scholarly journals Sulphated and undersulphated heparan sulphate proteoglycans in a Chinese hamster ovary cell mutant defective in N-sulphotransferase

1994 ◽  
Vol 303 (1) ◽  
pp. 81-87 ◽  
Author(s):  
K J Bame ◽  
L Zhang ◽  
G David ◽  
J D Esko
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Core Protein ◽  
Heparan Sulphate ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Cell Mutant ◽  
Heparan Sulphate Proteoglycans ◽  
Chain Polymerization

The Chinese hamster ovary cell mutant, pgsE-606, synthesizes undersulphated heparan sulphate glycosaminoglycans because of a deficiency in N-sulphotransferase activity [Bame and Esko (1989) J. Biol. Chem. 264, 8059-8065]. We compared the heparan sulphate proteoglycans synthesized by mutant and wild-type cells to determine what effect the undersulphation defect had on proteoglycan structure. The majority of heparan sulphate proteoglycans synthesized by pgsE-606 were undersulphated, but the mutant also synthesized a population of proteoglycans that were sulphated to the same extent as wild-type molecules. Anion-exchange analysis of the glycosaminoglycans in each proteoglycan population showed that they were all modified in the same way. The length of the glycosaminoglycans in each proteoglycan population were similar, suggesting that N-sulphation does not affect chain polymerization. To examine whether the sulphation state of the attached heparan sulphate glycosaminoglycans was dependent on the protein core, we purified syndecan-1 from mutant and wild-type cells using antibodies against the core protein. As with the unfractionated heparan sulphate proteoglycans, pgsE-606 synthesized both undersulphated and sulphated syndecan-1. Each pool contained either undersulphated or sulphated glycosaminoglycan chains respectively. Thus the modification of all heparan sulphate chains on a core protein occurs on a proteoglycan-wide basis (i.e. to the same extent).

Autoradiographic detection and characterization of a Chinese hamster ovary cell mutant deficient in fucoproteins

1982 ◽  
Vol 111 (3) ◽  
pp. 255-263 ◽  
Author(s):  
Carlos B. Hirschberg ◽  
Mary Perez ◽  
Martin Snider ◽  
Wendy L. Hanneman ◽  
Jeffrey Esko ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Cell Mutant ◽  
Autoradiographic Detection

scholarly journals Isolation and characterization of Chinese hamster ovary cell variants deficient in the expression of fibronectin receptor.

1989 ◽  
Vol 109 (6) ◽  
pp. 3157-3167 ◽  
Author(s):  
C L Schreiner ◽  
J S Bauer ◽  
Y N Danilov ◽  
S Hussein ◽  
M M Sczekan ◽  
...  
Keyword(s):  
Chinese Hamster Ovary Cell ◽  
Chinese Hamster Ovary ◽  
Plasma Membranes ◽  
Cdna Probe ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Wild Type ◽  
Fibronectin Receptor ◽  
Alpha Chain ◽  
Isolation And Characterization

Chinese hamster ovary cell populations were enriched for cells displaying low surface expression of the 140-kD integrin fibronectin receptor (FnR) by means of fluorescence-activated cell sorting using monoclonal anti-FnR antibodies. Selected cells were cloned by limiting dilution, and the resulting clones were screened for low cell surface FnR expression by ELISA. Two multiply sorted populations gave rise to variant clones possessing approximately 20 or 2% FnR expression, respectively, compared with wild-type cells. Growth rates of the "20%" and "2%" clones on serum-coated plastic dishes were similar to that of wild-type cells. Variant cells expressing 20% FnR could attach and spread on substrata coated with purified fibronectin, although somewhat more slowly than wild-type cells, while cells expressing 2% FnR could not attach or spread. Cells from all variant clones attached normally to vitronectin substrata, but some of the 2% clones displayed altered morphology on this type of substratum. Motility assays in blind well chambers showed a correlation of movement with level of expression of FnR. The number of cells migrating in response to fibronectin was greatly reduced compared with wild-type cells for the 20% FnR variant clones, while variant clones with 2% FnR showed virtually no migratory activity. Surface labeling with 125I and immunoaffinity purification of FnR showed reduced levels of intact FnR on the plasma membranes of variants with 20% FnR, while none was detected in variants expressing 2% FnR. Nevertheless, beta subunits were detected on the surfaces of all variant clones. Immunoblots of cell lysates from wild-type cells and from both types of variant clones showed substantial amounts of FnR beta chain as well as enhanced amounts of a pre-beta moiety in the variants. alpha chain was markedly reduced in the 20% variants and essentially absent in the 2% variants, indicating that failure to assemble intact FnR in these variants was due to deficiencies of alpha chain production. Dot blots of total mRNA from a representative clone expressing 20% FnR showed reduced levels of material hybridizing to an 0.97-kb hamster FnR alpha chain cDNA probe as compared with wild type, while mRNA from a representative clone expressing 2% FnR had no detectable hybridizable RNA; this seems to agree well with the results obtained by immunoblotting. Thus, the defect in the variant clones seems to be due to reduced levels of alpha chain mRNA leading to a deficit of mature FnR and consequent alterations in cell adhesion and motility on fibronectin substrata.

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