Thyroid-stimulating hormone: structure and function.
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Amino Acid Sequence↗
Biomedical subjects
J A Magner
Publications and source records attributed to J A Magner.
Assay of sulfotransferase in subcellular fractions of hypothyroid mouse pituitary and liver tissue.
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Animals↗
Concanavalin-A, lentil, and ricin lectin affinity binding characteristics of human thyrotropin: differences in the sialylation of thyrotropin in sera of euthyroid, primary, and central hypothyroid patients.
TSH from human serum was separated into classes by serial lectin affinity chromatography using Concanavalin-A (ConA), lentil, and ricin lectins. TSH from 10 euthyroid subjects, 40 patients with primary hypothyroidism, and 1 patient with central hypothyroidism was studied. The patterns of ConA and lentil affinity binding were similar for diverse patients; forms of TSH that bound firmly to ConA also tended to bind firmly to lentil. Differences in TSH-ricin binding suggested that there were differences in the sialylation of TSH in sera of euthyroid, primary, and central hypothyroidism patients. For euthyroid subjects, 16.1 +/- 5.4% (mean +/- SD) of the TSH bound to ricin, while after neuraminidase treatment, 38.4 +/- 5.4% bound. For patients with primary hypothyroidism, 23.5 +/- 6.0% of the TSH bound to the ricin, while after neuraminidase treatment, 65.7 +/- 8.8% bound. The increase in ricin binding induced by neuraminidase treatment was significantly higher for TSH from patients with primary hypothyroidism than in that from euthyroid subjects (42.3 +/- 7.6% vs. 22.3 +/- 4.4%; P less than 0.01) and was greater for long term than for short term hypothyroid patients (49.5 +/- 5.0% vs. 36.5 +/- 6.5%; P less than 0.01). While 30% of native TSH from the serum of the patient with central hypothyroidism bound to ricin, the amount bound increased only 17.6% after neuraminidase treatment. McKenzie bioassay of pituitary-derived TSH that was similarly fractionated using ricin failed to show detectable differences in bioactivity among the lectin column fractions. Thus, 1) circulating human TSH can be consistently separated into discrete classes using serial lectin affinity chromatography; 2) there is relatively more core fucosylation of the less processed high mannose and hybrid forms of TSH and less core fucosylation of more processed complex forms; 3) ConA and lentil binding of TSH in primary and central hypothyroidism is similar to that in the euthyroid state; 4) patients with primary hypothyroidism have more sialylated TSH than a patient with central hypothyroidism or euthyroid subjects; and 5) the degree of TSH sialylation increases with prolonged primary hypothyroidism.
Adult↗
Brefeldin A inhibits oligosaccharide processing of glycoproteins in mouse hypothyroid pituitary tissue at several subcellular sites.
We have studied the effects of brefeldin A (BFA) and monensin on the processing of the oligosaccharides of thyrotropin (TSH), free alpha-subunits, and cellular glycoproteins of mouse pituitary tissue to clarify the subcellular sites of action of BFA. BFA was previously shown to inhibit the translocation of glycoproteins from the rough endoplasmic reticulum to the Golgi apparatus but action at other sites was possible. Pituitaries from hypothyroid mice were incubated with [35S]methionine, [3H]mannose, [3H]galactose, [3H]fucose, N-[3H]acetylmannosamine, or [35S]sulfate for 2 hr in the absence or presence of 5 micrograms of BFA/ml or 2 microM monensin. TSH and free alpha-subunits were immunoprecipitated from tissue lysates and analyzed by sodium dodecyl sulfate-gel electrophoresis. The tryptic glycopeptides of TSH were separated using high-performance liquid chromatography. Total glycoproteins in cell lysates were precipitated using trichloroacetic acid. Labeled oligosaccharides were released from the tryptic glycopeptides of TSH and cellular glycoproteins by endoglycosidase H and they were analyzed by paper chromatography. Compared with control incubations, BFA caused the intracellular accumulation of glycoproteins having less than expected amounts of Man9GlcNAc2 units, but with excess Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2 units. There was a lesser accumulation of glucose-containing oligosaccharides, especially Glc1Man9GlcNAc2. Monensin also caused the accumulation of certain high mannose species, but the pattern differed from that seen for BFA, since Man9GlcNAc2 units were preserved and there was less excess of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2 units. BFA did not block the initial attachment of oligosaccharides at any of the three Asn-glycosylation sites of TSH, but caused the accumulation of Man5-8GlcNAc2 units at each site. Both monensin and BFA inhibited fucosylation, sulfation, and sialylation more markedly than mannose incorporation. Thus, in addition to its previously described action of inhibiting rough endoplasmic reticulum to Golgi transport, BFA appears to partially inhibit the glucose-trimming enzymes as well as some Golgi enzymes.
