Isolation of insoluble secretory product from bovine thyroid: extracellular storage of thyroglobulin in covalently cross-linked form.

Abstract

Abstract. ExtraceUular storage of thyroglobulin (TG)is an important prerequisite for maintaining constant levels of thyroid hormones in vertebrates. Storage of large amounts is made possible by compactation of TG in the follicle lumen with concentrations of at least 100-400 mg/ml. We recently observed that the luminal content from bovine thyroids can be isolated in an intact state and be separated from soluble TG. For this purpose, bovine thyroid tissue was homogenized and subjected to various steps of purification. This procedure resulted in a pellet of single globules measuring 20-120 #m in diameter. Scanning electron microscopy revealed a unique cobblestone-like surface pattern of isolated globules, showing in detail the impressions of the apical plasma membranes of thyrocytes which had formerly surrounded the luminal content before tissue homogenization. Isolated thyroid globules were rapidly digested by trypsin but extremely resistant to various protein solubilization procedures. Homogenization of isolated globules resulted in the release of ,,o 3 % of total protein, showing that only a minor proportion of TG was loosely incorporated in thyroid globules whereas ,,022% appeared to be interconnected with the globule matrix by disulfide bridges. Analysis by SDSgel electrophoresis and immunoblotting confirmed that the protein released by this procedure consisted of TG. The vast majority (•75%) of the globule matrix protein was found to be covalently cross-linked by nondisulfide bonds. TG in isolated globules was highly iodinated (*55 iodine atoms per 12-S TG subunit) suggesting that the covalent nondisulfide cross-linking occurs in part during the iodination of TG and that this process involves the formation of intermolecular dityrosine bridges. Mechanisms must exist which solubilize or disperse the insoluble luminal content prior to endocytosis of TG. CELLS with a regulated pathway of secretion are characterized by the ability to store the secretion product in granules and to release the granule content following specific signals for exocytosis. The secretory pathway in thyroid follicle cells is also regulated in that thyroglobulin (TG) t is temporarily stored within apically located export vesicles (11,53,54). However, the transport pathway of TG is unique in that additionally to the apical vesicles a large extracellular storage site exists within the lumina of thyroid follicles. Luminal TG provides a physiologically important reservoir which is regulated in size by the exocytotic addition of newly synthesized TG and by endocytic removal of TG from the lumen. EndocyticaUy removed TG is conveyed primarily to lysosomes for the proteolytic liberation of thyroid hormones. It can be concluded from numerous experiments Parts of this paper were reporlext at

Thyroid functions of mouse cathepsins B, K, and L

“…Human Tg globules consist of Tg mainly cross-linked by disulfide bridges (4,5). In addition to disulfide cross-links, isodipeptide bonds were detected in bovine (3,6), and dityrosine links in porcine Tg globules (31). Here, we have observed the occurrence of multilayered Tg in mouse thyroids, indicating that covalently cross-linked Tg exists also in the lumen of mouse thyroid follicles (Figure 8).…”

Identification of ERp29, an Endoplasmic Reticulum Lumenal Protein, as a New Member of the Thyroglobulin Folding Complex

“…It is worth noting that a similar absence of coimmunoprecipitating Tg, PDI, and BiP has been shown previously (25). Nevertheless, because the fate of Tg exported from the cultivated thyrocytes is quite different from the situation in the thyroid gland where secreted Tg is stored in the follicular lumen in form of compact, cross-linked aggregates (36), one cannot exclude the existence of the extracellular ERp29/Tg complexes in vivo.…”

Evidence for processing of compact insoluble thyroglobulin globules in relation with follicular cell functional activity in the human and the mouse thyroid

“…Covalent cross-links between Tg molecules has been suggested as a way to store Tg at a high concentration without increasing the osmolality (4,5). The present study shows that globules are mostly observed in follicles with a low Tg turnover, 'hypofunctioning' follicles in the normal human thyroid and 'cold' follicles in aged mice.…”

“…It has been suggested that Tg compaction is a mechanism to store the prohormone at high concentration (4,5). A morphological heterogeneity in the colloid aspect along with the presence of intrafollicular globules has also been observed on fixed histological sections (7,8).…”

“…Tg also exists in a multimerized insoluble form which consists of covalently cross-linked 19S Tg molecules. Such colloid globules have been isolated from bovine and human thyroids, with differences in size and cross-linking nature (4,5). Iodine content of isolated globules is high while hormones have not been detected (4,6).…”