Perlmutter D H, Joslin G, Nelson P, Schasteen C, Adams S P, Fallon R J
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1990 Oct 5;265(28):16713-6.
Alpha 1-Antitrypsin (alpha 1-AT) is similar to other members of the serine protease inhibitor (serpin) supergene family in that it undergoes structural rearrangement during the formation of a covalently stabilized inhibitory complex with its cognate enzyme, neutrophil elastase. We have recently demonstrated an abundant, high-affinity cell surface receptor on human hepatoma cells and human mononuclear phagocytes which recognizes a conformation-specific domain of the alpha 1-AT-elastase complex as well as of other serpin-enzyme complexes (Perlmutter, D. H., Glover, G. I., Rivetna, M., Schasteen, C. S., and Fallon, R. J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3753-3757). Binding to this serpin-enzyme complex (SEC) receptor activates a signal transduction pathway for increased expression of the alpha 1-AT gene and may be responsible for clearance of serpin-enzyme complexes. In this study, we show that there is time-dependent and saturable internalization of alpha 1-AT-elastase and alpha 1-AT-trypsin complexes in human hepatoma HepG2 cells. Internalization is mediated by the SEC receptor as defined by inhibition by synthetic peptides corresponding to residues 359-374 of alpha 1-AT. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of intracellular radioactivity demonstrated that intact 75- and 66-kDa alpha 1-AT-trypsin complexes were internalized. Kinetic analysis of internalization at 37 degrees C showed that a single cohort of 125I-alpha 1-AT-trypsin complexes, prebound to cells at 4 degrees C, disappeared from the cell surface and accumulated intracellularly within 5-15 min at 37 degrees C. The intracellular concentration of radiolabeled complexes then decreased rapidly coincident with appearance of acid-soluble degradation products in the extracellular culture fluid. Intracellular degradation was inhibited by internalization at 18 degrees C or by internalization at 37 degrees C in the presence of weak bases ammonium chloride, primaquine, and chloroquine, indicating that degradation is lysosomal. These results indicate that in addition to its role in signal transduction the SEC receptor participates in internalization and delivery of alpha 1-AT-protease complexes to lysosome for degradation.
α1 -抗胰蛋白酶（α1 - AT）与丝氨酸蛋白酶抑制剂（serpin）超基因家族的其他成员相似，即它在与其同源酶中性粒细胞弹性蛋白酶形成共价稳定的抑制复合物的过程中会发生结构重排。我们最近在人肝癌细胞和人单核吞噬细胞上发现了一种丰富的、高亲和力的细胞表面受体，该受体可识别α1 - AT -弹性蛋白酶复合物以及其他丝氨酸蛋白酶抑制剂 - 酶复合物的构象特异性结构域（珀尔马特，D. H.，格洛弗，G. I.，里韦特纳，M.，沙斯汀，C. S.，以及法伦，R. J.（1990年）《美国国家科学院院刊》87卷，3753 - 3757页）。与这种丝氨酸蛋白酶抑制剂 - 酶复合物（SEC）受体结合可激活一条信号转导途径，以增加α1 - AT基因的表达，并且可能负责丝氨酸蛋白酶抑制剂 - 酶复合物的清除。在本研究中，我们表明在人肝癌HepG2细胞中，α1 - AT -弹性蛋白酶和α1 - AT -胰蛋白酶复合物存在时间依赖性和饱和性内化。内化是由SEC受体介导的，这是通过与α1 - AT的359 - 374位残基对应的合成肽的抑制作用来定义的。对细胞内放射性的十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析表明，完整的75 kDa和66 kDa的α1 - AT -胰蛋白酶复合物被内化。在37℃下对内化进行动力学分析表明，在4℃下预先结合到细胞上的一批125I -α1 - AT -胰蛋白酶复合物在37℃下5 - 15分钟内从细胞表面消失并在细胞内积累。然后，放射性标记复合物的细胞内浓度迅速下降，与此同时细胞外培养液中出现酸溶性降解产物。在18℃下内化或在37℃下于弱碱氯化铵、伯氨喹和氯喹存在的情况下内化可抑制细胞内降解，这表明降解是溶酶体性的。这些结果表明，除了其在信号转导中的作用外，SEC受体还参与α1 - AT -蛋白酶复合物的内化并将其递送至溶酶体进行降解。
