the improvement of glioma immunotherapies. CD103(+) CD8 T cells dominate post-vaccine responses in human glioma patients for unknown reasons, but may be related to recent thymic emigrant (RTE) status. Importantly, CD8 RTE metrics correlated with beneficial immune responses in vaccinated glioma patients. METHODS: We show by flow cytometry that murine and human CD103(+) CD8 T cells respond better than their CD103(-) counterparts to tumor peptide-MHC I (pMHC I) stimulation in vitro and to tumor antigens on gliomas in vivo. RESULTS: Glioma responsive T cells from mice and humans both exhibited intrinsic de-sialylation-affecting CD8 beta. Modulation of CD8 T cell sialic acid with neuraminidase and ST3Gal-II revealed de-sialylation was necessary and sufficient for promiscuous binding to and stimulation by tumor pMHC I. Moreover, de-sialylated status was required for adoptive CD8 T cells and lymphocytes to decrease GL26 glioma invasiveness and increase host survival in vivo. Finally, increased tumor ST3Gal-II expression correlated with clinical vaccine failure in a meta-analysis of high-grade glioma patients. CONCLUSIONS: Taken together, these findings suggest that de-sialylation of CD8 is required for hyper-responsiveness and beneficial anti-glioma activity by CD8 T cells. Because CD8 de-sialylation can be induced with exogenous enzymes (and appears particularly scarce on human T cells), it represents a promising target for clinical glioma vaccine improvement.

Enzymatic engineering of polysialic acid on cells in vitro and in vivo using a purified bacterial polysialyltransferase.

In vertebrates, polysialic acid (PSA) is typically added to the neural cell adhesion molecule (NCAM) in the Golgi by PST or STX polysialyltransferase. PSA promotes plasticity, and its enhanced expression by viral delivery of the PST or STX gene has been shown to promote cellular processes that are useful for repair of the injured adult nervous system. Here we demonstrate a new strategy for PSA induction on cells involving addition of a purified polysialyltransferase from Neisseria meningitidis (PST(Nm)) to the extracellular environment. In the presence of its donor substrate (CMP-Neu5Ac), PST(Nm) synthesized PSA directly on surfaces of various cell types in culture, including Chinese hamster ovary cells, chicken DF1 fibroblasts, primary rat Schwann cells, and mouse embryonic stem cells. Similarly, injection of PST(Nm) and donor in vivo was able to produce PSA in different adult brain regions, including the cerebral cortex, striatum, and spinal cord. PSA synthesis by PST(Nm) requires the presence of the donor CMP-Neu5Ac, and the product could be degraded by the PSA-specific endoneuraminidase-N. Although PST(Nm) was able to add PSA to NCAM, most of its product was attached to other cell surface proteins. Nevertheless, the PST(Nm)-induced PSA displayed the ability to attenuate cell adhesion, promote neurite outgrowth, and enhance cell migration as has been reported for endogenous PSA-NCAM. Polysialylation by PST(Nm) occurred in vivo in less than 2.5 h, persisted in tissues, and then decreased within a few weeks. Together these characteristics suggest that a PST(Nm)-based approach may provide a valuable alternative to PST gene therapy.

Cell surface sialylation and fucosylation are regulated by the cell recognition molecule L1 via PLCgamma and cooperate to modulate embryonic stem cell survival and proliferation.

Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown. Using a panel of carbohydrate markers, we have shown that cell surface sialylation and fucosylation are upregulated in L1-transfected embryonic stem cells (L1-ESCs). Consistently, the mRNA levels of sialyltransferase ST6Gal1 and ST3Gal4, and fucosyltransferase FUT9 were significantly increased in L1-transfected ESCs. Activation of L1 signaling promoted cell survival and inhibited cell proliferation. ShRNAs knocking down FUT9, ST6Gal1 and ST3Gal4 blocked these effects. A phospholipase Cgamma (PLCgamma) inhibitor and shRNA reduced ST6Gal1, ST3Gal4 and FUT9 mRNA levels in the L1-ESCs. Thus, embryonic stem cell surface sialylation and fucosylation are regulated via PLCgamma by L1, with which they cooperate to modulate cell survival and proliferation.

Enterovirus 70 binds to different glycoconjugates containing alpha2,3-linked sialic acid on different cell lines.

