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

The primary S 1 subsite specificity of a recombinant cysteine proteinase, CPB2.8ΔCTE, of Leishmania mexicana was investigated in a systematic way using a series of peptides derived from Abz-KLRFSKQ-EDDnp in which Arg was substituted by all natural amino acids (where Abz is ortho-amino-benzoyl and ED...

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Published in:Molecular and biochemical parasitology 2001-08, Vol.116 (1), p.1-9
Main Authors: Alves, Lira C, Judice, Wagner A.S, Hilaire, Phaedria M.St, Meldal, Morten, Sanderson, Sanya J, Mottram, Jeremy C, Coombs, Graham H, Juliano, Luiz, Juliano, Maria A
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Animals
Cathepsin L
Cathepsins - metabolism
Cruzain
Cysteine Endopeptidases - genetics
Cysteine Endopeptidases - metabolism
Cysteine proteinase
Cysteine Proteinase Inhibitors - pharmacology
Humans
Inhibitors
Kininogens - metabolism
Leishmania
Leishmania mexicana
Leishmania mexicana - enzymology
Leishmania mexicana - genetics
Molecular Sequence Data
Peptide Fragments - metabolism
Protozoan Proteins - metabolism
Recombinant Proteins - metabolism
Substrate Specificity
Thiol protease
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Summary:The primary S 1 subsite specificity of a recombinant cysteine proteinase, CPB2.8ΔCTE, of Leishmania mexicana was investigated in a systematic way using a series of peptides derived from Abz-KLRFSKQ-EDDnp in which Arg was substituted by all natural amino acids (where Abz is ortho-amino-benzoyl and EDDnp is N-[2,4-dinitrophenyl]-ethylenediamine). The peptides from this series with charged side chain amino acids, Cys, Cys(SBzl), and Thr(OBzl) were well hydrolysed. All other substitutions resulted in peptides that were resistant or hydrolysed very slowly and inhibited the enzyme with K i values in the range of 9–400 nM. Looking for natural substrates for CPB2.8, we observed that the recombinant enzyme failed to release kinin from human kininogen, an activity earlier observed with cruzipain from Trypanosoma cruzi (Del Nery et al., J. Biol. Chem. 272 (1997) 25713.). This lack of activity seems to be a result of the resistance to hydrolysis of the sequence at the N-terminal site of bradykinin in the human kininogen. The preferences for 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 with Phe at P 1′ presented the highest affinity to the leishmanial enzyme. For comparison, some of the obtained peptides were also assayed with recombinant human cathepsin L and cruzain. The best substrates for CPB2.8ΔCTE 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 leishmanial enzyme.
ISSN:0166-6851
1872-9428
DOI:10.1016/S0166-6851(01)00290-0