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Nlend, Chabaud
The plasminogen-like molecule apically secreted by epithelial thyroid cells is sulfated.Giraud A, Chabaud O, Lejeune PJ, Barbaria J, Mallet B. Biochem Biophys Res Commun. 2006 Aug 4;346(3):746-50. Epub 2006 Jun 6. [abstract only]
"Plasminogen (Pl), a
circulating protease synthesized in the liver, is also present in
several tissues. In the thyroid gland a Pl-like protease was found in
the apical lumen where it is involved, through its proteolytic
activity, in luminal degradation of thyroglobulin (Tg). Here, we
showed for the first time that the Pl-like protease apically secreted
by epithelial thyroid cells is sulfated, both on tyrosine residue(s)
and on oligosaccharide side chains. The Pl molecule is composed of a
large N-terminal moiety made of five distinct Kringle domains (K1-K5)
separated by small peptidic fragments, and of a C-terminal domain with
serine protease activity. Using a software tool able to predict
tyrosine sulfation sites in protein sequences we localized the
potential tyrosine sulfation sites of Pl. Then, we became aware that,
whatever the species considered, at least three of the four potential
tyrosine sulfation sites of Pl were located on Kringle sites, and more
precisely, for K1, on the highly conserved binding domain of K1. We
determined with the same software tool which potential sulfation sites
were the most likely to be really sulfated. We hypothesize that the
sulfation of these sites modulates the binding properties of Pl."
Role of sulfated tyrosines of thyroglobulin in thyroid hormonosynthesis.Nlend MC, Cauvi DM, Venot N, Chabaud O. Endocrinology. 2005 Nov;146(11):4834-43. Epub 2005 Jul 21.
"Our previous studies showed that sulfated tyrosines (Tyr-S) are involved in thyroid hormone synthesis and that Tyr(5), the main hormonogenic site of thyroglobulin (Tg), is sulfated. In the present paper, we studied the role of Tyr-S in the formation and activity of the peroxidase-Tg complex. Results show that noniodinated (35)SO(3)-Tg specifically binds (Kd=1.758 microM) to immobilized lactoperoxidase (LPO) via Tyr-S linkage by using saturation binding and competition experiments. We found that NIFEY-S, a 15-amino acid peptide corresponding to the NH2-end sequence of Tg and containing the hormonogenic acceptor Tyr5-S, was a better competitor than cholecystokinin and Tyr-S. 35SO3-Tg, iodinated without peroxidase, bound to LPO with a Kd (1.668 microM) similar to that of noniodinated Tg, suggesting that 1) its binding occurs via Tyr-S linkage and 2) Tyr-S requires peroxidase to be iodinated, whereas nonsulfated Tyr does not. Iodination of NIFEY-S with [125I]iodide showed that Tyr5-S iodination increased with LPO concentration, whereas iodination of a nonsulfated peptide containing the donor Tyr130 was barely dependent on LPO concentration. Enzymatic hydrolysis of iodinated Tg or NIFEY-S showed that the amounts of sulfated iodotyrosines also depended on LPO amount. Sulfated iodotyrosines were detectable in the enzyme-substrate complex, suggesting they have a short life before the coupling reaction occurs. Our data suggest that after Tyr-S binding to peroxidase where it is iodinated, the sulfate group is removed, releasing an iodophenoxy anion available for coupling with an iodotyrosine donor."
Thyrotropin and iodide regulate sulfate concentration in thyroid cells. Relationship to thyroglobulin sulfation.Cauvi D, Venot N, Nlend MC, Chabaud OM. Can J Physiol Pharmacol. 2003 Dec;81(12):1131-8. [abstract only]
"Thyroglobulin (Tg), the
thyroid hormone precursor, is sulfated both on tyrosines and on
carbohydrates. We showed recently that sulfated tyrosines were
involved in thyroid hormone synthesis. Moreover, we also reported that
Tg sulfation is downregulated by thyrotropin (TSH), especially on
tyrosines. This control may occur at each step in the sulfation
process. In this paper, we studied the regulation of the concentration
of cytosolic inorganic sulfate, the first substrate, in porcine
thyroid cells stimulated by TSH with or without iodide. The amounts of
cytosolic sulfate and the cytosolic volumes measured showed that the
sulfate concentration depends only on cytosolic volume changes in
response to TSH and iodide treatment. After the cells were labelled
with [35S]-sulfate, the specific radioactivity (SRA) of cytosolic
sulfate was determined. When cells were treated with only TSH, the
concentration and SRA of cytosolic sulfate decreased by 30%, and by
about 15% when cells were incubated with both TSH and iodide. TSH
decreased more conspicuously the rate of [35S]-sulfate incorporation
into Tg (by 57% without iodide, by 43% with iodide) than the
concentration and SRA of cytosolic sulfate, while iodide altered these
parameters to the same extent (15%). These findings suggest that TSH
regulates other steps in the sulfation process, such as specific
substrate and enzyme levels, while iodide controls mainly the sulfate
concentration."
