Luisana Avilán

Enolase as a plasminogen binding protein in Leishmania mexicana

Enolase is a glycolytic and gluconeogenic enzyme also found on the surface of several eukaryotic and prokaryotic cells where it acts as plasminogen binding protein. Leishmania mexicana, one of the causative agents of Leishmaniasis, binds plasminogen and, in this parasite, enolase has been previously found associated with the external face of the plasma membrane. In this work, we show that the purified recombinant enolase has plasminogen binding activity indicating that, at the surface of the parasite, the protein may function as one of the plasminogen receptors. An internal motif 249AYDAERKMY257, similar to the nine amino-acid internal plasminogen-binding motif in Streptococcus pneumoniae enolase, is responsible for plasminogen interaction with the parasite enolase. Anti-enolase antibodies inhibited up to 60% of plasminogen binding on live parasites indicating that enolase act as a plasminogen receptor on the parasite. The fact that enolase acts as a possible plasminogen receptor in vivo makes this protein a promising target for therapy.

Molecular and biochemical characterization of hexokinase from Trypanosoma cruzi

The Trypanosoma cruzi hexokinase gene has been cloned, sequenced, and expressed as an active enzyme in Escherichia coli. Sequence analysis revealed 67% identity with its counterpart in Trypanosoma brucei but low similarity with all other available hexokinase sequences including those of human. It contains an N-terminal peroxisome-targeting signal (PTS-2) and has a calculated basic isoelectric point (pI = 9.67), a feature often associated with glycosomal proteins. The polypeptide has a predicted mass of approximately 50 kDa similar to that of many non-vertebrate hexokinases and the vertebrate hexokinase isoenzyme IV. The natural enzyme was purified to homogeneity from T. cruzi epimastigotes and appeared to exist in several aggregation states, an apparent tetramer being the predominant form. Its kinetic properties were compared with those of the purified recombinant protein. Higher K(m) values for glucose and ATP were found for the (His)(6)-tag-containing recombinant hexokinase. However, removal of the tag produced an enzyme displaying similar values as the natural enzyme (K(m) for glucose = 43 and 60 microM for the natural and the recombinant protein, respectively). None of these enzymes presented activity with fructose. As reported previously for hexokinases from several trypanosomatids, no inhibition was exerted by glucose 6-phosphate (G6-P). In contrast, a mixed-type inhibition was observed with inorganic pyrophosphate (PPi, K(i) = 0.5mM).

Enolase: A Key Player in the Metabolism and a Probable Virulence Factor of Trypanosomatid Parasites—Perspectives for Its Use as a Therapeutic Target

Glycolysis and glyconeogenesis play crucial roles in the ATP supply and synthesis of glycoconjugates, important for the viability and virulence, respectively, of the human-pathogenic stages of Trypanosoma brucei, Trypanosoma cruzi, and Leishmania spp. These pathways are, therefore, candidate targets for antiparasite drugs. The glycolytic/gluconeogenic enzyme enolase is generally highly conserved, with similar overall fold and identical catalytic residues in all organisms. Nonetheless, potentially important differences exist between the trypanosomatid and host enzymes, with three unique, reactive residues close to the active site of the former that might be exploited for the development of new drugs. In addition, enolase is found both in the secretome and in association with the surface of Leishmania spp. where it probably functions as plasminogen receptor, playing a role in the parasites invasiveness and virulence, a function possibly also present in the other trypanosomatids. This location and possible function of enolase offer additional perspectives for both drug discovery and vaccination.

Interaction of Leishmania mexicana promastigotes with the plasminogen–plasmin system

The binding of human plasminogen and plasmin to the promastigote form of Leishmania mexicana was investigated. L. mexicana was capable to bind both molecules, the binding being inhibited by epsilon-aminocaproic acid. Scatchard plot analysis revealed a dissociation constant (Kd) value of 2.4+/-0.8 microM and 0.9+/-0.1 x 10(4) binding sites per cell for plasminogen and a Kd value of 1.2+/-0.4 microM and 1.6+/-0.2 x 10(5) binding sites per cell for plasmin. C-terminal lysine residues are involved in plasminogen binding to cells, since carboxypeptidase B treatment reduced this binding by 34%. Ligand blotting analysis showed a group of proteins, with molecular masses between 105 and 115 kDa, capable to interact with plasminogen. Zymogram analysis showed that the protease activity acquired by L. mexicana, due to the interaction with either plasminogen or plasmin, comprises an important fraction of the total protease activity at pH 7.7. Plasminogen activation by tissue-type plasminogen activator (t-PA) was enhanced by the presence of L. mexicana promastigotes. These results raise the question whether the interaction of L. mexicana with components of the fibrinolytic system is involved in the virulence of the parasite.

