Sulfation of chondroitin sulfate secreted by baby hamster kidney cells and their polyoma virus-transformed counterparts

Sulfation of glycosaminoglycans (GAGs) secreted by baby hamster kidney (BHK) cells and the polyoma virus-transformants (PY-BHK) was investigated. It has been reported that chondroitin sulfate (CS) of cell membranes from PY-BHK cells is undersulfated compared to that from BHK cells (Cancer Res. 43, 2712-2717, 1983). In the first series of experiments of the present study, cells were incubated with [3H]glucosamine and [35S]sulfate, and GAGs isolated from the culture medium were examined. GAG composition was comparable between the BHK and PY-BHK cultures. Disaccharide analysis of the chondroitinase ACII digests of the hyaluronate lyase-resistant materials showed a high proportion (68% for BHK and 47% for PY-BHK) of delta Di-0S, with delta Di-4S (32% for BHK and 53% for PY-BHK) as the major sulfated disaccharide on the basis of 3H-radioactivities. The beta-D-xyloside treatment did not alter the degree of undersulfation of the CS of either culture. In the second series of experiments, disaccharide analysis of the chondroitinase ABC digests of unlabeled GAGs demonstrated similar disaccharide composition for the two cell types. The BHK and PY-BHK preparations showed 28 and 17% (mol percent) of delta Di-0S, 58 and 72% of delta Di-4S, and 14 and 11% of delta Di-6S, respectively. These results indicate a considerable degree of undersulfation of secretory CS from both cells, and a slightly higher degree, if any, of under-sulfation of secretory CS from BHK cells if compared between the two cell types, which is in contrast to the results reported for membrane CS.     

Glycopeptides prepared from mouse cerebrum inhibit protein synthesis and cell division in baby hamster kidney cells, but not in their polyoma virus-transformed analogs

Glycopeptides isolated from mouse cerebral cortex cell surfaces (BCSG) were shown to inhibit cell growth and protein synthesis in baby hamster kidney (BHK)-21 cells, whereas polyoma virus-transformed BHK-21 cells (pyBHK-21) were refractory to the inhibitory activity of the glycopeptides. Growth inhibition was shown to be reversible and non-lethal to BHK-21 cells. Despite that difference in sensitivity to the action of the glycopeptides, both cell lines could bind the inhibitor in a saturable fashion and in similar quantities. After trypsinization, BHK-21 cells appeared refractory to the inhibitor, whereas pyBHK-21 cells became sensitive. The data suggested the presence of a receptor for BCSG on the cell surface of both cell lines. Incubating BCSG with conditioned medium from pyBHK-21 cells resulted in loss of the glycopeptide's inhibitory activity. In contrast, medium conditioned by BHK-21 cells had no effect on the inhibitory activity of BCSG. We hypothesize that the refractoriness of pyBHK-21 cells to BCSG is related to their autonomous growth characteristics and failure to respond to topo-inhibitory growth control. BCSG may be a naturally occurring growth regulator whose function can be explored by use of the BHK-21/ pyBHK-21 model system.     

Release of the glucose-regulated protein 94 by baby hamster kidney cells

Glucose-regulated protein 94 (grp94) is a major component of the endoplasmic reticulum (ER) lumen of eukaryotic cells. We showed that grp94 is released from baby hamster kidney (BHK-21) cells into a serum-free medium. The exit of grp94 into the medium was not related to the protein discharge due to cell death and was independent of de novo protein synthesis. The treatment of cells with brefeldin A and monensin, the inhibitors of the classical pathway of protein secretion, did not decrease the extracellular level of grp94, indicating that the discharge of grp94 from cells does not occur through the ER/Golgi-dependent pathway. Exosomes, membrane vesicles secreted by several cell types, were not involved in the release of grp94 from cells. Methyl-β-cyclodextrin, a substance that disrupts the lipid raft organization, considerably reduced the extracellular level of grp94, indicating that lipid rafts are involved in the liberation of grp94 from BHK-21 cells. The results suggest that BHK-21 cells release grp94 into the serum-free medium via the nonclassical secretory pathway in which lipid rafts play an important role. Copyright ? 2012 John Wiley & Sons, Ltd.     

