Identification of a Heparin-binding Region of Rat Thyroglobulin Involved in Megalin Binding

Marinò, Michele; Friedlander, Joel A.; McCluskey, Robert T.; Andrews, David

doi:10.1074/jbc.274.43.30377

Cited by 43 publications

(67 citation statements)

References 44 publications

(38 reference statements)

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“…However, the Tg domains responsible for megalin binding (Zheng et al 1998, Marino et al 1999b have not yet been clearly identified. A heparin consensus sequence (SRRLKRP) involved in megalin binding was identified on rat Tg (Marino et al 1999a). The corresponding sequence in hTg (ARALKRS) is part of P1.…”

Section: Discussionmentioning

confidence: 99%

“…Thyroglobulin interactions with thyroid membranes have been observed for several years (Consiglio et al 1981). Several 'receptors' liable to mediate Tg binding to cell surfaces have been recently described: a receptor able to bind asialoagalacto Tg at acidic pH levels (Miquelis et al 1987); the protein disulfide isomerase (PDI) (Mezghrani et al 2000); histone H1 (Brix et al 1998); heparin (Marino et al 1999a); megalin (Zheng et al 1998, Marino et al 1999b; and rat hepatic lectin subunit of a rat asialoglycoprotein receptor (ASGPR) , Ulianich et al 1999. Some functions have been associated with various 'receptors': megalin was shown to be involved in transcytosis ; PDI was associated with Tg recycling (Miquelis et al 1987, Mezghrani et al 2000; histone H1 was involved in circulating Tg uptake by macrophages (Brix et al 1998); and a role was proposed for ASGPR in feedback regulation, ASGPR preventing thyroid-restricted gene expression under follicular Tg binding , Ulianich et al 1999.…”

Section: Introductionmentioning

confidence: 99%

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Identification of thyroglobulin domain(s) involved in cell-surface binding and endocytosis

Siffroi-Fernandez

Delom²,

Nlend³

et al. 2001

Journal of Endocrinology

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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

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of thyroglobulin domain(s) involved in cell-surface binding and endocytosis

Siffroi-Fernandez

Delom²,

Nlend³

et al. 2001

Journal of Endocrinology

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“…Tg was puri®ed from frozen rat thyroids by ammonium sulfate precipitation and column fractionation, as described elsewhere (10,27,28). Tg preparations were analyzed by western blotting, under both nonreducing and reducing conditions, using a rabbit antihuman Tg antibody cross-reactive with Tg from other species (Axle-Westbury, New York, NY, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Heparin (Sigma, St Louis, MO, USA) was used because it effectively releases megalin-bound Tg (15,28) and because rat Tg is a heparin binding protein (15,27,28). Wortmannin and cytochalasin D were obtained from Sigma.…”

Section: Methodsmentioning

confidence: 99%

Phosphoinositide 3-kinase inhibits megalin-mediated transcytosis of thyroglobulin across thyroid epithelial cells at a post-sorting level

Marinò

Chiovato

Lisi

et al. 2001

European Journal of Endocrinology

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Background: Phosphoinositide 3-kinase (PI3-K) is implicated in various cellular processes involving signaling, including intracellular traf®cking. PI3-K has been shown to play a part in both receptorand non-receptor-mediated transcytosis across cultured kidney cells and undifferentiated thyroid cells. Objective: To investigate the role of PI3-K in transcytosis of thyroglobulin (Tg) across differentiated cultured Fisher rat thyroid cells (FRTL-5 cells) ± a process known to be mediated by megalin, a member of the low-density lipoprotein receptor family. Design: We studied the effect of the microbial product wortmannin, a speci®c inhibitor of PI3-K, on transcytosis of Tg across FRTL-5 cells. Methods: Transcytosis experiments were performed using FRTL-5 cells cultured as tight layers on ®lters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper cell surface. Tg was added to the upper chamber and cells were incubated at 37 8C. Transcytosed Tg was measured in¯uids collected from the lower chamber. To study the role of PI3-K, cells were pre-incubated with wortmannin. Results: Pre-incubation of FRTL-5 cells with wortmannin did not affect Tg binding and uptake, but resulted in a considerable increase in Tg transcytosis (by 40±75%, depending on the concentration of wortmannin), suggesting that PI3-K exerts an inhibitory effect on Tg transcytosis. In experiments in which a monoclonal antibody against megalin was used to reduce Tg transcytosis, pre-incubation with wortmannin did not increase Tg transcytosis from its reduced levels, indicating that PI3-K is involved in the megalin-mediated pathway. Wortmannin did not affect the extent of release of tri-iodothyronine from exogenously added Tg by FRTL-5 cells, which was used as a measure of Tg degradation in the lysosomal pathway, indicating that the effect of PI3-K on transcytosis occurs after diversion of Tg from the lysosomal pathway. Conclusions: PI3-K exerts an inhibitory role on megalin-mediated Tg transcytosis across cultured thyroid cells. PI3-K action takes place at a post-sorting level, after Tg bypassing of the lysosomal pathway.

