Biomedical subjects

J L Franc

Publications and source records attributed to J L Franc.

11 recordsLinked to original sources

PubMed1992

Effects of deglycosylation of human thyroperoxidase on its enzymatic activity and immunoreactivity.

Thyroid peroxidase (TPO) is a glycoprotein enzyme which catalyses the iodination of thyroglobulin and the coupling of iodinated tyrosines. Human TPO (hTPO) is the microsomal antigen recognized by the autoantibodies in the serum of patients with autoimmune thyroid disease. An active detergent-solubilized immunoaffinity-purified hTPO was deglycosylated, either by peptide N-glycosidase F (PNGase F) or by endo-beta-N-acetylglucosaminidase H (endo H), and the enzymatic activity and immunoreactivity of the native and deglycosylated forms were compared. Electrophoretic controls and affinoblotting with concanavalin A showed that deglycosylation was not total and that it was more pronounced with endo H than with PNGase F. The enzymatic activity of hTPO was inhibited by endo H deglycosylation, but not by PNGase F deglycosylation; this inhibition was not due to aggregation and/or insolubilization of the molecule subsequent to deglycosylation. Immunoreactivity was monitored by enzyme-linked immunosorbent assay (ELISA) with 13 mouse monoclonal antibodies, rabbit polyclonal antibodies and antibodies from serum of patients with Hashimoto's thyroiditis. In contrast with enzymatic activity, immunoreactivity was not modified or was slightly enhanced (with four monoclonal antibodies) by deglycosylation. The results indicate that strong, if not total, deglycosylation induces a modification of the tertiary structure of hTPO, which affects the enzymatic site but does not modify markedly the epitopes implicated in the recognition of the molecule by the antibodies tested.

Amidohydrolases↗

PubMed1991

Effect of N-glycan removal on the enzymatic activity of porcine thyroid peroxidase.

Active porcine thyroid peroxidase (pTPO) has been purified either by deoxycholate extraction followed by immunoaffinity purification (pTPO A) or by trypsin/digitonin extraction followed by ion-exchange and gelfiltration chromatography (pTPO B); pTPO A appeared as a full-length molecule, while pTPO B appeared as peptide fragments. Purified pTPO were deglycosylated either by peptide N-glycosidase F (PNGase F) or by endo-beta-N-acetylglucosaminidase H (endo H) treatment. Electrophoretic controls and affinity blotting with concanavalin A indicated that deglycosylation was not total and that pTPO was more efficiently deglycosylated by endo H than by PNGase F. The enzymatic activity of pTPO A, checked by guaiacol and iodide oxidation, was inhibited by PNGase F and endo H deglycosylation, while that of pTPO B was not. After deglycosylation, the apparent Km of pTPO A for guaiacol and iodide increased, while the Vmax for both substrates decreased. The state of aggregation of pTPO A before and after deglycosylation was checked by sucrose density-gradient centrifugation. Results indicated that this inhibition was not due to a loss of pTPO A solubility. These observations suggest that deglycosylation induced a modification of the tertiary structure of pTPO A which affected the active-site domain of the enzyme.

Animals↗

PubMed1990

Inhibition of N-glycan processing affects iodide organification in porcine thyroid cells.

The N-glycan-processing inhibitors swainsonine (Sw) and deoxymannojirimycin (dMM) were used to study the influence of N-glycans on iodide organification in cultured porcine thyroid cells. Incubations with [125I]NaI were followed by determination of labeled trichloroacetic acid-insoluble material in culture media, follicular contents and cells. In controls, most of this material was in the follicular contents. With Sw and dMM, total acid-insoluble material was less than 10% of control. Iodide uptake was slightly inhibited and hydrogen peroxide release was not affected by inhibitors. Cell-surface thyroid peroxidase (TPO) activity, assayed by its ability to iodinate bovine serum albumin, was strongly inhibited. Pronase glycopeptide analysis indicated that with drugs the content in complex-type N-glycans was strongly decreased while that in hybrid or oligomannosidic type was increased. In conclusion, inhibition of N-glycan processing prevents iodide organification in cultured porcine thyroid cells by decreasing the recovery of cell-surface TPO activity.