Enterovirus 70 (EV70), the causative agent of acute hemorrhagic conjunctivitis, exhibits a restricted tropism for conjunctival and corneal cells in vivo but infects a wide spectrum of mammalian cells in culture. Previously, we demonstrated that human CD55 is a receptor for EV70 on HeLa cells but that EV70 also binds to sialic acid-containing receptors on a variety of other human cell lines. Virus recognition of sialic acid attached to underlying glycans by a particular glycosidic linkage may contribute to host range, tissue tropism, and pathogenesis. Therefore, we tested the possibility that EV70 binds to alpha2,3-linked sialic acid, like other viruses associated with ocular infections. Through the use of linkage-specific sialidases, sialyltransferases, and lectins, we show that EV70 recognizes alpha2,3-linked sialic acid on human corneal epithelial cells and on U-937 cells. Virus attachment to both cell lines is CD55 independent and sensitive to benzyl N-acetyl-alpha-D-galactosaminide, an inhibitor of O-linked glycosylation. Virus binding to corneal cells, but not U-937 cells, is inhibited by proteinase K, but not by phosphatidylinositol-specific phospholipase C treatment. These results are consistent with the idea that a major EV70 receptor on corneal epithelial cells is an O-glycosylated, non-glycosyl phosphatidylinositol-anchored membrane glycoprotein containing alpha2,3-linked sialic acid, while sialylated receptors on U-937 cells are not proteinaceous.

Differential enhancing effects of alpha2,8-sialyltransferase on the cell proliferation and mobility.

alpha2,8-Sialyltransferase (alpha2,8S-T, GD3 synthase) has been reported to be involved in the enhanced cell proliferation of malignant tumors. Using a cloned cDNA of alpha2,8S-T, transfectant cells were established and the effects of the gene expression on the cell phenotypes were analyzed. In contrast with PC12 cells, in which we reported marked growth enhancement based on the transfection of alpha2,8S-T, Swiss3T3 cells showed no enhancement in either cell proliferation or phosphorylation of MAP kinases after the transfection of alpha2,8S-T when treated with platelet-derived growth factor. Correspondingly, the receptor for platelet-derived growth factor also showed no increased phosphorylation upon the factor stimulation. However, in the wound-healing scratching assay, the Swiss3T3 transfectant cells demonstrated increased mobility as the PC12 transfectant cells. These results suggest that the enhancing effects of alpha2,8S-T on the proliferation and mobility are differential depending on the cell types, and ganglioside-associating molecules in the individual cell types need to be investigated.

Production of a complement inhibitor possessing sialyl Lewis X moieties by in vitro glycosylation technology.

Recombinant soluble human complement receptor type 1 (sCR1) is a highly glycosylated glycoprotein intended for use as a drug to treat ischemia-reperfusion injury and other complement-mediated diseases and injuries. sCR1-sLe(x) produced in the FT-VI-expressing mutant CHO cell line LEC11 exists as a heterogeneous mixture of glycoforms, a fraction of which include structures with one or more antennae terminated by the sialyl Lewis X (sLe(x)) [Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc]) epitope. Such multivalent presentation of sLe(x) was shown previously to effectively target sCR1 to activated endothelial cells expressing E-selectin. Here, we describe the use of the soluble, recombinant alpha2-3 sialyltransferase ST3Gal-III and the alpha1-3 fucosyltransferase FT-VI in vitro to introduce sLe(x) moieties onto the N-glycan chains of sCR1 overexpressed in standard CHO cell lines. The product (sCR1-S/F) of these in vitro enzymatic glycan remodeling reactions performed at the 10-g scale has approximately 14 N-glycan chains per sCR1 molecule, comprised of biantennary (90%), triantennary (8.5%), and tetraantennary (1.5%) structures, nearly all of whose antennae terminate with sLe(x) moieties. sCR1-S/F retained complement inhibitory activity and, in comparison with sCR1-sLe(x) produced in the LEC11 cell line, contained twice the number of sLe(x) moieties per mole glycoprotein, exhibited a twofold increase in area under the intravenous clearance curve in a rat pharmacokinetic model, and exhibited a 10-fold increase in affinity for E-selectin in an in vitro binding assay. These results demonstrate that in vitro glycosylation of the sCR1 drug product reduces heterogeneity of the glycan profile, improves pharmacokinetics, and enhances carbohydrate-mediated binding to E-selectin.