The hormonogenic tyrosine 5 of porcine thyroglobulin is sulfated.Venot N, Nlend MC, Cauvi D, Chabaud O. Biochem Biophys Res Commun. 2002 Oct 25;298(2):193-7. [abstract only]
"Our previous results showed that sulfated tyrosines of thyroglobulin (Tg), the molecular support of thyroid hormonosynthesis, are involved in the hormonogenic process. Moreover, the consensus sequence required for tyrosine sulfation is present in most of the hormonogenic sites. These observations suggest that tyrosine sulfation might play a critical role in the hormonogenic process. In this paper we studied the putative sulfation of tyrosine 5 contained in the preferential hormonogenic site. Porcine thyrocytes were cultured with thyrotropin but without iodide to preserve the sulfation state of tyrosine 5 and then incubated or not with [35S]sulfate. Secreted Tg was purified and submitted to peptide sequence analysis which confirmed the known peptide sequence of the NH(2) extremity of Tg:NIFEYQV. The treatment of [35S]sulfate-labeled Tg by leucine aminopeptidase, which sequentially digested its amino-terminal extremity, released the same amino acids and further analysis by thin layer chromatography showed that the tyrosine was sulfated. We concluded that tyrosine 5 is sulfated but the role of sulfate group in the hormonogenic process remains to be elucidated."
Identification of thyroglobulin domain(s) involved in cell-surface binding and endocytosis.Siffroi-Fernandez S, Delom F, Nlend MC, Lanet J, Franc JL, Giraud A. J Endocrinol. 2001 Jul;170(1):217-26.
"Thyroglobulin (Tg) binds to cell surfaces through various binding sites of high, moderate and low affinity. We have previously shown that binding with low to moderate affinity is pH dependent, selective, but not tissue specific. To identify the regions of Tg involved in this cell surface binding, we studied the binding of (125)I-labeled cyanogen bromide peptides from human Tg to cell surfaces of thyroid cells (inside-out follicles) and of CHO cells. Electrophoretic analysis of cell homogenates after binding of native or of reduced and alkylated (125)I-labeled peptides showed that three peptides, P1, P2 and P3, were always associated with the cells. Sequence analysis allowed the identification of P1 (Ser-2445 to Met-2596 or Met-2610) and P2 (Phe-2156 to Met-2306). P3 proved to be a mixture of several peptides among which two were identified: P3-1 (Cys-1306 to Met-1640) and P3-2 (Cys-2035 to Met-2413) which includes P2. P1, P2 and P3-2 are entirely (P1) or partly (P2 and P3-2) located in the C-terminal domain of Tg homologous with acetylcholinesterase. The smallest peptides, P1 and P2, were purified by preparative electrophoresis. They both displayed strong binding properties towards cell surfaces. Inhibition experiments of (125)I-labeled Tg binding by P1 or P2 indicated that they were involved in Tg binding to cell surfaces. All the other peptides tested for their binding abilities were either not or only poorly involved in Tg binding to cell surfaces, which suggested that P1 and P2 are major Tg sites of binding to cell surfaces. These two peptides are not involved in the binding of Tg to the known Tg 'receptors' described in the literature, to which recycling, transcytosis and regulation functions have been ascribed. Thus they are potential tools to identify cell surface components involved in the process of Tg endocytosis leading to lysosomal degradation."
Sulfate transport in porcine thyroid cells. Effects of thyrotropin and iodide.Cauvi D, Nlend MC, Venot N, Chabaud O. Am J Physiol Endocrinol Metab. 2001
Sep;281(3):E440-8. "In porcine thyroid cells, thyroglobulin sulfation is controlled by thyrotropin (TSH) and iodide, which contribute to regulating the intracellular sulfate concentration, as we previously established. Here, we studied the transport of sulfate and its regulation by these two effectors. Kinetic studies were performed after [(35)S]sulfate was added to either the basal or apical medium of cell monolayers cultured without any effectors, or with TSH with or without iodide. The basolateral uptake rates were about tenfold higher than the apical uptake rates. TSH increased the basolateral and apical uptake values (by 24 and 9%, respectively, compared with unstimulated cells), and iodide inhibited these effects of TSH. On the basis of results of the pulse-chase experiments, the basolateral and apical effluxes appeared to be well balanced in unstimulated cells and in cells stimulated by both TSH and iodide: approximately 40-50% of the intracellular radioactivity was released into each medium, whereas in the absence of iodide, 70% of the intracellular radioactivity was released on the basolateral side. The rates of transepithelial sulfate transport were increased by TSH compared with unstimulated cells, and these effects decreased in response to iodide. These results suggest that TSH and iodide may each control the sulfate transport process on two sides of the polarized cells, and that the absence of iodide in the TSH-stimulated cells probably results in an unbalanced state of sulfate transport."
