Sialyltransferases as specific cell surface probes of terminal and penultimate saccharide structures on living cells

Rat liver beta-galactoside alpha-2,6-sialyltransferase and Vibrio cholerae sialidase were used, in conjunction with CMP-N-acetyl-[3H]neuraminic acid, to probe the glycoconjugate distribution, sialylation state, and level of penultimate Gal beta 1-4GlcNAc residues on the surfaces of murine thymic lymphocytes. We report a detailed characterization of this sialyltransferase-mediated labeling system. Exogenous sialylation of intact cells is dependent on transferase, sugar nucleotide donor, cell number, and incubation time. Additionally, we have demonstrated that the system labeling the cell surface is noncytotoxic and nonmetabolic and is interacting with the entire cell population. Analysis of the exosialylated structures indicates that the sialyltransferase specifically produces an alpha 2-6 linkage on N-linked oligosaccharides. Using this labeling system, we have probed the cell surface saccharide structures of murine thymocytes and demonstrated that most Gal beta 1-4GlcNAc residues are sialylated in the native state. However, one antigen, T200 (Ly-5), is strikingly undersialylated when compared to other cell surface glycoproteins (e.g., Thy 1.2). Upon analysis of exogenously sialylated oligosaccharides, labeled sialic acid was found almost exclusively on monosialylated structures with the remainder on bisialylated oligosaccharides. This suggests that the purified sialyltransferase is very precise in its recognition of oligosaccharides present on the surface of living thymic lymphocytes. This paper illustrates the combined uses of specific glycosidases and glycosyltransferases and how they can be employed in the detailed study of selected cell surface saccharide structures on living nucleated cells.     

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.     

Branch specificity of bovine colostrum CMP-sialic acid: Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase. Sialylation of bi-, tri-, and tetraantennary oligosaccharides and glycopeptides of the N-acetyllactosamine type

Using 500-MHz 1H NMR spectroscopy we have investigated the branch specificity that bovine colostrum CMP-NeuAc:Gal beta 1----4GlcNAc-R alpha 2----6-sialyltransferase shows in its sialylation of bi-, tri-, and tetraantennary glycopeptides and oligosaccharides of the N-acetyllactosamine type. The enzyme appears to highly prefer the galactose residue at the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch for attachment of the 1st mol of sialic acid in all the acceptors tested. The 2nd mol of sialic acid becomes linked mainly to the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6 branch in bi- and triantennary substrates, but this reaction invariably proceeds at a much lower rate. Under the conditions employed, the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch is extremely resistant to alpha 2----6-sialylation. A higher degree of branching of the acceptors leads to a decrease in the rate of sialylation. In particular, the presence of the Gal beta 1----4GlcNAc beta 1----6Man alpha 1----6 branch strongly inhibits the rate of transfer of both the 1st and the 2nd mol of sialic acid. In addition, it directs the incorporation of the 2nd mol into tetraantennary structures toward the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch. In contrast, the presence of the Gal beta 1----4GlcNAc beta 1----4Man alpha 1----3 branch has only minor effects on the rates of sialylation and, consequently, on the branch preference of sialic acid attachment. Results obtained with partial structures of tetraantennary acceptors indicate that the Man beta 1----4GlcNAc part of the core is essential for the expression of branch specificity of the sialyltransferase. The sialylation patterns observed in vivo in glycoproteins of different origin are consistent with the in vitro preference of alpha 2----6-sialyltransferase for the Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3 branch. Our findings suggest that the terminal structures of branched glycans of the N-acetyllactosamine type are the result of the complementary branch specificity of the various glycosyltransferases that are specific for the acceptor sequence Gal beta 1----4GlcNAc-R.     

