The processing and activation of prolegumain were studied using the recombinant protein synthesized by cells that had been stably transfected with a human legumain cDNA construct. A cell line termed C13 was selected for the high-level expression of prolegumain. C13 cells produced primarily 56kDa prolegumain. The 56kDa form was enzymically inactive but stable at neutral pH, unlike the 35kDa mature pig legumain; it could be converted into a 46kDa active form by incubation at pH 4.5. The 56kDa pro-form and the 46kDa active form were found to have the same N-terminal amino acid sequence, indicating that cleavage at the N-terminus was not necessary for prolegumain activation, and that the decrease in molecular mass was due to a C-terminal cleavage. The C-terminal processing site was identified as Asn323. Replacement of Asn323 at the cleavage site with aspartate, serine, alanine or glutamate abolished the processing and activation of prolegumain. In contrast, mutation of other asparagine and aspartate residues near the cleavage site had no effect. These results demonstrate that Asn323 is essential for prolegumain activation.

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