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.
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
ARCARO A;GENTILE, Fabrizio
2006-01-01
Abstract
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