Stereoselective synthesis of phosphoramidate alpha(2-6)sialyltransferase transition-state analogue inhibitors.

The asymmetric synthesis of novel, potent phosphoramidate alpha(2-6)sialyltransferase transition-state analogue inhibitors such as (R)-9 (K(i)=68 microM) is described, via condensation of cytidine phosphitamide 6 with key chiral, non-racemic alpha-aminophosphonates, prepared in >98% ee by Mitsunobu azidation followed by Staudinger reduction of the corresponding chiral, non-racemic alpha-hydroxyphosphonates.

A ganglioside-specific sialyltransferase localizes to axons and non-Golgi structures in neurons.

To investigate the tissue distribution and subcellular localization of ST3GalV (CMP-NeuAc:lactosylceramide alpha2,3 sialyltransferase/GM3 synthase) in the adult mouse, we generated two antisera against mouse ST3GalV that were designated CS2 (directed against amino acids K227-I272) and CS14 (directed against amino acids D308-H359). We previously reported that CS2 antiserum stains medial and trans-Golgi cisternae in all cell types investigated. In neural tissue, however, CS14 antiserum reveals a subpopulation of ST3GalV with a subcellular distribution complementary to CS2 antiserum. CS14 antiserum strongly stains axons in cortical, cerebellar, brainstem, and spinal cord tissue sections. The subcellular localization of neuronal ST3GalV is maintained in primary cultures of rat hippocampal neurons and in PC12 cells. In PC12 cells, ST3GalV localization evolves during NGF-induced differentiation such that a pool of enzyme leaves the Golgi for a distal compartment in conjunction with neurite outgrowth. In PC12 cells transfected with an epitope-tagged form of ST3GalV, staining for the epitope tag coincides with expression of endogenous enzyme. The non-Golgi pool of ST3GalV does not colocalize with markers for the trans-Golgi network, endosome, or synaptic vesicles, nor is it detected on the cell surface. Distinct subpopulations of ST3GalV imply that ganglioside synthesis can occur outside of the Golgi or, alternatively, that a portion of the total ST3GalV pool subserves a nonenzymatic function. Significantly fewer transfected cells were found in PC12 cultures treated with plasmid encoding ST3GalV than in cultures treated with control plasmid, indicating that the expression of ST3GalV in excess of endogenous levels results in either cell death or a decreased rate of cell division.

Modification of sticholysin II hemolytic activity by free radicals.

Sticholysin II is a highly hemolytic toxin present in the caribbean sea anemone Stichodactyla helianthus. Pre-incubation of St II with 2,2'-azobis(2-amidinopropane), a source of peroxyl radicals in air saturated solution, readily reduces its hemolytic activity. Analysis of the amino acids present in the protein after its modification shows that only tryptophan groups are significantly modified by the free radicals. According to this, the loss of hemolytic activity correlates with the loss of the protein intrinsic fluorescence. The results indicate that, at high toxin concentrations, nearly a tryptophan residue and 0.2 toxin molecules are inactivated by each radical introduced into the system. Association of St II to multilamellar liposomes (egg yolk phosphatidyl choline:sphingomyelin 1:1) increases the toxin intrinsic fluorescence, indicating a more hydrophobic average environment of the five tryptophan groups of the protein. In agreement with this, incorporation of St II to the liposomes reduces the rate of fluorescence loss during its modification by free radicals, particularly at long incubation times. These results are explained in terms of two populations of tryptophans that are quenched at different rates by acrylamide and whose rates of inactivation by free radicals are also different.

Heat-stable antigen (CD24) as ligand for mouse P-selectin.

