Relationship of glycosylation to de novo thyroid-stimulating hormone biosynthesis and secretion by mouse pituitary tumor cells.

Weintraub, Bruce D.; Stannard, Bethel; Linnekin, Diana; Marshall, Merville C.

doi:10.1016/s0021-9258(19)70688-7

Cited by 102 publications

(9 citation statements)

References 46 publications

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“…On TSH, the same prominent intermediates Glc,Man9GlcNAc2, Man9GlcNAc2, and Man8GlcNAc2 were observed on both subunits, suggesting that the trimming of each subunit carbohydrate chain follows a similar if not identical pathway. Since 0-subunit synthesis has been suggested to be a rate-limiting step in hormone synthesis (Weintraub et al, 1980;Magner & Weintraub, 1982), it is likely that the fate of the 0 subunit chiefly reflects that of the combined hormone. Therefore, different rates of carbohydrate processing between immunoprecipitated a and 0 subunits may in fact reflect differences in the fate of combined vs. uncombined subunits.…”

Section: Discussionmentioning

confidence: 99%

“…Aliquots of lysates were precipitated with specific rabbit anti-bovine LHor anti-bovine TSH-jS, as indicated, by using a staphylococcal protein A method described previously (Weintraub et al, 1980). Each antisubunit antibody has been shown to precipitate both the respectively uncombined (free) subunit as well as the combined subunit in TSH.…”

Section: Methodsmentioning

confidence: 99%

“…(free ß); (ii) lysates were also first precipitated with anti-0 (TSH + ß) and the supernatants precipitated with anti-« (free a). These methods have been Validated previously (Weintraub et al, 1980) arid were useful in determining the subunit specificity of carbohydrate processing. However, because of limitations in material, it was not possible to analyze separately the carbohydrate structures of each free and combined subunit of TSH.…”

Section: Methodsmentioning

confidence: 99%

“…Studies from this laboratory and others [reviewed in Weintraub et al (1985)] have demonstrated that TSH subunit biosynthesis occurs through separate messenger RNA species producing precursor pre-and pre-/3 subunits containing amino-terminal signal peptides. Cotranslational cleavage of the signal peptide and glycosylation precede and appear necessary for the posttranslational a-/3-subunit combination, which begins in the rough endoplasmic reticulum (Weintraub et al, 1980(Weintraub et al, , 1983 Magner & Weintraub, 1982). We have recently demonstrated (Ronin et al, 1984) that in thyrotropic tumors, TSH subunit glycosylation, like that of other eukaryotic glycoproteins (Hubbard & Ivatt, 1981), occurs by transfer of preassembled oligosaccharides from lipid carriers.…”

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confidence: 99%

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

Ronin¹,

Stannard²,

Weintraub³

1985

Biochemistry

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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)

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Ronin¹,

Stannard²,

Weintraub³

1985

Biochemistry

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“…In certain glycoproteins, the carbohydrate may indeed by involved in the secretory process, whereas in others, it may serve an entirely different function. Weintraub et al (1980) have investigated the relationship of glycosylation to the synthesis of thyroid-stimulating hormone (TSH) in mouse pituitary tumor cells and found that a nonglycosylated a subunit was secreted in the presence of tunicamycin which was incompetent for combination with TSH-ß. They concluded that specific glycosylation may be required for -ß-subunit combination but not for secretion.…”

Section: Discussionmentioning

confidence: 99%

Synthesis of the glycoprotein hormone .alpha. subunit and placental alkaline phosphatase by HeLa cells: effect of tunicamycin, 2-deoxyglucose, and sodium butyrate

Cox¹

1981

Biochemistry

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The common α subunit of bovine glycoprotein hormones: Limited formation of native structure by the totally nonglycosylated polypeptide chain

Strickland

Thomason

Nilson

et al. 1985

J of Cellular Biochemistry

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The folding of the bovine glycoprotein hormone alpha subunit, synthesized in bacteria following insertion of the nucleotide sequence coding for this polypeptide, has been studied to determine the effect that a complete lack of carbohydrate has on this process. The bacterially derived alpha polypeptide (bac-alpha), extracted from E. coli in the presence of reductant and denaturant, had an estimated 0.2% native structure as determined by a conformationally sensitive radioimmunoassay. Upon reduction of disulfide bonds and reoxidation in air, the amount of native structure increased about 18-fold. Approximately 2% of the refolded bac-alpha preparation combines with the beta subunit of human chorionic gonadotropin (hCG beta) to form a complex that binds to the gonadotropin receptor and elicits a biological response. Since the correct folding (by immunological criteria) of bac-alpha (ca 3%) is significantly greater than expected from a random formation of disulfide bonds (0.1%), it appears that correct folding of alpha subunit can occur in the complete absence of carbohydrate, though in very low yield. Native bovine lutropin alpha subunit (LH alpha) and chemically deglycosylated LH alpha (which retains two asparagine-linked N-acetyl glucosamine residues per alpha oligosaccharide) were subjected to the same reduction/reoxidation regimen as the bacterially produced alpha subunit. As has been reported previously [Giudice LC, Pierce, JG, J Biol Chem 251: 6392, 1976] intact LH alpha fully regained its native structure. The partially deglycosylated LH alpha also refolds to a native-like structure in high yield as assessed by immunological assays and by its ability to combine with HCG beta to form a biologically active complex. The data show that carbohydrate, while not obligatory for correct folding, greatly facilitates the formation of functional alpha subunit.

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Relationship of glycosylation to de novo thyroid-stimulating hormone biosynthesis and secretion by mouse pituitary tumor cells.

Cited by 102 publications

References 46 publications

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

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

Synthesis of the glycoprotein hormone .alpha. subunit and placental alkaline phosphatase by HeLa cells: effect of tunicamycin, 2-deoxyglucose, and sodium butyrate

The common α subunit of bovine glycoprotein hormones: Limited formation of native structure by the totally nonglycosylated polypeptide chain

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