Animal cell mutants defective in glycosaminoglycan biosynthesis.

Esko, Jeffrey D.; Stewart, Tod E.; Taylor, W. Herman

doi:10.1073/pnas.82.10.3197

Cited by 543 publications

(587 citation statements)

References 32 publications

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“…However, KCP mutant Lys64Gln/Lys65Gln/Lys88Gln, that was defective in heparin binding, was not capable of binding to the cell surface. Also, KCP could not bind to CHO pgsA-745, which are deficient in xylosyltransferase and therefore do not produce glycosaminoglycans (Esko et al, 1985). The cell surface binding of KCP was ionic in nature, as shown by fig.…”

Section: Kcp Binds To Cell Surface Glycosaminoglycans Via the Heparinmentioning

confidence: 98%

“…CHO pgsA-745 cells, which are deficient in xylosyltransferase and do not produce glycosaminoglycans, (Esko et al, 1985) were received from Dr Dick Heinegård. The cells were propagated in F-12K Nutrient Mixture (Kaighn's Modification, GIBCO-Invitrogen, Carlsbad, USA) supplemented with 10% fetal calf serum and 50 U/ml pencillin and 50 µg/ml streptavidin.…”

Section: Cellsmentioning

confidence: 99%

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The Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) binds to heparin and cell surfaces via positively charged amino acids in CCP1–2

Mark

Lee

Spiller

et al. 2006

Molecular Immunology

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Section: Kcp Binds To Cell Surface Glycosaminoglycans Via the Heparinmentioning

confidence: 98%

Section: Cellsmentioning

confidence: 99%

The Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) binds to heparin and cell surfaces via positively charged amino acids in CCP1–2

Mark

Lee

Spiller

et al. 2006

Molecular Immunology

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“…XYLT2 encodes for xylosyltransferase II (XylT2), which when expressed in vitro failed to have xylosyltransferase activity (13,14), and therefore, its function is unknown. A highly useful tool for the study of PGs has been the xylosyltransferase-deficient Chinese hamster ovary cell (CHO) line pgsA-745 (15,16). This cell line was isolated from an ethylmethane sulfonate mutagenesis screen of CHO-K1 cells for sulfation incorporation mutants.…”

mentioning

confidence: 99%

Biosynthesis of Chondroitin and Heparan Sulfate in Chinese Hamster Ovary Cells Depends on Xylosyltransferase II

Cuellar

Chuong

Hubbell

et al. 2007

Journal of Biological Chemistry

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Xylosyltransferase (XylT) catalyzes the initial enzymatic reaction in the glycosaminoglycan assembly pathway for proteoglycan biosynthesis. Its activity is thought to be rate-limiting. Two xylosyltransferases have been found using genomic analyses, and one of these, XylT1, has been shown to have xylosyltransferase activity. On the other hand, the less studied XylT2 in recombinant form lacks xylosyltransferase activity and has no known function. Wild-type Chinese hamster ovary cells express abundant Xylt2 mRNA levels and lack detectable Xylt1 mRNA levels. Analysis of a previously described Chinese hamster ovary cell xylosyltransferase mutant (psgA-745) shows that it harbors an Xylt2 nonsense mutation and fails to assemble glycosaminoglycans onto recombinant biglycan. Transfection of this cell line with a murine Xylt2 minigene results in the production of recombinant chondroitin sulfate-modified biglycan core protein and restoration of fibroblast growth factor binding to cell surface-associated heparan sulfate. Expression analyses on 10 different human transformed cell lines detect exclusive XYLT2 expression in two and co-expression of XYLT1 and XYLT2 in the others but at disparate ratios where XYLT2 expression is greater than XYLT1 in most cell lines. These results indicate that XylT2 has a significant role in proteoglycan biosynthesis and that cell type may control which family member is utilized.Xylosyltransferase (UDP-D-xylose:protein ␤-D-xylosyltransferase, EC 2.4.2.26) (XylT) 3 activity was first isolated and studied in the early 1960s. This enzyme was one of the first glycosyltransferases isolated (1). It catalyzes the initial enzymatic reaction in the assembly of glycosaminoglycans to core proteins. This addition of a xylose to a designated serine is hypothesized to be rate-limiting for proteoglycan (PG) biosynthesis (2, 3). PGs are found on the cell surface and in the extracellular matrix. PGs are fundamental to cellular processes including enhancement of receptor binding of cytokines and growth factors and augmentation of enzyme-substrate interactions important in coagulation and lipid catabolism (4 -9). Extracellular matrix PGs maintain basement membrane integrity, augment growth factor and cytokine sequestration, and create morphogen gradients (4, 10 -12). Genomic analyses have identified two xylosyltransferase family members in mammals. XYLT1 encodes for xylosyltransferase I (XylT1), shown to have xylosyltransferase activity (13). XYLT2 encodes for xylosyltransferase II (XylT2), which when expressed in vitro failed to have xylosyltransferase activity (13,14), and therefore, its function is unknown. A highly useful tool for the study of PGs has been the xylosyltransferase-deficient Chinese hamster ovary cell (CHO) line pgsA-745 (15, 16). This cell line was isolated from an ethylmethane sulfonate mutagenesis screen of CHO-K1 cells for sulfation incorporation mutants. This cell line produces neither chondroitin nor heparan-sulfated PGs (15, 16). In our long term investigation of this enzymatic pathway an...

