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...

Foreign protein antigens must be broken down within endosomes or lysosomes to generate suitable peptides that will form complexes with class II major histocompatibility complex molecules for presentation to T cells. However, it is not known which proteases are required for antigen processing. To investigate this, we exposed a domain of the microbial tetanus toxin antigen (TTCF) to disrupted lysosomes that had been purified from a human B-cell line. Here we show that the dominant processing activity is not one of the known lysosomal cathepsins, which are generally believed to be the principal enzymes involved in antigen processing, but is instead an asparagine-specific cysteine endopeptidase. This enzyme seems similar or identical to a mammalian homologue of the legumain/haemoglobinase asparaginyl endopeptidases found originally in plants and parasites. We designed competitive peptide inhibitors of B-cell asparaginyl endopeptidase (AEP) that specifically block its proteolytic activity and inhibit processing of TTCF in vitro. In vivo, these inhibitors slow TTCF presentation to T cells, whereas preprocessing of TTCF with AEP accelerates its presentation, indicating that this enzyme performs a key step in TTCF processing. We also show that N-glycosylation of asparagine residues blocks AEP action in vitro. This indicates that N-glycosylation could eliminate sites of processing by AEP in mammalian proteins, allowing preferential processing of microbial antigens.

We have investigated the inhibition of the recently identified family C13 cysteine peptidase, pig legumain, by human cystatin C. The cystatin was seen to inhibit enzyme activity by stoichiometric 1:1 binding in competition with substrate. The K i value for the interaction was 0.20 nM, i.e. cystatin C had an affinity for legumain similar to that for the papain-like family C1 cysteine peptidase, cathepsin B. However, cystatin C variants with alterations in the N-terminal region and the "second hairpin loop" that rendered the cystatin inactive against cathepsin B, still inhibited legumain with K i values 0.2-0.3 nM. Complexes between cystatin C and papain inhibited legumain activity against benzoyl-AsnNHPhNO 2 as efficiently as did cystatin C alone. Conversely, cystatin C inhibited papain activity against benzoyl-Arg-NHPhNO 2 whether or not the cystatin had been incubated with legumain, strongly indicating that the cystatin inhibited the two enzymes with non-overlapping sites. A ternary complex between legumain, cystatin C, and papain was demonstrated by gel filtration supported by immunoblotting. Screening of a panel of cystatin superfamily members showed that type 1 inhibitors (cystatins A and B) and low M r kininogen (type 3) did not inhibit pig legumain. Of human type 2 cystatins, cystatin D was non-inhibitory, whereas cystatin E/M and cystatin F displayed strong (K i 0.0016 nM) and relatively weak (K i 10 nM) affinity for legumain, respectively. Sequence alignments and molecular modeling led to the suggestion that a loop located on the opposite side to the papain-binding surface, between the ␣-helix and the first strand of the main ␤-pleated sheet of the cystatin structure, could be involved in legumain binding. This was corroborated by analysis of a cystatin C variant with substitution of the Asn 39 residue in this loop (N39K-cystatin C); this variant showed a slight reduction in affinity for cathepsin B (K i 1.5 nM) but > >5,000-fold lower affinity for legumain (K i > >1,000 nM) than wild-type cystatin C.