Role of Cyclooxygenase-2 glycosylation in enzyme turnover and cell proliferation

Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in the biosynthesis of prostaglandins and thromboxanes by converting fatty acid arachidonic acid (AA) into prostaglandin H2. Consequently, COX-2 plays an important role in various physiological as well as pathophysiological conditions, such as cancer. COX-2 exists as two glycoforms-- 72 and 74 kDa-- with the 74 kDa form resulting from an additional oligosaccharide chain at residue Asn580. Since neither the biological significance nor the mechanism behind this differential glycosylation have been fully determined, the objective of this study was three-fold: (1) to determine if COX-2 glycosylation is affected by its substrate AA; (2) to elucidate the effect of glycosylation on COX-2 activity; and (3) to reveal any effects COX-2 glycosylation may have on cell division. Site-directed mutagenesis of the COX-2 gene at Asn580 was accomplished, and transient transfection of COS-1 cells with either the mutant or wild-type gene was achieved. COX-2 glycoforms were analyzed via Western blotting, while activity was determined using an ELISA that measured the presence of the downstream product prostaglandin E2. Finally, cell proliferation assays were performed on transfected and nontransfected cells. AA did not affect the expression of either the 72 or 74 kDa glycoforms. Cells transfected with the mutant COX-2 gene accumulated a greater amount of COX-2 protein-- expressed as the 72 kDa glycoform as well as an unexpected 70 kDa form-- and exhibited ~5-fold greater COX-2 activity compared to cells expressing both 72 and 74 kDa glycoforms. Cells unable to express the 74 kDa glycoform also tended to multiply at a significantly slower rate than cells expressing both glycoforms. In conclusion, this study demonstrated that COX-2 enzyme turnover depends upon glycosylation of the 72 kDa glycoform, and this glycosylation may subsequently affect the rate at which cells divide.