Role of sugar chains in the in vitro biological activity of human erythropoietin produced in recombinant Chinese hamster ovary cells

Abstract

Human erythropoietin contains three Asn-type and one mucin-type sugar chains. That the branching structure of the outer portion of Asn-type sugar chains is correlated to its biological activity in vivo has been reported recently (Takeuchi, M., Inoue, N., Strickland, T. W., Kubota, M., Wada, M., Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S., and Kobata, A. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7819-7822). In this study, the effect of trimming of sugar chains on the biological activity in vitro of this hormone was examined by using several glycosidases. Human erythropoietin produced by recombinant Chinese hamster ovary cells showed three times higher activity after desialylation. The activity was not changed significantly by further removal of the mucin-type sugar chain from the hormone, indicating no contribution of this type of sugar chain to the activity of erythropoietin in vitro. Sequential removal of galactose and N-acetylglucosamine from the outer chain moieties of the desialylated Asn-type sugar chains raised the activity of the hormone up to four and five times the intact erythropoietin, respectively. The activation effect was diminished slightly by further removing alpha-mannosyl residues and to a great extent by removing beta-mannosyl residues from the core portions of the Asn-type sugar chains. N-Glycanase digestion of intact erythropoietin resulted in almost complete loss of the activity in vitro. These results indicate that the core portion of the Asn-type sugar chains is necessary for erythropoietin to express its full biological activity in vitro and suggest that removal of the core portion of the sugar chains destroys the active conformation of erythropoietin.