Heat-stable antigen (HSA)/CD24 is a cell surface molecule expressed by many cell types in the mouse. The molecule has an unusual structure because of its small protein core and extensive glycosylation. In order to study the functional role of the HSA-associated glycoconjugates we have isolated different forms of HSA. Using lectin analysis we provide evidence for extensive heterogeneity in carbohydrate composition and sialic acid linkage. Several HSA forms were recognized by mouse P-selectin-IgG but not E-selectin-IgG in ELISA. As expected, P-selectin-IgG also bound to L2/HNK-1-positive neural glycoproteins (L2-glycoproteins) and sulfatides but not to gangliosides and other control glycoproteins. The binding of P-selectin-IgG to L2-glycoproteins and HSA required bivalent cations. The reactivity to HSA was sensitive to sialidase treatment whereas the binding to L2-glycoproteins was not. Studies with alpha 2-6 sialytransferase indicated that alpha 2-6 linked sialic acid was not involved in the P-selectin binding to HSA. Surprisingly, an L2/HNK-1 specific antibody was found to cross-react with some HSA glycoforms and its binding correlated with P-selectin-IgG reactivity. L2/HNK-1-positive or L2/HNK-1-negative HSA glycoforms were also analyzed after coating to polystyrene beads. Only the L2/HNK-1-positive HSA coated beads were reactive with P-selectin-IgG and could bind to activated bend3 endothelioma cells expressing P-selectin whereas the L2/HNK-1-negative HSA beads did not. It is suggested that in its L2/HNK-1 modified form the HSA molecule on leukocytes could represent a ligand for P-selectin on endothelial cells or platelets.

Actividad de sialiltransferasa en la membrana espermática y su relación con fertilidad y esterilidad humanas / Sialyltransferase activity on spermatic membrane and its relationship with human fertility and sterility

La actividad de la sialiltransferasa (ST) fue determinada en los espermatozoides (spz) de sujetos normospérmicos (>80 X 10 6 spz/ml y mn[as del 75% de movilidad) y en los spz de pacientes con problemas de fecundidad (oligospérmicos < 20 y 10 6 spz/ml y menos del 20% de movilidad y astenospérmicos > 40 X 10 6 spz/ml y menos del 10% de movilidad). La actividad de la ST se cuantifica mediante la transferencia de radiactividad de CMP -3 H- siálico hacia el aceptor exógeno (fetuina desializada). El complejo enzima sustrato formado, al ser colocado en presencia del ácido fosfotúrgstico da un producto de fosfotungstato insoluble, el cual es retenido en un filtro de fibra de vidrio. La actividad enzimática disminuye en los spz de oligospérmicos en un 62 + - 5% con respecto a los spz de normospérmicos. La disminución de la actividad de la ST en los spz de pacientes infértiles permite suponer que esta enzima participa probablemente como causa directa de su patología y que su disminución obedece a un daño en la integridad estructural de la membrana espermática.(au)

Surfaces of murine lymphocyte subsets differ in sialylation states and antigen distribution of a major N-linked penultimate saccharide structure.

Rat liver beta-galactoside alpha-2,6-sialyltransferase and Vibrio cholerae sialidase were used with cytidine-5'-monophospho-N-acetyl-[3H]neuraminic acid (CMP-[3H]NeuAc) to specifically probe the distribution and sialylation state of Gal beta 1-4GlcNAc residues on N-linked saccharides on the surfaces of murine lymphocytes. The relative extent of exogenous sialyltransferase-mediated sialylation (per cellular protein) was thymocytes greater than T-cells greater than T-cell lymphoma (EL-4) greater than B-cells greater than B-cell lymphoma (AKTB-1b) greater than splenocytes. Prior desialylation increased exogenous resialylation by 23.8-, 13.1-, 7.1-, 7.9-, 7.0-, and 5.3-fold for splenocytes, B-cells, T-cells, EL-4, AKTB-1b, and thymocytes, respectively. Though numerous glycoproteins were labeled, the majority of the Gal beta 1-4GlcNAc residues were detected on a relatively small number of cell surface proteins, many of which are well-defined lymphocyte antigens. Gal beta 1-4GlcNAc residues on thymocytes were found to exist in an undersialylated state on T200 but not on other antigens (e.g., Thy-1). T200 was found to be fully sialylated on mature cells (i.e., hydrocortisone-resistant thymocytes and splenic T-cells), suggesting that its sialylation state is developmentally regulated. These studies indicate that the number, sialylation state, and polypeptide distribution of the penultimate structure, Gal beta 1-4GlcNAc, differ on N-linked saccharides on the surfaces of different lymphocyte populations.

Influence of sialic acid on the binding activity of estrogen receptors.