Acetylglucosaminidase↗
Blockade by brefeldin A of intracellular transport of secretory proteins in mouse pituitary cells: effects on the biosynthesis of thyrotropin and free alpha-subunits.
We examined the effect of brefeldin A (BFA), a drug that inhibits the intracellular translocation of newly synthesized glycoproteins, on the biosynthesis of TSH and free alpha-subunits by pituitary tissue from hypothyroid mice. Incubation of tissue with 5 or 10 micrograms BFA/ml for 3.5 h caused marked dilatation of rough endoplasmic reticulum (RER) and mild swelling of Golgi in all pituitary cell types. As judged by incorporation of [35S]Met into acid-insoluble radioactivity, BFA at a concentration of 5 micrograms/ml did not substantially inhibit protein synthesis, but markedly reduced protein secretion. After a 2-h pulse with [35S]Met, followed by a 4-h chase, BFA at 5 micrograms/ml reduced the release of TSH and free alpha-subunits into the medium by 94% and 99%, respectively; subunits that accumulated within cells were forms with mol wt 2000-4000 less than normal. BFA also partially inhibited the release into the medium of TSH or free alpha-subunits labeled with [3H]fucose or [35S]SO4, but this effect was less marked than that for [35S]Met-labeled subunits. Both the morphological and the isotopic data suggest that BFA blocks transport of secretory proteins between rough endoplasmic reticulum and Golgi of pituitary cells, although transport within the Golgi may also be affected to some extent.
Animals↗
The effects of brefeldin-A on the high mannose oligosaccharides of mouse thyrotropin, free alpha-subunits, and total glycoproteins.
We have studied the effects of Brefeldin-A (BFA) on the processing of high mannose (Man) oligosaccharides of TSH. BFA is a drug that inhibits the intracellular translocation of newly synthesized glycoproteins and causes dilatation of the rough endoplasmic reticulum (RER) as well as mild swelling of the Golgi apparatus. Mouse pituitary thyrotropic tumor tissue was incubated with [3H]Man for a 2-h pulse, with and without a 3-h chase; BFA (5 micrograms/ml) was included during selected pulse and selected chase incubations. TSH and free alpha-subunits were obtained from detergent lysates of tissue by immunoprecipitation using specific antisera. Total glycoproteins were obtained by trichloroacetic acid precipitation. Endoglycosidase-H-released [3H]oligosaccharides were analyzed by paper chromatography. BFA inhibited carbohydrate processing of TSH, free alpha-subunits, and total glycoproteins, resulting in the accumulation of Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, and Man5GlcNAc2, especially during the chase period. Subcellular fractions enriched in RER, heavy (proximal) Golgi, and light (distal) Golgi were prepared by centrifugation in discontinuous sucrose gradients. [3H]Man-labeled oligosaccharides of TSH and total glycoproteins in the subcellular fractions were analyzed. In contrast to oligosaccharides with eight or nine Man residues found in control incubations, BFA caused the accumulation of oligosaccharides containing five to eight Man residues. These BFA-induced oligosaccharide alterations began in the RER and proximal Golgi with the 2-h pulse and extended into the distal Golgi during the chase incubations. Thus, BFA blocks the normal intracellular transport and processing of TSH, free alpha-subunits, and total glycoproteins within thyrotrophs, causing species with smaller than normal high Man oligosaccharides to appear in subcellular compartments as early as the RER. The translocation block between RER and Golgi produced by BFA may prevent the processing of Man8GlcNAc2 to Man5GlcNAc2 by Golgi (alpha,1-2)mannosidase I, yet the species retained within the RER may be subject to ongoing processing by endoplasmic reticulum (alpha,1-2)mannosidase, resulting in the accumulation of Man5-8GlcNAc2 within the RER.
