scholarly journals Partial purification of heparanase activities in Chinese hamster ovary cells: evidence for multiple intracellular heparanases

1998 ◽  
Vol 336 (1) ◽  
pp. 191-200 ◽  
Author(s):  
Karen J. BAME ◽  
Alan HASSALL ◽  
Crystal SANDERSON ◽  
Indumati VENKATESAN ◽  
Chao SUN
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Heparan Sulphate ◽  
Gel Filtration ◽  
Chinese Hamster ◽  
Basement Membranes ◽  
Partial Purification ◽  
Core Proteins ◽  
Exchange Resins

Heparanases are mammalian endoglycosidases that cleave heparan sulphate glycosaminoglycans from proteoglycan core proteins and degrade them into shorter chains. The enzymes have been proposed to act in a variety of cellular processes, including proteoglycan catabolism, remodelling of basement membranes and release of heparan sulphate-binding ligands from their extracellular storage sites. Additional functions for heparanases may be to generate short heparan sulphate chains that stabilize or activate other proteins. While heparanase activities have been described in a number of tissues and cell lines, it is not known how many different enzymes are responsible for these activities. Our recent studies characterizing the short glycosaminoglycans produced in Chinese hamster ovary (CHO) cells suggested that multiple heparanases are necessary for the formation of the short heparan sulphate chains [Bame and Robson (1997) J. Biol. Chem. 272, 2245–2251]. We examined whether this is the case by purifying heparanase activity from CHO cell homogenates. Based on their ability to bind ion-exchange resins and their elution from gel-filtration columns, four separate heparanase activities were partially purified. All four activities cleave free glycosaminoglycans over a broad pH range of 3.5–6.0 or 6.5, suggesting that they act in the endosomal/lysosomal pathway. The sizes of the short heparan sulphate chains generated by the partially purified heparanases ranged from 6 to 9 kDa, and for two of the activities the product size is pH-dependent. Three of the four activities degrade proteoglycans as well as the free glycosaminoglycan chain. Interestingly, all four enzymes generate short glycosaminoglycans with a sulphate-rich, modified domain at the non-reducing end of the newly formed chain. Since our previous studies showed that in CHO cells there is also a population of short heparan sulphates with a modified domain at the reducing end of the chain, this suggests that there may be another heparanase in CHO cells that was not purified. Alternatively, our findings suggest that the formation of short heparan sulphate glycosaminoglycans inside CHO cells may be a result of the concerted action of multiple heparanases, and may depend on the proportions of the different enzymes and the environment in which the chains are degraded.

Effect of lipoprotein lipase on binding of chylomicrons to LDL receptor-deficient Chinese hamster ovary cells

2001 ◽  
Vol 38 (2) ◽  
pp. 124-128 ◽  
Author(s):  
Junji Kobayashi ◽  
Jun Tashiro ◽  
Hideaki Bujo ◽  
Nobuhiro Morisaki
Keyword(s):  
Enzymatic Activity ◽  
Lipoprotein Lipase ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Specific Binding ◽  
Chinese Hamster Ovary Cells ◽  
Heparan Sulphate ◽  
Bovine Milk ◽  
Chinese Hamster ◽  
Carboxyl Terminal

The authors investigated the binding of human plasma 125I-labelled chylomicrons to Chinese hamster ovary (CHO) cells, i.e. native CHO cells are mutant ldl-A7 cells lacking the low-density lipoproteins receptor, in the absence and presence of exogenous bovine milk lipoprotein lipase (LPL) in the culture medium. Only a small amount of binding to either cell was observed in the absence of added LPL. Exogenously added LPL increased the specific binding of chylomicrons to ldlA7 cells, as well as to native CHO cells. The enhanced binding of chylomicrons to ldl-A7 cells or native CHO cells by LPL was inhibited by heparinase and a monoclonal antibody against LPL (5D2) which recognizes the carboxyl terminal of LPL. However, the enhanced binding was not inhibited by 1M NaCl, which abolishes the enzymatic activity of LPL in either ldl-A7 cell or native CHO cells. These results suggest that LPL enhances the binding of chylomicrons to heparan sulphate proteoglycans of CHO cells, and that it is the carboxyl terminal of LPL but not the enzymatic activity of LPL that is essential for LPL to mediate the binding of chylomicrons to CHO cells.

Review of the current methods of Chinese Hamster Ovary (CHO) cells cultivation for production of therapeutic protein

2020 ◽  
Vol 17 ◽  
Author(s):  
Shazid Md. Sharker ◽  
Md. Atiqur Rahman
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Mass Production ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Therapeutic Protein ◽  
Specific Productivity ◽  
Ovary Cells ◽  
Further Development ◽  
Interesting Area

Most of clinical approved protein-based drugs or under in clinical trial have a profound impact in the treatment of critical diseases. The mammalian eukaryotic cells culture approaches, particularly the CHO (Chinese Hamster Ovary) cells are mainly used in the biopharmaceutical industry for the mass-production of therapeutic protein. Recent advances in CHO cell bioprocessing to yield recombinant proteins and monoclonal antibodies have enabled the expression of quality protein. The developments of cell lines are possible to upgrade specific productivity. As a result, it holds an interesting area for academic as well as industrial researchers around the world. This review will concentrate on the recent progress of the mammalian CHO cells culture technology and the future scope of further development for the mass-production of protein therapeutics.

scholarly journals Cloning and characterization of small circular DNA from Chinese hamster ovary cells.

