Structure-function studies of oligosaccharides of recombinant human thyrotrophin by sequential deglycosylation and resialylation.

Recombinant human thyrotrophin (rhTSH) contains oligosaccharides terminating in -galactose-sialic acid, and had lower metabolic clearance and higher in vivo bioactivity compared to pituitary hTSH, which has oligosaccharides terminating predominantly in -N-acetylgalactosamine-sulphate. Previous studies using complete removal of the oligosaccharide chains showed an important role for the carbohydrate in the biological activity of the hormone. In the present study, we have determined the contribution of the individual monosaccharides to hormonal activity by sequential deglycosylation of rhTSH using exoglycosidases. We have also investigated the effect of resialylation of desialylated rhTSH using sialyltransferases. Sequential removal of sialic acid, galactose or N-acetylglucosamine resulted in a > 10-fold increase in the in vitro bioactivity of rhTSH. The metabolic clearance of the derivatives was faster than that of intact hormone, but agalacto-rhTSH was cleared slower than asialo-rhTSH. However, the in vivo bioactivity decreased progressively with each monosaccharide removal. The increased cyclic AMP-stimulating activity, increased metabolic clearance and the decreased in vivo biologic activity were all reversed by resialylation of the terminal galactose residues. These results indicate that the in vitro, as well as the in vivo, bioactivities of rhTSH are modulated by terminal sialylation. The effects of sequential deglycosylation on the in vitro activity of rhTSH are different from those reported earlier for human chorionic gonadotrophin. Thus, modification of the oligosaccharides by glycosidases and glycosyltransferases can be used as a powerful tool to delineate the function of carbohydrate in glycoproteins and to engineer more potent hormone analogues with a longer half-life and/or higher bioactivity.