Joslin G, Wittwer A, Adams S, Tollefsen D M, August A, Perlmutter D H
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1993 Jan 25;268(3):1886-93.
The serpin-enzyme complex (SEC) receptor recognizes a pentapeptide neo-domain of alpha 1-antitrypsin (alpha 1 AT)-elastase complexes and, in so doing, mediates internalization and intracellular catabolism of the macromolecular complex, mediates an increase in synthesis of alpha 1 AT, and elicits neutrophil chemotactic activity. In previous studies we have shown that this pentapeptide domain is highly conserved among members of the serpin family and that binding of a synthetic peptide corresponding to this region (125I-peptide 105Y, SIP-PEVKFNKPFVYLI, based on alpha 1 AT sequence 359-374) to HepG2 cells is blocked by several serpin-enzyme complexes. To determine whether the SEC receptor is the primary HepG2 cell surface binding site for these serpin-enzyme complexes, we examined the capacity for serpin-enzyme complexes to compete with each other for binding to the SEC receptor. The results indicate that binding of 125I-elastase-alpha 1 AT complexes is blocked by thrombin-antithrombin III (ATIII), thrombin-heparin cofactor II, and cathepsin G-alpha 1-antichymotrypsin (alpha 1 ACT) complexes. Moreover, unlabeled elastase-alpha 1 AT complexes compete for binding of 125I-thrombin-ATIII, 125I-thrombin-heparin cofactor II, and 125I-cathepsin G-alpha 1 ACT complexes. Preformed soluble tissue plasminogen activator-plasminogen activator inhibitor 1 complexes also compete for binding of elastase-alpha 1 AT complexes to the SEC receptor but do so to a less effective extent, probably because of a less favorable pentapeptide sequence for binding to the SEC receptor. Under conditions in which these serpin-enzyme complexes would be expected to bind to the SEC receptor there is an increase in synthesis of alpha 1 AT but not in synthesis of ATIII or alpha 1 ACT. Proteolytically modified alpha 1 AT also competes for binding of 125I-elastase-alpha 1 AT complexes to the SEC receptor and vice versa. The purified 51-kDa amino-terminal fragment of alpha 1 AT does not compete for binding of 125I-elastase-alpha 1 AT complexes, indicating that the pentapeptide neodomain in the 4-kDa carboxyl-terminal fragment is sufficient for binding to the SEC receptor.
丝氨酸蛋白酶抑制剂 - 酶复合物（SEC）受体可识别α1 - 抗胰蛋白酶（α1AT） - 弹性蛋白酶复合物的一个五肽新结构域，并借此介导该大分子复合物的内化和细胞内分解代谢，介导α1AT合成增加，并引发中性粒细胞趋化活性。在先前的研究中，我们已表明该五肽结构域在丝氨酸蛋白酶抑制剂家族成员中高度保守，且对应于该区域的合成肽（基于α1AT序列359 - 374的125I - 肽105Y，SIP - PEVKFNKPFVYLI）与HepG2细胞的结合会被几种丝氨酸蛋白酶抑制剂 - 酶复合物阻断。为了确定SEC受体是否是这些丝氨酸蛋白酶抑制剂 - 酶复合物在HepG2细胞表面的主要结合位点，我们研究了丝氨酸蛋白酶抑制剂 - 酶复合物相互竞争结合SEC受体的能力。结果表明，125I - 弹性蛋白酶 - α1AT复合物的结合会被凝血酶 - 抗凝血酶III（ATIII）、凝血酶 - 肝素辅因子II和组织蛋白酶G - α1 - 抗糜蛋白酶（α1ACT）复合物阻断。此外，未标记的弹性蛋白酶 - α1AT复合物会竞争125I - 凝血酶 - ATIII、125I - 凝血酶 - 肝素辅因子II和125I - 组织蛋白酶G - α1ACT复合物的结合。预先形成的可溶性组织纤溶酶原激活物 - 纤溶酶原激活物抑制剂1复合物也会竞争弹性蛋白酶 - α1AT复合物与SEC受体的结合，但效果较差，可能是因为其五肽序列对与SEC受体结合不太有利。在预期这些丝氨酸蛋白酶抑制剂 - 酶复合物会与SEC受体结合的条件下，α1AT的合成增加，但ATIII或α1ACT的合成未增加。经蛋白水解修饰的α1AT也会竞争125I - 弹性蛋白酶 - α1AT复合物与SEC受体的结合，反之亦然。纯化的α1AT的51 kDa氨基末端片段不会竞争125I - 弹性蛋白酶 - α1AT复合物的结合，这表明4 kDa羧基末端片段中的五肽新结构域足以与SEC受体结合。
