Chinese Hamster Ovary Cell Mutants Defective in Glycosaminoglycan Assembly and Glucuronosyltransferase I

Bai, Xiaomei; Ge, Wei; Sinha, Anjana; Esko, Jeffrey D.

doi:10.1074/jbc.274.19.13017

Cited by 103 publications

(96 citation statements)

References 86 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…cDNAs encoding human GalT-I (16,17) and human/mouse GalT-II (18) have been cloned based on the analysis of expressed sequence tags (EST). cDNAs for human and hamster GlcAT-I have been cloned based on the sequence homology to rat GlcAT-P that synthesizes the HNK-1 carbohydrate epitope on glycoproteins (19) and by expression cloning, respectively (20). Transcripts of the four genes are differentially yet broadly expressed in human tissues (15,17,18,21), consistent with the ubiquitous distribution of GAGs (Table 1).…”

Section: Formation Of the Glycosaminoglycan-protein Linkage Regionmentioning

confidence: 93%

Heparin and Heparan Sulfate Biosynthesis

2002

View full text Add to dashboard Cite

SummaryHeparan sulfate is one of the most informationally rich biopolymers in Nature. Its simple sugar backbone is variously modified to different degrees depending on the cellular conditions. Thus, it matures to have an enormously complicated structure, which most likely exhibits a considerable number of unique overlapping sequences with peculiar sulfation profiles. Such sequences are recognized by specific complementary proteins, which form a huge group of "heparin-binding proteins," and the sugar sequences in turn support unique functions of the respective proteins through specific interactions. The heparan sulfate sequences are not directly encoded by genes, but are created by elaborate biosynthetic mechanisms, which ensure the generation of these indispensable sequences. In heparan sulfate biosynthesis, the tetrasaccharide sequence (GlcAGal-Gal-Xyl-), designated the protein linkage region, is first assembled on a specific Ser residue at the glycosaminoglycan attachment site of a core protein. A heparan sulfate chain is then polymerized on this fragment by alternate additions of GlcNAc and GlcA through the actions of glycosyltransferases with overlapping specificities encoded by the tumor suppressor EXT family genes. Then follow various modifications by N-deacetylation and N-sulfation of glucosamine, C5-epimerization of GlcA and multiple O-sulfations of the component sugars. Recent studies have achieved purification of several, and molecular cloning of most, of the enzymes responsible for these reactions. Some of these enzymes are bifunctional. The availability of cDNA probes has facilitated elucidation of the crystal structures for two of the biosynthetic enzymes, demonstration of their intracellular location, and their occurrence in complexes to achieve rapid and efficient synthesis of complex sugar sequences. Genomic structure and transcript analysis have shown the existence of multiple isoforms for most of the sulfotransferases. Many aspects of the heparan sulfate biosynthetic scheme are shared by the structural analog heparin, which is synthesized in mast cells and some other mammalian cells and is several-fold higher degree of polymerization and more extensive modification than heparan sulfate.

show abstract

Section: Formation Of the Glycosaminoglycan-protein Linkage Regionmentioning

confidence: 93%

Heparin and Heparan Sulfate Biosynthesis

2002

View full text Add to dashboard Cite

show abstract

“…Flow Cytometry Analyses with FGF-2-This assay was performed as described previously (19). Biotinylated fibroblast growth factor 2 (FGF-2) was graciously provided by Dr. Jeff Esko of the University of California at San Diego.…”

Section: Northern Blot Rt-pcr and Real-time Rt-pcr-poly(a)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

View full text Add to dashboard Cite

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...

show abstract

“…Xylosyltransferase (2), galactosyltransferase I (GalTI) 1 (3,4), and glucuronosyltransferase (GlcATI) (5,6) have been cloned and partially characterized. In addition, mutant cell lines have been identified that contain defects in each of these enzymatic steps (7)(8)(9), and C. elegans mutants in GalTI (sqv3) and the GlcATI (sqv8) have been described, demonstrating the importance of these reactions and glycosaminoglycans in cellular processes and organismal development (10,11). The enzyme that transfers the second galactose unit, galactosyltransferase II (GalTII) has not yet been identified.…”

mentioning

confidence: 99%

Biosynthesis of the Linkage Region of Glycosaminoglycans

Bai

Zhou

Brown

et al. 2001

Journal of Biological Chemistry

Self Cite

162

View full text Add to dashboard Cite

A family of five ␤1,3-galactosyltransferases has been characterized that catalyze the formation of Gal␤1, 3GlcNAc␤ and Gal␤1,3GalNAc␤ linkages present in glycoproteins and glycolipids (␤3GalT1, -2, -3, -4, and -5). We now report a new member of the family (␤3GalT6), involved in glycosaminoglycan biosynthesis. The human and mouse genes were located on chromosomes 1p36.3 and 4E2, respectively, and homologs are found in Drosophila melanogaster and Caenorhabditis elegans. Unlike other members of the family, ␤3GalT6 showed a broad mRNA expression pattern by Northern blot analysis. Although a high degree of homology across several subdomains exists among other members of the ␤3-galactosyltransferase family, recombinant enzyme did not utilize glucosamine-or galactosamine-containing acceptors. Instead, the enzyme transferred galactose from UDP-galactose to acceptors containing a terminal ␤-linked galactose residue. This product, Gal␤1,3Gal␤ is found in the linkage region of heparan sulfate and chondroitin sulfate (GlcA␤1,3Gal␤1,3Gal␤1,4Xyl␤-O-Ser), indicating that ␤3GalT6 is the so-called galactosyltransferase II involved in glycosaminoglycan biosynthesis. Its identity was confirmed in vivo by siRNA-mediated inhibition of glycosaminoglycan synthesis in HeLa S3 cells.

show abstract

Chinese Hamster Ovary Cell Mutants Defective in Glycosaminoglycan Assembly and Glucuronosyltransferase I

Abstract: The proteoglycans of animal cells typically contain one or more heparan sulfate or chondroitin sulfate chains. These glycosaminoglycans assemble on a tetrasaccharide primer, -GlcA␤1,3Gal␤1,3Gal␤1,4Xyl␤-O-, attached to specific serine residues in the core protein.

Cited by 103 publications

References 86 publications

Heparin and Heparan Sulfate Biosynthesis

Heparin and Heparan Sulfate Biosynthesis

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

Biosynthesis of the Linkage Region of Glycosaminoglycans

Contact Info

Product

Resources

About