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C Ronin

Publications and source records attributed to C Ronin.

At least 37 records · Page 2Linked to original sources
PubMed1989

Differential effect of inhibitors of oligosaccharide processing on the secretion of thyrotropin from dispersed rodent pituitary cells.

We examined the effect of various inhibitors of oligosaccharide processing on the content and secretion of newly synthesized thyroid-stimulating hormone (TSH) from dispersed hypothyroid rodent pituitary cells. 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin, both inhibitors of glucosidases I and II, decreased intracellular TSH (to 60-76% of control) and secreted TSH (to 60-63% of control) after a 1-hour incubation (pulse) with [35S]methionine and an 8-hour incubation (chase) in isotope-free media. In contrast, deoxymannojirimycin and swainsonine, inhibitors of mannosidase I and II, respectively, increased both intracellular TSH (to 267-309% of control) and secreted TSH (to 192% of control) at 8 hours. TSH oligosaccharides synthesized in the presence of these glucosidase and mannosidase inhibitors were largely sensitive to endo-beta-N-acetylglucosaminidase H (endo H), confirming inhibition of processing. Despite differences in oligosaccharide structure, the in vitro bioactivities of these secreted TSH isoforms were nearly identical. These data confirm and extend previous work performed with 1-deoxynojirimycin suggesting that glucosylated high mannose forms of TSH are more susceptible to intracellular degradation. The novel finding that deoxymannojirimycin and swainsonine increase secreted and total TSH above control levels suggests that non-glucosylated high mannose forms as well as hybrid-type oligosaccharides may facilitate secretion and direct TSH away from a natural degradation pathway.

1-Deoxynojirimycin↗
PubMed1989

[Gonadotropin receptors].

The reproductive function of both the male and female is under the control of several pituitary hormones including lutropin (LH) and follitropin (FSH). These hormones are highly homologous glycoprotein dimers sharing a common subunit. Their polymorphism mainly resides in the microheterogeneity of their carbohydrate chains that is also responsible for the variations in biological activity. In the gonads, these hormones bind to specific receptors and regulate steroidogenesis and gametogenesis. LH receptors also bind the choriogonadotropin hormone and are located in Leydig cells in testis, interstitial and medulla cells in ovary. FSH receptors are present in Sertoli cells in the male while they control follicular maturation in the female. In both sexes, their activity is coupled to that of other receptors such as that of prolactin or one able to recognize GnRH. The purification of LH receptor has been successfully achieved in several animal species and showed a significant structural homology between the receptors of both sexes.

Animals↗
PubMed1988

Selective loss of binding sites for the iodinated alpha-neurotoxin I from Naja mossambica mossambica venom upon enzymatic deglycosylation of Torpedo electric organ membranes.

Removal of asparagine-linked carbohydrate chains from Torpedo marmorata electric organ membranes was found to inhibit the binding of the iodinated alpha-neurotoxin I from Naja mossambica mossambica snake venom to its receptor. Optimal deglycosylation of membranes by endoglycosidase F resulted in a 55% inhibition of alpha-neurotoxin-I-saturable binding. Under these conditions, up to 70% of concanavalin A binding was also lost, indicating an efficient removal of mannose-rich carbohydrate chains. Saturation binding experiments at equilibrium on membranes incubated in the absence of endoglycosidase F indicated, when analyzed by Scatchard plots, the presence of two classes of high-affinity binding sites for alpha-neurotoxin I (kd = 9 pM and 68 pM respectively) with capacities of 24 and 14 pmol/mg membrane proteins, respectively. After endoglycosidase F treatment, only the former class of binding sites (Kd = 11 pM) was recovered together with a 45% reduction in the number of total binding sites. Dissociation experiments further confirmed the presence of two types of toxin-receptor complexes in control membranes and the selective loss of the rapidly dissociating component upon deglycosylation. The binding of alpha-neurotoxin I to its receptor, deglycosylated or not, was totally inhibited by carbamoylcholine, d-tubocurarine or alpha-bungarotoxin. These findings show that the neurotoxin binding sites present on the acetylcholine receptor can be discriminated on the basis of their differential susceptibility to the removal of asparagine-linked carbohydrate chains.

Animals↗
PubMed1988

Differential carbohydrate processing and secretion of thyrotropin and free alpha subunit. Effects of 1-deoxynojirimycin.

