Affinity of galectin-8 and its carbohydrate recognition domains for ligands in solution and at the cell surface

Galectin-8 has two different carbohydrate recognition domains (CRDs), the N-terminal Gal-8N and the C-terminal Gal-8C linked by a peptide, and has various effects on cell adhesion and signaling. To understand the mechanism for these effects further, we compared the binding activities of galectin-8 in solution with its binding and activation of cells. We used glycan array analysis to broaden the specificity profile of the two galectin-8 CRDs, as well as intact galectin-8s (short and long linker), confirming the unique preference for sulfated and sialylated glycans of Gal-8N. Using a fluorescence anisotropy assay, we examined the solution affinities for a subset of these glycans, the highest being 50 nM for NeuAcalpha2,3Lac by Gal-8N. Thus, carbohydrate-protein interactions can be of high affinity without requiring multivalency. More importantly, using fluorescence polarization, we also gained information on how the affinity is built by multiple weak interactions between different fragments of the glycan and its carrier molecule and the galectin CRD subsites (A-E). In intact galectin-8 proteins, the two domains act independently of each other in solution, whereas at a surface they act together. Ligands with moderate or weak affinity for the isolated CRDs on the array are bound strongly by intact galectin-8s. Also galectin-8 binding and signaling at cell surfaces can be explained by combined binding of the two CRDs to low or medium affinity ligands, and their highest affinity ligands, such as sialylated galactosides, are not required.     

The effects of culture conditions on the glycosylation of secreted human placental alkaline phosphatase produced in Chinese hamster ovary cells

The effects of different culture conditions, suspension and microcarrier culture and temperature reduction on the structures of N-linked glycans attached to secreted human placental alkaline phosphatase (SEAP) were investigated for CHO cells grown in a controlled bioreactor. Both mass spectrometry and anion-exchange chromatography were used to probe the N-linked glycan structures and distribution. Complex-type glycans were the dominant structures with small amounts of high mannose glycans observed in suspension and reduced temperature cultures. Biantennary glycans were the most common structures detected by mass spectrometry, but triantennary and tetraantennary forms were also detected. The amount of sialic acid present was relatively low, approximately 0.4 mol sialic acid/mol SEAP for suspension cultures. Microcarrier cultures exhibited a decrease in productivity compared with suspension culture due to a decrease in both maximum viable cell density (15-20%) and specific productivity (30-50%). In contrast, a biphasic suspension culture in which the temperature was reduced at the beginning of the stationary phase from 37 to 33 degrees C, showed a 7% increase in maximum viable cell density, a 62% increase in integrated viable cell density, and a 133% increase in specific productivity, leading to greater than threefold increase in total productivity. Both microcarrier and reduced temperature cultures showed increased sialylation and decreased fucosylation when compared to suspension culture. Our results highlight the importance of glycoform analysis after process modification as even subtle changes (e.g., changing from one microcarrier to another) may affect glycan distributions.     

Calcium, asparagine and cAMP are required for ornithine decarboxylase activation in intact Chinese hamster ovary cells.

Asparagine specifically activated ornithine decarboxylase activity 5–7 fold by 7–8 h in confluent cultures maintained with a salts/glucose medium. When dibutyryl cAMP was added with asparagine, a 40–50 fold stimulation of ornithine decarboxylase activity was produced. Ornithine decarboxylase activation in the salts/glucose medium was not sensitive to actinomycin D. Omission of Ca⁺⁺ and Mg⁺⁺ from the medium abolished the ability of asparagine and/or dibutyryl cAMP to stimulate enzyme activity. Calcium was essential for the asparagine and dibutyryl cAMP mediated stimulation of ornithine decarboxylase activity.     

Pregnancy associated glycoprotein-1, -6, -7, and -17 are major products of bovine binucleate trophoblast giant cells at midpregnancy

Pregnancy associated glycoproteins (PAGs) are extensively glycosylated secretory proteins of ruminant trophoblast cells. In cattle placenta several PAG cDNAs are expressed, but the variety of correspondent proteins and their degree of glycosylation are not well characterized. Thus, we purified PAGs by using a protocol which included a lectin (Vicia villosa agglutinin) affinity chromatography. Due to their specific glycosylation pattern, PAGs derived from binucleate trophoblast giant cells were highly enriched by this protocol. PAGs were purified from cotyledons of 2 day 100 placentas and from a single placenta at day 155 and 180. In all samples three major bands (75; 66; 56 kDa) were detected by one-dimensional SDS-PAGE. Mass-spectrometric analysis identified the 75 kDa band as a mixture of PAG-7 and PAG-6, the 66 kDa band as PAG-1 and the 56 kDa band as PAG-17. N-terminal sequencing of the day 100 sample confirmed the mass spectrometric identifications. Enzymatic release of N-glycans with peptide-N-glycanase-F from PAGs reduced the molecular weight to approximately 37 kDa which corresponds to the theoretical molecular mass of PAGs. Limited peptide-N-glycanase-F treatment revealed that all four N-glycosylation sites are quantitatively occupied in PAG-1. Compared to PAG-1 the number of potential N-glycosylation sites is lower in PAG-17 (three sites) and higher in PAG-6 and -7 (five and six sites, respectively). This suggests that the number of attached N-glycans is the main determinant of molecular mass of bovine PAGs. The degree of glycosylation may be a major factor regulating the plasma half life of PAGs.     

