Kuznetsov G, Chen L B, Nigam S K
Harvard Medical School, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Jan 31;272(5):3057-63. doi: 10.1074/jbc.272.5.3057.
Thyroglobulin (Tg), the major protein secreted by thyroid epithelial cells and precursor of thyroid hormones, is a large dimeric glycoprotein with multiple disulfide bonds. The folding and assembly of this complex molecule begins in the endoplasmic reticulum (ER) and is likely to involve a variety of reactions catalyzed by molecular chaperones (Kuznetsov, G., Chen, L. B., and Nigam, S. K. (1994) J. Biol. Chem. 269, 22990-22995). By coimmunoprecipitation in rat thyroid cells, we were able to demonstrate that BiP, grp94, ERp72, and grp170, four proteins believed to function as specific molecular chaperones, complex with Tg during its maturation. The same complex of the four putative chaperones with Tg was observed in cells treated with tunicamycin, indicating that these four ER chaperones stably associate with Tg when it is misfolded/misassembled due to inhibition of its glycosylation. BiP, grp94, and ERp72 were also found to associate with Tg in cells in which misfolding was induced by perturbing ER calcium stores. To determine if the assembly of a complex between the four chaperones and Tg under conditions of misglycosylation was unique to the maturation of this particular secretory protein or a more general phenomenon, adenovirus-transformed rat thyroid cells that do not synthesize Tg were analyzed. In these transformed cells, the only protein these same four chaperones were found to complex with was a protein of approximately 200 kDa. This protein was subsequently identified as thrombospondin, which, like Tg, is a large oligomeric secreted glycoprotein with multiple disulfide bonds. We therefore propose that these ER chaperones complex together with a variety of large oligomeric secretory glycoproteins as they fold and assemble in the ER.
甲状腺球蛋白（Tg）是甲状腺上皮细胞分泌的主要蛋白质以及甲状腺激素的前体，是一种具有多个二硫键的大型二聚体糖蛋白。这种复杂分子的折叠和组装始于内质网（ER），可能涉及分子伴侣催化的多种反应（库兹涅佐夫，G.，陈，L.B.，和尼加姆，S.K.（1994年）《生物化学杂志》269，22990 - 22995）。通过在大鼠甲状腺细胞中的共免疫沉淀，我们能够证明BiP、grp94、ERp72和grp170这四种被认为作为特定分子伴侣发挥作用的蛋白质，在Tg成熟过程中与它形成复合物。在用衣霉素处理的细胞中也观察到了这四种假定的伴侣蛋白与Tg的相同复合物，这表明当Tg由于糖基化受到抑制而错误折叠/错误组装时，这四种内质网伴侣蛋白会与它稳定结合。在通过扰乱内质网钙储存诱导错误折叠的细胞中，还发现BiP、grp94和ERp72与Tg结合。为了确定在糖基化错误的条件下这四种伴侣蛋白与Tg之间复合物的组装是这种特定分泌蛋白成熟所特有的现象还是更普遍的现象，对不合成Tg的腺病毒转化大鼠甲状腺细胞进行了分析。在这些转化细胞中，发现这四种相同的伴侣蛋白与之形成复合物的唯一蛋白质是一种约200 kDa的蛋白质。该蛋白质随后被鉴定为血小板反应蛋白，它与Tg一样，是一种具有多个二硫键的大型寡聚体分泌糖蛋白。因此，我们提出这些内质网伴侣蛋白在各种大型寡聚体分泌糖蛋白在内质网中折叠和组装时会与之形成复合物。
