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

Abstract

Thyroid-stimulating hormone (TSH) alpha- and beta-subunit glycosylation was investigated in mouse thyrotropic tumor and in normal and hypothyroid pituitary cells for various periods of time in the presence of [3H]mannose or [3H]galactose. After sequential precipitation with anti-alpha and anti-beta sera, subunits were treated with Pronase followed by endo-beta-N-acetylglucosaminidase H (Endo H) and analyzed by paper chromatography. In primary cultures of thyrotropic tumor cells incubated for 60 min with [3H]mannose, primarily Man9GlcNAc and Man8GlcNAc were found on TSH + alpha subunits, whereas Glc1Man9GlcNAc and Man9GlcNAc were prominent on free beta subunits. After preincubation of cells for 16 h in the presence or absence of glucose followed by a 60-min pulse of [3H]mannose, there was an 8-fold increase in labeled TSH + alpha but only a minimal change in free beta or total proteins. In the absence of glucose, there was a selective accumulation of Man8GlcNAc on TSH + alpha but not on free beta or total proteins; however, there was no detectable accumulation of Endo H resistant forms during glucose starvation on TSH subunits or total proteins. Normal mouse and rat pituitary minces incubated for 60 min with either [3H]mannose or [3H]galactose showed no glucose-containing species on TSH subunits, but equal amounts of Man9GlcNAc and Man8GlcNAc on TSH + alpha, and mostly Man9GlcNAc on free beta subunits. In contrast, hypothyroid mouse and rat pituitaries exhibited an increase in Glc1Man9NAc and Glc1Man8GlcNAc on free beta but not on TSH + alpha or total proteins.(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).…”

Intrinsic bioactivity of thyrotropin in human serum is inversely correlated with thyroid hormone concentrations. Application of a new bioassay using the FRTL-5 rat thyroid cell strain.

“…We have also demonstrated that chemical deglycosylation of TSH greatly reduces its bioactivity and produces a form that acts as a competitive antagonist (36). Since thyroid hormones have been shown to affect TSH glycosylation in rodents (37,38), our present study suggests that they may also affect posttranslational TSH carbohydrate processing in man, either by a direct effect on the thyrotrophs or indirectly through TRH release and secretion.…”