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J. Biol. Chem., Vol. 281, Issue 31, 22200-22211, August 4, 2006

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

POTENTIAL INSIGHTS INTO THYROID HOMEOSTASIS AND AUTOIMMUNITY^*

Marisa Conte, Alessia Arcaro, Daniela D'Angelo, Ariele Gnata, Gianfranco Mamone^¶, Pasquale Ferranti^||, Silvestro Formisano, and Fabrizio Gentile¹

From the Dipartimento di Scienze per la Salute, Università del Molise, Via F. De Sanctis, 86100 Campobasso, the Dipartimento di Biologia e Patologia Cellulare e Molecolare, Università di Napoli Federico II, Via S. Pansini 5, 80131 Napoli, the ^¶Istituto di Scienze dell'Alimentazione del Consiglio Nazionale delle Ricerche, Via Roma 52a/c, 83100 Avellino, and the ^||Dipartimento di Scienza degli Alimenti, Università di Napoli Federico II, Parco Gussone, 80055 Portici, Italy

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-CS₀) 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 Ser²⁷³⁰ linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5 '-triiodothyronine (T₃) (171%) and 3,5,3',5'-tetraiodothyronine (T₄) (134%) than hTg-CS₀. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T₄ and, in particular, T₃ formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Lys²⁷¹⁴-Gly²⁷¹⁵ 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 T₄/T₃ 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.

Received for publication, December 16, 2005 , and in revised form, April 17, 2006.

^* This work was supported by PRIN 2004 Grant 2004062075 from the Ministero dell'Istruzione, Università e Ricerca, Rome, Italy (to F. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 and Tables 1 and 2.

¹ To whom correspondence should be addressed. Tel.: 39-0874-404852; Fax: 39-0874-404778; E-mail: gentilefabrizio{at}unimol.it.

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