Cysteine protease isoforms from Trypanosoma cruzi, cruzipain 2 and cruzain, present different substrate preference and susceptibility to inhibitors.
Cysteine-proteinases from parasitic protozoa have been recently characterized as factors of virulence and pathogenicity in several human and veterinary diseases. In Chagas' disease, the chronic infection caused by Trypanosoma cruzi, structure-functional studies on cysteine proteases were thus far limited to the parasite's major isoform, a cathepsin L-like lysosomal protease designated as cruzipain, cruzain or GP57/51. Encoded by a large gene family, cruzipain is efficiently targeted by synthetic inhibitors, which prevent parasite intracellular growth and differentiation. We have previously demonstrated that the multicopy cruzipain gene family includes polymorphic sequences, which could encode functionally different isoforms. We report here a comparative kinetic study between cruzain, the archetype of the cruzipain family, and an isoform, termed cruzipain 2, which is expressed preferentially by the mammalian stages of T. cruzi. Heterologous expression of the catalytic domain of cruzipain 2 in Saccharomyces cerevisae yielded an enzyme that differs markedly from cruzain with respect to pH stability, substrate specificity and sensitivity to inhibition by natural and synthetic inhibitors of cysteine proteases. We suggest that the structural-functional diversification imparted by genetic polymorphism of cruzipain genes may have contributed to T. cruzi adaptation to vertebrate hosts.
Lima
A P
AP
Laboratory of Molecular Immunology, Instituto de Biofísica Carlos Chagas Filho, Bloco G, CCS, UFRJ, Ilha do Fundão, RJ, CEP 21-944-900, Rio de Janeiro, Brazil. anapaula@biof.ufrj.br
dos Reis
F C
FC
Serveau
C
C
Lalmanach
G
G
Juliano
L
L
Ménard
R
R
Vernet
T
T
Thomas
D Y
DY
Storer
A C
AC
Scharfstein
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology* / Q000235:genetics
D000953:Antigens, Protozoan; D015853:Cysteine Proteinase Inhibitors; D007527:Isoenzymes; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; C074032:cruzain, Trypanosoma cruzi; C063214:cruzipain