Masumoto Kiyonari, Sakata Yasuhisa, Arima Kazuhiko, Nakao Isao, Izuhara Kenji
Division of Medical Biochemistry, Department of Biomolecular Sciences, Center for Comprehensive Community Medicine, Saga Medical School, 5-1-1, Nabeshima, Saga, 849-8501, Japan.
J Biol Chem. 2003 Nov 14;278(46):45296-304. doi: 10.1074/jbc.M307741200. Epub 2003 Aug 29.
The squamous cell carcinoma antigen (SCCA) 1 and its homologous molecule, SCCA2, belong to the ovalbumin-serpin family. Although SCCA2 inhibits serine proteinases such as cathepsin G and mast cell chymase, SCCA1 targets cysteine proteinases such as cathepsin S, K, L, and papain. SCCA1 is therefore called a cross-class serpin. The inhibitory mechanism of the standard serpins is well characterized; those use a suicide substrate-like inhibitory mechanism during which an acyl-enzyme intermediate by a covalent bond is formed, and this complex is stable against hydrolysis. However, the inhibitory mechanism of cross-class serpins remains unresolved. In this article, we analyzed the inhibitory mechanism of SCCA1 on a cysteine proteinase, papain. SCCA1 interacted with papain at its reactive site loop, which was then cleaved, as the standard serpins. However, gel-filtration analyses showed that SCCA1 did not form a covalent complex with papain, in contrast to other serpins. Interaction with SCCA1 severely impaired the proteinase activity of papain, probably by inducing conformational change. The decreased, but still existing, proteinase activity of papain was completely inhibited by SCCA1 according to the suicide substrate-like inhibitory mechanism; however, papain recovered its proteinase activity with the compromised level, when all of intact SCCA1 was cleaved. These results suggest that the inhibitory mechanism of SCCA1 is unique among the serpin superfamily in that SCCA1 performs its inhibitory activity in two ways, contributing the suicide substrate-like mechanism without formation of a covalent complex and causing irreversible impairment of the catalytic activity of a proteinase.
鳞状细胞癌抗原（SCCA）1及其同源分子SCCA2属于卵清蛋白-丝氨酸蛋白酶抑制剂家族。尽管SCCA2可抑制组织蛋白酶G和肥大细胞糜蛋白酶等丝氨酸蛋白酶，但SCCA1的作用靶点是组织蛋白酶S、K、L和木瓜蛋白酶等半胱氨酸蛋白酶。因此，SCCA1被称为跨类丝氨酸蛋白酶抑制剂。标准丝氨酸蛋白酶抑制剂的抑制机制已得到充分表征；它们采用类似自杀底物的抑制机制，在此过程中会形成通过共价键连接的酰基-酶中间体，并且该复合物对水解稳定。然而，跨类丝氨酸蛋白酶抑制剂的抑制机制仍未明确。在本文中，我们分析了SCCA1对半胱氨酸蛋白酶木瓜蛋白酶的抑制机制。SCCA1在其反应位点环与木瓜蛋白酶相互作用，随后该环被切割，这与标准丝氨酸蛋白酶抑制剂的情况相同。然而，凝胶过滤分析表明，与其他丝氨酸蛋白酶抑制剂不同，SCCA1未与木瓜蛋白酶形成共价复合物。与SCCA1的相互作用可能通过诱导构象变化严重损害了木瓜蛋白酶的蛋白酶活性。根据类似自杀底物的抑制机制，木瓜蛋白酶降低但仍存在的蛋白酶活性被SCCA1完全抑制；然而，当所有完整的SCCA1都被切割时，木瓜蛋白酶恢复了其受损水平的蛋白酶活性。这些结果表明，SCCA1的抑制机制在丝氨酸蛋白酶抑制剂超家族中是独特的，因为SCCA1通过两种方式发挥其抑制活性，一种是在不形成共价复合物的情况下促成类似自杀底物的机制，另一种是导致蛋白酶催化活性的不可逆损害。
