Figure 2.

Cystatins are legumain inhibitors as well as substrates. a) Binding of cystatin C (hCC) to legumain is pH dependent and reversible at pH 4.0. Both hCE and hCC are potent AEP inhibitors at pH 5.5. Subsequent incubation at pH 4.0 had no visible effect on hCE but led to regeneration of approximately 80 % AEP activity in the case of hCC, which is indicative of inhibitor release. b) Both cleaved and intact hCE bind to legumain. Size-exclusion chromatography revealed that both intact and processed hCE co-migrate with AEP following incubation at pH 4.0. Both cystatin species were also found in a separate, monomer peak. c) Processing of hCE by AEP is pH dependent. An excess of hCE could be processed at pH 4.0 but not at pH 5.5.