Animals↗
Rates of processing of the high mannose oligosaccharide units at the three glycosylation sites of mouse thyrotropin and the two sites of free alpha-subunits.
We have determined the structures of high mannose (Man) oligosaccharide units at individual glycosylation sites of mouse TSH. Mouse thyrotropic tumor tissue was incubated with D-[2-3H]Man with or without [14C]tyrosine ([14C] Tyr) for 2, 3, or 6 h, and for a 3-h pulse followed by a 2-h chase. TSH heterodimers or free alpha-subunits were obtained from homogenates using specific antisera. After reduction and alkylation, subunits were treated with trypsin. The tryptic fragments were then loaded on a reverse phase HPLC column to separate tryptic fragments bearing labeled oligosaccharides. The N-linked oligosaccharides were released with endoglycosidase-H and analyzed by paper chromatography. Man9GlcNac2 and Man8GlcNac2 units predominated at each time point and at each specific glycosylation site, but the processing of high Man oligosaccharides differed at each glycosylation site. The processing at Asn23 of TSH beta-subunits was slower than that at Asn56 or Asn82 of alpha-subunits. The processing at Asn82 was slightly faster than that at Asn56 for both alpha-subunits of TSH heterodimers and free alpha-subunits. The present study demonstrates that the early processing of oligosaccharides differs at the individual glycosylation sites of TSH and free alpha-subunits, perhaps because of local conformational differences.
Amino Acid Sequence↗
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.
Animals↗
Congestive heart failure and sudden death in a young woman with thyrotoxicosis.
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Adult↗
Structures of high-mannose oligosaccharides of mouse thyrotropin: differential processing of alpha- versus beta-subunits of the heterodimer.
We have determined the structures of high mannose (Man) oligosaccharide units of the alpha- and beta-subunits of mouse TSH heterodimers and of free alpha-subunits. Mouse thyrotropic tumor tissue, or pituitaries from euthyroid or hypothyroid mice, were incubated with D-[2-3H]Man, homogenized, and incubated in the presence or absence of pH 3 buffer to dissociate heterodimers. Highly enriched TSH beta-subunits, or TSH heterodimers, were obtained using anti-TSH beta serum and free alpha-subunits were subsequently obtained using anti-LH alpha-serum. High Man units released by endoglycosidase H were analyzed by paper chromatography. At 1 and 3 h oligosaccharides with 8 or 9 Man residues predominated in all subunits. In all three tissue types, units with 9 Man tended to accumulate in TSH beta-subunits, whereas Man-trimming from Man9GlcNAc to Man8GlcNAc proceeded more rapidly in TSH alpha-subunits, and in free alpha-subunits. The rate of TSH beta-subunit processing was also a function of tissue type; the rate in euthyroid pituitary tissue equaled that in tumor tissue and was greater than that in hypothyroid pituitary tissue, suggesting that processing may be physiologically regulated. Hypothyroid calf serum slowed TSH alpha-subunit processing in euthyroid pituitaries. Thus, the previously reported slower processing of Man9GlcNAc to Man8GlcNAc in heterodimers as compared to free alpha-subunits is in large part due to accumulation of Man9GlcNAc in TSH beta-subunits.
Animals↗
The subcellular sites of sulfation of mouse thyrotropin and free alpha subunits: studies employing subcellular fractionation and inhibitors of the intracellular translocation of proteins.