Cell surface sialic acid influences tumor cell recognition in the mixed lymphocyte reaction

The Ia+ B cell lymphoma, AKTB-1b, fails to stimulate thymic lymphocytes in a one-way mixed lymphocyte reaction unless pretreated with sialidase or inhibitors of N-linked oligosaccharide processing. A comparison of different sialidases and sialyltransferases suggests that the removal of only a subset of total surface sialic acid, rather than net desialylation of the cell surface, is required. Three sialidases were compared, including Vibrio cholerae (VC) and Clostridium perfringens (CP), which will cleave alpha 2-3, alpha 2-6, and alpha 2-8, sialic acid linkages, and Newcastle Disease virus (NDV), which will remove only alpha 2-3 and alpha 2-8 linked sialic acid. When treated with equivalent units of sialidase, CP-, VC-, and NDV-treated cells were 24-fold, sixfold, and threefold better stimulators than untreated cells. In contrast, VC released 1.3-fold and 2.5-fold more sialic acid per cell than did CP or NDV, respectively. Furthermore, VC was superior in reducing the levels of binding of the sialic acid-specific lectin, Limulus polyphemus agglutinin, in exposing Gal beta 1-3GalNAc and Gal beta 1-4GlcNAc residues, and in desialylating gangliosides. Two-dimensional gel analysis indicated that VC and CP were both equal and superior to NDV in the desialylation of iodinatable cell-surface proteins, including H-2Kk, I-A beta k, and a highly sialylated 65,000 dalton protein of unknown identity. Maximal resialylation of CP-treated cells with exogenously added CMP-NANA and either the alpha 2-3(Gal beta 1-3GalNAc) or alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase did not reduce the stimulatory capacity of these cells. However, resialylation of VC-treated cells with just CMP-NANA alone resulted in 49% reversal of their stimulatory capacity, and no additional reversal could be achieved with either of the sialyltransferases. Although the alpha 2-6(Gal beta 1-4GlcNAc) sialyltransferase was capable of adding back approximately 10% of the sialic acid removed, the endogenous activity added back approximately 0.1% of the total sialic acid removed. SDS-PAGE gels of the sialylated cells indicated that the exogenously added sialyltransferase labeled many different proteins, whereas the endogenous activity labeled far fewer proteins, predominantly in 46,000 and 25,000 m.w. range. Both the desialylation and resialylation data suggest that the sialidase-dependent stimulation is due to the desialylation of specific membrane structures. Together with previous studies, these data suggest that the sialic acids involved are probably alpha 2-6 linked to N-linked glycosyl moieties.     

Differential sialylation of cell surface glycoconjugates in a human B lymphoma cell line regulates susceptibility for CD95 (APO-1/Fas)-mediated apoptosis and for infection by a lymphotropic virus.

Sialic acid, as a terminal saccharide residue on cell surface glycoconjugates, plays an important role in a variety of biological processes. In this study, we investigated subclones of the human B lymphoma cell line BJA-B for differences in the glycosylation of cell surface glycoconjugates, and studied the functional implications of such differences. With respect to the expression level of most of the tested B cell-associated antigens, as well as the presence of penultimate saccharide moieties on oligosaccharide chains, subclones were phenotypically indistinguishable. Marked differences among subclones, however, were found in the overall level of glycoconjugate sialylation, involving both alpha-2,6 and alpha-2,3-linked sialic acid residues. Accordingly, subclones were classified as highly- (group I) or hyposialylated (group II). The function of two sialic acid-dependent receptor-mediated processes is correlated with the sialylation status of BJA-B subclones. Susceptibility to and binding of the B lymphotropic papovavirus (LPV) was dependent on a high sialylation status of host cells, suggesting that differential sialylation in BJA-B cells can modulate LPV infection via its alpha-2,6-sialylated glycoprotein receptor. CD95-mediated apoptosis, induced by either the human CD95 ligand or a cytotoxic anti-CD95 monoclonal antibody, was drastically enhanced in hyposialylated group II cells. An increase in endogenous sialylation may be one antiapoptotic mechanism that converts tumor cells to a more malignant phenotype. To our knowledge, this is the first report demonstrating that differential sialylation in a clonal cell line may regulate the function of virus and signal-transducing receptors.     

