Differential susceptibility to N-glycanase at the individual glycosylation sites of mouse thyrotropin and free alpha-subunits.
We have studied the differential susceptibility to N-glycanase (peptide-N4-[N-acetyl-beta-glucosaminyl]asparagine amidase) of oligosaccharides at the individual glycosylation sites of mouse TSH and free alpha-subunits. Mouse thyrotropic tumor tissue or hypothyroid pituitary tissue were incubated with D-[2-3H]mannose for 6 h. [3H]Mannose-labeled TSH or free alpha-subunits were obtained from homogenates using specific antisera and were digested with N-glycanase in their native state or after heat denaturation and reduction in the absence or presence of detergents. Tryptic fragments of the digestion products were then analyzed by reverse phase HPLC so that the effects of N-glycanase at the individual glycosylation sites could be determined. N-Glycanase treatment of native molecules did not cleave oligosaccharides efficiently at Asn56 of alpha-subunits and Asn23 of TSH beta, whereas oligosaccharides at Asn82 of alpha-subunits were more susceptible regardless of whether the alpha-subunits were combined with TSH beta. Heat denaturation, reduction, and the presence of detergents did not substantially increase the cleavage by N-glycanase of the protected oligosaccharides, suggesting that the primary structures of the TSH subunits influenced efficiency at specific sites. Pretreatment of free alpha-subunits with trypsin failed to enable N-glycanase to work fully, as oligosaccharides at Asn56 were cleaved less effectively than those at Asn82. Thus, the susceptibility to N-glycanase differs at the individual glycosylation sites of mouse TSH and free alpha-subunits, and these differences may result from effects of the primary structures of the TSH subunits.
Miura
Y
Y
Division of Endocrinology, Michael Reese Hospital, University of Chicago, Illinois 60616.
Perkel
V S
VS
Magner
J A
JA
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005260:Female; D015292:Glycoprotein Hormones, alpha Subunit / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D006358:Hot Temperature; D007037:Hypothyroidism / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D051379:Mice; D009844:Oligosaccharides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010902:Pituitary Gland / Q000378:metabolism; D010911:Pituitary Neoplasms / Q000378:metabolism; D011489:Protein Denaturation; D013972:Thyrotropin / Q000378:metabolism*; D014357:Trypsin
D015292:Glycoprotein Hormones, alpha Subunit; D009844:Oligosaccharides; D010446:Peptide Fragments; D013972:Thyrotropin; D006026:Glycoside Hydrolases; C020668:glycanase; D014357:Trypsin; D008358:Mannose