Activation of human prolegumain by cleavage at a C-terminal asparagine residue

The processing and activation of prolegumain were studied using the recombi nant protein synthesized by cells that had been stably transfected with a h uman legumain cDNA construct. A cell line termed C13 was selected for the h igh-level expression of prolegumain. C13 cells produced primarily 56 kDa pr olegumain. The 56 kDa form was enzymically inactive but stable at neutral p H, unlike the 35 kDa mature pig legumain; it could be converted into a 46 k Da active form by incubation at pH 4.5. The 56 kDa pro-form and the 46 kDa active form were found to have the same N-terminal amino acid sequence, ind icating that cleavage at the N-terminus was not necessary for prolegumain a ctivation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn(323). Replac ement of Asn(323) at the cleavage site with aspartate, serine, alanine or g lutamate abolished the processing and activation of prolegumain. In contras t, mutation of other asparagine and aspartate residues near the cleavage si te had no effect. These results demonstrate that Asn(323) is essential for prolegumain activation.