Carbohydrate chains of human thyrotropin are differentially susceptible to endoglycosidase removal on combined and free polypeptide subunits

The accessibility of the asparagine-linked carbohydrate chains of human thyrotropin (hTSH) and free α and β subunits was investigated by their susceptibility to endoglycosidases H and F as well as to peptide:N-glycosidase F. Iodinated hTSH or subunits were incubated with a commercial enzyme preparation containing both endoglycosidase F and N-glycosidase F activities and further analyzed by sodium dodecyl sulfate gel electrophoresis followed by quantitative autoradiography. The authors show that, working at the optimum of the N-glycosidase activity, the relative amount of endoglycosidase required for half-deglycosylation was 20-fold higher for native hTSH than for the reduced and dissociated subunits. Under nondenaturing conditions, the 18K β subunit of hTSH could be readily deglycosylated to a 14K species while the 22K α subunit was largely resistant. However, both subunits were converted to an apoprotein of similar apparent molecular weight of 14K following reduction of disulfide bonds. In contrast, the free α subunit of human choriogonadotropin appeared fully sensitive to carbohydrate removal under nonreducing conditions despite the presence of a partially deglycosylated 18K intermediate at low concentration of endoglycosidase. Similarly, both hTSH-α and hTSH-β could be completely deglycosylated after acid dissociation of the native hormone. While all three carbohydrate chains of hTSH are sensitive to pure peptide:N-glycosidase F, only one on α and the single oligosaccharide present on β in hTSH appeared to be cleaved by pure endoglycosidase F. These findings indicate that while the carbohydrate chain on β is not involved in αΒ association, the oligosaccharides on α are hindered when hTSH subunits are combined