1-Deoxynojirimycin↗

PubMed1990

Role of sialic acid residues in crossed immuno-affinoelectrophoresis of alpha 1-proteinase inhibitor.

We have investigated the importance of the degree of sialylation when an acute phase glycoprotein, alpha 1-proteinase inhibitor (alpha 1-Pi), was analysed both by affinity chromatography on concanavalin A (Con A)-Sepharose and by crossed immuno-affinoelectrophoresis (CIAE) using Con A in the first dimension. Human alpha 1-Pi was isolated by immunosorption chromatography and then more or less desialylated. On Con A-Sepharose chromatography no significant difference was observed in the percentage of the two fractions (retained or not retained) whatever the degree of desialylation. In contrast by CIAE this degree was largely involved in the separation of the different isoforms obtained in the first dimension.

Chromatography, Affinity↗

PubMed1990

Characterization of the two oligosaccharides present in the preferential hormonogenic domain of human thyroglobulin.

The N-terminal fragment of human thyroglobulin (residues 1 to 171) contains the preferential hormonogenic site of the molecule and 2 potential sites of N-glycosylation (Asn57 and Asn91). This fragment was isolated from a human thyroglobulin purified from a single goiter. The tryptic peptides bearing the glycosylation sites were separated by Bio-Gel P-30 and HPLC columns. The oligosaccharides borne at each site were analyzed, after tritium labeling, by concanavalin A-Sepharose and HPLC. At both sites the structures observed are heterogenous, with a majority of biantennary complex type structures.

Chromatography, High Pressure Liquid↗

PubMed1990

Effects of deoxymannojirimycin and castanospermine on the polarized secretion of thyroglobulin.

In order to better explore a possible role of oligosaccharide structures in the polarized secretion of thyroglobulin (Tg), we labeled cultured porcine thyroid cells with L-[3,4,5-3H]leucine or D-[2-3H]mannose in the presence or absence of the following inhibitors of N-linked oligosaccharide processing: deoxymannojirimycin (dMM), an inhibitor of mannosidase I expected to give rise to high mannose units exclusively, thus abolishing the formation of lactosaminyl branches, and castanospermine (Cs), an inhibitor of glucosidases which is expected to produce glucosylated high mannose oligosaccharides. [3H]Leucine pulse-chase experiments were performed to study the rate of Tg secretion in the presence and absence of dMM (1 mM) or Cs (1.6 mM). After a 15-h chase period, dMM and Cs did not modify the relative proportions of released Tg (65% in the follicular content, 16% in the medium) and cellular Tg (19%). In contrast, Cs led to a lower rate of secretion in both secretory pathways (t1/2 increased from 42 min to 105 min in the apical pathway and from 108 min to 138 min in the basal pathway). After a long-term labeling (16 h) with [3H]leucine or [3H]mannose, drugs did not notably affect the relative proportions of labeled Tg in the different compartments (follicular content, medium, and cell). The structures of N-glycans borne by Tg, with and without drugs, were checked. After Tg immunoprecipitation, pronase-glycopeptides were fractionated on concanavalin A-Sepharose 4B. The glycopeptides tightly bound to the lectin were treated by endo-beta-N-acetylglucosaminidase H and oligosaccharides separated by HPLC. With dMM, complex-type glycans of Tg were totally replaced by high mannose-type glycans, Man8-9GlcNAc; Cs induced the accumulation of glucosylated high mannose-type structures, Glc3Man7-9GlcNAc and Glc2Man8-9GlcNAc, but the action of this inhibitor was not total. In conclusion the correct secretion of Tg does not require the presence of the sialyllactosaminyl structure; the presence of glucose residues on high mannose-type structures lowers the rate of exocytosis.

1-Deoxynojirimycin↗

PubMed1989

Thyroglobulin structure and function: recent advances.