Stimulation of sialyltransferase activity of melanoma cells by retinoic acid

Retinoic acid (RA) treatment of murine S91-C2 melanoma cells has been found to augment the activity of glycoprotein: sialyltransferase in a dose-dependent and time-dependent process. The enzymatic activity in cells treated with 10 microM RA reached a maximal level, 3-fold higher than in untreated cells, 72 h after initiation of treatment. In contrast, the addition of RA directly into the reaction mixture had no stimulatory effect on sialyltransferase. The endogenous glycoproteins to which sialic acid is transferred from cytidine monophosphate (CMP)-[14C] sialic acid by the action of sialyltransferase have been identified by fluorography after polyacrylamide gel electrophoresis. One of these acceptors, a glycoprotein of Mr 160 000, comigrated in gel electrophoresis with a cell surface sialoglycoprotein that can be labeled by the periodate-tritiated borohydrate procedure more intensely on intact RA-treated than on untreated cells. Removal of sialic acid residues exposed on the surface of either control or RA-treated cells enhanced 2- to 3-fold the transfer of sialic acid to endogenous acceptors. These results suggest that the increased sialyltransferase activity in RA-treated melanoma cells may be responsible for the enhanced sialylation of certain cell surface glycoproteins. RA treatment of several other tumor cell lines also resulted in stimulation of sialyltransferase activity indicating that this effect of RA is not limited to the S91-C2 melanoma cells.     

A specific cell surface glycoconjugate controlling cell motility: evidence by functional monoclonal antibodies that inhibit cell motility and tumor cell metastasis

The biochemical basis of cell motility has been viewed as a complex process involving cell surface membrane proteins, integrin receptors, growth factors and their receptors, and cytoskeletal components [Rosen & Goldberg (1989) In Vitro 25, 1079]. The possible involvement of glycoconjugates at the cell surface in controlling cell motility has not been systematically investigated. We addressed this question using functional monoclonal antibodies (MAbs), which inhibit cell motility and the metastatic potential of tumor cells, as probes. Two such MAbs, derived from two independent processes of immunization and selection, were found to directed to a common specific carbohydrate structure, Fuc alpha 1----2Gal beta 1----R. MAb MIA-15-5 was established after immunization of mice with small cell lung carcinoma line PC7 and selected on the basis of inhibition of U937 and HEL cell migration. MAb MIA-22-20 was established after immunization with lung adenocarcinoma line MAC-10 and selected on the basis of inhibition of MAC-10 cell migration. These two MAbs were both IgM and were consistently reactive with the Fuc alpha 1----2Gal beta 1----R structure, regardless of the identity of the R group. Various other anti-H MAbs, specific to carrier isotype, did not affect cell motility. MAb MIA-15-5 reacted with 30-40% of high-metastatic variant BL6 of mouse melanoma B16 line but with only less than 5% of low-metastatic variant F1. Metastatic deposition to lung after injection of BL6 cells was inhibited if MAb MIA-15-5 was injected within 3 h but was not inhibited by injection of other anti-H antibodies under the same conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Soyasaponin I decreases the expression of alpha2,3-linked sialic acid on the cell surface and suppresses the metastatic potential of B16F10 melanoma cells

The transfer of sialic acids to the non-reducing terminal positions on sugar chains of glycoconjugates is catalyzed by sialyltransferases (STs). Increased sialylation is correlated with oncogenic transformation and metastatic potential. ST inhibitors may be potentially valuable as anti-cancer and anti-metastatic agents. In this study, we evaluated the effects of soyasaponin I (Ssa I), a known inhibitor of STs, on tumor metastasis through studying a highly metastatic cancer cell line B16F10. Ssa I specifically inhibited the expression of alpha2,3-linked sialic acids without affecting other glycans on the B16F10 cell surface. We also found that Ssa I decreased the migratory ability of cells, enhanced cell adhesion to extracellular matrix proteins. Finally, a pulmonary metastasis assay demonstrated that alteration of glycosylation in this way significantly reduced the ability of tumor cells to distribute to the lungs of mice. Collectively, these findings suggested that alpha2,3-linked sialic acids may play an important role in metastasis potential of B16F10 cells.     

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.     

Biosynthesis of the O-glycosidically linked oligosaccharide chains of fetuin. Indications for an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase with a narrow acceptor specificity in fetal calf liver

Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.     

The generation and characterization of a rat neural cell line overexpressing the α2,6(N) sialyltransferase

In order to examine the effects of altered protein sialylation on neural cell function, B104 rat neuroblastoma cells were stably transfected with the cDNA coding for 2,6(N) sialyltransferase (ST(6)N). Lectin blot analysis of the clones demonstrated an increase in staining of the Sambucus nigra lectin, which detects 2,6 linked sialic acid, in parallel with enzyme activity. There was a concomitant decrease in staining by the Maackia amurensis lectin which labels 2,3-linked sialic acid, indicating that the individual sialyltransferase enzymes may compete for penultimate galactose acceptor sites. While there was an initial increase in protein-bound sialic acid in parallel with enzyme activity, the sialylation of the cells was demonstrated to be saturable. There was an inverse relationship between cell adhesion to a fibronectin substrate and ST(6)N activity suggesting that the negatively charged sugar acts to modulate cell-substrate interaction. These cells will provide an ideal model system with which to further investigate the effect of altered sialic acid on neural cell function.     

Specificity of ovine submaxillary-gland sialyltransferases. Application of high-pressure liquid chromatography in the identification of sialo-oligosaccharide products.

High-pressure liquid chromatography was used to identify the sialo-oligosaccharide products obtained after sialylation of [14C]Gal-GalNAc-protein in vitro by an ovine submaxillary-gland microsomal fraction. Among other products, two isomeric trisaccharides could be identified. NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol and Gal beta 1 leads to 3-(NeuAc alpha 2 leads to 6)GalNAcol respectively, indicating that ovine submaxillary gland contains two sialyltransferases acting on mucin-type acceptors, a beta-galactoside alpha 2 leads to 3 sialyltransferase and a N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase. This conclusion was fully supported by methylation analysis of the two trisaccharide products.     

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.     

Engineering the sialic acid in organs of mice using N-propanoylmannosamine

Sialic acids play an important role during development, regeneration and pathogenesis. The precursor of most physiological sialic acids, such as N-acetylneuraminic acid is N-acetyl-D-mannosamine. Application of the novel N-propanoylmannosamine leads to the incorporation of the new sialic acid N-propanoylneuraminic acid into cell surface glycoconjugates. Here we analyzed the modified sialylation of several organs with N-propanoylneuraminic acid in mice. By using peracetylated N-propanoylmannosamine, we were able to replace in vivo between 1% (brain) and 68% (heart) of physiological sialic acids by N-propanoylneuraminic acid. The possibility to modify cell surfaces with engineered sialic acids in vivo offers the opportunity to target therapeutic agents to sites of high sialic acid concentration in a variety of tumors. Furthermore, we demonstrated that application of N-propanoylmannosamine leads to a decrease in the polysialylation of the neural cell adhesion molecule in vivo, which is a marker of poor prognosis for some tumors with high metastatic potential.     

The evolution of galactose α2,3-sialyltransferase: <Emphasis Type="Italic">Cionaintestinalis</Emphasis> ST3GAL I/II and <Emphasis Type="Italic">Takifugu rubripes</Emphasis> ST3GAL II sialylate Galβ1,3GalNAc structures on glycoproteins but not glycolipids

Sialyltransferases are a family of enzymes catalyzing the transfer of sialic acid residues to terminal non-reducing positions of oligosaccharide chains of glycoproteins and glycolipids. Although expression of sialic acid is well documented in animals of the deuterostomian lineage, sialyltransferases have been predominantly described for relatively recent vertebrate lineages such as birds and mammals. This study outlines the characterization of the only sialyltransferase gene found in the tunicate Ciona intestinalis, the first such report of a non-vertebrate deuterostomian sialyltransferase, which has been discussed as a possible orthologue of the common ancestor of galactose α2,3-sialyltransferases. We also report for the first time the characterization of a ST3Gal II gene from the bony fish Takifugu rubripes. We demonstrate that both genes encode functional α2,3-sialyltransferases that are structurally and functionally related to the ST3Gal family of mammalian sialyltransferases. However, characterization of the recombinant, purified forms of both enzymes reveal novel acceptor substrate specificities, with sialylation of the disaccharide Galβ1-3GalNAc and asialofetuin, but not GM1 or GD1b observed. This is in contrast to the mammalian ST3Gal II that predominantly sialylates gangliosides. Taken together the ceramide binding/recognition site previously proposed for the mouse ST3Gal II might represent a unique feature of mammalian ST3Gal II that is missing in the evolutionary more distant fish and tunicate species reported here. This suggests that during the evolution of the ST3Gal II, probably following the separation of the teleosts, a significant shift in substrate specificity enabling the sialylation of gangliosides took place.     

Cell adhesion in a dynamic flow system as compared to static system. Glycosphingolipid-glycosphingolipid interaction in the dynamic system predominates over lectin- or integrin-based mechanisms in adhesion of B16 melanoma cells to non-activated endothelial cells.

Initial adhesion of B16 melanoma variants to non-activated endothelial cells is mediated through specific interaction between GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----Cer) expressed on melanoma cells and lactosylceramide (LacCer, Gal beta 1----4Glc beta 1----Cer) expressed on endothelial cells. This adhesion is predominant over integrin- or lectin-mediated adhesion in a dynamic flow experimental system employing a parallel plate laminar flow chamber (Lawrence, M. B., Smith, C. W., Eskin, S. G., and McIntire, L. V. (1990) Blood 75, 227-237). In this system, a tumor cell suspension flows over a glass plate coated with glycosphingolipid, lectin, or fibronectin, and adhesion is recorded on videotape. These conditions were designed to mimic the microvascular environment in which tumor metastatic deposition takes place. In contrast, lectin- and fibronectin-based mechanisms are predominant in previously used static adhesion systems. Under static conditions, the relative degree of adhesion of the four B16 variants to endothelial cells or to LacCer-coated plates was the same as their relative degree of GM3 expression (i.e. BL6 approximately F10 greater than F1 greater than WA4), and adhesion was inhibited in the presence of methyl-beta-lactoside, or liposomes containing LacCer or GM3. Adhesion was also inhibited by pretreatment of B16 cells with anti-GM3 antibody DH2 or sialidase and by pretreatment of endothelial cells with anti-LacCer antibody T5A7. Under dynamic flow conditions, WA4 cells did not adhere to mouse endothelial cells at high shear stress (greater than 2.5 dynes/cm2) but did adhere at lower shear stress. In contrast, BL6 and F10 cells adhered strongly at both low and high shear stress. BL6 cell adhesion to endothelial cells at both low and high shear stress was inhibited in the presence of antibody DH2, ethyl-beta-lactoside, or lactose, as well as by pretreatment of BL6 cells with sialidase. Thus, some clear differences, as well as similarities, in cell adhesion under static versus dynamic conditions are demonstrated. These findings suggest that melanoma cell adhesion to endothelial cells, based on GM3/LacCer interaction, initiates metastatic deposition, which may trigger a series of "cascade" reactions leading to activation of endothelial cells and expression of Ig family or selectin receptors, thereby promoting adhesion and migration of tumor cells.     

Protein sialylation by sialyltransferase involves radiation resistance

Previously, we identified beta-galactoside alpha(2,6)-sialyltransferase (ST6Gal I) as a candidate biomarker for ionizing radiation. The expression of ST6Gal I and the level of protein sialylation increased following radiation exposure in a dose-dependent manner. Radiation induced ST6Gal I cleavage and the cleaved form of ST6Gal I was soluble and secreted. Sialylation of integrin beta1, a glycosylated cell surface protein, was stimulated by radiation exposure and this increased its stability. Overexpression of ST6Gal I in SW480 colon cancer cells that initially showed a low level of ST6Gal I expression increased the sialylation of integrin beta1 and also increased the stability of the protein. Inhibition of sialylation by transfection with neuraminidase 2 or neuraminidase 3 or by treatment with short interfering RNA targeting ST6Gal I reversed the effects of ST6Gal I overexpression. In addition, ST6Gal I overexpression increased clonogenic survival following radiation exposure and reduced radiation-induced cell death and caspase 3 activation. However, removal of sialic acids by neuraminidase 2 or knockdown of expression by short interfering RNA targeting ST6Gal I restored radiation-induced cell death phenotypes. In conclusion, radiation exposure was found to increase the sialylation of glycoproteins such as integrin beta1 by inducing the expression of ST6Gal I, and increased protein sialylation contributed to cellular radiation resistance.     

小鼠肝癌及肝正常组织B4GalT糖基转移酶家族mRNA表达差异及其对细胞膜相关糖链的影响

探讨肝癌模型鼠与正常小鼠肝组织B4GalT(β-1,4-半乳糖转移酶)家族mRNA表达差异以及对细胞膜相关糖链的影响.采用RT-PCR方法检测肝癌模型鼠和正常对照小鼠肝癌组织中B4GalT家族7个成员以及唾液酸α-2,3转移酶ST3GalⅢ、ST3GalⅣ、ST3GalⅥ、α-1,6-岩藻糖转移酶FUT8 mRNA表达差异,应用凝集素芯片检测细胞膜表面半乳糖、岩藻糖、唾液酸表达情况.结果显示:与正常对照组相比,肝癌模型鼠肝组织中B4GalT-1和B4GalT-3、ST3GalⅣ和ST3GalⅥ、FUT8呈现高表达,肝癌细胞膜半乳糖、岩藻糖、唾液酸类型糖链增加,提示B4GalT-1和B4GalT-3与肝癌细胞膜半乳糖链增加相关.由于细胞Galβ-1,4-GlcNAc糖表位在ST3GalⅢ、ST3GalⅣ或ST3GalⅥ催化下与唾液酸α-2,3连接生成s-lewis x抗原前体,本实验中B4GalT-1和B4GalT-3与ST3GalⅣ、ST3GalⅤ、FUT8 mRNA表达具有相关性,提示B4GalT-1和B4GalT-3可能与ST3GalⅣ、ST3GalⅥ以及FUT4协同作用,导致肝癌细胞膜半乳糖、岩藻糖、唾液酸类型糖链增加.     

Enhancement of sialyltransferase in two melanoma cell lines that are growth-inhibited by retinoic acid results in increased sialylation of different cell-surface glycoproteins

Previous studies have demonstrated the ability of retinoic acid (RA) to inhibit the growth of two spontaneous murine melanoma cell lines (B16-F1 and S91-C2) and to augment both sialyltransferase activity and the sialylation of an Mr 160,000 cell-surface glycoprotein. The present study examined the effects of RA on an ultraviolet irradiation-induced murine melanoma cell line K-1735P. Like the two spontaneous melanomas, the uv-induced melanoma exhibited susceptibility to the growth-inhibitory action of RA. Both the anchorage-dependent and the anchorage-independent growths of the K-1735P cells were suppressed by RA, with IC50 values of 5 X 10(-9) and 3 X 10(-12) M, respectively. Sialyltransferase activity in both S91-C2 and K-1735P cells treated with 10(-6) or 10(-5) M RA increased two- and three-fold, respectively, as compared with untreated cells. In contrast, cell-surface sialo- and galactoglycoproteins, revealed by labeling with periodate and tritiated borohydrate or with neuraminidase, galactose oxidase, and tritiated borohydrate, respectively, varied between the S91-C2 and the K-1735P cells, and each cell line's modulation by RA was also distinct. These findings suggest that although RA can increase the activity of sialyltransferase in different melanoma cells, this increased activity may, in turn, result in an increased sialylation of distinct cell-surface glycoproteins.     

To sialylate,or not to sialylate: that is the question

Most oropharyngeal pathogens express sialic acid units on their surfaces, mimicking the sialyl-rich mucin layer coating epithelial cells and the glycoconjugates present on virtually all host cell surfaces and serum proteins. Unlike the host's cells, which synthesize sialic acids endogenously, several microbial pathogens use truncated sialylation pathways. How microorganisms regulate sialic acid metabolism to ensure an adequate supply of free sugar for surface remodeling is a new area of research interest to basic scientists and those focused on the clinical outcome of the host-pathogen interaction.