The influence of sialidase and sialyltransferase on the binding of 3H-estradiol to estrogen receptors in baboon uterus was investigated to ascertain if sialylation was involved. Specific binding capacity increased approximately 37% in the presence of sialidase, although Kd values essentially remained unchanged. 3H-Estradiol binding was correlated with free sialic acid in the presence of either sialidase or sialyltransferase. As sialidase concentrations were increased, 3H-estradiol binding and free sialic acid concentration increased linearly (r = 0.937, p less than 0.001). Incubation of 22 x 10(-5) U sialidase with its inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, decreased binding capacity and sialic acid concentration (r = 0.929, p less than 0.001). Although a decrease in binding capacity and free sialic acid concentration was observed in the presence of increasing amounts of sialyltransferase, a positive correlation was found between these two parameters (r = 0.839, p less than 0.035). A negative trend that was statistically insignificant was observed between binding capacity and sialic acid concentration when 2 x 10(-4) U sialyltransferase was incubated with the inhibitor, acetylsalicylic acid (r = -0.571, p = 0.195). The sialic acid concentration increased, while the 3H-estradiol binding capacity decreased. Collectively, these results show that both sialidase and sialyltransferase affect the binding of estradiol to its receptor in opposite directions. We suggest that biological activities of estrogen receptors in target cells may be regulated by the extent of sialylation of the receptor molecule itself. This posttranslational alteration may represent a new type of control mechanism for estrogen action.

Structure of the carbohydrate moiety of human interferon-beta secreted by a recombinant Chinese hamster ovary cell line.

The carbohydrate structure of the major oligosaccharide of human interferon-beta (IFN-beta) synthesized by a genetically engineered Chinese hamster ovary cell line has been determined. Analysis of the glycopeptidase F-released carbohydrates by sequential exoglycosidase treatment, methylation analysis, and fast atom bombardment-mass spectrometry revealed that 95% of the IFN-beta oligosaccharides had the following structure: (Formula: see text). The remaining 5% of the carbohydrates are probably tri- or higher antennary oligosaccharide chains. The major oligosaccharide of the recombinant IFN-beta is remarkably homogeneous with respect to terminal galactose sialylation. NeuAc, which is alpha 2-3-linked to galactose in the human IFN-beta secreted by Chinese hamster ovary cells, can be re-incorporated with an alpha 2-6 linkage in vitro, into enzymatically desialylated IFN-beta using rat liver Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase. The sugar chain is important for maintaining protein solubility as shown by the fact that IFN-beta protein precipitates after deglycosylation with glycopeptidase F.

Sialylation of glycoprotein oligosaccharides with N-acetyl-, N-glycolyl-, and N-O-diacetylneuraminic acids.

Four common sialic acids (Sia), NeuAc, N-glycolyl-neuraminic acid (NeuGc), 4-O-acetyl-N-acetylneuraminic acid (4-O-Ac-NeuAc), and 9-O-Ac-NeuAc were examined for activation to their corresponding CMP-sialic acid conjugates and subsequently for their transfer to glycoprotein oligosaccharides by purified mammalian sialyltransferases. CMP-sialic acid synthetases from calf brain and from bovine and equine submaxillary glands were found to convert NeuAc, NeuGc, and 9-O-Ac-NeuAc to their corresponding CMP-sailic acids. In contrast, no conversion of 4-O-Ac-NeuAc to CMP-4-O-Ac-NeuAc was observed for any of the three synthetases examined. A new procedure for the preparation of CMP-9-O-Ac-NeuAc, CMP-NeuGc, and CMP-NeuAc in high yield and purity was developed, using the calf brain CMP-sialic acid synthetase. Each of these derivatives was tested as donor substrates for six mammalian sialyltransferases purified from porcine, rat, and bovine tissues, including a bovine GalNAc alpha 2,6 sialyltransferase whose purification is described in this report. The sialyltransferases examined represent those which form the Sia alpha 2,6Gal beta 1,4-GlcNAc-, Sia alpha 2,3Gal beta 1,3(4)GlcNAc-, Sia alpha 2,3Gal beta 1,3-GalNAc- and Sia alpha 2,6GalNAc- sequences found on N-linked and O-linked oligosaccharides of glycoproteins. CMP-NeuAc and CMP-NeuGc were equally good donor substrates for all six sialyltransferases. However, transfer of 9-O-Ac-NeuAc from CMP-9-O-Ac-NeuAc varied from only 10% to nearly 70% that of the transfer of NeuAc from CMP-NeuAc. Results are viewed to define the relative roles of direct transfer of these sialic acids and modification of glycosidically bound NeuAc in glycoproteins.