In pulse-chase experiments we compared the kinetics of early carbohydrate processing and subsequent secretion of thyroid-stimulating hormone (TSH) and free alpha subunit under control conditions and after treatment with 1-deoxynojirimycin, an inhibitor of glucosidases I and II. Under control conditions TSH achieved resistance to endo-beta-N-acetylglucosaminidase H (endo H) more rapidly than free alpha (t1/2 0.3 h versus 0.9 h); however, free alpha was secreted more rapidly than TSH (t1/2 2.2 h versus 3.4 h). With 1-deoxynojirimycin, oligosaccharides co-migrating with G3Man9GlcNAc and G2Man9GlcNAc were demonstrated on TSH for the first time, suggesting that previous pulse-chase studies did not disclose these intermediates due to rapid removal of glucose residues from the common G3Man9GlcNAc2 precursor. 1-Deoxynojirimycin delayed the rate of attainment of endo H resistance for both TSH and free alpha, but there was no effect on subunit combination. With 5 mM 1-deoxynojirimycin the amount of secreted free alpha was reduced to 65% of control; secreted TSH was reduced markedly to 17% of control without intracellular accumulation, suggesting increased intracellular degradation. There was no significant toxicity from these doses of 1-deoxynojirimycin on the production or secretion of the two major nonglycosylated pituitary proteins, growth hormone and prolactin, or on at least 10 other secretory proteins. Basal differences in the relative rates of TSH and free alpha processing and secretion as well as differential sensitivity to 1-deoxynojirimycin suggest separate secretory pathways for these two closely related proteins.

1-Deoxynojirimycin↗
PubMed1988

Polymorphism of prolactin secreted by human prolactinoma cells: immunological, receptor binding, and biological properties of the glycosylated and nonglycosylated forms.

Multiple forms of PRL differing in their physicochemical and biological characteristics have been described. We have analyzed the molecular forms of human (h) PRL released in culture by pure hPRL-secreting tumors with a particular attention to glycosylated hPRL. The prolactinoma cells from six different tumors released, in serum-free conditions, 10-28 mg hPRL. The combination of polyacrylamide gel electrophoresis and immunoblotting techniques using a [125I]anti-hPRL monoclonal antibody allowed qualitative and quantitative analysis of the hPRL variants. The ratio of the glycosylated 25,000-mol wt form (G-hPRL) to the 23,000-mol wt nonglycosylated monomeric hPRL (NG-hPRL) varied from 0.13 to 0.25. Under the conditions of our studies, cleaved forms of the hormone (19,000 and 15,000 mol wt) accounted for less than 5% of the total immunoreactivity. G- and NG-hPRL were subsequently purified by gel filtration and lectin affinity chromatography. G-hPRL appeared fully sensitive to endoglycosidase F digestion, further supporting the presence of a freely accessible N-linked carbohydrate chain. When assayed for their ability to react with polyclonal antibodies directed against hPRL in a competitive RIA, G-hPRL was 3 times less immunoreactive than NG-hPRL. However, both types of hPRL exhibited superimposable displacement curves when tested in an immunoassay using an anti-hPRL monoclonal antibody. In binding studies using crude rabbit mammary gland membranes G-hPRL was half as potent as NG-hPRL. In stimulating the growth of the Nb2 lymphoma cell line, G-hPRL was 50% less active than NG-hPRL. Thus 1) under basal conditions, hPRL undergoes partial and variable glycosylation; 2) glycosylation of the hormone may modulate its immunoreactivity; 3) glycosylation of hPRL not only lowers its mammary gland receptor binding capacity but also its growth-promoting activity.

Adenoma↗
PubMed1987

Carbohydrate chains of human thyrotropin are differentially susceptible to endoglycosidase removal on combined and free polypeptide subunits.

The accessibility of the asparagine-linked carbohydrate chains of human thyrotropin (hTSH) and free alpha and beta subunits was investigated by their susceptibility to endoglycosidases H and F as well as to peptide:N-glycosidase F. Iodinated hTSH or subunits were incubated with a commercial enzyme preparation containing both endoglycosidase F and N-glycosidase F activities and further analyzed by sodium dodecyl sulfate gel electrophoresis followed by quantitative autoradiography. We show that, working at the optimum of the N-glycosidase activity, the relative amount of endoglycosidase required for half-deglycosylation was 20-fold higher for native hTSH than for the reduced and dissociated subunits. Under nondenaturing conditions, the 18K beta subunit of hTSH could be readily deglycosylated to a 14K species while the 22K alpha subunit was largely resistant. However, both subunits were converted to an apoprotein of similar apparent molecular weight of 14K following reduction of disulfide bonds. In contrast, the free alpha subunit of human choriogonadotropin appeared fully sensitive to carbohydrate removal under nonreducing conditions despite the presence of a partially deglycosylated 18K intermediate at low concentration of endoglycosidase. Similarly, both hTSH-alpha and hTSH-beta could be completely deglycosylated after acid dissociation of the native hormone. While all three carbohydrate chains of hTSH are sensitive to pure peptide:N-glycosidase F, only one on alpha and the single oligosaccharide present on beta in hTSH appeared to be cleaved by pure endoglycosidase F. Interestingly, one of the two carbohydrate chains present on alpha was also found to be susceptible to endoglycosidase H.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrates↗
PubMed1986

Immunochemical evidence for a role of complex carbohydrate chains in thyroglobulin antigenicity.

