Structure--function studies of oligosaccharides of recombinant human thyrotrophin by sequential deglycosylation and resialylation |
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Authors: | Thotakura, N.Rao Szkudlinski, Mariusz W. Weintraub, Bruce D. |
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Affiliation: | Molecular and Cellular Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Bethesda, MD 20892, USA |
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Abstract: | Recombinant human thyrotrophin (rhTSH) contains oligosaccharidesterminating in -galactose-sialic acid, and had lower metabolicclearance and higher in vivo bioactivity compared to pituitaryhTSH, which has oligosaccharides terminating predominantly in-N-acetylgalactosamine-sulphate. Previous studies using completeremoval of the oligosaccharide chains showed an important rolefor the carbohydrate in the biological activity of the hormone.In the present study, we have determined the contribution ofthe individual monosaccharides to hormonal activity by sequentialdeglycosylation of rhTSH using exoglycosidases. We have alsoinvestigated the effect of resialylation of desialylated rhTSHusing sialyltransferases. Sequential removal of sialic acid,galactose or N-acetylglucosamine resulted in a > 10-foldincrease in the in vitro bioactivity of rhTSH. The metabolicclearance of the derivatives was faster than that of intacthormone, but agalacto-rhTSH was cleared slower than asialo-rhTSH.However, the in vivo bioactivity decreased progressively witheach monosaccharide removal. The increased cyclic AMP-stimulatingactivity, increased metabolic clearance and the decreased invivo biologic activity were all reversed by resialylation ofthe terminal galactose residues. These results indicate thatthe in vitro, as well as the in vivo, bioactivities of rhTSHare modulated by terminal sialylation. The effects of sequentialdeglycosylation on the in vitro activity of rhTSH are differentfrom those reported earlier for human chorionic gonadotrophin.Thus, modification of the oligosaccharides by glycosidases andglycosyltransferases can be used as a powerful tool to delineatethe function of carbohydrate in glycoproteins and to engineermore potent hormone analogues with a longer half-life and/orhigher bioactivity. deglycosylation exoglycosidases oligosaccharides recombinant thyrotrophin sialyltransferases |
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