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PubMed1996

Reciprocal regulation of Gs alpha by palmitate and the beta gamma subunit.

Hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase, promotes dissociation of alpha s from G beta gamma, accelerates removal of covalently attached palmitate from the G alpha subunit, and triggers release of a fraction of alpha s from the plasma membrane into the cytosol. To elucidate relations among these three events, we assessed biochemical effects in vitro of attached palmitate on recombinant alpha s prepared from Sf9 cells. In comparison to the unpalmitoylated protein (obtained from cytosol of Sf9 cells, treated with a palmitoyl esterase, or expressed as a mutant protein lacking the site for palmitoylation), palmitoylated alpha s (from Sf9 membranes, 50% palmitoylated) was more hydrophobic, as indicated by partitioning into TX-114, and bound beta gamma with 5-fold higher affinity. beta gamma protected GDP-bound alpha s, but not alpha s-GTP[gamma S], from depalmitoylation by a recombinant esterase. We conclude that beta gamma binding and palmitoylation reciprocally potentiate each other in promoting membrane attachment of alpha s and that dissociation of alpha s.GTP from beta gamma is likely to mediate receptor-induced alpha s depalmitoylation and translocation of the protein to cytosol in intact cells.

Animals↗

PubMed1998

Variable and tissue-specific hormone resistance in heterotrimeric Gs protein alpha-subunit (Gsalpha) knockout mice is due to tissue-specific imprinting of the gsalpha gene.

Albright hereditary osteodystrophy (AHO), an autosomal dominant disorder characterized by short stature, obesity, and skeletal defects, is associated with heterozygous inactivating mutations of GNAS1, the gene encoding the heterotrimeric G protein alpha-subunit (Gsalpha) that couples multiple receptors to the stimulation of adenylyl cyclase. It has remained unclear why only some AHO patients present with multihormone resistance and why AHO patients demonstrate resistance to some hormones [e.g., parathyroid hormone (PTH)] but not to others (e.g., vasopressin), even though all activate adenylyl cyclase. We generated mice with a null allele of the mouse homolog Gnas. Homozygous Gs deficiency is embryonically lethal. Heterozygotes with maternal (m-/+) and paternal (+/p-) inheritance of the Gnas null allele have distinct phenotypes, suggesting that Gnas is an imprinted gene. PTH resistance is present in m-/+, but not +/p-, mice. Gsalpha expression in the renal cortex (the site of PTH action) is markedly reduced in m-/+ but not in +/p- mice, demonstrating that the Gnas paternal allele is imprinted in this tissue. Gnas is also imprinted in brown and white adipose tissue. The maximal physiological response to vasopressin (urinary concentrating ability) is normal in both m-/+ and +/p- mice and Gnas is not imprinted in the renal inner medulla (the site of vasopressin action). Tissue-specific imprinting of Gnas is likely the mechanism for variable and tissue-specific hormone resistance in these mice and a similar mechanism might explain the variable phenotype in AHO.

Alleles↗

PubMed2003

Constitutive signaling of the human cytomegalovirus-encoded receptor UL33 differs from that of its rat cytomegalovirus homolog R33 by promiscuous activation of G proteins of the Gq, Gi, and Gs classes.

The human cytomegalovirus (HCMV) UL33 gene is conserved among all beta-herpesviruses and encodes a protein that shows sequence similarity with chemokine receptors belonging to the family of G protein-coupled receptors. Here, we show that HCMV UL33 is predominantly transcribed as a spliced mRNA of which the 5' terminus is localized 55 bp upstream of the start codon. Like its homolog from rat cytomegalovirus (RCMV), R33, UL33 activates multiple signaling pathways in a ligand-independent manner. Although both receptors constitutively activate phospholipase C via G(q/11), and partially via G(i/o)-mediated pathways, they exhibit profound differences in the modulation of cAMP-responsive element (CRE) activation. R33 constitutively inhibits, whereas UL33 constitutively enhances CRE-mediated transcription. For R33, the inhibition of CRE-driven transcription is entirely G(i/o)-mediated. For UL33, however, CRE-mediated transcription is modulated not only through coupling to Galpha(i/o) but also through coupling to Galphas. In addition, UL33 was found to enhance CRE activation through the Rho/p38 pathway, via Gbetagamma. Interestingly, by studying chimeric UL33/R33 proteins, we found the C-terminal cytoplasmic tail of UL33, but not that of R33, to be responsible for the activation of G(i/o) proteins. A UL33-deficient variant of HCMV was generated to analyze UL33-signaling properties in a physiologically relevant model system. Data obtained with infected cells show that HCMV induces CRE activation, and this effect is, at least in part, dependent on UL33 expression. Taken together, our data indicate that constitutive signaling of UL33 differs from that of R33 by promiscuous activation of G proteins of the Gq, G(i/o), as well as Gs class. Thus, HCMV may effectively use UL33 to orchestrate multiple signaling networks within infected cells.

