Summary
We have studied the differential susceptibility to endoglycosidase F and H of oligosaccharides
at the individual glycosylation sites of mouse TSH and free α-subunits. Mouse thyrotropic
tumor tissue was incubated with D-[2-3H]mannose for 6h. [3H]Man-labeled TSH and free α-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 α-subunits showed
slight preferential cleavage at Asn 82 of α-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 α-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 α-subunits, and these small differences could be due to properties
of either the enzyme or substrates.
Key words
Thyrotropin - TSH - Deglycosylation - Oligosaccharides - Endoglycosidase H - Endoglycosidase
F