Thyroglobulin secreted by porcine thyroid cells in serum-free culture was previously found (Ronin, C., Fenouillet, E., Hovsepian, S., Fayet, G., and Fournet, B. (1986) J. Biol. Chem. 261, 7287-7293) to contain more highly branched complex carbohydrate chains than the thyroid-derived molecules. When assayed for their ability to react with polyclonal antibodies directed against the natural prohormone in competitive radioimmunoassays, using the porcine antigen as iodinated tracer and standard competitor, in vitro thyroglobulin appeared to be 4-fold less immunoreactive than the in vivo molecule. However, both types of thyroglobulin exhibited superimposable incomplete displacement curves after peripheral deglycosylation using a mixture of neuraminidase, alpha-, and beta-galactosidases while they behave as good competitors as the native antigen after removal of the majority of the carbohydrate chains by Endo-beta-N-acetylglucosaminidase F. Solid-phase binding assays revealed that in vitro thyroglobulin was able to bind less antibodies than its thyroid-derived counterpart before as well as after treatment with exo- or endoglycosidases. Furthermore, only 20% of thyroglobulin biosynthetically labeled with glucosamine could be precipitated with specific antibodies followed by addition of staphylococcal-protein A, whereas up to 61% of the labeling was specifically bound when the antibodies were preincubated with protein A. After pronase digestion, both thyroglobulin-like material displayed different carbohydrate structures as judged by concanavalin A-Sepharose analysis. Thus, substituting multiantennary complex carbohydrate chains to the usual biantennary and high mannose structures present on thyroid-derived thyroglobulin had a profound effect on the prohormone immunoreactivity.

Animals↗
PubMed1986

Regulation of thyroglobulin glycosylation. A comparative study of the thyroglobulins from porcine thyroid glands and follicles in serum-free culture.

Porcine thyroid cells were cultured in serum-free medium and thyrotropin was or was not added at day 4 and [3H]glucosamine at day 6 for 24 h. The major glycoprotein secreted outside the follicles proved to be thyroglobulin by immunoprecipitation, polyacrylamide gel electrophoresis, and amino acid composition. Thyroglobulin glycopeptides were analyzed by sequential affinity chromatography on immobilized lectins and compared to chemically labeled carbohydrate chains released from thyroid-derived thyroglobulin by hydrazinolysis. 82% and 85% of the glucosamine-labeled oligosaccharides of thyroglobulin from control and stimulated cells, respectively, were unretained on concanavalin A (ConA)-Sepharose compared to 46% only for in vivo thyroglobulin. 35-42% and 33-35% of the ConA-unbound glycopeptides were retarded on erythrophytohemagglutinin and leukophytohemagglutinin under basal or stimulatory conditions, respectively, while none of the triantennary structures of in vivo thyroglobulin was. Moreover, binding to Bandieraea-agarose showed that 20% of these complex structures contained alpha-linked galactose in thyroglobulin secreted by control cells, but only 10% in the molecules derived from thyroid. When analyzed on ricin-agarose after neuraminidase treatment, the ConA-unbound glycopeptides were retained to an extent of 65% for those from control cells and 98% for those from stimulated cells. Furthermore, 15% of desialylated ConA-unbound glycopeptides from cellular origin were also found to bind to wheat germ agglutinin. Carbohydrate composition, gel chromatography, and exoglycosidase treatment further demonstrated that thyroglobulin carbohydrate chains synthesized under serum-free cell culture were essentially composed of heterogeneous multiantennary structures instead of usual biantennary and high mannose type species. Under thyrotropin stimulation, 85% of the carbohydrate chains of thyroglobulin was shown to be sialylated by high performance liquid chromatography analysis instead of 65% under basal conditions, suggesting that thyrotropin may shift terminal glycosylation of thyroglobulin from alpha-galactose to sialic acid.

Animals↗
PubMed1985

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

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)

Animals↗
PubMed1984

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↗
PubMed1984

Carbohydrate processing of thyrotropin differs from that of free alpha-subunit and total glycoproteins in microsomal subfractions of mouse pituitary tumor.