Amino Acid Sequence↗

PubMed1996

Activation-induced subcellular redistribution of Gs alpha.

We have examined the subcellular distribution of alpha s, the alpha subunit of the heterotrimeric G protein Gs, by using immunofluorescence microscopy. In transiently transfected HEK293 cells, wild-type alpha s localizes to the plasma membrane. However, a mutationally activated alpha s (alpha sR201C) is diffusely distributed throughout the cytoplasm. Similarly, cholera toxin activation of alpha s causes it to redistribute from the plasma membrane to cytoplasm in stably transfected cells. In HEK293 cells stably transfected with alpha s and the beta 2-adrenergic receptor (beta-AR), stimulation of the beta-AR by the agonist isoproterenol also causes a translocation of alpha s from the plasma membrane to cytoplasm. Replacing the agonist with antagonist allows alpha s to return to the plasma membrane, demonstrating the reversibility of alpha s translocation. Receptor-activated alpha s does not colocalize with internalized beta-AR at endosomes. Incubation of cells in hypertonic sucrose to inhibit clathrin-coated pit-mediated endocytosis of agonist-activated beta-AR failed to block agonist-stimulated redistribution of alpha s. These findings demonstrate that activated alpha s reversibly undergoes a translocation from the plasma membrane to cytoplasm and begin to address the relationship between regulated trafficking of a seven-transmembrane receptor and its cognate G protein.

Adrenergic beta-Agonists↗

PubMed2004

Genetic variants of the extra-large stimulatory Gs protein alpha-subunit and risk of thrombotic and haemorrhagic disorders.

A polymorphism of the gene encoding the extra-large stimulatory G-protein alpha-subunit (XLalphas), originally identified in three patients with a bleeding tendency, involved a 36-bp insertion and two missense changes. A paternally-inherited insertion displayed a moderate platelet Gsalpha over-expression, which lead to platelet hypo-reactivity. These data prompted us to investigate the genetic, functional and clinical relevance of this polymorphism in the Mediterranean population. We included 414 healthy subjects and three case/control studies: 263 consecutive patients with a first episode of primary intracerebral haemorrhage, 195 patients with deep venous thrombosis, and 104 patients with cerebrovascular disease. Controls were selected by approximating criteria to match selected risk factors to patients. Moreover, we performed studies of platelet function. We developed a simple method to determine the methylated allele, by digestion of genomic DNA with Sma I before polymerase chain reaction amplification. We identified two new rare variants, resulting from the loss of repeat units 7 and 5. The AB genotype was present in 3.6% of healthy population and the prevalence of the B allele was similar among cases and controls. Accordingly, the non-methylated B allele did not modify either the expression of platelet Gsalpha or the platelet response to Gs-agonists. Thus, our study suggests a minor functional role of XLalphas polymorphism in thrombotic or in haemorrhagic disorders.

Aged↗

PubMed1997

Recurrent ovarian cyst and mutation of the Gs alpha gene in ovarian cyst fluid cells: what is the link with McCune-Albright syndrome?

Isolated peripheral precocious puberty due to recurrent ovarian cysts evokes a McCune-Albright syndrome (MAS). This syndrome associates endocrine dysfunction such as precocious puberty, polyostotic fibrous dysplasia, and "café-au-lait" skin lesions. We report the case of a 3-y-old girl who presented with peripheral puberty with extremely elevated oestradiol level, low LH and FSH levels, and an ovarian cyst that quickly resolved. Skeletal X-rays were normal and she had no café-au-lait spots. GnRH analogue treatment was ineffective. A second ovarian cyst appeared and was completely drained under ultrasonographic guidance. Molecular biological analysis performed on fluid cells revealed the Arg201-->His mutation of the Gs alpha gene described in MAS. Percutaneous aspiration of simple ovarian cyst to detect "MAS" mutation is of interest in the diagnosis of recurrent ovarian cyst.

Child, Preschool↗

PubMed1996

Associative learning disrupted by impaired Gs signaling in Drosophila mushroom bodies.

