Philip G. Strange

Constraints on the transport and glycosylation of recombinant IFN-γ in Chinese hamster ovary and insect cells

In this study we compare intracellular transport and processing of a recombinant glycoprotein in mammalian and insect cells. Detailed analysis of the N-glycosylation of recombinant human IFN-gamma by matrix-assisted laser-desorption mass spectrometry showed that the protein secreted by Chinese hamster ovary and baculovirus-infected insect Sf9 cells was associated with complex sialylated or truncated tri-mannosyl core glycans, respectively. However, the intracellular proteins were predominantly associated with high-mannose type oligosaccharides (Man-6 to Man-9) in both cases, indicating that endoplasmic reticulum to cis-Golgi transport is a predominant rate-limiting step in both expression systems. In CHO cells, although there was a minor intracellular subpopulation of sialylated IFN-gamma glycoforms identical to the secreted product (therefore associated with late-Golgi compartments or secretory vesicles), no other intermediates were evident. Therefore, anterograde transport processes in the Golgi stack do not limit secretion. In Sf9 insect cells, there was no direct evidence of post-ER glycan-processing events other than core fucosylation and de-mannosylation, both of which were glycosylation site-specific. To investigate the influence of nucleotide-sugar availability on cell-specific glycosylation, the cellular content of nucleotide-sugar substrates in both mammalian and insect cells was quantitatively determined by anion-exchange HPLC. In both host cell types, UDP-hexose and UDP-N-acetylhexosamine were in greater abundance relative to other substrates. However, unlike CHO cells, sialyltransferase activity and CMP-NeuAc substrate were not present in uninfected or baculovirus-infected Sf9 cells. Similar data were obtained for other insect cell hosts, Sf21 and Ea4. We conclude that although the limitations on intracellular transport and secretion of recombinant proteins in mammalian and insect cells are similar, N-glycan processing in Sf insect cells is limited, and that genetic modification of N-glycan processing in these insect cell lines will be constrained by substrate availability to terminal galactosylation.

Chinese hamster ovary cell growth and interferon production kinetics in stirred batch culture.

SummaryRecombinant human interferon-λ production by Chinese hamster ovary cells was restricted to the growth phase of batch cultures in serum-free medium. The specific interferon production rate was highest during the initial period of exponential growth but declined subsequently in parallel with specific growth rate. This decline in specific growth rate and interferon productivity was associated with a decline in specific metabolic activity as determined by the rate of glucose uptake and the rates of lactate and ammonia production. The ammonia and lactate concentrations that had accumulated by the end of the batch culture were not inhibitory to growth. Glucose was exhausted by the end of the growth phase but increased glucose concentrations did not improve the cell yield or interferon production kinetics. Analysis of amino acid metabolism showed that glutamine and asparagine were exhausted by the end of the growth phase, but supplementation of these amino acids did not improve either cell or product yields. When glutamine was omitted from the growth medium there was no cell proliferation but interferon production occurred, suggesting that recombinant protein production can be uncoupled from cell proliferation.

Activation of microtubule-associated protein kinase (Erk) and p70 S6 kinase by D2 dopamine receptors

Abstract: The ability of human and rat D2(short) and D2(long) dopamine receptors to activate microtubule‐associated protein (MAP) kinase (Erk1/2) and p70 S6 kinase has been investigated in recombinant cells expressing these receptors. In cells expressing the D2(short) receptor, dopamine activated both enzymes in a transient manner but with very different time courses, with activation of Erk being much quicker. Activation of both enzymes by dopamine was dose‐dependent and could be prevented by a range of selective dopamine antagonists. Excellent correlations were observed between the potencies of the antagonists for blocking enzyme activation and their affinities for the D2 dopamine receptor. Activation of Erk and of p70 S6 kinase via the D2 dopamine receptors was prevented by pretreatment of the cells with pertussis toxin, indicating the involvement of G proteins of the Gi or Go family. Inhibitors of phosphatidylinositol 3‐kinase (PI 3‐kinase) were found to block substantially, but not completely, activation of p70 S6 kinase by dopamine, suggesting the involvement of PI 3‐kinase‐dependent and ‐independent signalling pathways in its control by dopamine. p70 S6 kinase activation was completely blocked by rapamycin. In the case of Erk, activation was partially blocked by wortmannin or LY294002, indicating a possible link with PI 3‐kinase.

Effect of Multiple Serine/Alanine Mutations in the Transmembrane Spanning Region V of the D2 Dopamine Receptor on Ligand Binding

Abstract: Three conserved serine residues (Ser193, Ser194, and Ser197) in transmembrane spanning region (TM) V of the D2 dopamine receptor have been mutated to alanine, individually and in combination, to explore their role in ligand binding and G protein coupling. The multiple Ser → Ala mutations had no effect on the binding of most antagonists tested, including [3H]spiperone, suggesting that the multiple mutations did not affect the overall conformation of the receptor protein. Double or triple mutants containing an Ala197 mutation showed a decrease in affinity for domperidone, whereas Ala193 mutants showed an increased affinity for a substituted benzamide, remoxipride. However, dopamine showed large decreases in affinity (>20‐fold) for each multiple mutant receptor containing the Ser193 Ala mutation, and the high‐affinity (coupled) state of the receptor (in the absence of GTP) could not be detected for any of the multiple mutants. A series of monohydroxylated phenylethylamines and aminotetralins was tested for their binding to the native and multiple mutant D2 dopamine receptors. The results obtained suggest that Ser193 interacts with the hydroxyl of S‐5‐hydroxy‐2‐dipropylaminotetralin (OH‐DPAT) and Ser197 with the hydroxyl of R‐5‐OH‐DPAT. We predict that Ser193 interacts with the hydroxyl of R‐7‐OH‐DPAT and the 3‐hydroxyl (m‐hydroxyl) of dopamine. Therefore, the conserved serine residues in TMV of the D2 dopamine receptor are involved in hydrogen bonding interactions with selected antagonists and most agonists tested and also enable agonists to stabilise receptor‐G protein coupling.

