Figure 5.

Active form of Cat V is expressed in human thymus. (a) Identification of human Cats V, L, and S in human thymus extracts. Equal amounts (7.5 μg) of lysosomal fractions from human thymus were subjected to SDS-PAGE (10% gel), followed by Western blot analysis using antihuman Cat V antibody, antihuman Cat S antibody (1:2000 dilution), and mAb33/1 and mAb33/2 recognizing human Cat L and human Cats L and V, respectively. (b) Inhibition of recombinant human Cat S using LHVS inhibitor. Cats V and S (10 ng) were incubated with 1 nM LHVS at 37°C for the indicated time before labeling with DCG-04 as described in Methods. Activity of Cat S was completely blocked by incubation with 1 nM LHVS at 37°C for 20 minutes, but that of Cat V was not. Recombinant human Cat V is present in two isoforms distinguished by differential glycosylation as described in 14. (c) Selective inhibition of Cat S but not of Cat V in thymus extracts. Lysosomal fractions (1.5 μg) from human thymus were incubated with or without LHVS (1 nM or 1 μM) at 37°C for 20 minutes before labeling with DCG-04. At 1 nM LHVS concentration, the remaining band represents Cat V, whereas at 1 μM LHVS all cathepsins are inhibited and thus not labeled by DCG-04.