Substrate specificity of recombinant cysteine proteinase, CPB, of Leishmania mexicana

The primary S-1 subsite specificity of a recombinant cysteine, proteinase, CPB2.8 Delta CTE, of Leishmania mexicana was investigated in a systematic w ay using a series of peptides derived from Abz-KLRFSKQ-EDDnp in which Arg w as substituted by all natural amino acids (where Abz is oi-tho-amino-benzoy l and EDDnp is N-[2,4-dinitrophenyl]-ethylenediamine). The peptides from th is series with charged side chain amino acids, Cys, Cys(SBzl), and Thr(OBzl ) were well hydrolysed. All other substitutions resulted in peptides that w ere resistant or hydrolysed very slowly and inhibited the enzyme with Ki va lues in the range of 9-400 nM. Looking for natural substrates for CPB2.8, w e observed that the recombinant enzyme failed to release kinin from human k ininogen, an activity earlier observed with cruzipain from Trypanosoma cruz i (Del Nery et al., J. Biol. Chem. 272 (1997) 25713.). This lack of activit y seems to be a result of the resistance to hydrolysis of the sequence at t he N-terminal site of bradykinin in the human kininogen. The preferences fo r the S-3, S-2 and S-1' -S-3' for some amino acids were also examined using substrates derived from Abz-KLRFSKQ-EDDnp with variations at Lys, Leu, Phe , Ser and Lys, using the amino acids Ala, Phe, Leu, His or Pro. Peptides wi th Phe at P-1' presented the highest affinity to the leishmanial enzyme. Fo r comparison, some of the obtained peptides were also assayed with recombin ant human cathepsin L and cruzain. The best substrates for CPB2.8 Delta CTE were also well hydrolysed by cathepsin L, however, the best inhibitors of the parasite enzyme have low affinity to cathepsin L. These promising data provide leads for the design of anti-parasitic drugs directed against the l eishmanial enzyme. (C) 2001 Elsevier Science B.V. All rights reserved.