3H]nemonapride and [3H]spiperone label equivalent numbers of D2 and D3 dopamine receptors in a range of tissues and under different conditions

Abstract: [3H]Nemonapride and [3H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [3H]nemonapride and [3H]spiperone give markedly different Bmax values for preparations of D2 dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D2(short), D2(long), and D3] in different buffers. Bmax values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [3H]spiperone and [3H]nemonapride do not differentiate between different forms or populations of D2‐like receptors.

Dopamine receptors: Studies on structure and function

Publisher Summary This chapter considers the information that has emerged recently on dopamine receptors from the application of the techniques of molecular biology to the dopamine receptors. It considers the mechanism of ligand binding to dopamine receptors from a theoretical and experimental standpoint. It should be clear throughout that the knowledge gained with molecular biological techniques has revolutionized the ability to study and conceptualize dopamine receptors. The application of biochemical, pharmacological, and physiological techniques to the study of dopamine receptors showed that there are multiple receptors for dopamine, and it was proposed that there were two subtypes of dopamine receptor (D1 and D2). These had distinct biochemical and pharmacological properties and a summary of the properties of these two subtypes has been given. Hydrophobic interactions with aromatic residues provide an important contribution to the free energy of binding. The contributions of these interactions to the overall free energy of the ligand binding process have been estimated and the experimental observations reported are consistent with the need for several favorable interactions between ligand and receptor to compensate for the unfavorable process of ligand binding and to lead to receptor activation.

Receptors for neurotransmitters and related substances

Advances in techniques for cloning neurotransmitter receptors have revealed new targets for selective drug design. Cell systems for more efficient expression of cloned receptor genes have also been developed. Knowledge of the nature of ligand-binding sites is now becoming available and this should aid in the design of better drugs with fewer side effects.

Changes in the Glycosylation of Interferon-γ during Culture

We have shown in previous studies that recombinant human interferon-γ (IFN-γ) expressed in CHO cells displays macroheterogeneity caused by variable N-glycosylation (Curling et al., 1990). In this paper, FAB-Mass Spectrometry of IFN-γ glycopeptides was used to assign the N-glycosylation preference of individual asparagine (Asn) sites within the IFN-γmolecule. Whilst both Asn28 and Asn100 N-glycosylation sites were used in doubly-glycosylated IFN-γ, a clear preference for the Asn28 site was apparent in the singly-glycosylated IFN-γ glycoform. The extent of glycosylation at the Asn28 site remained relatively stable during batch culture, whereas the percentage of Asn100 sites glycosylated decreased from 60% to 23% over the same period. These results demonstrate that marked differences exist in the capacity of CHO cells to glycosylate at individual Asn sites during cell culture.

Epitope Determination for Antibodies Raised Against Recombinant Human Interferon-Gamma

Oligosaccharides, like peptides, make excellent antigens due to their hydrophilicity and complexity and can also mask potential antigenic sites on the peptide backbone. Human interferon-gamma (IFN-γ) has been purified by immunoaffinity chromatography from the supernatant of batch cultures of recombinant Chinese hamster ovary cells to determine its glycosylation profile. To validate this purification procedure and the quantification of BFN-γ by enzyme-linked immunosorbent assay (ELISA) with mouse monoclonal antibody 20B8, the effects of glycosylation upon antibody recognition were investigated. Preliminary experiments with glycosylated and deglycosylated IFN-γ demonstrated that there was no difference in antibody binding by ELISA. To confirm this result, IFN-γ was digested with trypsin and the resulting peptides separated by reverse-phase high-performance liquid chromatography, the isolated peptides being identified by matrix-assisted laser desorption/ionisation mass spectrometry. Glycopeptides containing the Asn25 and ASH97 glycosylation sites were not recognised by anti-IFNγ 20B8 in ELISA studies. Its epitopes were located on non-glycosylated regions of IFN-γ, 20B8 recognising protein epitopes rather than the carbohydrate moieties. The purification and quantification of IFN-γ by ELISA with this antibody are therefore independent of the glycosylation status and are not selecting a sub-population of IFN-γ molecules.

Atypical antipsychotics — serotonergic mechanisms but don't forget dopamine

Busatto and Kerwin point out that for many of the currently used antipsychotic drugs there is an excellent correlation between the therapeutic doses used and the affinities of the drugs at the group of D2-like dopamine receptors (which includes D2, D3 and D4). No such general correlation is observed for affinities at other receptors including serotonin receptors. Therefore it seems reasonable to suppose that these drugs are achieving their therapeutic actions via blockade of D2-like receptors. These drugs would be considered to be typical (or classical) antipsychotic drugs in that they can induce extrapyramidal side-effects (EPS) in addition to their therapeutic antipsychotic effect. For atypical antipsychotic drugs such as clozapine, it is frequently assumed that the mechanism of action cannot be primarily via dopamine receptor antagonism. This is based on the observation that the affinity at D2 receptors is too low for there to be high levels of binding at the levels of free clozapine observed in the plasma at therapeutic doses. Also, it has been reported that positron emission tomography (PET) scans show that clozapine occupies lower levels of dopamine receptors in the basal ganglia than do typical antipsychotics. We wish to point out that there is still considerable uncertainty over these