Thyroglobulin Degradation by Thyroidal Proteases: Action of Thiol Endopeptidases in Vitro*

Abstract

We have obtained evidence of thiol endopeptidases in the thyroid which are active in thyroglobulin degradation in vitro. Four pepstatin-insensitive endopeptidase fractions were distinguished in extracts of rabbit thyroids by gel filtration on Bio-Gel A-0.5m. An enzyme from one fraction was obtained in highly purified form and was found to be identical to cathepsin B described in other tissues. Endopeptidases in the three remaining fractions were designated as cathepsins 180K, 110K, and 45K, respectively, on the basis of their estimated molecular size. These were partially purified by either organomercurial affinity chromatography or DEAE-cellulose chromatography. They are identified as thiol endopeptidases on the basis of their sensitivity to inhibition by both leupeptin and the thiol-blocking agent iodoacetic acid and by their activation with the reducing agent glutathione. Each is distinguished from cathepsin B on the basis of molecular size and limited ability to hydrolyze benzoylarginine-2-naphthylamide. The action of the thiol endopeptidases on [125I]thyroglobulin was analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate or in sodium dodecyl sulfate and urea. In each instance, the initial peptide fragments were approximately 40-45K and 30K, with iodothyronine contents similar to or less than that of intact thyroglobulin. Later products of digestion than that of intact thyroglobulin. Later products of digestion included first, 20K peptides, which showed a low iodothyronine content, and finally, peptides of approximately 10K, which showed a 1.5-fold enrichment of T4 and T3 over that of intact thyroglobulin. Each of the thiol endopeptidases had a synergistic effect when incubated with cathepsin D and [125I]thyroglobulin. Among the products of such incubations were small iodopeptides, which were iodothyronine-enriched, and free T4, itself. The results show that thiol endopeptidases are present in the thyroid gland and are collectively as important as cathepsin D in the hydrolysis of thyroglobulin in vitro. The action of these enzymes must be considered along with that of cathepsin D in understanding thyroglobulin hydrolysis in vivo.

The type‐1 repeats of thyroglobulin regulate thyroglobulin degradation and T3, T4 release in thyrocytes

“…In step A (Fig. 2), hormone-containing Tg (shown as a monomer for clarity) in endocytic vesicles is naked, ready to meet lysosomal proteases (espe-cially cathepsins [5][6][7][8][9]). In step B, a few minutes later, early thyroid lysosomes are formed.…”

“…Intensive Tg degradation is a delayed process, requiring several hours [4]. In spite of numerous reports on Tg proteolytic attacks by thyroid proteases [5][6][7][8][9], the physiological mechanisms for selective hormone release and sequential processing remain unclear. The amino acid sequence of Tg [10,11] encloses three kinds of cysteine-rich repetitive regions.…”

Purification of a cysteine endopeptidase which is secreted with bioactive peptides from the epidermal glands of Xenopus laevis

“…For example, mammalian hepatic cathepsins B and L cleave SucAla-Phe-Lys-Amc with a K, < 10 pM [38], whereas XSCEPI exhibits a K, > 200 pM for this substrate. Cathepsin L is a very poor catalyst of Z-Arg-Arg-Amc cleavage : its activity with this substrate being difficult to detect, but it can catalyse the cleavage of Z-Phe-Arg-Amc rapidly ( K , < 3 pM) [39,40].…”

Physiological role of thiol proteases in thyroid hormone secretion

“…Earlier studies (see Dunn & Dunn 1982a) indicated that the acid protease, cathepsin D, was of primary importance in degrading Tg. More recently, another class of proteolytic enzymes, the thiol proteases, has been implicated in Tg proteolysis (Nakagawa et al 1981a,b;Dunn & Dunn 1982b). The biochemical evidence supporting the role of these proteases in thyroid hormone secretion was formerly based entirely on studies with cell-free incubation sys¬ tems.…”

“…Several different thiol proteases have been de¬ scribed in thyroid tissue (Dunn & Dunn 1982b;Nakagawa & Ohtaki 1984, and further studies are required to determine their specific roles in Tg proteolysis. Of particular interest in this connection is a recent report by Nakagawa 8c Ohtaki (1985) describing a thiol protease that releases T4 very efficiently from a naturally occur¬ ring 19 amino acid T4-containing fragment of Tg originally described by Rawitch et al (1983), but which displays only little activity in liberating T4 from intact Tg.…”