Kininogenase activity by the major cysteinyl proteinase (cruzipain) from Trypanosoma cruzi.
The major isoform of Trypanosoma cruzi cysteinyl proteinase (cruzipain) has generated Lys-bradykinin (Lys-BK or kallidin), a proinflammatory peptide, by proteolysis of kininogen. The releasing of this peptide was demonstrated by mass spectrometry, radioimmunoassay, and ileum contractile responses. The kinin-releasing activity was immunoabsorbed selectively by monoclonal antibodies to the characteristic COOH-terminal domain of cruzipain. To determine the hydrolysis steps that account for the kininogenase activity of cruzipain, we synthesized a fluorogenic peptide (o-aminobenzoyl-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-S er-Pro-Phe-Arg389-Ser390-Ser-Arg-Ile-NH2) based on the sequence Leu373 to Ile393 of the human high molecular weight kininogen. The hydrolysis products from this peptide were isolated by high performance liquid chromatography, and Lys-BK was characterized as the major released kinin by mass spectrometry. Intramolecularly quenched fluorogenic peptides spanning the Met379-Lys380 and Arg389-Ser390 bradykinin-flanking sequences were then used to assess the substrate specificity requirements of the parasite-derived protease compared with two COOH-terminal truncated recombinant isoforms (cruzain and cruzipain 2). In contrast to the high catalytic efficiency of parasite-derived cruzipain, the recombinant proteinases cleaved the bradykinin-flanking sites at markedly different rates. In addition, we also demonstrated that cruzipain activates plasmatic prekallikrein, which would be a second and indirect way of the parasite protease to release bradykinin.
Del Nery
E
E
Department of Biophysics, Universidade Federal de São Paulo, Escola Paulista de Medicina, Rua Três de Maio, 100, São Paulo, 04044-020, Brazil.
Juliano
M A
MA
Lima
A P
AP
Scharfstein
J
J
Juliano
L
L
D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000378:metabolism; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D006023:Glycoproteins / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D007609:Kallidin / Q000378:metabolism; D007610:Kallikreins / Q000378:metabolism*; D007700:Kinetics; D007704:Kininogens / Q000378:metabolism; D008969:Molecular Sequence Data; D015800:Protozoan Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D000953:Antigens, Protozoan; D006023:Glycoproteins; D007704:Kininogens; D015800:Protozoan Proteins; D011994:Recombinant Proteins; D007609:Kallidin; D007610:Kallikreins; D003546:Cysteine Endopeptidases; C063214:cruzipain