1984 ◽  
Vol 4 (1) ◽  
pp. 173-180 ◽  
Author(s):  
S W Stanfield ◽  
D R Helinski
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Single Copy ◽  
Chromosomal Dna ◽  
Cho Cell ◽  
Ovary Cells ◽  
Chromosomal Sequences

Small polydisperse circular (spc) DNA was isolated and cloned, using BglII from Chinese hamster ovary (CHO) cells. The properties of 47 clones containing at least 43 different BglII fragments are reported. The majority of the clones probably contain entire sequences from individual spcDNA molecules. Most of the clones were homologous to sequences in CHO cell chromosomal DNA, and many were also homologous to mouse LMTK- cell chromosomal sequences. The majority of homologous CHO cell chromosomal sequences were repetitive, although a few may be single copy. Only a small fraction of cloned spcDNA molecules were present in every cell; most occurred less frequently than once in 15 cells. Localization studies indicated that at least a portion of spcDNA is associated with the nucleus in CHO cells.

Expression of human Mn SOD in Chinese hamster ovary cells confers protection from oxidant injury

1993 ◽  
Vol 264 (6) ◽  
pp. L598-L605
Author(s):  
B. Warner ◽  
R. Papes ◽  
M. Heile ◽  
D. Spitz ◽  
J. Wispe
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Lethal Dose ◽  
Chinese Hamster ◽  
Eukaryotic Expression ◽  
Wild Type ◽  
Oxidant Injury ◽  
Sod Activity ◽  
Recombinant Cho Cells

Manganese superoxide dismutase (Mn SOD) is an important component of antioxidant defense in aerobic cells because of its location in the mitochondria, a significant source of oxygen radicals and an important target of oxidant injury. To test the hypothesis that increased mitochondrial Mn SOD protects from oxidant injury, Chinese hamster ovary (CHO) cells were transfected with a eukaryotic expression vector containing the human Mn SOD cDNA. In recombinant CHO cells, Mn SOD activity was increased threefold over wild-type controls. Acute survival during paraquat exposure (0–500 microM) was significantly improved in CHO cells expressing human Mn SOD, with 71% of recombinant CHO cells surviving at the 50% lethal dose (LD50) for wild-type CHO controls. Cell growth following exposure to paraquat (100 microM) was also significantly improved in recombinant CHO cells. CHO cells expressing human Mn SOD continued to grow and divide after paraquat exposure, whereas growth of wild-type CHO cells was negligible. Protection against oxidant-induced injury was directly related to increased Mn SOD, occurring in the absence of changes in other antioxidant enzymes including catalase, Cu,Zn SOD, and glutathione associated cellular antioxidant mechanisms. We conclude that increased expression of human Mn SOD in vitro directly confers protection against oxidant injury.

scholarly journals Continued initiation of DNA synthesis in arginine-deprived Chinese hamster ovary cells.

1977 ◽  
Vol 73 (1) ◽  
pp. 200-205 ◽  
Author(s):  
A S Weissfeld ◽  
H Rouse
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Multiplication ◽  
Ovary Cells ◽  
Cell Cycle Phases

When exponentially growing CHO cells were deprived of arginine (Arg), cell multiplication ceased after 12 h, but initiation of DNA synthesis continued: after 48 h of starvation with continuous [3H]thymidine exposure, 85% of the population had incorporated label, as detected autoradiographically. Consideration of the distribution of exponential cells in the various cell cycle phases leads to a calculation that most cells in G1 at the time that Arg was removed, as well as those in S, engaged in some DNA synthesis during starvation. In contrast, isoleucine (Ile)-starved cells did not initiate DNA synthesis, as has been reported by others. Experiments with cells synchronized by mitotic selection confirmed this difference in Arg- and Ile- deprived behavior, but also showed that cells which underwent the mitosis leads to G1 transition during Arg starvation remained arrested in G1 (G0?). The results suggest that Arg-deprived cells continue to maintain some proliferative function(s) while Ile-deprived cells do not.

scholarly journals Kinetics of endosome acidification in mutant and wild-type Chinese hamster ovary cells.

