Susceptibility to endoglycosidase F and H at the individual glycosylation sites of mouse thyrotropin and free alpha-subunits.
We have studied the differential susceptibility to endoglycosidase F and H of oligosaccharides at the individual glycosylation sites of mouse TSH and free alpha-subunits. Mouse thyrotropic tumor tissue was incubated with D-[2-3H]mannose for 6 h. [3H]Man-labeled TSH and free alpha-subunits were obtained from homogenates using specific antisera and were digested with endoglycosidase F and H in their native states or after heat-denaturation and reduction in the presence of detergents. Tryptic fragments of the digestion products were then analyzed by reverse phase HPLC so that effects of endoglycosidase at the individual glycosylation sites could be determined. There was very little preferential cleavage by endoglycosidase H and F among the glycosylation sites of TSH subunits. Endoglycosidase F treatment of native free alpha-subunits showed slight preferential cleavage at Asn 82 of alpha-subunits after a 4 h incubation, whereas endoglycosidase H cleaved oligosaccharides equally well at Asn 56 and Asn 82. The Asn 82 oligosaccharide of native TSH heterodimers was also slightly preferentially cleaved by endoglycosidase F, but endoglycosidase H cleaved oligosaccharides equally well at all TSH glycosylation sites. Heat denaturation, reduction and the presence of detergent did not alter this slight preferential cleavage by endoglycosidase F at Asn 82 of alpha-subunits, suggesting that the primary structures of the TSH subunits in part influenced the efficiency of enzyme action at specific sites. Thus, the susceptibility to endoglycosidase F differs very slightly at the individual glycosylation sites of mouse TSH and free alpha-subunits, and these small differences could be due to properties of either the enzyme or substrates.
Miura
Y
Y
Division of Endocrinology, Micheal Reese Hospital, University of Illinois, Chicago.
Perkel
V S
VS
Magner
J A
JA
D000118:Acetylglucosaminidase / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D006026:Glycoside Hydrolases / Q000032:analysis / Q000378:metabolism*; D006031:Glycosylation; D006596:Hexosaminidases / Q000378:metabolism*; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D013972:Thyrotropin / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology
D013972:Thyrotropin; D006026:Glycoside Hydrolases; D006596:Hexosaminidases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D014357:Trypsin