Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase

Jinq May Chen, Pam M. Dando, Neil D. Rawlings, Molly A. Brown, Nina E. Young, Richard A. Stevens, Eric Hewittt, Colin Watts, Alan J. Barrett (Lead / Corresponding author)

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    312 Citations (Scopus)

    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 (trans-epoxysuccinyl-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.

    Original languageEnglish
    Pages (from-to)8090-8098
    Number of pages9
    JournalJournal of Biological Chemistry
    Volume272
    Issue number12
    DOIs
    Publication statusPublished - 21 Mar 1997

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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