Disruptions in mushroom body (MB) or central complex (CC) brain structures impair Drosophila associative olfactory learning. Perturbations in adenosine 3',5' monophosphate signaling also disrupt learning. To integrate these observations, expression of a constitutively activated stimulatory heterotrimeric guanosine triphosphate-binding protein alpha subunit (Galphas*) was targeted to these brain structures. The ability to associate odors with electroshock was abolished when Galphas* was targeted to MB, but not CC, structures, whereas sensorimotor responses to these stimuli remained normal. Expression of Galphas* did not affect gross MB morphology, and wild-type Galphas expression did not affect learning. Thus, olfactory learning depends on regulated Gs signaling in Drosophila MBs.

Adenylyl Cyclases↗

PubMed2005

Gs and Gi coupling of adrenomedullin in adult rat ventricular myocytes.

Adrenomedullin (ADM) acts as an autocrine or a paracrine factor in the regulation of cardiac function. The intracellular mechanisms involved in the direct effect of ADM on adult rat ventricular myocytes (ARVMs) are still to be elucidated. In ARVMs from normal rats, ADM produced an initial (< 30 min) increase in cell shortening and Ca2+ transients and a marked decrease in both on prolonged incubation (> 1 h). Both effects were sensitive to ADM antagonist ADM-(22-52). Treatment with SQ-22536, an inhibitor of adenylate cyclase, blocked the positive inotropic effect of ADM and potentiated its negative inotropic effect. The negative inotropic effect was sensitive to inhibition by pertussis toxin (PTX), an inhibitor of Gi proteins and KT-5720, an inhibitor of PKA. The observations suggest a switch from Gs-coupled to PTX-sensitive, PKA-dependent Gi coupling by ADM in ARVMs. The ADM-mediated Gi-signaling system involves cAMP-dependent pathways because SQ-22536 further increased the negative inotropic actions of ADM. Also, because ADM is overproduced by ARVMs in our rat model of septic shock, ARVMs from LPS-treated rats were subjected to treatment with ADM-(22-52) and PTX. The decrease in cell shortening and Ca2+ transients in LPS-treated ARVMs could be reversed back with ADM-(22-52) and PTX. This indicates that ADM plays a role in mediating the negative inotropic effect in LPS-treated ARVM through the activation of Gi signaling. This study delineates the intracellular pathways involved in ADM-mediated direct inotropic effects on ARVMs and also suggests a role of ADM in sepsis.

Adrenomedullin↗

PubMed1997

Somatic mutations in the thyrotropin receptor gene and not in the Gs alpha protein gene in 31 toxic thyroid nodules.

Studies on frequency and distribution pattern of TSH receptor (TSHR) and Gs alpha protein (gsp) mutations in toxic thyroid nodules (TTNs) reported conflicting results, most likely also related to the different screening methods applied and the investigation of only part of exon 10 of the TSHR. Therefore, we screened a consecutive series of 31 TTNs for both TSHR and gsp mutations by direct sequencing of exon 9 and the entire exon 10 of the TSHR gene and exons 7-10 of the gsp gene. Somatic TSHR mutations were identified in 15 of 31 TTNs. TSHR mutations were localized in the third intracellular loop (Asp619Gly and Ala623Val), the sixth transmembrane segment (Phe631Leu and Thr632Ile, Asp633Glu) and the second extracellular loop (Ile568Thr). One mutation was found in the extracellular TSHR domain (Ser281Asn). Two new TSHR mutations were identified. One involves codon 656 in the third extracellular loop (Val656Phe). The other new mutation is a 27-bp deletion in the third intracellular loop resulting in deletion of 9 amino acids at codons 613-621. Transient expression of the new TSHR mutations in COS-7 cells demonstrated their constitutive activity. No mutation was found in exons 7-10 of the gsp gene. This finding was confirmed by an allele-specific PCR for mutations in gsp codons 201 (Arg-->His, Cys) and 227 (Gln-->His, Arg). Our data indicate that constitutively activating TSHR mutations can be found in 48% of TTNs and thus currently represent the most frequent molecular mechanism known in the etiopathogenesis of TTNs. Moreover, the absence of gsp mutations in our series argues for an only minor role of these mutations in TTNs. Constitutive activation of the TSHR by a deletion in a region that might be involved in G protein coupling of the TSHR offers new insights into TSHR activation.

Amino Acid Sequence↗

PubMed1998

Pronostic and therapeutic consequences of Gs alpha mutations in somatotroph adenomas.