A hydroxyproline-containing glycopeptide released from the walls of sycamore tissue-culture cells by hydrazinolysis

Degradation of the sycamore cell wall by hydrazinolysis yielded soluble glycopeptides. The peptide portion of the predominant glycopeptide was shown to be cyclic (4-trans-hydroxy-l-prolyl)-4-trans-hydroxy- l-proline. From this structure and the composition of the major glycopeptide it was deduced that only short oligosaccharides are attached to the hydroxyprolyl residues of the wall glycoprotein. Detailed evidence for the structure of the peptide is given in a supplementary paper that has been deposited as Supplementary Publication SUP 50020, at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.     

Mutator genes of baby hamster kidney cells.

Two mutator genes of mammalian cells were demonstrated. One was associated with the ribonucleoside diphosphate reductase, and the other was associated with an extreme adenosine sensitivity.     

Thermal stability of ribosomal ribonucleic acid from baby hamster kidney cells

1. RNA has been prepared from baby hamster kidney cells by extraction with a phenol–EDTA mixture and further purified by passing through a column of Sephadex G-25 that had been equilibrated with water. 2. Aging of the total RNA extracts at 4° or heating at 95° followed by rapid cooling caused a conversion of 28s RNA into material sedimenting in sucrose gradients at approx. 18s. 3. When heated RNA was re-extracted with phenol the sedimentation profile was not returned to that of the unheated RNA. 4. The 28s and 18s RNA fractions were collected separately from sucrose gradients by precipitation with 2vol. of ethanol and passed through a Sephadex G-25 column equilibrated with water. 5. Heat treatment of purified 28s RNA at 95° caused the sedimentation coefficient to increase to approx. 40s, whereas similar treatment of 18s RNA caused no significant increase. If the RNA was heated before the Sephadex G-25 treatment the sedimentation coefficient of the 28s and 18s RNA decreased to approx. 12s and 8s. 6. Heating mixtures of purified 28s and 18s RNA at 95° caused some aggregation of 18s material with the 28s fraction.     

Isolation and characterization of norleucine-resistant baby hamster kidney cells

Variants resistant to low and high levels of the methionine analogue norleucine were isolated in baby hamster kidney cells and in two clonal sublines, B1 and TG2. Clones resistant to high levels of norleucine were observed only after chemical mutagenesis, whereas clones capable of growing in low concentrations of norleucine occurred with equal frequency spontaneously and after mutagenesis. The variants were characterized with respect to uptake of ¹⁴C-norleucine and ¹⁴C-methionine. Five clones were found to be deficient in ¹⁴C-norleucine uptake, and of these, four showed reduced ¹⁴C-methionine uptake. The variants were tested also for increased activity of N₅-methyl-tetrahydrofolate: homocysteine methyltransferase, the enzyme which catalyses the terminal reaction in methionine biosynthesis. In four clones, higher levels of the methyltransferase were present than in the wild-type cells, suggesting overproduction or stabilization of this enzyme.     

Enzymatic basis for the structural changes of asparagine-linked sugar chains of membrane glycoproteins of baby hamster kidney cells induced by polyoma transformation

Previous studies indicated that enrichment of the GlcNAc beta 1----6Man alpha 1---- group with concomitant decrease of the GlcNAc beta 1----4Man alpha 1---- group occurs in the complex-type asparagine-linked sugar chains of the membrane glycoproteins of baby hamster kidney cells transformed by polyoma virus. The enzymatic basis of the chemical change is reported in this paper. By using oligosaccharides isolated from the urine of patients with a variety of exoglycosidase deficiencies, beta-N-acetylglucosaminyltransferases in the cell homogenate were successfully assayed separately. Both baby hamster kidney cells and their polyoma transformants contain beta-N-acetylglucosaminyltransferases I, II, IV, V, and VI, but not beta-N-acetylglucosaminyltransferase III. The beta-N-acetylglucosamine residue added by each beta-N-acetylglucosaminyltransferase (GnT) is shown below. (formula see text) Comparative studies of the specific activities of the five beta-N-acetylglucosaminyltransferases in the two cell lines revealed that the value of beta-N-acetylglucosaminyltransferase V in the polyoma transformant was twice of that in the normal cells, while those of the other four transferases in the two cell lines were not significantly different. Therefore the increase in beta-N-acetylglucosaminyltransferase V may be the direct cause of the changes found in the sugar chains of surface glycoproteins in baby hamster kidney cells transformed by polyoma virus.     

Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.     

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.     