Studies on adenosine 3′,5′-phosphate-binding and adenosine 3′,5′-phosphate-dependent protein kinase activities associated with subcellular fractions of Chinese hamster ovary cells

Both cyclic AMP-binding and cyclic AMP-dependent protein kinase activities exists in Chinese hamster ovary cell extract. Competition experiments demonstrate that the binding is specific for cyclic AMP. All cellular elements including nucleus, mitochondria, plasma membrane, microsome, ribosome and cytosol contain both activities. Binding activity is highest in the cytosol and lowest in the nucleus. Each fraction contains endogenous protein kinase activity which is insensitive to cyclic AMP activation. When histone was used as a substrate, protein kinase activity in all fractions was stimulated by cyclic AMP (with the highest in cytosol and lowest in the nucleus) and inhibited by Walsh's protein kinase inhibitor.     

Development of a mathematical model for evaluating the dynamics of normal and apoptotic Chinese hamster ovary cells

A metabolic flux based methodology was developed for modeling the metabolism of a Chinese hamster ovary cell line. The elimination of insignificant fluxes resulted in a simplified metabolic network which was the basis for modeling the significant metabolites. Employing kinetic rate expressions for growing and non-growing subpopulations, a logistic model was developed for cell growth and dynamic models were formulated to describe culture composition and monoclonal antibody (MAb) secretion. The model was validated for a range of nutrient concentrations. Good agreement was obtained between model predictions and experimental data. The ultimate goal of this study is to establish a comprehensive dynamic model which may be used for model-based optimization of the cell culture for MAb production in both batch and fed-batch systems.     

Effect of chemical chaperone addition on production and aggregation of recombinant flag-tagged COMP-angiopoietin 1 in Chinese hamster ovary cells

To investigate the effect of chemical chaperones on the production and aggregation of flag-tagged cartilage oligomeric matrix protein-Angiopoietin1 (FCA1) in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in serum-free media with various chemical chaperones, 1 mM 4-phenylbutyrate (4-PBA), 200 mM proline, 3% glycerol, 2% dimethyl sulfoxide (DMSO), and without chemical chaperone as control. The addition of chemical chaperones enhanced FCA1 production and specific FCA1 productivity, q(FCA)(1). For example, the q(FCA)(1) at 200 mM proline was fourfold higher than that at control. Unlike q(FCA)(1), the aggregation of FCA1 was strongly affected by which chemical chaperone was added. The addition of 2% DMSO and 200 mM proline significantly reduced the proportion of aggregates, but the addition of 1 mM 4-PBA and 3% glycerol was hardly effective. The proportions of aggregates were 29.5 and 55.6% at 2% DMSO and 200 mM proline, respectively, whereas it was 79.6% at control. The exact mechanism how chemical chaperones affected the aggregation of FCA1 was not investigated in this study, and therefore, more extensive works will be needed to clarify why different chemical chaperones behaved differently in reducing the aggregation of FCA1. Among chemical chaperones tested, DMSO was the most effective one in regard to enhancing the production and reducing the aggregation of FCA1 in CHO cells.     

Observations on the influence of glutamine,asparagine and peptone on growth and t-PA production of Chinese hamster ovary (CHO) cells

When a transfected CHO cell, that produces tissue-type Plasminogen Activator, t-PA, was transferred from a medium based on 5% Fetal Calf Serum, FCS, to a medium based on 0.8% casein peptone with variable glutamine and asparagine content, it was observed, that the growth of the cells changed from anchorage dependant to suspension culture giving more reproducible cultivations. In the FCS culture t-PA was unstable, observed as a decline in t-PA concentration after 250 h. This decline in t-PA concentration was not observed in the serum free culture, although there was a decline in productivity after 200 h. This change in production profile may be attributed to either no proteolytic attack from serum or by scavenging of proteolytic activities produced by the cells from the peptone peptides. Increasing amounts of glutamine/asparagine gave higher production of t-PA in synchrony with an increasing production of ammonia/ammonium ions. Ammonia inhibition does not seem to be a key factor for this cell line as seen with many others.     