Thyroglobulin is a large-size iodoglycoprotein specific to thyroid tissue and is the substrate for the synthesis of thyroid hormones, thyroxine and 3,5,3'-triiodothyronine. Recent studies, which greatly benefited from recombinant DNA methodologies, improved the knowledge of several structural features of this dimeric protein and permitted insights into some structure-function relationships. Analysis-function of the primary structure of the human thyroglobulin monomer revealed several main characteristics: 1) 3 types of internal homologies; 2) extensive homology with the bovine thyroglobulin monomer and known partial sequences in the thyroglobulins of other mammalian species; 3) significant homologies with 2 other non-thyroid proteins (acetylcholinesterase and the invariant chain of the Ia class II histocompatibility antigen); 4) a terminal localization of the hormonogenic sites at both ends of the monomer. Current studies aim at determining conformational characteristics, understanding the molecular mechanisms of thyroid hormone formation and unraveling those interactions which in the thyroid cell and the thyroid follicle will permit this large pro-hormone to synthesize and release a few small thyroid hormone molecules. A more precise knowledge of this molecule in higher vertebrates and during evolution would impart valuable information concerning thyroid pathology, since thyroglobulin has been implicated in some genetic and in autoimmune thyroid diseases.

Amino Acid Sequence↗

PubMed1989

Effects of tunicamycin and N-linked oligosaccharide-processing inhibitors on the morphology of cultured porcine thyroid cells.

The effects of tunicamycin and of N-linked oligosaccharide-processing inhibitors on the ability of cultured porcine thyroid cells to adhere to a plastic support and to form organized structures were examined. The culture conditions used allowed the epithelial cells to adhere to the support and to form either a monolayer (no thyrotropin) or follicles (thyrotropin 4 mU/ml). The follicles thus obtained tend to disappear after 8 to 9 days, giving rise to a monolayer. Tunicamycin prevented both cell adhesion to the support and formation of organized structures. Swainsonine, an inhibitor of mannosidase II, had no obvious effect. Deoxymannojirimycin, an inhibitor of mannosidase I, did not prevent cell adhesion to the support and formation of monolayers or follicles, but it favored the maintenance of follicles at a time when they were no longer present in controls. It also led to the appearance of some follicles in cultures without thyrotropin. Castanospermine, an inhibitor of glucosidase I, did not prevent cell adhesion but slowed cell spreading, thus delaying monolayer formation. Pronase glycopeptides prepared from cell-surface glycoproteins were examined with respect to their behavior on concanavalin A-Sepharose. The glycopeptides from control cells displayed complex and high-mannose glycans. The content in complex glycans was decreased in inhibitor-treated cells, while that in hybrid or high-mannose glycans was increased, indicating that the inhibitors modify the N-glycan structures. In conclusion, N-glycosylation of glycoproteins is necessary for cellular adhesion to the support. Complex structures do not seem necessary for cell adhesion monolayer or follicle formation. High-mannose structures favor follicular organization, while glucoses on the high mannose structures hinder cell spreading.

1-Deoxynojirimycin↗

PubMed1988

Nuclear T3 receptor: depletion by tunicamycin despite the absence of N-linked glycan units in a murine preadipocyte cell line and rat liver.

In experiments designed to evaluate the possible presence of N-glycan units in the nuclear T3 receptor, tunicamycin markedly depleted the nuclear T3 receptor sites when added to the culture medium of the T3 responsive ob 17 preadipocyte cell line under conditions which almost totally abolished protein N-glycosylation without significant alteration of protein synthesis. The affinity for T3 was unchanged. However, no significant interaction could be detected between the T3 receptor solubilized from the ob 17 cells, or from rat liver, and several insolubilized lectins of different specificities. Furthermore, treatment of the cells with swainsonine, a Golgi mannosidase II inhibitor, did not lead to any significant interaction of the receptor with concanavalin A as it would have occurred if the receptor had contained complex glycan units unrecognized by this lectin. These results are strong arguments against the presence of N-glycosidic moieties in the nuclear T3 receptor from mouse adipose cells and rat liver. The receptor sites depletion after tunicamycin treatment may most probably reflect an indirect effect through other glycoproteins which could be on the one hand required for either receptor stabilization or localization in the chromatin, or on the other hand involved in receptor level regulation.

