A non-glycosylated extracellular superoxide dismutase variant.
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.
Edlund
A
A
Symbicom AB, Umeå, Sweden.
Edlund
T
T
Hjalmarsson
K
K
Marklund
S L
SL
Sandström
J
J
Strömqvist
M
M
Tibell
L
L
D000818:Animals; D016466:CHO Cells; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D005110:Extracellular Space / Q000201:enzymology; D006031:Glycosylation; D006493:Heparin / Q000378:metabolism; D008970:Molecular Weight; D010449:Peptide Mapping; D011485:Protein Binding; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism / Q000493:pharmacokinetics; D012995:Solubility; D013329:Structure-Activity Relationship; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000378:metabolism / Q000493:pharmacokinetics
D011994:Recombinant Proteins; D006493:Heparin; D013482:Superoxide Dismutase