Regulation of thyroid cell volumes and fluid transport: opposite effects of TSH and iodide on cultured cells.Cauvi D, Penel C, Nlend MC, Venot N, Allasia C, Chabaud O. Am J Physiol Endocrinol Metab. 2000 Sep;279(3):E546-53.
"Cell volume regulation by thyrotropin (TSH) and iodide, the main effectors involved in thyroid function, was studied in cultured thyroid cells. The mean cell volume, determined by performing 3-D reconstitution on confocal microscopy optical slices from living octadecylrhodamine-labeled cells cultured with both TSH and iodide (control cells), was 3.73 +/- 0.06 pl. The absence of iodide resulted in cell hypertrophy (136% of control value) and the absence of TSH in cell shrinkage (81%). These changes mainly affected the cell heights. The effect of TSH on cell volume was mediated by cAMP. The proportion of cytosolic volume (3-O-methyl-D-glucose space vs. total volume) decreased in the absence of iodide (85% of control value) and increased in the absence of TSH (139%), whereas protein content showed the opposite changes (121 and 58%, respectively). The net apical-to-basal fluid transport was also inversely controlled by the two effectors. Iodide thus antagonizes TSH effects on cell volumes and fluid transport, probably via adenylylcyclase downregulation mechanisms. The absence of either iodide or TSH may mimic the imbalance occurring in pathological thyroids."
Sulfated tyrosines of thyroglobulin are involved in thyroid hormone synthesis.Nlend MC, Cauvi D, Venot N, Chabaud O. Biochem Biophys Res Commun. 1999 Aug 19;262(1):193-7. [abstract only]
"Thyroid hormone synthesis
is under the control of thyrotropin (TSH), which also regulates the
sulfation of tyrosines in thyroglobulin (Tg). We hypothesized that
sulfated tyrosine (Tyr[S]) might be involved in the hormonogenic
process, since the consensus sequence required for tyrosine sulfation
to occur was observed at the hormonogenic sites. Porcine thyrocytes,
cultured with TSH but without iodide in the presence of
[(35)S]sulfate, secreted Tg which was subjected to in vitro
hormonosynthesis with increasing concentrations of iodide. A 63%
consumption of Tyr[S] (1 residue) was observed at 40 atoms of iodine
incorporated into Tg, corresponding to a 40% hormonosynthesis
efficiency. In addition, hyposulfated Tg secreted by cells incubated
with sodium chlorate was subjected to in vitro hormonosynthesis. With
0.5 Tyr[S] residue (31% of the initial content), the efficiency of the
hormonosynthesis was 29%. In comparison, when hormonosynthesis was
performed by cells, with only 0.25 Tyr[S] residue (16% of the initial
content), the hormonosynthesis efficiency fell to 18%. These results
show that there exists a close correlation between the sulfated
tyrosine content of Tg and the production of thyroid hormones."
Thyrotropin regulates tyrosine sulfation of thyroglobulin.Nlend MC, Cauvi D, Venot N, Desruisseau S, Chabaud O. Eur J Endocrinol. 1999 Jul;141(1):61-9.
"OBJECTIVE: To study the regulation of thyroglobulin sulfation by thyrotropin (TSH) and iodide. Sulfation, a widespread post-translational modification of proteins, is involved in various biological activities. Thyroglobulin has been reported to be sulfated but, to date, the role of sulfate residues in the metabolism and function of thyroglobulin is not known; moreover, the regulation of thyroglobulin sulfation has not been yet investigated.
METHODS: The effect of TSH on thyroglobulin sulfation was studied in porcine thyroid cells cultured on porous collagen-coated filters. Cells cultured with or without TSH and with or without iodide (KI) were incubated for 4 days with radioactive sulfate. The specific radioactivity of thyroglobulin subunit (330kDa) was determined from apical media analyzed by electrophoresis. Enzymatic hydrolysates of the purified thyroglobulin were separated by oligosaccharide affinity chromatography and thin-layer chromatography; alkaline hydrolysates were analyzed only by thin-layer chromatography.
RESULTS: Thyroglobulin secreted by TSH-stimulated cells incorporated about twofold less radioactive sulfate. Iodide slightly modified this incorporation. Enzymatic hydrolysates of purified thyroglobulin showed sulfate residues bound essentially to complex oligosaccharide units. Alkaline hydrolysis was necessary to release all sulfated amino acids (tyrosine and serine). In the absence of TSH the proportion of tyrosine sulfate was dramatically increased: 24% compared with 7% (+KI) or 5% (-KI). The ratio of specific radioactivity of thyroglobulin to the specific radioactivity of intracellular inorganic sulfate (determined in each culture condition) gave the number of sulfated residues incorporated: 46 (-TSH) and 31 (+TSH) per mol thyroglobulin. From this distribution, we deduced the number of residues bound to complex oligosaccharide units and to tyrosine. Thus TSH decreased the number of sulfate residues on tyrosine from 11 to 2 per mol thyroglobulin.
CONCLUSIONS: TSH regulates the binding of sulfate groups to tyrosine residues. Iodide exerts a slight control over this process."
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