Adipose Tissue↗

PubMed1987

Effects of severe burns on glycan microheterogeneity of four acute phase proteins.

In serum from 8 severely burned patients, haptoglobin (Hp), alpha 1-acid glycoprotein (AG) and alpha 1-antitrypsin (AT) were found to be increased by factors of 5, 6 and 2 respectively. Ceruloplasmin (Cp) was slightly decreased. In order to appreciate possible modifications to the structure of their attached N-glycans, whole sera were fractionated on concanavalin A (Con A)-Sepharose and respective glycoproteins measured by laser nephelometry using a monospecific antiserum. In the serum from normal as well as burned patients Hp was almost entirely bound to the immobilized lectin (but eluted with 300 mmol/l alpha 1-methylglucoside) and Cp was bound at about 92%. For AG, in contrast, the fraction without affinity for Con A, 25% in normal serum, decreased to 5% in patients, whereas the retained species increased in proportion. A very weakly reactive fraction (which was only retarded and eluted without alpha-methylglucoside) amounted to 72% in both types of serum. When reduced and alkylated, this intermediate fraction gave rise to both non-retained and retained species always in a proportion of about 1/3. On the whole one concludes that there is a significant shift for AG in burned patients towards species enriched in bi-antennary (Con A-reactive) glycans. For AT a minor part was not recognized by the lectin and about 27% was retarded. The latter, which increased in burned patients, gave rise mainly to retained species after reduction and alkylation. This again suggests a shift to bi-antennary glycans.

Acute-Phase Proteins↗

PubMed1986

Inhibition of N-linked oligosaccharide processing does not prevent the secretion of thyroglobulin. A study with swainsonine and deoxynojirimycin.

The effects of two drugs, swainsonine (SW) and deoxynojirimycin (dNM), on synthesis and export of thyroglobulin were studied in folliculized porcine thyroid cells cultured in a serum-free medium. These drugs were expected to alter N-linked glycans in thyroglobulin. Newly synthesized thyroglobulin labeled with [2-3H]mannose or [4,5-3H]leucine was obtained by immunoprecipitation from the follicular contents, culture media and cell extracts; the first two compartments, containing secreted thyroglobulin, were sometimes analyzed together. Leucine incorporation was not inhibited by SW and only slightly by dNM. In contrast dNM strongly decreased mannose incorporation (by up to 50-75% at 1-3 mM). However after 16-h mannose labelings, SW and/or dNM at 2.5 microM and 3 mM respectively did not significantly modify the relative proportions of radioactive thyroglobulin in the above-mentioned compartments. Pronase glycopeptides prepared from these thyroglobulins were examined with respect to behaviour on concanavalin-A-Sepharose and position on Bio-Gel P-4. Oligosaccharides released by endoglucosaminidase H and with high affinity for the lectin, i.e. high-mannose and certain hybrids, were further characterized by various exoglycosidase treatments. Thyroglobulin from control cells displayed complex and high-mannose glycans comparable in size and proportion to those attributed to tissue-extracted porcine thyroglobulin. After treatment with SW (an inhibitor of alpha-mannosidase II), complex glycans were almost totally replaced by sialylated hybrid glycans. In contrast to this nearly total suppression, dNM (an inhibitor of the trimming glucosidases) caused only a 30% decrease in labeling of complex units and an about 50% increase in high-mannose glycans, covered to some degree by glucose. Finally a [3H]leucine pulse-chase study was performed on thyroglobulin secretion in the absence or presence of both SW and dNM. Though a slowdown was detectable in the first few hours, this study revealed no change in the long-term export of thyroglobulin.

1-Deoxynojirimycin↗