Animal cell mutants defective in glycosaminoglycan biosynthesis.
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Journal: 1985/June - Proceedings of the National Academy of Sciences of the United States of America
ISSN: 0027-8424
PUBMED: 3858816
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 microgram of cell protein by a factor of 6-60 compared to the wild type. Some of the mutants incorporated [6-3H]glucosamine into glycosaminoglycans to the same extent as the wild type, suggesting that sulfate addition was specifically altered. In contrast, five strains failed to generate 3H-labeled glycosaminoglycans normally. In four of these, the initiation of glycosaminoglycan assembly was specifically altered, since the addition of p-nitrophenyl-beta-xyloside restored sulfation to normal. Enzymatic assay of the xylosyltransferase in extracts prepared from these mutants revealed that one of the strains, S745, contained less enzyme activity by a factor of 15 than the wild type. This mutant provides genetic evidence that the xylosyltransferase assayed in vitro is responsible for the initiation of chondroitin sulfate and heparan sulfate biosynthesis in vivo.
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Proc Natl Acad Sci U S A 82(10): 3197-3201
PMID: 3858816

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 microgram of cell protein by a factor of 6-60 compared to the wild type. Some of the mutants incorporated [6-3H]glucosamine into glycosaminoglycans to the same extent as the wild type, suggesting that sulfate addition was specifically altered. In contrast, five strains failed to generate 3H-labeled glycosaminoglycans normally. In four of these, the initiation of glycosaminoglycan assembly was specifically altered, since the addition of p-nitrophenyl-beta-xyloside restored sulfation to normal. Enzymatic assay of the xylosyltransferase in extracts prepared from these mutants revealed that one of the strains, S745, contained less enzyme activity by a factor of 15 than the wild type. This mutant provides genetic evidence that the xylosyltransferase assayed in vitro is responsible for the initiation of chondroitin sulfate and heparan sulfate biosynthesis in vivo.

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Selected References

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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 microgram of cell protein by a factor of 6-60 compared to the wild type. Some of the mutants incorporated [6-3H]glucosamine into glycosaminoglycans to the same extent as the wild type, suggesting that sulfate addition was specifically altered. In contrast, five strains failed to generate 3H-labeled glycosaminoglycans normally. In four of these, the initiation of glycosaminoglycan assembly was specifically altered, since the addition of p-nitrophenyl-beta-xyloside restored sulfation to normal. Enzymatic assay of the xylosyltransferase in extracts prepared from these mutants revealed that one of the strains, S745, contained less enzyme activity by a factor of 15 than the wild type. This mutant provides genetic evidence that the xylosyltransferase assayed in vitro is responsible for the initiation of chondroitin sulfate and heparan sulfate biosynthesis in vivo.
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