INHIBITION OF TRYPANOSOMAL CYSTEINE PROTEINASES BY THEIR PROPEPTIDES

The ability of the prodomains of trypanosomal cysteine proteinases to inhibit their active form was studied using a set of 23 overlapping 15 -mer peptides covering the whole prosequence of congopain, the major c ysteine proteinase of Trypanosoma congolense. Three consecutive peptid es with a common 5-mer sequence YHNGA were competitive inhibitors of c ongopain. A shorter synthetic peptide consisting of this 5-mer sequenc e flanked by two Ala residues (AYHNGAA) also inhibited purified congop ain. No residue critical for inhibition was identified in this sequenc e, but a significant improvement in K-i value was obtained upon N-term inal elongation. Procongopain-derived peptides did not inhibit lysosom al cathepsins B and L but did inhibit native cruzipain (from Dm28c clo ne epimastigotes), the major cysteine proteinase of Trypanosoma cruzi, the proregion of which also contains the sequence YHNGA. The position ing of the YHNGA inhibitory sequence within the prosegment of trypanos omal proteinases is similar to that covering the active site in the pr osegment of cysteine proteinases, the three-dimensional structure of w hich has been resolved. This strongly suggests that trypanosomal prote inases, despite their long C-terminal extension, have a prosegment tha t folds similarly to that in related mammal and plant cysteine protein ases, resulting in reverse binding within the active site, Such revers e binding could also occur for short procongopain-derived inhibitory p eptides, based on their resistance to proteolysis and their ability to retain inhibitory activity after prolonged incubation. In contrast, h omologous peptides in related cysteine proteinases did not inhibit try panosomal proteinases and were rapidly cleaved by these enzymes.