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“…The pgsA-745 is a mutant cell line deficient in xylosyltransferase, and was established from CHO-K1 cells (wild type) [22]. In the biosynthesis of CS and HS, a xylosyltransferase initiates the formation of the tetrasaccharide linkage region by transferring a Xyl residue to a specific Ser residue of the core protein.…”

Section: Analysis Of the Interactions Of 4e1/d6 With Cho Cell Lines Dmentioning

confidence: 99%

“…The CHO mutant cells, which are deficient in the glycosyltransferases that synthesize the linkage region tetrasaccharide core, cannot synthesize either CS/DS or HS/Hep, suggesting the same glycosyltransferases synthesize the linkage region common to both types of the GAG chains [22,47,48]. Therefore, the types of GAG chains to be selectively assembled on the linkage region tetrasaccharide are determined after or during the construction of the linkage region by these enzymes.…”

Section: Galactosaminoglycans (Cs/ds) and Glucosaminoglycans (Hs/hep)mentioning

confidence: 99%

Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage

et al. 2010

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Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage region, GlcA1-3Gal1-3Gal1-4Xyl1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans, and one was characterized in detail. This antibody bound hexa-and pentasaccharide-peptides more strongly than tetrasaccharide-peptides, suggesting the importance of 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 Xyl-Ser linkage completely abolished the binding activity, suggesting the importance of the Xy-Ser linkage for the binding. Furthermore, the antibody stained wild-type CHO cells, but not mutant cells deficient in xylosyltransferase required for the synthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc-GlcA-Gal-Gal-Xyl-Ser that is modified by 6-O-sulfation on GalNAc and/or Gal. The antibody will be a useful tool for investigating the significance of the linkage region in the biosynthesis and/or intracellular transport of different GAG chains.

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Animal cell mutants defective in glycosaminoglycan biosynthesis.

Abstract: We have obtained Chinese hamster ovary cell mutants defective in the biosynthesis of glycosaminoglycans by screening replicate colonies immobilized on polyester cloth. Depending upon the strain, the mutants accumulated less 35S-labeled glycosaminoglycans per ,ug of cell protein by a

Cited by 543 publications

References 32 publications

The Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) binds to heparin and cell surfaces via positively charged amino acids in CCP1–2

The Kaposi's sarcoma-associated herpesvirus complement control protein (KCP) binds to heparin and cell surfaces via positively charged amino acids in CCP1–2

Biosynthesis of Chondroitin and Heparan Sulfate in Chinese Hamster Ovary Cells Depends on Xylosyltransferase II

Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage

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