To determine the subcellular sites of sulfation of thyrotropin (TSH) and free alpha-subunits, mouse thyrotropic tumor minces were incubated simultaneously with [3H]Met and [35S]SO4 for 1 or 3h, homogenized, and fractionated by discontinuous sucrose gradient ultracentrifugation. Dual-labeled TSH or free alpha-subunits were immunoprecipitated, and analyzed by SDS-gel electrophoresis. Endoglycosidase F released all [35S], but little [3H], from the dual-labeled species, indicating that [35S]SO4 was incorporated into oligosaccharides of TSH and free alpha-subunits. Both [35S]TSH and [35S] free alpha-subunits were predominantly in Golgi fractions at 1 and 3 h, but small amounts were also detected in fractions enriched in rough endoplasmic reticulum (RER). Similar distributions of [35S]SO4-labeled species were noted in cell fractions prepared from mouse pituitaries. Pituitaries from hypothyroid mice were incubated with [3H]Met and [35S]SO4 for 2 h, then chased for 4 or 16 h in the absence or presence of 2 uM monensin (Mon) or 10 uM carboxyl cyanide m-chlorophenylhydrazone (CCCP). At 4h, release into the medium of [3H]TSH was inhibited 59% and 86% by Mon and CCCP, respectively; release of [35S]TSH was inhibited 28% and 46%. At 4h, release of [3H]free alpha-subunits was inhibited 58% and 81% by these drugs, respectively; release of [35S]free alpha-subunits was inhibited 6% and 50%. Thus, Mon and CCCP inhibited the release of each [3H] species more than the [35S] species, indicating that most sulfation occurred in Golgi.
Animals↗
Familial generalized resistance to thyroid hormones: report of three kindreds and correlation of patterns of affected tissues with the binding of [125I] triiodothyronine to fibroblast nuclei.
We here report three kindreds with a total of 19 persons affected with central and peripheral resistance to thyroid hormones: one kindred with 10 affected persons is the largest reported to date. Male to male transmission of the syndrome was evident in two kindreds, consistent with an autosomal dominant mode of inheritance. During several years of follow up, the degree of resistance to thyroid hormones did not ameliorate. Within a given kindred, a given tissue or tissues was consistently more resistant to thyroid hormone than other tissues. The pattern of tissues most affected in one kindred differed from that of another kindred, perhaps reflecting the inherited underlying molecular defects. Members of kindred A frequently had bone involvement, and several had learning disabilities and recurring infections, while most members of kindreds B and C had little bone involvement, but marked hepatic and cardiac resistance to thyroid hormones. Kinetic studies of the binding of [125I] triiodo-L-thyronine to nuclei from skin fibroblasts from affected patients from each of the kindreds demonstrated decreased maximum binding as compared to normal fibroblasts, but there was no correlation between this parameter and other features of the disease. Four of the 19 patients had previously been treated inappropriately with antithyroid therapies, demonstrating how the syndrome may be readily confused with Graves' disease by some clinicians. Behavior or school performance improved in all children treated with thyroid hormones, and a growth spurt was documented in six children, but objective improvement in IQ scores was not demonstrated, suggesting that initiation of hormone therapy at an early age may be important for maximum benefit.
Adult↗
The role of glycosylation in the molecular conformation and secretion of thyroxine-binding globulin.