Human pituitary somatotroph adenomas can be associated with mutations of the s alpha-subunit of G proteins. However, the impact of the gsp mutations on the tumoral phenotype is not well understood at present. This study aims to determine whether the detection of this mutation could impact on the management of acromegalic patients. We examined 30 acromegalic patients; 8 were gsp positive, and 22 were gsp negative. The gsp-positive adenomas appeared to secrete significantly more when the ratio of basal GH level/tumor size was considered. A better octreotide sensitivity of mutated adenomas was clearly shown under in vivo (short and long term) and in vitro conditions. During the acute octreotide test, the GH nadir was significantly lower in the gsp-positive adenomas (85% of maximal inhibition vs. 52%). Eighteen patients were treated with octreotide (300 micrograms/day) for at least 3 months before surgery: the percent inhibition of GH hypersecretion was higher in gsp-positive adenomas (76% vs. 47%). In cell culture, the octreotide-induced inhibition of GH release was significantly higher in gsp-positive adenomas (71% vs. 30%). Finally, during 2 yr of postoperative follow-up, GH hypersecretion was controlled in all patients with gsp mutation even in those in whom tumoral tissue remained after surgery. On the contrary, in the gsp-negative group, octreotide treatment was unable to control hypersecretion in 4 patients bearing tumoral remnants. The Gs alpha mutation could, therefore, be a new marker to foresee the susceptibility of the tumor to be controlled by somatostatin analogs, which improves prognosis.

Acromegaly↗

PubMed2001

Study of the multiple endocrine neoplasia type 1, growth hormone-releasing hormone receptor, Gs alpha, and Gi2 alpha genes in isolated familial acromegaly.

Familial acromegaly may occur as an isolated pituitary disorder or as a feature of hereditary syndromes, such as multiple endocrine neoplasia type 1 (MEN1) or the Carney complex. Herein, we characterized a newly identified kindred with isolated acromegaly and searched for germline mutation in genes that have been associated with endocrine tumors [i.e. MEN1, Gs alpha (GNAS1), and Gi2 alpha (GNAI2)], as well as the GHRH receptor (GHRH-R) gene. Genomic DNA was used to amplify exons 2-10 of MEN1, followed by dideoxy fingerprinting mutation analysis and direct sequencing. The GHRH-R gene was analyzed via direct sequencing of PCR-amplified fragments representing the coding exons and intron-exon junctions. To exclude mutation at hot spot areas of GNAS1 and GNAI2, exons 8 and 9 of GNAS1 and exons 5 and 6 of GNAI2 were amplified and screened for mutation via denaturing gradient gel electrophoresis. No mutations were detected in any of the four genes. The present data extend prior reports of the absence of mutation in MEN1, GHRH-R, and GNAS1 and describe the first family with isolated acromegaly in which germline mutation in GNAI2 has been searched.

Acromegaly↗

PubMedDate not supplied

Odontogenic myxomas are not associated with activating mutations of the Gs alpha gene.

BACKGROUND: Myxoma is a rare bone tumor of the mandible and maxillary sinus whose etiology and underlying molecular mechanisms remain unknown. Mutations that inhibit the GTPase activity of the a subunit of the stimulating G protein (Gsa) have been demonstrated in the myocardium of patients with McCune-Albright syndrome. The histopathological similarities shared by cardiac and jaw myxomas coupled with the paucity of reported candidate genes involved in jaw tumor pathogenesis, prompted us to investigate for the presence of gsp mutations in 23 sporadic jaw myxomas. MATERIALS AND METHODS: We used the polymerase chain reaction (PCR) to amplify the appropriate genomic fragments, followed by denaturing gradient gel electrophoresis (DGGE) analysis. RESULTS: No gsp mutations could be demonstrated in any of tumors analyzed, while the technique has a proven capability to detect these specific mutations. CONCLUSIONS: We conclude that mutations of the Gs alpha gene rarely, if ever, are associated with sporadic jaw myxomas tumorigenesis.

Biopsy↗

PubMed2000

Transglycosylation by Streptococcus mutans GS-5 glucosyltransferase-D: acceptor specificity and engineering of reaction conditions.

The acceptor specificity of Streptococcus mutans GS-5 glucosyltransferase-D (GTF-D) was studied, particular the specificity toward non-saccharide compounds. Dihydroxy aromatic compounds like catechol, 4-methylcatechol, and 3-methoxycatechol were glycosylated by GTF-D with a high efficiency. Transglycosylation yields were 65%, 50%, and 75%, respectively, using 40 mM acceptor and 200 mM sucrose as glucosyl donor. 3-Methoxylcatchol was also glycosylated, though at a significantly lower rate. A number of other aromatic compounds such as phenol, 2-hydroxybenzaldehyde, 1,3-dihydroxybenzene, and 1, 2-phenylethanediol were not glycosylated by GTF-D. Consequently GTF-D aromatic acceptors appear to require two adjacent aromatic hydroxyl groups. In order to facilitate the transglycosylation of less water-soluble acceptors the use of various water miscible organic solvents (cosolvents) was studied. The flavonoid catechin was used as a model acceptor. Bis-2-methoxyethyl ether (MEE) was selected as a useful cosolvent. In the presence of 15% (v/v) MEE the specific catechin transglucosylation activity was increased 4-fold due to a 12-fold increase in catechin solubility. MEE (10-30% v/v) could also be used to allow the transglycosylation of catechol, 4-methylcatechol, and 3-methoxycatechol at concentrations (200 mM) otherwise inhibiting GTF-D transglycosylation activity.

