Pig kidney legumain: an asparaginyl endopeptidase with restricted specificity

Legumain was recently discovered as a lysosomal endopeptidase in mammals [C hen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (199 7) J. Biol. Chem. 272, 8090-8098], having been known previously only from p lants and invertebrates. It has been shown to play a key role in processing of the C fragment of tetanus toxin for presentation by the MHC class-II sy stem [Manoury, Hewitt, Morrice, Dando, Barrett and Watts (1998) Nature (Lon don) 396, 695-699]. We examine here the specificity of the enzyme from pig kidney by use of protein, oligopeptide and synthetic arylamide substrates, all determinations being made at pH 5.8. In proteins, only about one in ten of the asparaginyl bonds were hydrolysed, and these were mostly predicted to be located at turns on the protein surface. Bonds that were not cleaved in tetanus toxin were cleaved when presented in oligopeptides, sometimes fa ster than an equivalent oligopeptide based on a bond that was cleaved in th e protein. Legumain cleaved the bait region of rat alpha(1)-macroglobulin a nd was 'trapped' by the macroglobulin, as most other endopeptidases are, bu t did not interact with human alpha(1)-macroglobulin, which contains no asp aragine residue in its bait region. Glycosylation of asparagine totally pre vented hydrolysis by legumain. Specificity for arylamide substrates was eva luated with reference to benzyloxycarbonyl-Ala-Ala-Asn-aminomethylcoumarin and the preference for the P3-position amino acid was Ala > Tyr (tertiary b utyl) > Val > Pro > Phe = Tyr > Leu = Gly. There was no hydrolysis of subst rate analogues containing mono- or di-N-methylasparagines, L-2-amino-3-urei dopropionic acid or citrulline in the P1 position. We conclude that mammali an legumain appears to be totally restricted to the hydrolysis of asparagin yl bonds in sutbstrates of all kinds. There seem to be no strong preference s for particular amino acids in other subsites, and yet there are still uni dentified factors that prevent hydrolysis of many asparaginyl bonds in prot eins.