scholarly journals Functional analysis of Chinese hamster phosphatidylserine synthase 1 through systematic alanine mutagenesis

2004 ◽  
Vol 381 (3) ◽  
pp. 853-859 ◽  
Author(s):  
Tomoko OHSAWA ◽  
Masahiro NISHIJIMA ◽  
Osamu KUGE
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Sequences ◽  
Amino Acid Residues ◽  
Intact Cells ◽  
Base Exchange ◽  
Ovary Cells

PtdSer (phosphatidylserine) synthesis in mammalian cells occurs through the exchange of L-serine with the base moieties of phosphatidylcholine and phosphatidylethanolamine, which is catalysed by PSS (PtdSer synthase) 1 and 2 respectively. PtdSer synthesis in intact cells and an isolated membrane fraction was inhibited by exogenous PtdSer, indicating that feedback control is involved in the regulation of PtdSer biosynthesis. PSS 1 and 2 are similar in amino acid sequence, with an identity of 32%; however, due to a lack of homology with other known enzymes, their amino acid sequences do not provide information on their catalytic and regulatory mechanisms. In the present study, to identify amino acid residues crucial for the activity and/or regulation of PSS 1, we systematically introduced mutations into a Chinese hamster PSS 1 cDNA clone; namely, each of the 66 polar amino acid residues common to PSS 2 was replaced with an alanine residue. On analysis of Chinese hamster ovary cells transfected with each of the alanine mutant clones, we identified eight amino acid residues (His-172, Glu-197, Glu-200, Asn-209, Glu-212, Asp-216, Asp-221 and Asn-226) as those crucial for the enzyme reaction or the maintenance of the correct structure required for serine base-exchange activity. Among these residues, Asn-209 was suggested to be involved in the recognition and/or binding of free L-serine. We also identified six amino acid residues (Arg-95, His-97, Cys-189, Arg-262, Gln-266 and Arg-336) as those important for regulation of PSS 1. In addition, we found that the alanine mutations at Tyr-111, Asp-166, Arg-184, Arg-323, and Glu-364 affected the production and/or stability of PSS 1 in Chinese hamster ovary cells.

scholarly journals Characterisation of Recombinant Human Erythropoietin Obtained from CHOpE—Erythropoietin Producing Strain of Chinese Hamster Ovary Cells

2020 ◽  
Vol 20 (2) ◽  
pp. 116-125
Author(s):  
I. F. Radaeva ◽  
V. A. Ternovoy ◽  
A. O. Sementsova ◽  
N. B. Dumchenko ◽  
E. A. Nechaeva
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
High Molecular Weight ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Amino Acid Sequences ◽  
Producer Strain ◽  
Ovary Cells ◽  
Related Substances

Development and implementation in clinical practice of recombinant human erythropoietins (rhEPOs) remain a priority task today. Additional studies were performed in order to obtain clinical trial authorisation for rhEPO tablets for oral use. The studies were aimed to demonstrate the suitability of the erythropoietin producer strain based on Chinese hamster ovary cells (CHOpE) for the production of rhEPO, and the compliance of the substance characteristics with the requirements for erythropoietin (EPO).The aim of the study was to characterise the rhEPO substance obtained from the CHOpE strain cells in accordance with the requirements for EPO.Materials and methods: the rhEPO substance was obtained by culturing the strain of Chinese hamster ovary cells—CHOpE. The expression construct of the producer strain was evaluated using methods for determination of nucleotide and amino acid sequences. The Sanger method was used to perform sequencing of the nucleotide sequence encoding the human EPO gene. The amino acid sequences of the rhEPO molecule C- and N-termini were determined by the Edman method. The copy number of the EPO gene in CHOpE cells was determined by real-time polymerase chain reaction. The properties of the rhEPO substance were evaluated in accordance with the requirements for EPO. Isoelectric focusing, peptide mapping, and polyacrylamide gel electrophoresis were used for identification of the rhEPO substance. The ratio of isoform composition was determined by capillary electrophoresis. Dimer impurities and high molecular weight related substances were determined by high-pressure liquid chromatography. The content of protein and residual nucleic acids was determined by spectrophotometry. The concentration of the rhEPO substance was assessed by enzyme immunoassay.The results of the study confirmed genetic stability of the CHOpE producer strain and demonstrated identity of N- and C-terminal amino acid sequences of the rhEPO molecule to those of the natural EPO. The CHOpE producer strain was used to obtain a rhEPO substance which is homogenous and does not contain impurities of EPO oligomeric forms. Dimers and high molecular weight related substances account for less than 0.5%. The rhEPO molecular weight ranges from 32 to 38 kDa, and the isoelectric point is within 2.8—4.15. The study identified the peaks of isoforms 1–8, the isoform composition of the rhEPO substance corresponds to that of EPO. It was determined that 1 mol of the substance contains 13.75 mols of sialic acids.Conclusions: the study confirmed the suitability of the CHOpE producer strain for the production of rhEPO. The obtained rhEPO substance meets requirements for EPO. 

