TY - JOUR
T1 - Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase
AU - Chen, Jinq May
AU - Dando, Pam M.
AU - Rawlings, Neil D.
AU - Brown, Molly A.
AU - Young, Nina E.
AU - Stevens, Richard A.
AU - Hewittt, Eric
AU - Watts, Colin
AU - Barrett, Alan J.
PY - 1997/3/21
Y1 - 1997/3/21
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0030992979&partnerID=8YFLogxK
U2 - 10.1074/jbc.272.12.8090
DO - 10.1074/jbc.272.12.8090
M3 - Article
C2 - 9065484
AN - SCOPUS:0030992979
SN - 0021-9258
VL - 272
SP - 8090
EP - 8098
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 12
ER -