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

Abstract

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)

Thyroid-Stimulating Hormone: Biosynthesis, Cell Biology, and Bioactivity

“…These species are generally similar to lipid-linked oligosaccharides extracted from hepatocytes and other cell types (62,63). After short 10-min incubations of thyrotropes with [ 3 H] mannose, Glc 3 Man 9 GlcNAc 2 oligosaccharides were detected bound to some cellular glycoproteins, but this very early glucose-containing precursor was not found linked to nascent TSH subunits (65)(66)(67), suggesting either that TSH subunits were initially glycosylated with a nonglycosylated precursor, or that the glucose residues were very rapidly trimmed from the TSH subunits. When Stannard et al (68) employed the glucosidase inhibitor 1-deoxynojirimycin in 1988 it became clear that very rapid glucose trimming was the more likely explanation.…”

“…When Stannard et al (68) employed the glucosidase inhibitor 1-deoxynojirimycin in 1988 it became clear that very rapid glucose trimming was the more likely explanation. In the earlier studies (65)(66)(67) it was also apparent that there was differential processing of the high-mannose units of TSH as compared to free a-subunits, which also differed from that of the bulk of glycoproteins in these cells. The rate of trimming of a mannose residue from Man 9 GlcNAc 2 to produce Man 8 GlcNAc 2 units appeared to be much faster for free a-subunits; after a 60-min incubation with [ 3 H]mannose, Man 8 GlcNAc 2 units predominated in free a-subunits, whereas Man 9 GlcNAc 2 units still predominated in TSH heterodimers and in other cellular glycoproteins.…”

“…Pulse-chase labeling studies employing [ 3 H] mannose, instead of radiolabeled amino acids, have provided new information about TSH high-mannose oligosaccharide biosynthesis and processing (65)(66)(67). After incubation of thyrotropic tumor cells with [ 3 H]mannose for 60 min, the lipid-linked oligosaccharides were extracted from the cells; these precursor oligosaccharides bound to the dolichol carrier were found to be predominantly Glc 3 Man 9 GlcNAc 2 , Man 9 -8 GlcNAc 2 , and Man 5 GlcNAc 2 (66).…”

Subcellular Localization of Fucose Incorporation into Mouse Thyrotropin and Freeα-Subunits: Studies Employing Subcellular Fractionation and Inhibitors of the Intracellular Translocation of Proteins*

“…As an extension of prior studies of TSH processing by thyrotrophs (5,11,35,36,(38)(39)(40)(41)(42) and in light of an interesting recent study of hCG which employed ionophore drugs (30), we chose to investigate the subcellular sites of TSH processing in the stimulated thyrotrophs of hypothyroid mice. Our goal was to study a more physiological nontumorous model despite the potential difficulties of detecting small amounts of TSH precursors in cell fractions.…”

Differential processing and regulation of thyroid-stimulating hormone subunit carbohydrate chains in thyrotropic tumors and in normal and hypothyroid pituitaries

“…Whether the late maturation of TSH carbohydrate chains is influenced by their early processing is still speculative, but this is possible since TSH assembly selectively proceeds on mannose-rich a and p subunits . Specifically, the increase of Glc,-containing intermediates caused by hypothyroidism or TRH may result from posttranslational addition of glucose which could alter the later action of a-mannosidases and glycosyltransferases (Ronin & Caseti, 1981;Parodi et al, 1983).…”