Biotechnology↗

PubMed2001

Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells.

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.

Amidohydrolases↗

PubMed2001

The response of GS-NS0 myeloma cells to pH shifts and pH perturbations.

The perceived sensitivity of animal cells to hydrodynamic shear has limited agitation and aeration at large-scale. This makes it difficult to ensure adequate mixing of the vessel contents and may lead to inhomogeneities in operational parameters such as temperature, dissolved oxygen concentration, and especially pH. The effect of pH shifts and pH perturbations on the cellular responses, in batch culture, of a GS-NS0 mouse myeloma cell line, expressing a recombinant antibody, was investigated. In addition, the effect of extreme pH on the structure of the purified antibody product was studied using isoelectric focusing. The fermentation pH value was shifted abruptly from pH 7.3 to pH values ranging from 6.5 to 9.0. Culture pH was maintained at this new value for the remainder of the fermentation. All pH shifts of above 0.2 units caused a transient increase in apoptosis. However, cultures shifted to pH values between 7.0 and 8.0 continued to grow and the apoptotic fraction returned to initial levels. Cultures shifted to pH values above pH 8.0 and below pH 7.0 did not recover resulting in culture death. For example, a shift to pH 8.5 caused accumulation of cells in the G(2)/M phase of the cell cycle followed by apoptotic death. After the pH shift, maximum specific growth rate was observed over the range pH 7.3 to 7.5 and maximum viable cell number was seen at pH 7.3. Maximum volumetric antibody production, resulting from increased culture longevity, was seen at pH 7.0. It was also observed that glucose consumption increased with increasing pH. In a separate set of experiments cells were subjected to a single pH perturbation ranging in duration from 0 to 600 minutes. Exposure of cells to a pH value greater than 8.5 for more than 10 minutes caused a decrease in the proportion of viable cells and induced a lag in cell growth. At very low pH (6.5) similar effects were seen, but only for extended perturbations (600 min). However, after recovery from the pH perturbation, growth, product secretion and metabolism all returned to original levels. Incubation of the antibody, at the range of pH values investigated, indicated no alterations in the structure of the antibody as determined by the isoelectric focusing pattern.

Animals↗

PubMed2007

Protein-free fed-batch culture of non-GS NS0 cell lines for production of recombinant antibodies.

Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.

Animals↗

PubMed2007

Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies.

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.

Animals↗

PubMed1991

Activating mutations in the NH2- and COOH-terminal moieties of the Gs alpha subunit have dominant phenotypes and distinguishable kinetics of adenylyl cyclase stimulation.

The alpha subunit polypeptides of the G proteins Gs and Gi2 stimulate and inhibit adenylyl cyclase, respectively. The alpha s and alpha i2 subunits are 65% homologous in amino acid sequence but have highly conserved GDP/GTP binding domains. Previously, we mapped the functional adenylyl cyclase activation domain to a 122 amino acid region in the COOH-terminal moiety of the alpha s polypeptide (Osawa et al: Cell 63:697-706, 1990). The NH2-terminal half of the alpha s polypeptide encodes domains regulating beta gamma interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH2- or COOH-terminal coding sequence of alpha s substituted with the corresponding alpha i2 sequence were used to introduce multi-residue non-conserved mutations in different domains of the alpha s polypeptide. Mutation of either the amino- or carboxy-terminus results in an alpha s polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated alpha s polypeptides having mutations in either the NH2- or COOH-terminus demonstrate an enhanced rate of GTP gamma S activation of adenylyl cyclase. In membrane preparations from cells expressing the various alpha s mutants, COOH-terminal mutants, but not NH2-terminal alpha s mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTP gamma S and fluoride ion. Neither mutation at the NH2- nor COOH-terminus had an effect on the GTPase activity of the alpha s polypeptides. Thus, mutation at NH2- and COOH-termini influence the rate of alpha s activation, but only the COOH-terminus appears to be involved in the regulation of the alpha s polypeptide activation domain that interacts with adenylyl cyclase.

Adenylyl Cyclases↗