INFLUENCE OF GLYCOSYLATION ON THE FUNCTIONAL PROPERTIES OF HUMAN THERAPEUTIC PLASMA PROTEINS

Abstract

Comparative analysis of the carbohydrate structure of plasma antithrombin III and recombinant antithrombin III synthesized in Chinese Hamster Ovary cells revealed differences in the linkage of NeuAc, the presence of higher than biantennary structures and the presence of proximal fucose. Treatment of the carbohydrate part of antithrombin III from both sources with glycopeptidase F or sialidase had a strong negative effect on the serum half-life. In order to analyze the effects of elimination of individual carbohydrate side chains on the pharmacokinetic and functional properties of AT III the four N-linked glycosylation sites of the recombinant molecule were altered individually or in combination by site directed mutagenesis of Asn to Gln. All mutants showed a shorter serum half-life compared to natural antithrombin II]. However molecules modified at residues Asn 135, Asn 155 and Asn 192 showed higher heparin affinity and/or maximal stimulation at lower heparin concentrations. As in the case of antithrombin III the three N-glycosylation sites of tissue plasminogen activator mutated individually or in combination. Whereas the specific activities of single glycosylation mutants were unaltered, simultaneous mutation of two (Asn 117 and Asn 184) or three Asn residues to Gln resulted in molecules with 2-3 fold higher specific activities.