The role of carbohydrate in thyrotropin action assessed by a novel approach using enzymatic deglycosylation.
Deglycosylation of thyrotropin (TSH) and gonadotropins by chemical methods virtually abolishes their biological activity without impairing receptor binding activity. Recent reports have suggested that enzymatic deglycosylation, using endoglycosidases caused a much smaller decrease, if any, in the potency of the glycoprotein hormones without altering the Vmax. However, in these studies complete removal of the carbohydrate chains from the hormones was not unequivocally documented. We have prepared completely deglycosylated bovine TSH by endoglycosidase F digestion of its subunits, which were more readily deglycosylated than the intact hormone. The deglycosylated subunits were separated from any incompletely digested subunits by concanavalin A affinity chromatography. Carbohydrate compositional analysis, using a highly sensitive pulsed amperometric detection method coupled to ion-exchange high performance liquid chromatography, was performed to ascertain the complete removal of the glycan moieties from the subunits. The deglycosylated subunits thus prepared were recombined to obtain deglycosylated TSH dimer. Receptor binding activity of bTSH was minimally affected by the carbohydrate removal. In an in vitro bioassay using stimulation of cyclic AMP production in FRTL-5 cells, deglycosylated bTSH showed reduced activity with a potency 5-10-fold lower than that of control, although the Vmax remained unaltered. In contrast, the deglycosylated bTSH showed a reduction in Vmax, when assayed for its adenylyl cyclase stimulating activity in bovine thyroid membranes. Previous reports using chemical methods have apparently overestimated the effects of deglycosylation, probably because of altered protein conformation, while those using endoglycosidases have apparently underestimated these effects, probably because of incomplete deglycosylation.
Thotakura
N R
NR
Molecular Cellular and Nutritional Endocrinology Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
LiCalzi
L
L
Weintraub
B D
BD
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002460:Cell Line; D002846:Chromatography, Affinity; D003208:Concanavalin A; D000242:Cyclic AMP / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D046911:Macromolecular Substances; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008970:Molecular Weight; D011869:Radioligand Assay; D051381:Rats; D011989:Receptors, Thyrotropin / Q000378:metabolism; D013961:Thyroid Gland / Q000187:drug effects / Q000378:metabolism; D013972:Thyrotropin / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*
D002241:Carbohydrates; D046911:Macromolecular Substances; D011989:Receptors, Thyrotropin; D003208:Concanavalin A; D013972:Thyrotropin; D000242:Cyclic AMP; D006026:Glycoside Hydrolases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D000262:Adenylyl Cyclases