Autocatalytic cleavage of prolegumain occurs in trans. SDS–PAGE of C189S prolegumain incubated with super-activated wild-type legumain at pH values as indicated (4 h, 310 K). M, molecular-weight marker (labelled in kDa); band 1, C189S prolegumain; band 2, C-terminal propeptide cleaved (cleavage after Asn323); band 3, N-terminal and C-terminal propeptides cleaved (cleavage after Asp25 and Asn323); band 4, C-terminal propeptide.