T4-binding globulin (TBG), the principal carrier of thyroid hormone in serum, is a glycoprotein containing 20% carbohydrate. The importance of the carbohydrate moiety has been previously studied by enzymatic deglycosylation, which showed that deglycosylated TBG retains its original immunological an T4-binding properties. However, the structure and properties of TBG before glycosylation and the steps involved in carbohydrate addition have not been explored. In the present report, we used a human hepatoma cell line (Hep G2) which synthesizes and secretes TBG into the medium. This TBG binds T4 and possesses immunoreactivity and microheterogeneity identical to those of native TBG (nTBG) from serum. Cells were pulsed with [35S]methionine, [3H]mannose, and [3H]glucosamine in the absence or presence of 5 micrograms tunicamycin/ml medium. Materials from cells and media were immunoprecipitated with antibodies specific for nTBG and denatured TBG molecule. They were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells incubated with [35S]methionine contained two forms of labeled TBG, with apparent mol wt of 60K (TBG1) and 54K (TBG2). Medium contained only the TBG1 form, which is identical to nTBG in serum. In contrast, in the presence of tunicamycin, the predominant intracellular form of TBG had an apparent mol wt of 44K (TBG3). At no time was this material detected in the medium. [3H]Mannose and [3H]glucosamine labeled both TBG1 and TBG2, but not TBG3. TBG1 and TBG2 reacted with anti-nTBG serum, whereas TBG3 reacted only with anti-dentured TBG serum, specific for the unfolded TBG molecule. Intracellular TBG was rich (80-90%) in high mannose (seven to nine mannose residues) oligosaccharides and was relatively poor (10-20%) in complex-type species, resistant to endoglycosidase H. These results indicate that 1) the precursor nonglycosylated 44K TBG3 is glycosylated to produce TBG2 (54K) and TBG1 (60K); 2) TBG2 contains oligosaccharides rich in mannose and appears to be a major intracellular intermediate in the synthesis of TBG1; 3) only the endoglycosidase H-resistant TBG1 is secreted; and 4) prior glycosylation of TBG appears to be required for the molecule to assume its tertiary structure and, ultimately, for its secretion. However, once the peptide chain is folded, removal of the carbohydrate moieties does not alter the tertiary structure.
Carbohydrate Conformation↗
Biosynthesis, glycosylation, and secretion of rat luteinizing hormone alpha- and beta-subunits: differential effects of orchiectomy and gonadotropin-releasing hormone.
We have studied the de novo biosynthesis and secretion of LH subunits in pituitary quarters from orchiectomized and intact control adult male rats and their regulation by GnRH. After labeling with [35S]cystine ([35S]Cys), [35S]methionine, or [3H]glucosamine ([3H]GlcN) in the presence or absence of 10(-8) M GnRH, tissue lysates and media were immunoprecipitated with antisera to LH beta, then LH alpha (after removal of TSH by immunoprecipitation with anti-TSH beta), and the products were analyzed by sodium dodecyl sulfate gradient gel electrophoresis. During a 12-min pulse labeling with [35S]methionine, three forms of immunoreactive alpha were labeled at 21,000, 18,000, and 12,000 mol wt. After a 30-min chase with excess unlabeled methionine, the 12,000 form decreased from 10% to 3% of total radioactivity, while the 21,000 form increased from 57% to 69%, implying a precursor-product relationship. Neither orchiectomy nor GnRH had any effect on [35S]Cys or [3H]GlcN incorporation into intracellular or secreted total proteins. After a 6-h continuous labeling, incorporation of [35S]Cys into intracellular combined LH alpha in castrates was 158% of the control value, combined LH beta was 304%, and free alpha was 466%. The [3H]GlcN to [35S]Cys ratio, reflecting relative glycosylation, was unchanged in castrates for total proteins or LH alpha and somewhat decreased for LH beta and free alpha. Orchiectomy increased [35S]Cys-labeled secreted LH beta and free alpha to 183% and 231% of control values, respectively. Relative glycosylation of secreted LH alpha, LH beta, and free alpha was unchanged in castrates. Incorporation of [35S]Cys into intracellular combined LH alpha, LH beta and free alpha-subunit was unaffected by GnRH in pituitaries from intact rats. In castrates, LH alpha was unchanged, but LH beta and free alpha were slightly increased. Incorporation of [3H]GlcN into intracellular combined LH alpha, LH beta, and free alpha was increased with GnRH in both intacts and castrates, such that the 3H to 35S ratio, reflecting relative glycosylation, was also increased with GnRH. In castrates, the ratios, as a percentage of the control, were, respectively 250%, 250%, and 223% for LH alpha, LH beta, and free alpha. In intact animals, the ratios were 221%, 281%, and 143%, respectively. Incorporation of both [35S]Cys and [3H]GlcN into secreted subunits was increased in most instances, such that the 3H to 35S ratio was increased only for LH beta.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Differential sulfation and sialylation of secreted mouse thyrotropin (TSH) subunits: regulation by TSH-releasing hormone.