The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes

The fluorescent probes pyrene, pyrene butyric acid and $\text{N-}\text{phenyl}$ 1-naphthylamine have been used to investigate the changes that accompany in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe $\text{N-}\text{phenyl}$ 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrne probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.     

Fibronectin-mediated binding and phagocytosis of polystyrene latex beads by baby hamster kidney cells

The binding and phagocytosis of fibronectin (pFN)-coated latex beads by baby hamster kidney (BHK) cells was studied as a function of fibronectin concentration and bead diameter. Cells were incubated with radioactive pFN-coated beads, and total bead binding (cell surface or ingested) was measured as total radioactivity associated with the cells. Of the bound beads, those that also were phagocytosed were distinguished by their insensitivity to release from the cells by trypsin treatment. In continuous incubations, binding of pFN-coated beads to cells occurred at 4 degrees C or 37 degrees C, but phagocytosis was observed only at 37 degrees C. In addition, degradation of 3H-pFN from ingested beads occurred at 37 degrees C, as shown by the release of trichloroacetic acid-soluble radioactivity into the incubation medium. When the fibronectin density on the beads was varied, binding at 4 degrees C and ingestion at 37 degrees C were found to have the same dose-response dependencies, which indicated that pFN densities that permitted bead binding were sufficient for phagocytosis to occur. The fibronectin density for maximal binding of ingestion was approximately 250 ng pFN/cm2. When various sized beads (0.085-1.091 micron), coated with similar densities of pFN, were incubated with cells at 4 degrees C, no variation in binding as a function of bead size was observed. Under these conditions, the absolute amount of pFN ranged from less than 100 molecules on the 0.085-micron beads to greater than 15,000 molecules on the 1.091-micron beads. Based upon these results it can be concluded that the critical parameter controlling fibronectin-mediated binding of latex beads by BHK cells is the spacing of the pFN molecules on the beads. Correspondingly, it can be suggested that the spacing between pFN receptors on the cell surface that is optimal for multivalent interactions to occur is approximately 18 nM. When phagocytosis of various sized beads was compared, it was found that the largest beads were phagocytosed slightly better (two fold) than the smallest beads. This occurred both in continuous incubations of cells with beads and when the beads were prebound to the cells. Finally, the kinetic constants for the binding of 0.085 microM pFN-coated beads to the cells were analyzed. There appeared to be approximately 62,000 binding sites and the KD was 4.03 X 10(-9) M. Assuming a bivalent interaction, it was calculated that BHK cells have approximately 120,000 pFN receptors/cell and the binding affinity between pFN and its receptor is approximately 6 X 10(-5) M.     

Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a K_m value for dCMP of 0.91mm. dCTP activation required the presence of Mg²⁺ or Mn²⁺ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg²⁺ or Mn²⁺ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).     

Functional integrins from normal and glycosylation-deficient baby hamster kidney cells. Terminal processing of asparagine-linked oligosaccharides is not correlated with fibronectin-binding activity.

We have examined the properties of the alpha 5 beta 1 integrin of baby hamster kidney (BHK) cells, a ricin-resistant variant Ric14 lacking N-acetylglucosaminyl transferase I, and hence unable to complete assembly of hybrid- or complex-type N-glycans, and BHK cells treated with 1-deoxymannojirimycin (dMM), an inhibitor of Golgi mannosidases involved in the initial processing of N-glycan precursors. Comparable amounts of alpha 5 beta 1 integrin were isolated from these cells by chromatography of detergent extracts on a fibronectin cell-binding fragment affinity column and elution with EDTA. The alpha 5 beta 1 integrin obtained from normal BHK cells by fibronectin affinity chromatography contained mainly endoglycosidase H-resistant oligosaccharides, whereas in RicR14 cells or dMM-treated BHK cells these were entirely endoglycosidase H-sensitive. Analysis of lactoperoxidase labeled or long term biosynthetically 35S-labeled proteins from cultures of normal or glycosylation deficient cells showed similar steady state levels of alpha 5 beta 1 integrin and expression at the cell surface. Pulse-chase experiments in normal BHK cells showed rapid conversion of the alpha 5 subunit into a mature form containing oligosaccharides resistant to endoglycosidase H and slower maturation of a precursor beta 1 subunit, as in other cell types. In Ric14 cells the precursor beta 1 subunit was found to carry glycans larger than the fully processed Man5GlcNAc2 glycan of the mature subunit, indicating that the bulk precursor pool had not been translocated into the cis-Golgi compartment containing mannosidase I. We conclude that in BHK cells terminal oligosaccharide processing of alpha 5 beta 1 integrin subunits is not required for dimer formation, surface expression, and fibronectin binding, and that expression of the glycosylation defect of Ric14 cells on the alpha 5 beta 1 integrin does not account for the reduced adhesiveness of these cells on fibronectin compared with normal and dMM-treated BHK cells.     

