Dataset Information

Subsite specificity (S3, S2, S1', S2' and S3') of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity.

ABSTRACT: We characterized the extended substrate binding site of recombinant oligopeptidase B enzymes from Trypanosoma cruzi (Tc-OP) and Trypanosoma brucei (Tb-OP), evaluating the specificity of their S3, S2, S1', S2' and S3' subsites. Five series of internally quenched fluorescent peptides based on the substrate Abz-AGGRGAQ-EDDnp [where Abz is o -aminobenzoic acid and EDDnp is N -(2,4-dinitrophenyl)ethylenediamine] were designed to contain amino acid residues with side chains of a minimum size, and each residue position of this substrate was modified. Synthetic peptides of different lengths derived from the human kininogen sequence were also examined, and peptides of up to 17 amino acids were found to be hydrolysed by Tc-OP and Tb-OP. These two oligopeptidases were essentially arginyl hydrolases, since for all peptides examined the only cleavage site was the Arg-Xaa bond. We also demonstrated that Tc-OP and Tb-OP have a very specific carboxypeptidase activity for basic amino acids, which depends on the presence of at least of a pair of basic amino acids at the C-terminal end of the substrate. The peptide with triple Arg residues (Abz-AGRRRAQ-EDDnp) was an efficient substrate for Tc-OP and Tb-OP: the Arg-Ala peptide bond was cleaved first and then two C-terminal Arg residues were successively removed. The S1' subsite seems to be an important determinant of the specificity of both enzymes, showing a preference for Tyr, Ser, Thr and Gln as hydrogen donors. The presence of these amino acids at P1' resulted in substrates that were hydrolysed with K (m) values in the sub-micromolar range. Taken together, this work supports the view that oligopeptidase B is a specialized protein-processing enzyme with a specific carboxypeptidase activity. Excellent substrates were obtained for Tb-OP and Tc-OP (Abz-AMRRTISQ-EDDnp and Abz-AHKRYSHQ-EDDnp respectively), which were hydrolysed with remarkably high k (cat) and low K (m) values.

SUBMITTER: Hemerly JP

PROVIDER: S-EPMC1223545 | biostudies-other | 2003 Aug

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:BackgroundChagas disease, also known as American Trypanosomiasis, is a chronic parasitic disease caused by the flagellated protozoan Trypanosoma cruzi that affects about 8 million people around the world where more than 25 million are at risk of contracting the infection. Despite of being endemic on 21 Latin-American countries, Chagas disease has become a global concern due to migratory movements. Unfortunately, available drugs for the treatment have several limitations and they are generally administered during the chronic phase of the infection, when its efficacy is considered controversial. Thus, prophylactic and/or therapeutic vaccines are emerging as interesting control alternatives. In this work, we proposed Trypanosoma cruzi 80 kDa prolyl oligopeptidase (Tc80) as a new antigen for vaccine development against Chagas disease.Methodology/principal findingsIn a murine model, we analyzed the immune response triggered by different immunization protocols based on Tc80 and evaluated their ability to confer protection against a challenge with the parasite. Immunized mice developed Tc80-specific antibodies which were able to carry out different functions such as: enzymatic inhibition, neutralization of parasite infection and complement-mediated lysis of trypomastigotes. Furthermore, vaccinated mice elicited strong cell-mediated immunity. Spleen cells from immunized mice proliferated and secreted Th1 cytokines (IL-2, IFN-γ and TNF-α) upon re-stimulation with rTc80. Moreover, we found Tc80-specific polyfunctional CD4 T cells, and cytotoxic T lymphocyte activity against one Tc80 MHC-I peptide. Immunization protocols conferred protection against a T. cruzi lethal challenge. Immunized groups showed a decreased parasitemia and higher survival rate compared with non-immunized control mice. Moreover, during the chronic phase of the infection, immunized mice presented: lower levels of myopathy-linked enzymes, parasite burden, electrocardiographic disorders and inflammatory cells.Conclusions/significanceConsidering that an early control of parasite burden and tissue damage might contribute to avoid the progression towards symptomatic forms of chronic Chagas disease, the efficacy of Tc80-based vaccines make this molecule a promising immunogen for a mono or multicomponent vaccine against T. cruzi infection.

