Glycosylation and processing of high-mannose oligosaccharides of thyroid-stimulating hormone subunits: comparison to nonsecretory cell glycoproteins

• Published in: Biochemistry (Easton) Vol. 23; no. 20; pp. 4503 - 4510
• Main Authors: Ronin, Catherine, Stannard, Bethel S, Rosenbloom, Isabel L, Magner, James A, Weintraub, Bruce D
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 25.09.1984
• Subjects: alpha-Mannosidase; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Galactose - metabolism; Glucose - metabolism; Glycoproteins - biosynthesis; Glycoproteins - genetics; Macromolecular Substances; Mannose - metabolism; Mannosidases; Mice; Oligosaccharides - biosynthesis; Oligosaccharides - isolation & purification; Pituitary Neoplasms - metabolism; Protein hormones. Growth factors. Cytokines; Protein Processing, Post-Translational; Proteins; Thyrotropin - biosynthesis; Thyrotropin - genetics; Thyrotropin - isolation & purification; Tritium; Translation; Oligosaccharide; Rodentia; TSH; Glycosylation; Glycoprotein hormone; Subunit; Processing; Vertebrata; Regulation(control); Mammalia; Mouse; Thyreotropic cell; Pituitary diseases; Pituitary gland; Tumor
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00315a001

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.