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.

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.

Interactions of bovine viral diarrhoea virus glycoprotein Erns with cell surface glycosaminoglycans

Microbiology ◽  
2000 ◽  
Vol 81 (2) ◽  
pp. 451-459 ◽  
Author(s):  
Munir Iqbal ◽  
Helen Flick-Smith ◽  
John W. McCauley
Keyword(s):  
Cho Cells ◽  
Chondroitin Sulphate ◽  
Heparan Sulphate ◽  
Flow Cytometric Analysis ◽  
Chinese Hamster ◽  
Bovine Viral Diarrhoea Virus ◽  
Bovine Viral Diarrhoea ◽  
High Concentration ◽  
Mammalian Cell Lines ◽  
Diarrhoea Virus

Recombinant Erns glycoprotein of bovine viral diarrhoea virus (BVDV) has been tagged with a marker epitope or linked to an immunoglobulin Fc tail and expressed in insect and mammalian cell lines. The product was shown to be functional, both having ribonuclease activity and binding to a variety of cells that were permissive and non-permissive for replication of BVDV. Addition of soluble Erns to the medium blocked replication of BVDV in permissive cells. Binding of epitope-tagged Erns to permissive calf testes (CTe) cells was abolished and virus infection was reduced when cells were treated with heparinases I or III. Erns failed to bind to mutant Chinese hamster ovary (CHO) cells that lacked glycosaminoglycans (pgsA-745 cells) or heparan sulphate (pgsD-677 cells) but bound to normal CHO cells. Erns also bound to heparin immobilized on agarose and could be eluted by heparin and by a high concentration of salt. Flow cytometric analysis of Erns binding to CTe cell cultures showed that glycosaminoglycans such as heparin, fucoidan and dermatan sulphate all inhibit binding but dextran sulphate, keratan sulphate, chondroitin sulphate and mannan fail to inhibit binding. The low molecular mass polysulphonated inhibitor suramin also inhibited binding to CTe cells but poly-l-lysine did not. Furthermore, suramin, the suramin analogue CPD14, fucoidan and pentosan polysulphate inhibited the infectivity of virus. It is proposed that binding of Erns to cells is through an interaction with glycosaminoglycans and that BVDV may bind to cells initially through this interaction.

scholarly journals Recombinant human sulphamidase: expression, amplification, purification and characterization

1998 ◽  
Vol 329 (1) ◽  
pp. 145-150 ◽  
Author(s):  
Julie BIELICKI ◽  
J. John HOPWOOD ◽  
L. Elizabeth MELVILLE ◽  
S. Donald ANSON
Keyword(s):  
Cell Lines ◽  
Cho Cells ◽  
Expression System ◽  
Heparan Sulphate ◽  
Chinese Hamster ◽  
Lysosomal Storage ◽  
Dhfr Gene ◽  
Enzyme Replacement ◽  
High Level ◽  
Mps Iiia

Mucopolysaccharidosis type IIIA (MPS IIIA, Sanfilippo A syndrome) is a lysosomal storage disease that causes a profound neurological deterioration. The disorder is caused by a deficiency of the lysosomal enzyme sulphamidase which is a requisite for the degradation of heparan sulphate. To facilitate the development of enzyme-replacement strategies for MPS IIIA patients, we have constructed a high-level expression system for recombinant human sulphamidase in Chinese hamster ovary (CHO) cells. An expression construct containing a methotrexate-resistant dihydrofolate reductase (DHFR) gene allowed amplification of expression levels from less than 1 mg of sulphamidase per litre of culture medium to approx. 15 mg/l. Unlike many cell lines made by gene amplification in DHFR-deficient CHO cells, and utilizing the normal DHFR gene, these cell lines appeared to be stable in the absence of selective pressure. Recombinant human sulphamidase was purified from unamplified and amplified cell lines. The native enzyme was found to be a dimer of 115 kDa. Denaturing and reducing SDS/PAGE revealed a subunit size of 62 kDa. Kinetic analysis demonstrated that the recombinant enzyme had broadly similar kinetic characteristics to sulphamidase purified from liver. Recombinant human sulphamidase was able to correct the storage phenotype of MPS IIIA fibroblasts after endocytosis via the mannose-6-phosphate receptor.

scholarly journals Identification of a calicivirus isolate of unknown origin

2003 ◽  
Vol 84 (10) ◽  
pp. 2837-2845 ◽  
Author(s):  
Angelika Oehmig ◽  
Mathias Büttner ◽  
Frank Weiland ◽  
William Werz ◽  
Klaus Bergemann ◽  
...  
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Unknown Origin ◽  
Open Reading Frames ◽  
Coding Region ◽  
Electron Microscopic ◽  
Significant Similarity ◽  
Western Blots ◽  
Reading Frame ◽  
A Minor

Chinese hamster ovary (CHO) cells manifesting striking cytopathogenic changes in culture were investigated to determine the causative agent. Electron microscopic analyses revealed viral particles of about 40 nm in diameter, displaying typical calicivirus morphology. To date, this virus, designated isolate 2117, exclusively replicates in CHO cells, achieving only moderate titres. After cloning, the coding region of 7928 nucleotides, the 3′ non-coding region and the poly(A) tail were sequenced. The genome consists of three open reading frames (ORFs), with the first and second ORF having the same reading frame. The overall genomic organization as well as the nucleotide sequence of isolate 2117 is most similar to that of a recently described canine calicivirus, but also shows significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus. In Western blots, using antibodies against the viral protease, a stable, unprocessed 3CD protein of 68 kDa was identified in homogenates of 2117-infected CHO cells. Furthermore, antibodies raised against ORF 3 reacted with the respective protein in 2117-virions, demonstrating that this predicted 9 kDa protein is a minor structural component of the virion. In addition, an RT-PCR assay was established to detect 2117 viral RNA in biological products such as foetal bovine serum, which will aid the discovery of the origin and host of the virus.

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.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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 Recombinant γY278H Fibrinogen Showed Normal Secretion from CHO Cells, but a Corresponding Heterozygous Patient Showed Hypofibrinogenemia

2021 ◽  
Vol 22 (10) ◽  
pp. 5218
Author(s):  
Tomu Kamijo ◽  
Takahiro Kaido ◽  
Masahiro Yoda ◽  
Shinpei Arai ◽  
Kazuyoshi Yamauchi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cho Cells ◽  
Fibrinogen Level ◽  
Culture Media ◽  
Chinese Hamster ◽  
Plasma Fibrinogen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cho Cell ◽  
Accelerated Degradation ◽  
Heterozygous Patient

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients’ plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and “D:D” interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient’s hepatocytes but then underwent accelerated degradation by plasmin in the circulation.

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