To determine whether sulfate and/or sialic acid are present on secreted mouse TSH, thyrotropic tumor minces and hypothyroid pituitaries were incubated with [3H]methionine and [35S]sulfate, or [35S]methionine and [3H]N-acetylmannosamine. The metabolically labeled TSH and free alpha-subunits were then analyzed by gel electrophoresis. [3H]N-Acetylmannosamine was a specific precursor (greater than 80%) for the sialic acid [3H]N-acetylneuraminic acid, as established by HPLC characterization of tritium label released by acid hydrolysis. Each of the three secreted subunits (TSH alpha, TSH beta, and free alpha) incorporated both sulfate and sialic acid. The incorporation of these labels was confirmed by the release of [35S]sulfate by endoglycosidase F and of [3H]N-acetylneuraminic acid by neuraminidase. Differential labeling of newly synthesized secreted TSH subunits was observed. In secreted TSH dimer, TSH beta incorporated 1.3 times more [35S]sulfate (P less than 0.05) and 2.5 times more [3H] N-acetylmannosamine (P less than 0.02) per carbohydrate chain than did TSH alpha. Secreted free alpha-subunit incorporated more [3H]N-acetylmannosamine, but less [35S]sulfate, then did secreted TSH alpha. To investigate the effect of TRH on TSH sulfation and sialylation, thyrotropic tumor minces and hypothyroid pituitaries were incubated with [35S]sulfate or [3H]N-acetylmannosamine, with or without 10(-7) M TRH; labeling was then normalized in each case to incorporation of [3H]mannose, a marker of the inner core sugars. TSH secreted in the presence of TRH had a lower sulfate to mannose ratio [28 +/- (+/- SE) 4% of control; P less than 0.05] and a lower sialic acid to mannose ratio (63 +/- 8% of control; P less than 0.05). TSH alpha and TSH beta were affected equally. No change was seen in the labeling of non-TSH secretory proteins. Differential glycoprotein sulfation and sialylation may, in part, explain the previously observed variability in isoelectric point, bioactivity, and MCR of TSH in different physiological states and may represent a point of regulation by TRH.
Animals↗
Subcellular localization of fucose incorporation into mouse thyrotropin and free alpha-subunits: studies employing subcellular fractionation and inhibitors of the intracellular translocation of proteins.
To determine the subcellular sites of fucose incorporation into TSH subunits, pituitaries from hypothyroid mice were incubated with [3H]fucose and fractionated by sucrose gradient centrifugation. To assess potential molecular cross-contamination between subcellular fractions enriched in rough endoplasmic reticulum (RER) or Golgi elements, trace amounts of exogenous [35S]methionine-labeled proteins or [125I]rat TSH were added before tissue homogenization. Particulate contamination of fractions was monitored by electron microscopy. TSH subunits were immunoprecipitated from fractions and analyzed by gel electrophoresis. After both a 2-h pulse incubation and a 2-h pulse, 3-h chase incubation, about half (range, 44-71%) of the [3H]fucose-labeled TSH subunit precursors present in microsomes were in the RER (amounts in excess of estimated contamination by nonspecific readsorption of molecules to vesicles or the presence of Golgi vesicles in the RER fractions); [3H]fucose-labeled free alpha-subunits were also detected in RER as well as in Golgi fractions. During chase incubations, both monensin and carboxyl cyanide m-chlorophenylhydrazone inhibited the appearance of [35S]methionine- or [3H]fucose-labeled TSH subunits in medium in a dose-dependent manner, suggesting that [3H] fucose was added to subunits, in part, early in the secretory pathway. Free alpha-subunits were more fucosylated than was TSH; in TSH heterodimers, beta-subunits were richer in fucose than were alpha-subunits. Thus, the fucosylation of TSH and free alpha-subunits in pituitaries of hypothyroid mice appears to begin at an unusually early stage of intracellular transport and may represent an adaptation to special posttranslational processing requirements.
Acetylglucosaminidase↗
Glycosylation and posttranslational processing of thyroid-stimulating hormone: clinical implications.
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Animals↗
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)
Animals↗