Molecular, functional and structural properties of the prolyl oligopeptidase of Trypanosoma cruzi (POP Tc80), which is required for parasite entry into mammalian cells.
We have demonstrated that the 80 kDa POP Tc80 (prolyl oligopeptidase of Trypanosoma cruzi) is involved in the process of cell invasion, since specific inhibitors block parasite entry into non-phagocytic mammalian host cells. In contrast with other POPs, POP Tc80 is capable of hydrolysing large substrates, such as fibronectin and native collagen. In this study, we present the cloning of the POPTc80 gene, whose deduced amino acid sequence shares considerable identity with other members of the POP family, mainly within its C-terminal portion that forms the catalytic domain. Southern-blot analysis indicated that POPTc80 is present as a single copy in the genome of the parasite. These results are consistent with mapping of POPTc80 to a single chromosome. The active recombinant protein (rPOP Tc80) displayed kinetic properties comparable with those of the native enzyme. Novel inhibitors were assayed with rPOP Tc80, and the most efficient ones presented values of inhibition coefficient Ki < or = 1.52 nM. Infective parasites treated with these specific POP Tc80 inhibitors attached to the surface of mammalian host cells, but were incapable of infecting them. Structural modelling of POP Tc80, based on the crystallized porcine POP, suggested that POP Tc80 is composed of an alpha/beta-hydrolase domain containing the catalytic triad Ser548-Asp631-His667 and a seven-bladed beta-propeller non-catalytic domain. Docking analysis suggests that triple-helical collagen access to the catalytic site of POP Tc80 occurs in the vicinity of the interface between the two domains.
Bastos
Izabela M D
IM
Laboratório Multidisciplinar de Pesquisa em Doença de Chagas (CP 04536), Universidade de Brasília, 70919-970, Brasília, DF, Brazil.
Grellier
Philippe
P
Martins
Natalia F
NF
Cadavid-Restrepo
Gloria
G
de Souza-Ault
Marian R
MR
Augustyns
Koen
K
Teixeira
Antonio R L
AR
Schrével
Joseph
J
Maigret
Bernard
B
da Silveira
José F
JF
Santana
Jaime M
JM
D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002448:Cell Adhesion; D002460:Cell Line; D004791:Enzyme Inhibitors; D008969:Molecular Sequence Data; D000085822:Prolyl Oligopeptidases; D017434:Protein Structure, Tertiary; D015800:Protozoan Proteins; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000378:metabolism; D014349:Trypanosoma cruzi / Q000201:enzymology*
D004791:Enzyme Inhibitors; D015800:Protozoan Proteins; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases; C000705288:Tc80 prolyl oligopeptidase, Trypanosoma cruzi