Release of the glucose‐regulated protein 94 by baby hamster kidney cells

Glucose‐regulated protein 94 (grp94) is a major component of the endoplasmic reticulum (ER) lumen of eukaryotic cells. We showed that grp94 is released from baby hamster kidney (BHK‐21) cells into a serum‐free medium. The exit of grp94 into the medium was not related to the protein discharge due to cell death and was independent of de novo protein synthesis. The treatment of cells with brefeldin A and monensin, the inhibitors of the classical pathway of protein secretion, did not decrease the extracellular level of grp94, indicating that the discharge of grp94 from cells does not occur through the ER/Golgi–dependent pathway. Exosomes, membrane vesicles secreted by several cell types, were not involved in the release of grp94 from cells. Methyl‐β‐cyclodextrin, a substance that disrupts the lipid raft organization, considerably reduced the extracellular level of grp94, indicating that lipid rafts are involved in the liberation of grp94 from BHK‐21 cells. The results suggest that BHK‐21 cells release grp94 into the serum‐free medium via the nonclassical secretory pathway in which lipid rafts play an important role. Copyright © 2012 John Wiley & Sons, Ltd.     

Growth of baby hamster kidney cells in media containing neuraminidase

An established line of baby hamster kidney cells, BHK 21/C13 grows in media containing high concentrations of Vibrio cholerae neuraminidase at the same initial exponential rate as control cells grown in the absence of the enzyme. Glycoprotein fractions removed from the surface of cells grown with neuraminidase contain less than 4% of the sialic acid present in similar fractions removed from control cells. The significance of these results are discussed in relation to the role suggested for sial glycoproteins in growth control. A small but significant increase was observed in the density of confluent cells in media containing neuraminidase compared with control cultures.     

The study of rous sarcoma virus-transformed baby hamster kidney cells using fluorescent probes.

The fluorescent probes pyrene, pyrene butyric acid and N-phenyl 1-naphthylamine have been used to investigate the changes that accompany in vitro transformation of a baby hamster kidney cell line using Rous sarcoma virus. The fluorescent probes which reside in the membrane were used to compare the changes in microviscosity and polarity of the membranes of normal cells with two transformed cell lines. The spectrofluorimetric data indicate that following transformation the probe N-phenyl 1-naphthylamine resides in a more polar environment. However, using the probe pyrene, the yield of excimer indicates decreased mobility of this probe in the membrane of transformed cells. The data also indicate differences between the two transformed cell lines. Laser photolysis was used to study the lifetime of the pyrene probes and the quenching of the pyrene fluorescence in the membrane by several different quenching molecules. The data indicate differences between the three cell lines and suggest that transformation decreases movement within the membrane.     

Recovery of intact 2-aminobenzamide-labeled O-glycans released from glycoproteins by hydrazinolysis

The initial step in quantitative analysis of O-linked glycans of glycoproteins is to release them in high yield, nonselectively, unmodified, and with a free reducing terminus. In contrast to other techniques, hydrazinolysis can meet these criteria. However, when analyzing pools of O-linked glycans as described in the accompanying article by L. Royle et al. (2002, Anal. Biochem. 304), some peeling of the glycans was observed. Critical steps in the sample preparation and glycan recovery were therefore evaluated by analyzing and identifying both intact O-glycans and degraded products. Synthetic O-glycopeptides were characterized by mass spectrometry. Released glycans were identical to those on the glycopeptide. O-Linked glycans from a range of glycoproteins of increasing complexity, namely, bovine serum fetuin, glycophorin A, and previously uncharacterized glycopeptides isolated from human salivary mucin Muc5B, were also analyzed. Quantitative analysis of the glycan profile confirmed that there was <2% peeling of O-glycans released by hydrazinolysis conditions of 60 degrees C for 6 h, and recovered using the optimised procedure now described. This demonstrated that O-glycans can be prepared by hydrazinolysis without degradation and, as part of an analytical strategy, makes the analysis of O-glycans attached to low-microgram levels of naturally occurring glycoproteins feasible.