scholarly journals A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

1995 ◽  
Vol 6 (2) ◽  
pp. 135-150 ◽  
Author(s):  
N T Ktistakis ◽  
C Y Kao ◽  
R H Wang ◽  
M G Roth
Keyword(s):  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Secretory Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Brefeldin A ◽  
Secretory Activity ◽  
Specific Marker ◽  
Ovary Cells ◽  
Fluorescent Lipid

The use of reporter proteins to study the regulation of secretion has often been complicated by posttranslational processing events that influence the secretion of certain proteins, but are not part of the cellular mechanisms that specifically regulate secretion. This has been a particular limitation for the isolation of mammalian secretion mutants, which has typically been a slow process. To provide a reporter of secretory activity independent of protein processing events, cells were labeled with the fluorescent lipid analogue C5-DMB-ceramide (ceramide coupled to the fluorophore boron dipyrromethene difluoride) and its secretion was followed by fluorescence microscopy and fluorescence-activated cell sorting. Brefeldin A, which severely inhibits secretion in Chinese hamster ovary cells, blocked secretion of C5-DMB-ceramide. At high temperature, export of C5-DMB-ceramide was inhibited in HRP-1 cells, which have a conditional defect in secretion. Using C5-DMB-ceramide as a reporter of secretory activity, several different pulse-chase protocols were designed that selected mutant Chinese hamster ovary cells that were resistant to the drug brefeldin A and others that were defective in the transport of glycoproteins to the cell surface. Mutant cells of either type were identified in a mutagenized population at a frequency of 10(-6). Thus, the fluorescent lipid C5-DMB-ceramide can be used as a specific marker of secretory activity, providing an efficient, general approach for isolating mammalian cells with defects in the secretory pathway.

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells

1986 ◽  
Vol 6 (6) ◽  
pp. 1926-1935
Author(s):  
P J Mitchell ◽  
G Urlaub ◽  
L Chasin
Keyword(s):  
Amino Acid ◽  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Splice Sites ◽  
Ovary Cells ◽  
Splicing Mutations ◽  
Intron 4

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

Choline Chloride Improves the Desiccation Tolerance of Chinese Hamster Ovary Cells

2010 ◽  
Author(s):  
Nilay Chakraborty ◽  
Michael A. Menze ◽  
Heidi Elmoazzen ◽  
Steve C. Hand ◽  
Mehmet Toner
Keyword(s):  
Chinese Hamster Ovary ◽  
Desiccation Tolerance ◽  
Mammalian Cells ◽  
Cell Injury ◽  
Chinese Hamster Ovary Cells ◽  
Choline Chloride ◽  
Chinese Hamster ◽  
Ovary Cells ◽  
Sodium Toxicity ◽  
Ionic Imbalance

Recently there has been much interest in using sugars such as trehalose to preserve mammalian cells in a dry state as an alternative to cryopreservation (1–5). However, some studies indicate that sugars alone may not be sufficient to prevent cell injury during drying. Other factors like sodium toxicity, ionic imbalance and pH excursions during dehydration are a few of the mechanisms that have been hypothesized to decrease the viability of mammalian cells. In the present study, we investigated whether or not substituting sodium chloride with choline chloride (2-hydroxy-N, N,N-trimethylethanaminium chloride) in the preservation medium improves desiccation tolerance of Chinese Hamster Ovary (CHO) cells.

scholarly journals cDNA cloning and characterization of guinea-pig leukotriene B4 receptor

1999 ◽  
Vol 342 (1) ◽  
pp. 79-85 ◽  
Author(s):  
Kazuyuki MASUDA ◽  
Takehiko YOKOMIZO ◽  
Takashi IZUMI ◽  
Takao SHIMIZU
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Leukotriene B4 ◽  
Xenopus Laevis Oocytes ◽  
Amino Acid Residues ◽  
Ovary Cells ◽  
Hek 293 ◽  
Leukotriene B4 Receptor

