Chen J M, Dando P M, Rawlings N D, Brown M A, Young N E, Stevens R A, Hewitt E, Watts C, Barrett A J
Medical Research Council Peptidase Laboratory, Department of Immunology, The Babraham Institute, Babraham Hall, Babraham, Cambridgeshire CB2 4AT, United Kingdom.
J Biol Chem. 1997 Mar 21;272(12):8090-8. doi: 10.1074/jbc.272.12.8090.
Legumain is a cysteine endopeptidase that shows strict specificity for hydrolysis of asparaginyl bonds. The enzyme belongs to peptidase family C13, and is thus unrelated to the better known cysteine peptidases of the papain family, C1 (Rawlings, N. D., and Barrett, A. J. (1994) Methods Enzymol. 244, 461-486). To date, legumain has been described only from plants and a blood fluke, Schistosoma mansoni. We now show that legumain is present in mammals. We have cloned and sequenced human legumain and part of pig legumain. We have also purified legumain to homogeneity (2200-fold, 8% yield) from pig kidney. The mammalian sequences are clearly homologous with legumains from non-mammalian species. Pig legumain is a glycoprotein of about 34 kDa, decreasing to 31 kDa on deglycosylation. It is an asparaginyl endopeptidase, hydrolyzing Z-Ala-Ala-Asn-7-(4-methyl)coumarylamide and benzoyl-Asn-p-nitroanilide. Maximal activity is seen at pH 5.8 under normal assay conditions, and the enzyme is irreversibly denatured at pH 7 and above. Mammalian legumain is a cysteine endopeptidase, inhibited by iodoacetamide and maleimides, but unaffected by compound E64 (trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane). It is inhibited by ovocystatin (cystatin from chicken egg white) and human cystatin C with Ki values < 5 nM. We discuss the significance of the discovery of a cysteine endopeptidase of a new family and distinctive specificity in man and other mammals.
天冬酰胺内肽酶是一种半胱氨酸内肽酶，对天冬酰胺键的水解具有严格的特异性。该酶属于肽酶家族C13，因此与木瓜蛋白酶家族C1中更为人熟知的半胱氨酸肽酶无关（罗林斯，N. D.，和巴雷特，A. J.（1994年）《酶学方法》244卷，461 - 486页）。迄今为止，仅在植物和一种血吸虫——曼氏血吸虫中发现了天冬酰胺内肽酶。我们现在证明天冬酰胺内肽酶存在于哺乳动物中。我们已经克隆并测序了人天冬酰胺内肽酶以及部分猪天冬酰胺内肽酶。我们还从猪肾中纯化出了均一的天冬酰胺内肽酶（纯化了2200倍，产率8%）。哺乳动物的序列与非哺乳动物物种的天冬酰胺内肽酶明显同源。猪天冬酰胺内肽酶是一种约34 kDa的糖蛋白，去糖基化后降至31 kDa。它是一种天冬酰胺内肽酶，能水解Z - 丙氨酰 - 丙氨酰 - 天冬酰胺 - 7 -（4 - 甲基）香豆素酰胺和苯甲酰 - 天冬酰胺 - 对硝基苯胺。在正常测定条件下，pH 5.8时活性最高，该酶在pH 7及以上会不可逆地变性。哺乳动物天冬酰胺内肽酶是一种半胱氨酸内肽酶，受碘乙酰胺和马来酰亚胺抑制，但不受化合物E64（反式环氧琥珀酰 - L - 亮氨酰胺 -（4 - 胍基）丁烷）影响。它受卵类胱抑素（来自鸡蛋白的胱抑素）和人胱抑素C抑制，抑制常数Ki值<5 nM。我们讨论了在人和其他哺乳动物中发现一个新家族的半胱氨酸内肽酶及其独特特异性的意义。
