Cloning, Isolation, and Characterization of Mammalian Legumain, an Asparaginyl Endopeptidase

Abstract

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 (transepoxysuccinyl-L-leucylamido-(4-guanidino)butane). It is inhibited by ovocystatin (cystatin from chicken egg white) and human cystatin C with K i 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.Cysteine peptidases form one of the major groups of proteolytic enzymes, and can be divided into about 30 separate families on the basis of their molecular structures (reviewed in Refs. 1 and 2). Three families of cysteine endopeptidases have been known to be represented in mammals. The most numerous are those of the papain family (C1), which include cathepsins B, H, L, S, and others. These are predominantly lysosomal enzymes, responsible for proteolysis in the lysosomal/endosomal system and also are secreted to act extracellularly. In the cytosolic fraction of the cell, there are members of the other two families of cysteine endopeptidases: the families of calpain (family C2) and caspase (previously interleukin 1␤-converting enzyme; C14). These peptidases mediate limited proteolysis of cytosolic substrates. We now report that the legumain family (C13) can be added to the list of mammalian cysteine endopeptidases.Legumain is the name that was given by Kembhavi et al. (3) to an endopeptidase that is present in many leguminous and other seeds, after they had isolated and characterized the enzyme from Vigna aconitifolia (moth bean). Legumain is specific for the hydrolysis of asparaginyl bonds. The amino acid sequence of legumain from Ricinus communis (castor bean) showed it to be homologous with an enzyme from the fluke Schistosoma mansoni (4). At that time, the fluke enzyme was of unknown specificity, but it has now been shown also to be an asparaginyl endopeptidase (5), active on the test substrate that had been introduced by Kembhavi et al.The appearance of sequences homolog...

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