The cDNA for leukotriene B4 (LTB4) receptor (BLT) was cloned from a guinea-pig leucocyte cDNA library. The cloned receptor cDNA encodes 348 amino acid residues and shares 73% identity with the amino acid sequence of human BLT. Northern blot analysis showed the highest expression of the receptor mRNA in leucocytes, followed by lung and spleen. The membrane fractions of HEK-293 and Cos-7 cells transfected with the cDNA showed specific LTB4-binding activities, with Kd values of 0.27 and 0.17 nM respectively. Xenopus laevis oocytes injected with the cRNA of guinea-pig BLT showed LTB4-induced Cl- currents, indicating that the cloned receptor is functional. LTB4 is metabolized to 20-hydroxy-LTB4 and then to 20-carboxy-LTB4, a transformation considered as a major inactivation pathway of the compound. Using the cloned receptor, we analysed the agonistic effects of LTB4 and these two metabolites. 20-Carboxy-LTB4 is a much weaker agonist, with a Kd value higher than that of LTB4 by three orders of magnitude, corresponding to a much weaker chemotactic activity. Although 20-hydroxy-LTB4 is as potent as LTB4 in inhibiting [3H]LTB4 binding and cAMP formation, it is less potent than LTB4 in the mobilization of intracellular Ca2+ and the chemotaxis of Chinese hamster ovary cells expressing the guinea-pig BLT. The present study demonstrated that although LTB4 and 20-hydroxy-LTB4 bind to the receptor with similar affinities, they do differ in activating intracellular signalling.

Purification of phosphatidylglycerophosphate synthase from Chinese hamster ovary cells

2001 ◽  
Vol 354 (1) ◽  
pp. 9-15 ◽  
Author(s):  
Kiyoshi KAWASAKI ◽  
Osamu KUGE ◽  
Yoshio YAMAKAWA ◽  
Masahiro NISHIJIMA
Keyword(s):  
Chinese Hamster Ovary ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mitochondrial Fraction ◽  
Ovary Cells ◽  
Protein Levels ◽  
Defective Mutant ◽  
Sds Page

Phosphatidylglycerophosphate (PGP) synthase catalyses the committed step in the biosynthesis of phosphatidylglycerol and cardiolipin in mammalian cells. Recently we isolated a Chinese hamster ovary (CHO) PGS1 cDNA encoding PGP synthase. In the present study we purified this PGP synthase to near-homogeneity from the mitochondrial fraction of CHO-K1 cells; the final enzyme preparation gave a single 60kDa protein on SDS/PAGE. Polyclonal antibodies raised against a recombinant CHO PGS1 protein cross-reacted with the purified 60kDa protein and with CHO membrane proteins of 60kDa and 62kDa that increased after transfection with the PGS1 cDNA. The 60 and 62kDa protein levels in a PGP synthase-defective mutant of CHO-K1 cells were markedly lower than those in CHO-K1 cells. These results indicated that the purified 60kDa protein was PGP synthase encoded by the PGS1 gene. In addition we found that the purified PGP synthase had no PGP phosphatase activity, indicating that phosphatidylglycerol was produced from CDP-diacylglycerol through two steps catalysed by distinct enzymes, PGP synthase and PGP phosphatase.

scholarly journals Induction of calreticulin expression in response to amino acid deprivation in Chinese hamster ovary cells

1998 ◽  
Vol 329 (2) ◽  
pp. 389-394 ◽  
Author(s):  
Richard HEAL ◽  
John McGIVAN
Keyword(s):  
Amino Acid ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Asparagine Synthetase ◽  
Mrna Levels ◽  
Amino Acid Deprivation ◽  
Ovary Cells ◽  
Protein Levels

The role of calreticulin as a stress-induced molecular chaperone protein of the endoplasmic reticulum is becoming more apparent. We characterize here the induction of calreticulin in response to complete amino acid deprivation in Chinese hamster ovary cells. Amino acid deprivation caused a 4-fold increase in calreticulin protein levels over a period of 4-10 h. In addition to an overall increase in protein levels, the glycosylation of calreticulin was increased. This glycosylation event was blocked by tunicamycin and was not required for the increase in calreticulin protein levels. Immunofluorescence studies localized calreticulin to the ER of CHO cells, and no significant change was observed after amino acid deprivation. Northern-blot analysis showed that calreticulin mRNA levels were increased approx. 10-fold in response to complete amino acid deprivation. The response was sensitive to actinomycin D and α-amanitin, implying that regulation is primarily at the level of transcription. These results are similar to the large increases in asparagine synthetase mRNA observed in response to amino acid deprivation, but the amino acid-deprivation-response element identified to be involved in asparagine synthetase induction is absent from the calreticulin promoter.

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