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

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. 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...

Reduced sulfation of chondroitin sulfate in thyroglobulin derived from human papillary thyroid carcinomas

“…demonstrated that CS oligosaccharide units are a main source of the molecular microheterogeneity of human Tg. ( 9 ) Thus, our results should be considered to represent the average total sulfation levels of CS in human Tg extracted from normal thyroid and carcinoma tissues.…”

“…( 7 ) Human Tg is unique in that it has O ‐glycosidic‐linked oligosaccharides that have not been found in other animals. ( 8 ) Recently, the CS side chain of Tg was reported to affect the proteolytic accessibility and hormone‐liberating qualities of Tg; ( 9 ) to date, no detailed analysis of CS in carcinoma‐derived Tg has been reported.…”

Identification of Chondroitin Sulfate Linkage Region Glycopeptides Reveals Prohormones as a Novel Class of Proteoglycans

“…Intriguingly, the ratio of T 3 and T 4, which is derived from human thyroglobulin, was affected by a CSchain linked onto a subset of the protein. This effect was likely related to the blocking of specific proteolytic cleavage sites (30). Whether CS-modifications influence the processing of the above identified prohormones in a similar way remains to be determined.…”

“…Prohormones are known to undergo extensive post-translational modifications, such as phosphorylation, tyrosine sulfation, and glycosylations (30). These modifications may influence the action of proprotein convertases and thereby contribute to the functional processing of the prohormones.…”

Hot Spots for Protein Partnerships at the Surface of Cholinesterases and Related α/β Hydrolase Fold Proteins or Domains—A Structural Perspective

“…In human TG, the major epitopes for autoimmune thyroid disease-related antibodies involve the surface loop between helices α3(7,8) and α4(7,8), the C-terminal helix α10 and strands β9 and β10, all being located in the C-terminal part of the subunit, suggesting that antibody binding could destabilize or disrupt the dimer [ 50 , 51 ] ( Figure 1 ). In contrast, presence of a bulky chondroitin 6-sulfate oligosaccharide moiety linked downstream to helix α10, and reported to enhance hormone formation and limit proteolytic accessibility of the C-terminus [ 52 ], may contribute to dimer stability ( Figure 1 ).…”