1987 ◽  
Vol 105 (6) ◽  
pp. 2713-2721 ◽  
Author(s):  
D J Yamashiro ◽  
F R Maxfield
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Ph Value ◽  
Ph Regulation ◽  
Chinese Hamster Ovary Cells ◽  
Mutant Cell ◽  
Chinese Hamster ◽  
Wild Type ◽  
Cell Biol ◽  
Endocytic Compartments

Acidification of endocytic compartments is necessary for the proper sorting and processing of many ligands and their receptors. Robbins and co-workers have obtained Chinese hamster ovary (CHO) cell mutants that are pleiotropically defective in endocytosis and deficient in ATP-dependent acidification of endosomes isolated by density centrifugation (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308). In this and the following paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2723-2733) we describe detailed studies of endosome acidification in the mutant and wild-type CHO cells. Here we describe a new microspectrofluorometry method based on changes in fluorescein fluorescence when all cellular compartments are equilibrated to the same pH value. Using this method we measured the pH of endocytic compartments during the first minutes of endocytosis. We found in wild-type CHO cells that after 3 min, fluorescein-labeled dextran (F-Dex) was in endosomes having an average pH of 6.3. By 10 min, both F-Dex and fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) had reached acidic endosomes having an average pH of 6.0 or below. In contrast, endosome acidification in the CHO mutants DTG 1-5-4 and DTF 1-5-1 was markedly slowed. The average endosomal pH after 5 min was 6.7 in both mutant cell lines. At least 15 min was required for F-Dex and F-alpha 2M to reach an average pH of 6.0 in DTG 1-5-4. Acidification of early endocytic compartments is defective in the CHO mutants DTG 1-5-4 and DTF 1-5-1, but pH regulation of later compartments on both the recycling pathway and lysosomal pathway is nearly normal. The properties of the mutant cells suggest that proper functioning of pH regulatory mechanisms in early endocytic compartments is critical for many pH-mediated processes of endocytosis.

scholarly journals Chinese hamster ovary cells replicate adenovirus deoxyribonucleic acid.

1981 ◽  
Vol 1 (3) ◽  
pp. 208-215 ◽  
Author(s):  
M Longiaru ◽  
M S Horwitz
Keyword(s):  
Dna Synthesis ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Deoxyribonucleic Acid ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Adenovirus Type ◽  
Viral Dna ◽  
Nuclear Extracts

Chinese hamster ovary (CHO) cells infected with adenovirus type 2 (Ad2) produced amounts of viral deoxyribonucleic acid (DNA) equal to that synthesized in permissively infected HeLa cells. However, there was 6,000-fold less virion produced in CHO cells. Since the structural viral polypeptides were not detected by pulse-labeling CHO cells at various times postinfection, the block in virion formation is located between the synthesis of viral DNA and late proteins. Extracts of CHO cells could also function in a recently reported in vitro Ad2 DNA synthesis system which is dependent upon the addition of exogenous Ad2 DNA covalently linked to a 5'-terminal protein (Ikeda et al., Proc. Natl. Acad. Sci. U.S.A. 77:5827-5831, 1980). Extracts of infected CHO cytoplasm were able to complement uninfected CHO nuclear extracts to synthesize viral DNA on Ad2 templates. This in vitro replication system has the potential to probe host DNA synthesis requirements as well as viral factors.

scholarly journals Characterization of a novel intracellular heparanase that has a FERM domain

2002 ◽  
Vol 364 (1) ◽  
pp. 265-274 ◽  
Author(s):  
Karen J. BAME ◽  
Indumati VENKATESAN ◽  
Jean DEHDASHTI ◽  
Jeffrey McFARLANE ◽  
Rebecca BURFEIND
Keyword(s):  
Cho Cells ◽  
Heparan Sulphate ◽  
Chinese Hamster ◽  
Protein Activity ◽  
Western Blots ◽  
Ferm Domain ◽  
Erm Proteins ◽  
Cytoplasmic Proteins ◽  
Core Proteins ◽  
Major Species

The catabolism of cell-surface heparan sulphate proteoglycans is initiated by endosomal heparanases, which are endoglycosidases that cleave the glycosaminoglycans off core proteins and degrade them to shorter oligosaccharides. We have purified previously four intracellular heparanase activities from Chinese hamster ovary (CHO) cells [Bame, Hassall, Sanderson, Venkatesan and Sun (1998) Biochem. J. 336, 191–200], and in the present study we characterize further the most abundant activity (C1A heparanase). This enzyme purifies as a family of 37–48kDa proteins from both CHO cells and the rat liver, with the major species being 37 and 40kDa. Amino acid sequence analysis shows the purified C1A heparanase protein is highly homologous with the N-terminal domain, or FERM domain, of the ≈80kDa proteins ezrin, radixin and moesin (ERM proteins, after ezrin-radixin-moesin). This domain, which is also found in erythrocyte protein 4.1, links cytoplasmic proteins to membranes. Antibodies against the FERM domain recognize all the C1A heparanase proteins on Western blots, suggesting that the smaller species are derived from a larger protein. Activity binds to, and is affected by, molecules known to interact with FERM domains, supporting the hypothesis that the intracellular C1A heparanase is the purified FERM domain protein. Since bacterially expressed FERM domains of radixin and moesin lack heparanase activity, and some tryptic peptides generated from the enzyme do not have a match in any ERM protein, it appears that, rather than being derived from ezrin, radixin or moesin, C1A heparanase may be a new member of the FERM domain family.

Partial purification of centrosomes from Chinese hamster ovary cells

1978 ◽  
Vol 113 (1) ◽  
pp. 183-187 ◽  
Author(s):  
Gary R. Blackburn ◽  
Marny D. Barrau ◽  
William C. Dewey
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Partial Purification ◽  
Ovary Cells
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