We have determined the structures of high mannose (Man) oligosaccharide units of TSH, free alpha-subunit, and non-TSH-related total glycoproteins (TP) within microsomal subfractions of mouse thyrotropic tumor. Tumor minces were incubated with D-[2-3H]Man, homogenized, and subfractionated into rough endoplasmic reticulum (RER) as well as proximal and distal smooth endoplasmic reticulum/Golgi apparatus. TSH subunits and TP were precipitated from these fractions, and high Man units released by endoglycosidase H were analyzed by paper chromatography. Glc3Man9GlcNAc2 (Glc = glucose; GlcNAc = N-acetylglucosamine) was not detected in TSH subunit precursors in any fraction, but was detected in TP. Glc1Man9GlcNAc2 accumulated in TSH with chase, but only small amounts were detected in free alpha-subunit and TP. Trimming of Man9GlcNAc2 to Man8GlcNAc2 began in RER before 1 h for all species, but the rate of Man trimming was rapid for free alpha-subunit, moderate for TSH, and slow for TP. Man8GlcNAc2 was a rate-limiting step in processing for all species in the RER. Man5GlcNAc2 was a second major rate-limiting step in processing of free alpha-subunits only and accumulated in distal smooth endoplasmic reticulum/Golgi apparatus. Other studies in progress suggest that a function of the thyrotroph is to modulate the structure of TSH oligosaccharide units during biosynthesis to achieve mature hormone with physiologically appropriate metabolic clearance and intrinsic biological activity. The present study supports this notion, since we found that the qualitative nature and kinetics of processing differ for oligosaccharide units of TSH, free alpha-subunit and TP within the microsomes.

Animals↗
PubMed1981

Transfer of glucose in the biosynthesis of thyroid glycoproteins. I. Inhibition of glucose transfer to oligosaccharide lipids by GDP-mannose.

Thyroid rough microsomes catalyzed the synthesis of glucose-containing oligosaccharide lipids which were compared to those extracted from labeled thyroid cells and were found to be largely similar. Glucose transfer to these oligosaccharide lipids in the microsomal system was shown to be markedly depressed by an addition of GDPmannose. This sugar nucleotide, already at 1 microM, blocked dolichol-P-glucose synthesis, thus restraining further glucosylation of oligosaccharide lipids. Using this concentration of radioactive GDPmannose in the incubation medium lead to the detection of three glucose containing mannose-labeled oligosaccharide lipids. Double labeling experiments suggested a precursor-product relationship between them. Previously labeled oligosaccharide lipids, containing glucose or not were compared in their efficiency to act as donors of their oligosaccharide chain to an exogenous synthetic Asn-X-Thr containing peptide. It was found that the presence of glucose did not significantly influence the transfer. Free glucose was released during the reaction when using the glucose-labeled oligosaccharide lipid.

Animals↗
PubMed1981

Transfer of glucose in the biosynthesis of thyroid glycoproteins. II. Possibility of a direct transfer of glucose from UDP-glucose to proteins.

When thyroid rough microsomes are incubated with labeled UDPglucose, extensive labeling occurs on endogenous protein. The present study reports various time-course experiments of glucose incorporation from UDPglucose either into endogenous acceptors or into exogenous synthetic peptide, whether or not conditions are used which depress dolichol-P-glucose synthesis. The results are not compatible with the presently accepted idea of oligosaccharide lipids as sole donors of labeled glucose to proteins. Some structural properties of glucose-labeled oligosaccharides released from proteins by endo-beta-N-acetylglucosaminidase H were also investigated. They appeared to be of a single species and less degradable by alpha-mannosidase than their lipid-linked counterpart. Since glucose-labeled glycopeptides were found susceptible to a beta-glucosidase, it is proposed that some labeled glucose residues are involved in beta-linkages. The possibility is raised that glucose might be directly transferred from UDPglucose to endogenous glycoproteins.

Animals↗
PubMed1981

Synthetic substrates for thyroid oligosaccharide transferase. Effects of peptide chain length and modifications in the Asn-Xaa-Thr-region.

The N-glycosylation of proteins is initiated by transfer of preassembled oligosaccharides from lipid carriers to an asparagine residue in the sequence Asn-Xaa-Ser(Thr). Various synthetic peptides were previously shown to act as effective acceptors when supplied to microsomal membranes. The present work was undertaken to delineate further within such peptides the structural determinants required for the N-glycosylation process. Several new peptides were synthesized to evaluate the effects of the chain length and of the modification on the asparagine, Xaa or threonine residues: asparagine was replaced by glutamine, Xaa by proline or N-methylated alanine, threonine by an homoserine residue. Furthermore, both side chains of asparagine and threonine were simultaneously replaced by those of glutamine and homoserine respectively to restore a putative hydrogen bonding between the carbonyl and hydroxyl groups. The assays were performed with a solubilized form of the oligosaccharide transferase and the acceptor capacities of the peptides expressed by the ratio V/Km of their apparent kinetic parameters V and Km. Results showed that N-acetyl-tripeptide amides are excellent substrates for the enzyme. All modifications in the -Asn-Xaa-Thr- region resulted in a loss of acceptor capacity. The importance of the conformational aspect is discussed.

Amino Acid Sequence↗