A non-glycosylated extracellular superoxide dismutase variant
| Autor: | M Strömqvist, Anders Edlund, Stefan L. Marklund, Karin Hjalmarsson, Lena A. E. Tibell, Thomas Edlund, Jan Sandström |
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| Rok vydání: | 1992 |
| Předmět: |
Glycosylation
CHO Cells Peptide Mapping Biochemistry law.invention Superoxide dismutase Structure-Activity Relationship chemistry.chemical_compound law Cricetinae Animals Molecular Biology Chromatography High Pressure Liquid chemistry.chemical_classification Expression vector biology Molecular mass Heparin Superoxide Dismutase Chinese hamster ovary cell Mutagenesis Cell Biology Molecular biology Recombinant Proteins Molecular Weight Enzyme Solubility chemistry biology.protein Recombinant DNA Rabbits Extracellular Space Research Article Protein Binding |
| Zdroj: | Biochemical Journal. 288:451-456 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj2880451 |
| Popis: | The secretory tetrameric extracellular superoxide dismutase (EC-SOD) is the only glycosylated SOD isoenzyme. The importance of the carbohydrate moiety for the properties of the enzyme is unknown. An expression vector defining nonglycosylated EC-SOD (ngEC-SOD) was constructed by mutagenesis of the codon for Asn-89 into a codon for Gln. The vector was transfected into Chinese hamster ovary DXB-11 cells and ngEC-SOD was isolated to 70% purity from the culture media of selected clones. The absence of glycosylation was established by the lack of affinity for various lectins, the absence of staining with the periodic acid-Schiff reagent, the change in mobility and composition of the tryptic peptide containing the mutated glycosylation site, and the reduction in apparent molecular mass upon SDS/PAGE and size-exclusion chromatography. The tetrameric state was retained. The heparin affinity, a fundamental and distinguishing property of EC-SOD, was found to be slightly increased. The enzymic activity was essentially retained. The major difference from native glycosylated enzyme in physical properties was a marked reduction in solubility. Like glycosylated EC-SOD, ngEC-SOD was, after intravenous injection into rabbits, rapidly sequestered by the vessel endothelium, and was promptly released into plasma after injection of heparin. The only difference from glycosylated EC-SOD in this behaviour, was a slightly more rapid elimination of the mutant enzyme from the vasculature. It is concluded that no specific biological role for the EC-SOD carbohydrate moiety could be revealed. |
| Databáze: | OpenAIRE |
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