Project description:BackgroundThe trypanosomatids Leishmania major, Trypanosoma brucei and Trypanosoma cruzi cause some of the most debilitating diseases of humankind: cutaneous leishmaniasis, African sleeping sickness, and Chagas disease. These protozoa possess complex life cycles that involve development in mammalian and insect hosts, and a tightly coordinated cell cycle ensures propagation of the highly polarized cells. However, the ways in which the parasites respond to their environment and coordinate intracellular processes are poorly understood. As a part of an effort to understand parasite signaling functions, we report the results of a genome-wide analysis of protein kinases (PKs) of these three trypanosomatids.ResultsBioinformatic searches of the trypanosomatid genomes for eukaryotic PKs (ePKs) and atypical PKs (aPKs) revealed a total of 176 PKs in T. brucei, 190 in T. cruzi and 199 in L. major, most of which are orthologous across the three species. This is approximately 30% of the number in the human host and double that of the malaria parasite, Plasmodium falciparum. The representation of various groups of ePKs differs significantly as compared to humans: trypanosomatids lack receptor-linked tyrosine and tyrosine kinase-like kinases, although they do possess dual-specificity kinases. A relative expansion of the CMGC, STE and NEK groups has occurred. A large number of unique ePKs show no strong affinity to any known group. The trypanosomatids possess few ePKs with predicted transmembrane domains, suggesting that receptor ePKs are rare. Accessory Pfam domains, which are frequently present in human ePKs, are uncommon in trypanosomatid ePKs.ConclusionTrypanosomatids possess a large set of PKs, comprising approximately 2% of each genome, suggesting a key role for phosphorylation in parasite biology. Whilst it was possible to place most of the trypanosomatid ePKs into the seven established groups using bioinformatic analyses, it has not been possible to ascribe function based solely on sequence similarity. Hence the connection of stimuli to protein phosphorylation networks remains enigmatic. The presence of numerous PKs with significant sequence similarity to known drug targets, as well as a large number of unusual kinases that might represent novel targets, strongly argue for functional analysis of these molecules.

Project description:In the recent X-ray crystallographic structural models of photosystem II, Glu189 of the D1 polypeptide is assigned as a ligand of the oxygen-evolving Mn(4) cluster. To determine if D1-Glu189 ligates a Mn ion that undergoes oxidation during one or more of the S(0) --> S(1), S(1) --> S(2), and S(2) --> S(3) transitions, the FTIR difference spectra of the individual S-state transitions in D1-E189Q and D1-E189R mutant PSII particles from the cyanobacterium Synechocystis sp. PCC 6803 were compared with those in wild-type PSII particles. Remarkably, the data show that neither mutation significantly alters the mid-frequency regions (1800-1200 cm(-)(1)) of any of the FTIR difference spectra. Importantly, neither mutation eliminates any specific symmetric or asymmetric carboxylate stretching mode that might have been assigned to D1-Glu189. The small spectral alterations that are observed are similar in amplitude to those that are observed in wild-type PSII particles that have been exchanged into FTIR analysis buffer by different methods or those that are observed in D2-H189Q mutant PSII particles (the residue D2-His189 is located >25 A from the Mn(4) cluster and accepts a hydrogen bond from Tyr Y(D)). The absence of significant mutation-induced spectral alterations in the D1-Glu189 mutants shows that the oxidation of the Mn(4) cluster does not alter the frequencies of the carboxylate stretching modes of D1-Glu189 during the S(0) --> S(1), S(1) --> S(2), or S(2) --> S(3) transitions. One explanation of these data is that D1-Glu189 ligates a Mn ion that does not increase its charge or oxidation state during any of these S-state transitions. However, because the same conclusion was reached previously for D1-Asp170, and because the recent X-ray crystallographic structural models assign D1-Asp170 and D1-Glu189 as ligating different Mn ions, this explanation requires that (1) the extra positive charge that develops on the Mn(4) cluster during the S(1) --> S(2) transition be localized on the Mn ion that is ligated by the alpha-COO(-) group of D1-Ala344 and (2) any increase in positive charge that develops on the Mn(4) cluster during the S(0) --> S(1) and S(2) --> S(3) transitions be localized on the one Mn ion that is not ligated by D1-Asp170, D1-Glu189, or D1-Ala344. An alternative explanation of the FTIR data is that D1-Glu189 does not ligate the Mn(4) cluster. This conclusion would be consistent with earlier spectroscopic analyses of D1-Glu189 mutants, but would require that the proximity of D1-Glu189 to manganese in the X-ray crystallographic structural models be an artifact of the radiation-induced reduction of the Mn(4) cluster that occurred during the collection of the X-ray diffraction data.

Dataset Information

Subsite specificity (S3, S2, S1', S2' and S3') of oligopeptidase B from Trypanosoma cruzi and Trypanosoma brucei using fluorescent quenched peptides: comparative study and identification of specific carboxypeptidase activity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets