Glycosyl transferases of baby-hamster-kidney (BHK) cells and ricin-resistant mutants. N-glycan biosynthesis

Five cell lines of ricin-resistant BHK cells have been assayed for gross carbohydrate analysis of cellular glycoproteins, for the activities of several glycosidases and of specific glycosyl transferases active in assembly of N-glycans of glycoproteins. The latter enzymes include sialyl transferase using asialofetuin as glycosyl acceptor, fucosyl transferases using asialofetuin and asialoagalactofetuin acceptors, galactosyl transferases using ovalbumin, ovomucoid and N-acetylglucosamine as acceptors and N-acetylglucosaminyl transferases using ovalbumin and glycopeptides as acceptors. Cell line RicR14, binding less ricin than normal BHK cells, contains reduced amounts of sialic acid, galactose and N-acetylglucosamine in cellular glycoproteins and lacks almost completely N-acetylglucosamine transferase I, an essential enzyme in assembly of ricin-binding carbohydrate sequences of N-glycans. These cells also contain reduced levels of N-acetylglucosamine transferase II active on a product of N-acetylglucosamine transferase I action. Sialyl transferase activity is severely depressed while fucose-(alpha 1 leads to 6)-N-acetylglucosamine fucosyl transferase activity is increased. Cell lines RicR15, 17, 19 and 21 showed partial deficiencies in galactosyl and N-acetylglucosaminyl transferases. A hypothesis is put forward to account for the different carbohydrate compositions and ricin binding properties of glycoproteins synthesised by these cells in terms of the determined enzyme defects, the normal level of sialyl transferases detected in RicR15 and RicR21 cells and the elevated levels of sialyl and fucosyl transferases detected in RicR17 and 19 cells. None of the above changes in glycosyl transfer reactions in the RicR cell lines are due to enhanced glycosidase or sugar nucleotidase activities in the mutant cells.     

Some factors affecting the production, by cultured baby-hamster kidney cells, of BHK glycoprotein I which cross-reacts immunologically with Tamm-Horsfall glycoprotein.

Cultured baby-hamster kidney cells (BHK-21/C13), which are adapted to grow in suspension (strain 2P), roduce a glycoprotein, termed BHK glycoprotein I, which cross-reacts immunologically with hamster urinary (Tamm-Horsfall glycoprotein. BHK glycoprotein I was isolated in an electrophoretically (sodium dodecyl sulphate/polyacrylamide gel) homogeneous form by application of affinity chromatography to the medium in which cells had been cultured. Insolubilized anti-(Tamm-Horsfall glycoprotein immunoglobulin G) was used as the adsorbent. The amount of BHK glycoprotein I associated with the cultured cells was found by both radioimmunoassay and immunofluorescence to be related to the amount of Ca2+ in the medium and to the particular stage of the cell cycle. 5'-Nucleotidase was also shed by the cells into the culture medium in amounts related to the stage of the cell cycle. The turnover of hamster Tamm-Horsfall glycoprotein in vivo appeared to be considerably more rapid than can be accounted for by cell turnover. Hamster Tamm-Horsfall glycoprotein was shown to be ineffective in inhibiting agglutination of chicken erythrocytes caused by influenza virus.     

Analysis by lectin affinity chromatography of N-linked glycans of BHK cells and ricin-resistant mutants.

Normal baby hamster kidney (BHK) fibroblasts and ricin-resistant (RicR) mutants of BHK cells derived from them were labelled metabolically with [3H]mannose or [3H]fucose. Glycopeptides obtained by digestion of disrupted cells with Pronase were separated by affinity chromatography on concanavalin A-Sepharose. In the normal BHK cells major glycopeptide fractions were obtained consisting of tetra- and tri-antennary sialylated complex glycans, bi-antennary sialylated glycans, and neutral oligomannosidic chains. The majority of bi-antennary chains were shown to contain a fucosyl-(alpha 1-6)-N-acetylglucosaminyl sequence in the core region by their ability to bind to a lentil lectin affinity column. All of the mutant cell lines examined were found to accumulate oligomannosidic glycans in cellular glycoproteins: complex sialylated glycans were either absent or greatly reduced in amount. Analysis of fractions isolated from concanavalin A-Sepharose by Bio-Gel P-4 chromatography and glycosidase degradation indicated that the glycans accumulating in RicR14 cells have the general structure: (formula; see text) and derivatives having fewer alpha-mannosyl units. We have also analysed the glycopeptides released by trypsin treatment from the surface of the normal and mutant cells, as well as those obtained by proteolysis of fibronectin isolated from the medium. The glycopeptide profiles of the cell-surface-derived material and of fibronectin showed for the mutant cells a marked accumulation of oligomannosidic chains at the expense of complex oligosaccharide chains. Hence, the alterations in glycan structure detected in bulk cellular glycoproteins of RicR cells are expressed also in cell surface glycoproteins and in fibronectin, a secreted glycoprotein.     

Asparagine-linked oligosaccharides of BHK cells treated with inhibitors of oligosaccharide processing.

Baby-hamster kidney (BHK) cells were labelled with [2-3H]mannose for 1-2 days in media containing 1-deoxynojirimycin, N-methyl-1-deoxynojirimycin or 1-deoxymannojirimycin. Glycopeptides obtained by Pronase digestion of disrupted cells were analysed by lectin affinity chromatography, by Bio-Gel P4 gel filtration and by paper chromatography of oligosaccharides released by endo-beta-N-acetylglucosaminidase H. Biosynthesis of complex-type oligosaccharides was diminished but not abolished, the greatest effect being obtained by continuous culture of cells with 1-deoxymannojirimycin. Under these conditions cells contained only 20-30% of the concentration of complex-type chains found in control cells and correspondingly increased amounts of oligomannose-type chains. Similar concentrations of asparagine-linked Man6-GlcNAc2 and Man5GlcNAc2 were present in 1-deoxymannojirimycin-treated cells and control cells, indicating that the inhibition of complex-type chain formation was not related simply to an inability of inhibitor-treated cells to carry out extensive mannosidase-catalysed processing. N-Methyl-1-deoxynojirimycin induced accumulation of oligomannose-type chains containing three glucose residues, and cells treated with 1-deoxynojirimycin contained oligosaccharides with one to three glucose residues. Cells cultured in the presence of the inhibitors retained sensitivity towards the galactose-binding lectins ricin and modeccin.     

Assembly of asparagine-linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases

We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases.     

Swainsonine, an inhibitor of glycoprotein processing, enhances mitogen induced interleukin 2 production and receptor expression in human lymphocytes

Swainsonine, an inhibitor of mannosidase II, enhanced Con A induced lymphocyte IL-2 receptor expression, IL-2 production, and proliferation. Mitogen activated lymphocytes treated with swainsonine and subsequently restimulated with IL-2 showed a three-fold increase in proliferation. Castanospermine, 1-deoxynojirimycin, bromoconduritol and 1-deoxymannojirimycin, inhibitors of glucosidase 1, glucosidases 1 and II, glucosidase II, and mannosidase 1, respectively, did not exhibit any immunoenhancing activity. These results indicate that specific inhibition of mannosidase II during glycoprotein processing can enhance IL-2 mediated lymphocyte mitogenesis.     

Effect of swainsonine, an inhibitor of glycoprotein processing, on cultured mammalian cells

Swainsonine is an indolizidine alkaloid that inhibits glycoprotein processing by inhibiting mannosidase II. Thus, cells grown in the presence of this alkaloid exhibit a decreased amount of complex types of oligosaccharides at their cell surface, and instead have hybrid types of structures. Since this compound could be useful for studying functional roles of glycoproteins, it was important to determine whether it affected the growth of mammalian cells in culture, and whether it was cytotoxic to these cells. At levels of up to 1 μg/ml, swainsonine did not affect the growth rate of Madin-Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO), simian virus-181 (SV-101), B-16 melanoma, or intestine 407 cells, as measured by the increase in cell numbers over a 5-day period. There was also no apparent change in cell size or cell shape in cells grown in the presence of this inhibitor. Swainsonine also did not appear to be cytotoxic, nor to cause alterations in cell morphology, as evidenced by comparison of thin sections of normal and swainsonine-grown cells in the electron microscope. Since alterations in the oligosaccharide chains of cell surface glycoproteins could greatly affect cell surface properties, we examined the binding of various lectins and bacteria to cells grown in swainsonine as a measure of changes in their cell surface carbohydrates. Thus, when MDCK cells, CHO cells, or B-16 melanoma cells were grown for several days in the presence of swainsonine (100–500 ng/ml), these cells showed a 50–100% increase in their ability to bind [³H]concanavalin A, and a substantial decrease in the binding of [³H]wheat germ agglutinin. These alterations suggested an increase in high-mannose (or hybrid) types of receptors and a decrease in the complex types. The adhesion of E. coli B-886, a bacterium that binds to high-mannose glycoproteins, was also increased 1.5-to twofold, in cells grown in swainsonine. However, the binding of E. coli SS-142, another bacterial strain that does not bind to high-mannose receptors, was not altered by growth in swainsonine. In addition to the decrease in wheat germ agglutinin binding, another indication of a decrease in complex chains was the finding that CHO cells grown in swainsonine were more resistant to the toxic effects of the lectin, ricin. This increased resistance could be measured microscopically by the decrease in the number of cells remaining attached to the plates, or by the inhibition of amino acid incorporation, at various ricin concentrations. The effect of swainsonine on the incorporation of amino acids and sugars into protein was also examined. When MDCK cells were grown overnight in swainsonine (1 μg/ml), or were incubated in the alkaloid for several hours before the start of the experiment, there was no alteration in the incorporation of [³H]leucine or [³H]proline into protein. There was, however, a significant inhibition in the incorporation of [³H]fucose, [³H]glucosamine, and [³H]galactose caused by this alkaloid. Fucose incorporation was decreased by about 40%, glucosamine by about 40 or 50%, and galactose by about 50%. In many cases (but not all), the incorporation of mannose was enhanced about 20–30% in cells grown in swainsonine.     

An endogenous carbohydrate-binding agglutinin of BHK cells. Purification, specificity and interaction with normal and ricin-resistant cell lines

Several lectin-like activities were detected on the surface of unfixed, viable BHK cells by reaction with FITC-labelled glycosylated albumin derivatives. A prominent surface staining was obtained with the beta-lactosyl, beta-N-acetylgalactosaminyl, alpha-mannosyl and beta-N-acetylglucosaminyl derivatives. Endogenous lectin-like activities were also detected in BHK cell homogenates by haemagglutination of glutaraldehyde-fixed rabbit erythrocytes. Haemagglutinating activity was purified by chromatography of sodium deoxycholate-extracts of BHK cell microsomal fractions on Sepharose 4B and asialofetuin-Sepharose 4B. A purified agglutinin was eluted from the latter column with 0.2 M thiodigalactoside. The haemagglutination mediated by the purified factor was inhibited by thiodigalactoside, N-acetylgalactosamine, galactosyl-beta 1-4-N-acetylglucosamine and several glycoproteins. The purified agglutinin agglutinated trypsinised, fixed normal BHK cells more readily than several ricin-resistant cell lines. By contrast, a mannose-binding lectin from rabbit serum reacted equally well with normal and mutant cells. These results are in general agreement with models of cell-cell adhesion involving the interaction of surface located lectins with carbohydrate sequences of normal BHK cell surface glycoproteins.     

Comparison of N-acetylglucosaminyltransferase V activities in Rous sarcoma-transformed baby hamster kidney (RS-BHK) and BHK cells

Recent studies have demonstrated that Rous sarcoma virus-transformed baby hamster kidney (RS-BHK) cells express twofold higher levels of those N-linked oligosaccharides that contain the sequence [GlcNAc-beta(1,6)Man (1,6)] compared to nontransformed parental BHK cells (Pierce and Arango, J. Biol.Chem. 261, 10772 [1986]). We have investigated in RS-BHK and BHK cells the activity of UDP-GlcNAc:alpha-D-mannoside beta(1,6)N-acetylglucosaminyltransferase V, the enzyme that begins the synthesis of the sequence that is increased in the RS-BHK cells. We have measured GnT V activity using UDP-[3H]-GlcNAc and a synthetic oligosaccharide acceptor, GlcNAc beta(1,2)Man alpha(1,6)Man beta-O-(Ch2)8COOCH3, separating the radioactive product by a newly devised reverse-phase chromatographic technique. Assayed under optimal conditions, the specific activity of GnT V is about fourfold higher in RS-BHK sonicates than in BHK sonicates, suggesting that this increase in activity may be the primary mechanism that causes the increase in [GlcNAc beta(1,6)Man] sequences in the RS-BHK cells. The apparent Km values of the enzymes in RS-BHK and BHK cell sonicates for UDP-GlcNAc and the synthetic acceptor are similar, as are the pH optima. These results suggest that the increase in GnT V-specific activity in RS-BHK cells is not caused by the presence in these cells of a GnT V with markedly different kinetic properties.     

Swainsonine, an inhibitor of mannosidase II during glycoprotein processing, enhances concanavalin A-induced T cell proliferation and interleukin 2 receptor expression exclusively via the T cell receptor complex.

The T cell receptor (TCR) is a disulfide-linked heterodimer consisting of both complex and high-mannose types of N-linked oligosaccharides. The objective of the present investigation was to examine the effect of altered oligosaccharide structure on the expression and function of the TCR. Human mononuclear lymphocytes (MNL) were treated with castanospermine (CAST) or swainsonine (SW), inhibitors of glucosidase I or mannosidase II, respectively. Treatment with these inhibitors does not prevent glycosylation, but results in synthesis of glycoproteins with high-mannose or hybrid types of oligosaccharides. Treatment of MNL with CAST (1000-10 microM) or SW (100-1 microM) for up to 72 hr had no effect on cell surface expression of of the TCR. SW potentiated Con A-induced T cell proliferation without effecting anti-CD3 (OKT3) or alloantigen-induced proliferation. CAST had no effect on Con A, anti-CD3, or alloantigen-induced T cell proliferation. The T cell proliferative response to Con A in the presence of SW was completely eliminated in the presence of monoclonal anti-TCR antibodies. Monoclonal anti-CD2, -CD3, -CD4, -CD8, or isotypic control monoclonal antibodies had no effect on SW enhancement of T cell proliferation. SW treatment potentiated Con A-induced MNL expression of both the alpha and beta subunits of the IL 2R. This effect was also specifically blocked by anti-TCR monoclonal antibodies. These results demonstrate that selective changes in the glycosylation state of the TCR complex can alter mitogen recognition and subsequent cellular activation.     

Cell-surface mucosubstances from trypsin disaggregation of normal and virus-transformed lines of baby-hamster kidney cells (Short Communication)

Cell disaggregation by trypsin solubilizes significantly less mucosubstance from the surface of polyoma-virus-transformed baby-hamster kidney cells than from the same non-transformed cell line. The mucosubstance, which consists of both acid mucopolysaccharides and mucoproteins, also differs qualitatively in the two cell lines.     

Oligodendrocyte-myelin glycoprotein (OMgp) is an inhibitor of neurite outgrowth

A protein fraction purified from bovine brain myelin, previously called arretin because of its ability to inhibit neurite outgrowth, has been identified as consisting predominantly of oligodendrocyte-myelin glycoprotein (OMgp). We show that it is a potent inhibitor of neurite outgrowth from rat cerebellar granule and hippocampal cells; from dorsal root ganglion explants in which growth cone collapse was observed; from rat retinal ganglion neurons; and from NG108 and PC12 cells. OMgp purified by a different procedure from both mouse and human myelin behaves identically in all bioassays tested.     

Acetylation of spermidine and methylglyoxal bis(guanylhydrazone) in baby-hamster kidney cells (BHK-21/C13).

Treatment of BHK-21/C13 cells with methylglyoxal bis(guanylhydrazone) (MGBG) induced the cytosolic form of spermidine N1-acetyltransferase. It stabilized the enzyme against proteolytic degradation, but the drug did not affect the enzyme activity in vitro. MGBG was itself acetylated by BHK-21/C13 cells, but at only one-tenth the rate at which spermidine was acetylated. Acetylation occurred almost exclusively in the nuclear fraction. The product was identified as N-acetyl-MGBG by h.p.l.c., by using [3H]acetyl-CoA and [14C]MGBG as co-substrates. The results suggest that the acetylation of MGBG by BHK-21/C13 cells occurs by a different acetyltransferase enzyme from that which acetylates spermidine.     

Resynthesis of sphingomyelin from plasma-membrane phosphatidylcholine in BHK cells treated with Staphylococcus aureus sphingomyelinase.

About 60-65% of the total sphingomyelin in intact BHK cells is in a readily accessible pool which is rapidly degraded by Staphylococcus aureus sphingomyelinase. No more sphingomyelin is broken down in cells which have been fixed with glutaraldehyde or lysed with streptolysin O, suggesting that all the sphingomyelin which is available to the enzyme is on the cell surface. The inaccessible pool of sphingomyelin does not equilibrate with the plasma-membrane pool, even after prolonged incubation. Experiments using [3H]-choline show that much more phosphocholine is released from the intact cells treated with sphingomyelinase than can be accounted for by breakdown of the original cell-surface pool of sphingomyelin; the excess appears to be a consequence of the breakdown of sphingomyelin newly resynthesized at the expense of a pool of phosphatidylcholine which represents about 8% of total cell phosphatidylcholine and may reside in the plasma membrane. This would be consistent with resynthesis of cell-surface sphingomyelin by the phosphatidylcholine: ceramide phosphocholinetransferase pathway, which has previously been shown to be localized in the plasma membrane. However, in [3H]palmitate-labelled cells there appeared to be no accumulation of the diacylglycerol expected to be produced by this reaction, and no enhanced synthesis of phosphatidate or phosphatidylinositol; instead there was an increased synthesis of triacylglycerol. A similar increase in labelling of triacylglycerol was seen in enzyme-treated cells where the sphingomyelinase was subsequently removed, allowing resynthesis of sphingomyelin which occurred at a rate of about 25% of total sphingomyelin/h. Treatment of BHK cells with sphingomyelinase caused no change in the rates of fluid-phase endocytosis or exocytosis as measured with [3H]inulin.     

Localization of Tamm-Horsfall-glycoprotein-like immunoreactivity in cultured baby-hamster kidney cells, shown by immunofluorescence and by light- and electron-microscopic immunoperoxidase techniques.

Tamm-Horsfall glycoprotein was isolated from hamster urine, and antiserum against it was produced in rabbits. IgG was isolated from the antiserum. Immunocytochemical methods were used to localize Tamm-Horsfall-like immunoreactivity in three substrains of baby-hamster kidney (BHK) cells. Indirect immunofluorescence techniques showed that, in two substrains (BHK-21/C13/2P and BHK-21/C13/3P), a proportion of the cells fluoresced brilliantly, whereas those of the third substrain (BHK-21/ICRF) were totally negative. Related findings were obtained by the immunoperoxidase optical-microscopic technique. From the results of immunoperoxidase techniques using the electron microscope, it was concluded that the substance was present in association with the plasma membranes of the reacting cells. Our data suggest that the line of baby-hamster kidney cells, BHK-21/C13, may contain cells of renal-tubular epithelial origin, and that the proportion of these may be variable from one subculture to another.