Differential glycosylation regulates turnover of cyclooxygenase‐2

Cyclooxygenase‐2 (COX‐2) converts arachidonic acid (AA) into prostaglandin H 2 and thus plays a key role in various physiological and pathophysiological conditions. COX‐2 exists as a 72 or 74 kD glycoform, with the latter resulting from an additional oligosaccharide chain at residue Asn 580 . Since neither the significance nor mechanism behind this differential glycosylation have been fully determined, the objective of this study was three‐fold: (1) to elucidate the effect of glycosylation on COX‐2; (2) to determine if AA influences glycosylation; and (3) to reveal any effects COX‐2 glycosylation may have on cells. Site‐directed mutagenesis of the COX‐2 gene at Asn 580 was achieved, and COS‐1 cells were transfected with either the mutant or wild‐type gene. Glycoforms were analyzed via Western blotting, and activity was determined using an ELISA. Microscopic analyses and cell proliferation assays were also performed. Cells expressing the mutant COX‐2 gene accumulated a greater amount of COX‐2 protein— expressed as the 72 kD glycoform and as an unexpected 70 kD form— and exhibited ~5‐fold greater COX‐2 activity compared to cells with the wild‐type gene. AA did not affect the expression of either glycoform. Cells unable to express the 74 kD glycoform also proliferated at a significantly slower rate than cells expressing both glycoforms. No morphological differences between the two cell groups were observed. In conclusion, COX‐2 turnover is regulated by the glycosylation of the 72 kD glycoform, and this glycosylation might then affect the rate at which cells divide. Supported by VA Merit Review and NASA NCC‐2‐1361 grants