Dunn A D, Crutchfield H E, Dunn J T
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908.
Endocrinology. 1991 Jun;128(6):3073-80. doi: 10.1210/endo-128-6-3073.
The release of T4 and T3 from the prohormone thyroglobulin (Tg) occurs in thyroid lysosomes. To examine the role of cathepsin-B, -D, and -L, the three major endopeptidases in this process, we incubated rabbit [125I]Tg, labeled in vivo, with lysosomal extracts from human thyroids. Iodopeptide formation was evaluated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate after short term incubations (20-45 min), while iodoamino acid release was assessed by paper chromatography after long term incubations (8 and 24 h). Using pepstatin to inhibit cathepsin D, Z-Phe-Ala-CHN2 to inhibit both cathepsin B and L, and Z-Phe-Phe-CHN2 to selectively inhibit cathepsin L, we obtained the following results: 1) blocking of all three endopeptidases reduced both iodopeptide formation in short term experiments and iodoamino acid release in long term experiments by 80-90%; 2) iodopeptide formation was reduced by 85% with Z-Phe-Ala-CHN2, by 56% with Z-Phe-Phe-CHN2, and by 26% with pepstatin; 3) iodoamino acid release was reduced by 60-80% with Z-Phe-Ala-CHN2 and by 40-50% with either Z-Phe-Phe-CHN2 or pepstatin at 8 h, but by less than 20% at 24 h; pepstatin and Z-Phe-Phe-CHN2 together reduced iodoamino acid release by 80% and 60% at 8 and 24 h, respectively. Limited hydrolysis of Tg by lysosomal enzymes produced at least eight peptide fragments of less than 100,000 mol wt. Three of these, together representing 32% of the 125I released, resulted from cleavages in the C-terminal region of Tg corresponding to residues 2487, 2393, and 2390 of cDNA-derived human Tg. Several other peptides, together containing 38% of the 125I released, included the N-terminus of Tg. These C-terminal and N-terminal fragments contained three of Tg's four major hormonogenic sites, but none of the cleavage sites fell close to the hormone sites themselves. We conclude that 1) the formation of discrete iodopeptides precedes the release of iodothyronines and iodotyrosines from Tg; 2) the cysteine proteinases are more important than cathepsin D in this process; and 3) these endopeptidases selectively cleave Tg to favor the production of hormone-containing intermediates for subsequent processing by exopeptidases.
甲状腺球蛋白（Tg）前激素释放T4和T3发生在甲状腺溶酶体中。为了研究组织蛋白酶B、D和L这三种主要内肽酶在此过程中的作用，我们将体内标记的兔[125I]Tg与人甲状腺溶酶体提取物一起孵育。短期孵育（20 - 45分钟）后，通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳评估碘肽的形成，而长期孵育（8和24小时）后，通过纸色谱法评估碘氨基酸的释放。使用胃蛋白酶抑制剂抑制组织蛋白酶D，Z - 苯丙氨酸 - 丙氨酸 - CHN2抑制组织蛋白酶B和L，Z - 苯丙氨酸 - 苯丙氨酸 - CHN2选择性抑制组织蛋白酶L，我们得到以下结果：1）在短期实验中，三种内肽酶的阻断使碘肽形成减少80 - 90%，在长期实验中使碘氨基酸释放减少80 - 90%；2）Z - 苯丙氨酸 - 丙氨酸 - CHN2使碘肽形成减少85%，Z - 苯丙氨酸 - 苯丙氨酸 - CHN2使碘肽形成减少56%，胃蛋白酶抑制剂使碘肽形成减少26%；3）在8小时时，Z - 苯丙氨酸 - 丙氨酸 - CHN2使碘氨基酸释放减少60 - 80%，Z - 苯丙氨酸 - 苯丙氨酸 - CHN2或胃蛋白酶抑制剂使碘氨基酸释放减少40 - 50%，但在24小时时减少不到20%；胃蛋白酶抑制剂和Z - 苯丙氨酸 - 苯丙氨酸 - CHN2共同作用，在8小时和24小时时分别使碘氨基酸释放减少80%和60%。溶酶体酶对Tg进行有限水解产生了至少八个分子量小于100,000的肽片段。其中三个片段共占释放的125I的32%，它们是由Tg对应于cDNA衍生的人Tg的2487、2393和2390位残基的C末端区域的切割产生的。其他几个肽片段共包含释放的125I的38%，包括Tg的N末端。这些C末端和N末端片段包含Tg四个主要激素生成位点中的三个，但没有一个切割位点靠近激素位点本身。我们得出结论：1）离散碘肽的形成先于碘甲状腺原氨酸和碘酪氨酸从Tg的释放；2）在此过程中，半胱氨酸蛋白酶比组织蛋白酶D更重要；3）这些内肽酶选择性切割Tg，有利于产生含激素的中间体，以便随后由外肽酶进行加工。