Effect of the phytoestrogen, genistein-8-C-glucoside, on Chinese hamster ovary cells in vitro

Genistein-8-C-glucoside (G8CG) belongs to isoflavones, which are a subclass of flavonoids, a large group of polyphenolic compounds widely distributed in plants. A number of studies on flavonoids show their cardioprotective and antiosteoporosis properties in in vitro and in vivo models. As a phytoestrogen, genistein has recently generated interest as a potential anticancer and antiatherogenic agent. Several flavonoids are known as antioxidants and scavengers of free oxygen radicals. In the current investigation we used glycosylated genistein (genistein-8-C-glucoside) from flowers of lupine (Lupinus luteus L.). Many authors have found that the action of genistein is not so simple, although many reports conducted in vitro have demonstrated that it is cytotoxic and genotoxic. Therefore, the cytotoxic and genotoxic effects of this compound in Chinese hamster ovary cells (line CHO) were studied. A colorimetric MTT assay to assess cytotoxicity and a Comet assay for the detection of DNA damage were used. Apoptosis was determined by the Hoechst 33258/propidium iodide staining technique. We have also demonstrated antioxidant properties of G8CG. The level of reactive oxygen species generated by G8CG alone and/or H2O2 was evaluated with fluorescence probes: dichlorofluorescein-diacetate (DCFDA) by flow cytometry. The cells were exposed to various concentrations of genistein-8-C-glucoside (1-290 microM) and hydrogen peroxide (10-130 microM) and the effect of G8CG alone or in combination with H2O2 was determined. The results reveal that G8CG at concentrations higher than 10 microM significantly reduced cell viability, induced apoptosis and DNA damage. However at lower concentrations (5 and 7.5 microM), G8CG showed antioxidant properties, but had no cytotoxic or genotoxic activity.     

Galectin-loaded cells as a platform for the profiling of lectin specificity by fluorescent neoglycoconjugates: a case study on galectins-1 and -3 and the impact of assay setting

The involvement of galectins as pleiotropic regulators of cell adhesion and growth in disease progression explains the interest to define their ligand-binding properties. Toward this end, it is desirable to approach in vivo conditions to attain medical relevance. In order to simulate physiological conditions with cell surface glycans as recognition sites and galectins as mediators of intercellular contacts we developed an assay using galectin-loaded Raji cells. The extent of surface binding of fluorescent neoglycoconjugates depended on the lectin presence and the type of lectin, the nature of the probes' carbohydrate headgroup and the density of unsubstituted beta-galactosides on the cell surface. Using the most frequently studied galectins-1 and -3, application of this assay led to rather equal binding levels for linear and branched oligomers of N-acetyllactosamine. A clear preference of galectin-3 for alpha1-3-linked galactosylated lactosamine was noted. In parallel, a panel of 24 neoglycoconjugates was tested as inhibitors of galectin binding from solution to N-glycans of surface-immobilized asialofetuin. These two assays differ in presentation of the galectin and ligand, facilitating identification of assay-dependent properties. Under the condition of the cell assay, selectivity among oligosaccharides for the lectins was higher, and extraordinary affinity of galectin-1 to 3'-O-sulfated probes in a solid-phase assay was lost in the cell assay. Having introduced and validated a cell assay, the comprehensive profiling of ligand binding to cell-surface-presented galectins is made possible.     

Systematic development of temperature shift strategies for Chinese hamster ovary cells based on short duration cultures and kinetic modeling

Temperature shift (TS) to a hypothermic condition has been widely used during protein production processes that use Chinese hamster ovary (CHO) cells. The effect of temperature on cell growth, metabolites, protein titer and quality depends on cell line, product, and other bioreactor conditions. Due to the large numbers of experiments, which typically last 2–3 weeks each, limited systematic TS studies have been reported with multiple shift temperatures and steps at different times. Here, we systematically studied the effect of temperature on cell culture performance for the production of two monoclonal antibodies by industrial GS and DG44 CHO cell lines. Three 2–8 day short-duration methods were developed and validated for researching the effect of many different temperatures on CHO cell culture and quality attributes. We found that minor temperature differences (1–1.5 °C) affected cell culture performance. The kinetic parameters extracted from the short duration data were subsequently used to compute and predict cell culture performance in extended duration of 10–14 days with multiple TS conditions for both CHO cell lines. These short-duration culture methods with kinetic modeling tools may be used for effective TS optimization to achieve the best profiles for cell growth, metabolites, titer and quality attributes. Although only three short-duration methods were developed with two CHO cell lines, similar short-duration methods with kinetic modeling may be applied for different hosts, including both microbial and other mammalian cells.     

Inhibition of the adhesion of Chinese hamster ovary cells by the naphthylsulfonamides dansylcadaverine and N-(6-aminohexyl)-5-chloro-1-naphthylenesulfonamide (W7)

Treatment of Chinese hamster ovary cells with dansylcadaverine or N-(6-aminohexyl)-5-chloro-1-naphthylenesulfonamide (W7) reduced cell attachment in a reversible, dose-dependent manner. The concentration of dansylcadaverine required to produce 50% inhibition of adhesion was significantly higher than that of W7, 300 μM and 50 μM, respectively. Concentrations of dansylcadaverine and W7 which produced decreased adhesion also antagonized calmodulin-dependent activation of phosphodiesterase. Chlorpromazine, another calmoldulin antagonist also decreased cell attachment. Dansylcadaverine and W7 both interfere with cellular transglutaminase activity, but several other transglutaminase antagonists, such as methylamine, butylamine, putrescine and bacitracin, had no effect on CHO cell attachment. We conclude that naphthylsulfonamides such as dansylcadaverine and W7 may inhibit the attachment of CHO cells by a mechanism which could involve inhibition of calmodulin-dependent processes, although further studies are required to show a direct role of calmodulin in cell adhesion.