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 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 CPB2.8 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 leishmanial enzyme.
Alves
L C
LC
Department of Biophysics, Escola Paulista de Medicina, Universidade Federal de São Paulo, Rua Tres de Maio 100, 04044-020 São Paulo, Brazil.
Judice
W A
WA
St Hilaire
P M
PM
Meldal
M
M
Sanderson
S J
SJ
Mottram
J C
JC
Coombs
G H
GH
Juliano
L
L
Juliano
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D007704:Kininogens / Q000378:metabolism; D007894:Leishmania mexicana / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D015800:Protozoan Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D015853:Cysteine Proteinase Inhibitors; D007704:Kininogens; D010446:Peptide Fragments; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D002403:Cathepsins; D003546:Cysteine Endopeptidases; C074032:cruzain, Trypanosoma cruzi; C534281:CTSL protein, human; D056668:Cathepsin L