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“…A part of Tg, internalized via the endocytic receptor megalin, bypasses lysosomes and is transcytosed across cells, to be secreted from the basolateral pole (43). Tg binding to megalin is facilitated by accessory interactions with heparan-sulfate proteoglycans on the surface of thyroid cells (44,45). Experiments are in progress to determine whether chondroitin 6-sulfate units may interfere with the binding of hTg to megalin.…”

mentioning

confidence: 99%

A Single Chondroitin 6-Sulfate Oligosaccharide Unit at Ser-2730 of Human Thyroglobulin Enhances Hormone Formation and Limits Proteolytic Accessibility at the Carboxyl Terminus

Conte

Arcaro

D'Angelo

et al. 2006

Journal of Biological Chemistry

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We localized the site of type D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg). hTg was chromatographically separated into chondroitin 6-sulfate-containing (hTg-CS) and chondroitin 6-sulfate-devoid (hTg-CS 0 ) molecules on the basis of their D-glucuronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6%. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondroitin 6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxyl-terminal region, starting at residue 2514. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as LTAGXGLRE (residues 2726 -2734, X being Ser 2730 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5-triiodothyronine (T 3 ) (171%) and 3,5,3,5-tetraiodothyronine (T 4 ) (134%) than hTg-CS 0 . Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T 4 and, in particular, T 3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Lys 2714 -Gly 2715 from proteolysis, during the limited digestion of hTg-CS with trypsin. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T 4 /T 3 ratio in hTg. The potential implications in the ability of hTg to function as an autoantigen and into the pathogenesis of thyroidal and extra-thyroidal manifestations of autoimmune thyroid disease are discussed. Thyroglobulin (Tg),2 the molecular site of thyroid hormone formation, is a large homodimeric glycoprotein with an M r of 660,000. It is also a major antigen, involved in the pathogenesis of thyroid autoimmunity. In fact, experimental autoimmune thyroiditis (EAT) can be induced in genetically susceptible mice by immunization with human thyroglobulin (hTg) in complete adjuvant, and autoantibodies to hTg are found in the blood of humans affected with autoimmune thyroid disease (AITD), even though their pathogenic significance is unclear (1). Several post-translational modifications contribute to the molecular microheterogeneity of hTg, including iodination, glycosylation, phosphorylation, and sulfation (reviewed in Ref. 2). Iodine addition and hormone formation at specific sites have the most obvious implications for thyroid function, but the effects of glycosylation on the hormone-forming efficiency at specific sites and on the antigenicity of Tg have also been documented (3,4). hTg is modified with the addition of several oligosaccharide units of different kinds, among which the N-linked type A (high-mannose) and type B (complex) units have been characterized best as to their composition and localization (5-7). Type C units are linked to serine and th...

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Identification of a Heparin-binding Region of Rat Thyroglobulin Involved in Megalin Binding

Cited by 43 publications

References 44 publications

Identification of thyroglobulin domain(s) involved in cell-surface binding and endocytosis

Identification of thyroglobulin domain(s) involved in cell-surface binding and endocytosis

Phosphoinositide 3-kinase inhibits megalin-mediated transcytosis of thyroglobulin across thyroid epithelial cells at a post-sorting level

A Single Chondroitin 6-Sulfate Oligosaccharide Unit at Ser-2730 of Human Thyroglobulin Enhances Hormone Formation and Limits Proteolytic Accessibility at the Carboxyl Terminus

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