Cloning, isolation, and characterization of mammalian legumain, an asparaginyl endopeptidase.
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.
Chen
J M
JM
Medical Research Council Peptidase Laboratory, Department of Immunology, The Babraham Institute, Babraham Hall, Babraham, Cambridgeshire CB2 4AT, United Kingdom.
Dando
P M
PM
Rawlings
N D
ND
Brown
M A
MA
Young
N E
NE
Stevens
R A
RA
Hewitt
E
E
Watts
C
C
Barrett
A J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D018076:DNA, Complementary; D006031:Glycosylation; D006801:Humans; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D010940:Plant Proteins; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine; D000099240:Asparaginyl Endopeptidase
D003546:Cysteine Endopeptidases; D015853:Cysteine Proteinase Inhibitors; D018076:DNA, Complementary; D010940:Plant Proteins; D000099240:Asparaginyl Endopeptidase