Eakin A E, Mills A A, Harth G, McKerrow J H, Craik C S
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
J Biol Chem. 1992 Apr 15;267(11):7411-20.
The complete sequence of the gene encoding the major cysteine protease from Trypanosoma cruzi is reported. The amino acid sequence predicted from the gene sequence aligns well with members of the papain family of cysteine proteases, suggesting the name cruzain. The sequence is most closely related to the cysteine protease of Trypanosoma brucei (59.3%) and the murine cathepsin L (42.2%). At least six copies of the gene are present in the genome and are organized in a tandem array of copies which are identical in all restriction endonuclease sites tested. The gene appears to be expressed in all developmental stages of T. cruzi with mRNA levels approximately 2-fold higher in the intracellular amastigote form. A copy of the T. cruzi gene was expressed in bacteria as an inactive, insoluble fusion polypeptide to approximately 5% of the total cell protein. The fusion protein was readily purified, solubilized in urea, and successfully refolded to produce a polyprotein which processed autocatalytically to yield approximately 1 mg of active protease per 3 g of wet cell paste. The processed form of the recombinant protease has an NH2-terminal sequence identical to that of the mature form of the protease purified from T. cruzi (Murta, A. C. M., Persechini, P. M., Souto-Padrón, T., de Souza, W., Guimaraes, J. A., and Scharfstein, J. (1990) Mol. Biochem. Parasitol. 43, 27-38; Cazzulo, J. J., Couso, R., Raimondi, A., Wernstedt, C., and Hellman, U. (1989) Mol. Biochem. Parasitol. 33, 33-42). This suggests that the recombinant protease possesses the requisite specificity and activity to correctly process the proform of the protease in vitro. Kinetic assays with peptide substrates demonstrate that the substrate specificity and kinetic parameters for the recombinant protease are consistent with those of the endogenous protease. The proteolytic activity of the recombinant protease is enhanced by dithiothreitol, inhibited by leupeptin, N alpha-p-tosyl-L-lysine chloromethyl ketone and trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane (E-64) but is unaffected by phenylmethylsulfonyl fluoride, pepstatin, and 1,10-phenanthroline. More specifically, the recombinant enzyme was inhibited by benzyloxycarbonyl-Phe-Arg-fluoromethyl ketone, which inhibits replication and differentiation of T. cruzi within mammalian cells in culture.
本文报道了克氏锥虫主要半胱氨酸蛋白酶编码基因的完整序列。根据该基因序列预测的氨基酸序列与半胱氨酸蛋白酶木瓜蛋白酶家族成员高度匹配，因而将其命名为克氏锥虫蛋白酶（cruzain）。该序列与布氏锥虫的半胱氨酸蛋白酶（相似度为59.3%）以及小鼠组织蛋白酶L（相似度为42.2%）最为接近。基因组中至少存在该基因的六个拷贝，它们以串联阵列的形式排列，在所有测试的限制性内切酶位点均完全相同。该基因似乎在克氏锥虫的所有发育阶段均有表达，在细胞内无鞭毛体形式中的mRNA水平约高2倍。克氏锥虫基因的一个拷贝在细菌中表达为一种无活性的不溶性融合多肽，约占总细胞蛋白的5%。该融合蛋白易于纯化，可在尿素中溶解，并成功复性，产生一种多蛋白，该多蛋白可自动催化加工，每3克湿细胞糊可产生约1毫克活性蛋白酶。重组蛋白酶的加工形式具有与从克氏锥虫纯化的蛋白酶成熟形式相同的NH2末端序列（Murta, A. C. M., Persechini, P. M., Souto-Padrón, T., de Souza, W., Guimaraes, J. A., and Scharfstein, J. (1990) Mol. Biochem. Parasitol. 43, 27 - 38; Cazzulo, J. J., Couso, R., Raimondi, A., Wernstedt, C., and Hellman, U. (1989) Mol. Biochem. Parasitol. 33, 33 - 42）。这表明重组蛋白酶具有在体外正确加工蛋白酶前体形式所需的特异性和活性。用肽底物进行的动力学分析表明，重组蛋白酶的底物特异性和动力学参数与内源性蛋白酶一致。重组蛋白酶的蛋白水解活性可被二硫苏糖醇增强，被亮抑酶肽、Nα - 对甲苯磺酰 - L - 赖氨酸氯甲基酮和反式 - 环氧琥珀酰 - L - 亮氨酰胺（4 - 胍基）丁烷（E - 64）抑制，但不受苯甲基磺酰氟、胃蛋白酶抑制剂和1,10 - 菲咯啉影响。更具体地说，重组酶被苄氧羰基 - 苯丙氨酸 - 精氨酸氟甲基酮抑制，该物质可抑制克氏锥虫在培养的哺乳动物细胞内的复制和分化。
