Glycosylation and processing of high-mannose oligosaccharides of thyroid-stimulating hormone subunits: comparison to nonsecretory cell glycoproteins.
Thyroid-stimulating hormone (TSH) subunit glycosylation was compared to that of total cell glycoproteins in mouse thyrotropic tumors. Lipid-linked oligosaccharides, total cell glycoproteins, and TSH subunits were labeled with either [3H]mannose, [3H]galactose, or [3H]glucose in pulse and pulse-chase experiments. The various oligosaccharides were isolated respectively by lipid extraction and mild acid hydrolysis, by selective immunoprecipitation, or by acid precipitation followed by trypsin and endoglycosidase H treatment. The nature of the oligosaccharides was assessed by their migration in paper chromatography, their relative incorporation of different precursors, and also their resistance to alpha-mannosidase. At 60 min, lipid-linked oligosaccharides were found to be composed of Glc3-2Man9GlcNAc2, Man9-8GlcNAc2, and Man5GlcNAc2. At 10 or 60 min of labeling, total cell proteins contained Glc3Man9GlcNAc2, Glc1Man9GlcNAc2, Man9GlcNAc2, Glc1Man8GlcNAc2, Man8GlcNAc2, and Man7GlcNAc2. The largest oligosaccharide, Glc3Man9GlcNAc2, had an unusually long half-life of about 2 h. In contrast, no Glc3Man9GlcNAc2 was found either on TSH + alpha subunits or on free beta subunits isolated either by immunoprecipitation or by sodium dodecyl sulfate gel electrophoresis. Instead, primarily Man9GlcNAc2 was found after a 10-min pulse both on TSH + alpha subunits and on beta subunits. When the pulse was followed by a chase up to 2 h, there was a progressive increase in Man8GlcNAc2 in higher amounts on TSH + alpha-subunit carbohydrate chains than on beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D046911:Macromolecular Substances; D008358:Mannose / Q000378:metabolism*; D008361:Mannosidases; D051379:Mice; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000302:isolation & purification; D010911:Pituitary Neoplasms / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013972:Thyrotropin / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014316:Tritium; D043323:alpha-Mannosidase
D006023:Glycoproteins; D046911:Macromolecular Substances; D009844:Oligosaccharides; D014316:Tritium; D013972:Thyrotropin; D008361:Mannosidases; D043323:alpha-Mannosidase; D005947:Glucose; D008358:Mannose; D005690:Galactose