Translocation of solutes and proteins across the glycosomal membrane of trypanosomes; possibilities and limitations for targeting with trypanocidal drugs.

Glycosomes are specialized peroxisomes found in all kinetoplastid organisms. The organelles are unique in harbouring most enzymes of the glycolytic pathway. Matrix proteins, synthesized in the cytosol, cofactors and metabolites have to be transported across the membrane. Recent research on Trypanosoma brucei has provided insight into how these translocations across the membrane occur, although many details remain to be elucidated. Proteins are imported by a cascade of reactions performed by specialized proteins, called peroxins, in which a cytosolic receptor with bound matrix protein inserts itself in the membrane to deliver its cargo into the organelle and is subsequently retrieved from the glycosome to perform further rounds of import. Bulky solutes, such as cofactors and acyl-CoAs, seem to be translocated by specific transporter molecules, whereas smaller solutes such as glycolytic intermediates probably cross the membrane through pore-forming channels. The presence of such channels is in apparent contradiction with previous results that suggested a low permeability of the glycosomal membrane. We propose 3 possible, not mutually exclusive, solutions for this paradox. Glycosomal glycolytic enzymes have been validated as drug targets against trypanosomatid-borne diseases. We discuss the possible implications of the new data for the design of drugs to be delivered into glycosomes.

Exploring CP12 binding proteins revealed aldolase as a new partner for the phosphoribulokinase/glyceraldehyde 3‐phosphate dehydrogenase/CP12 complex – purification and kinetic characterization of this enzyme from Chlamydomonas reinhardtii

Possible binding proteins of CP12 in a green alga, Chlamydomonas reinhardtii, were investigated. We covalently immobilized CP12 on a resin and then used it to trap CP12 partners. Thus, we found an association between CP12 and phosphoribulokinase (EC 2.7.1.19), glyceraldehyde 3‐phosphate dehydrogenase (EC 1.2.1.13) and aldolase. Immunoprecipitation with purified CP12 antibodies supported these data. The dissociation constant between CP12 and fructose 1,6‐bisphosphate (EC 4.1.2.13) aldolase was measured by surface plasmon resonance and is equal to 0.48 ± 0.05 μm and thus corroborated an interaction between CP12 and aldolase. However, the association is even stronger between aldolase and the phosphoribulokinase/glyceraldehyde 3‐phosphate dehydrogenase/CP12 complex and the dissociation constant between them is equal to 55±5 nm. Moreover, owing to the fact that aldolase has been poorly studied in C. reinhardtii, we purified it and analyzed its kinetic properties. The enzyme displayed Michaelis–Menten kinetics with fructose 1,6‐bisphosphate and sedoheptulose 1,7‐bisphosphate, with a catalytic constant equal to 35 ± 1 s−1 and 4 ± 0.1 s−1, respectively. The Km value for fructose 1,6‐bisphosphate was equal to 0.16 ± 0.02 mm and 0.046 ± 0.005 mm for sedoheptulose 1,7‐bisphosphate. The catalytic efficiency of aldolase was thus 219 ± 31 s−1·mm−1 with fructose 1,6‐bisphosphate and 87 ± 9 s−1·mm−1 with sedoheptulose 1,7‐bisphosphate. In the presence of the complex, this parameter for fructose 1,6‐bisphosphate increased to 310 ± 23 s−1·mm−1, whereas no change was observed with sedoheptulose 1,7‐bisphosphate. The condensation reaction of aldolase to form fructose 1,6‐bisphosphate was also investigated but no effect of CP12 or the complex on this reaction was observed.

Parasitism in optima forma: Exploiting the host fibrinolytic system for invasion

The interaction of pathogenic bacteria with the host fibrinolytic system through the plasminogen molecule has been well documented. It has been shown, using animal models, to be important in invasion into the host and establishment of the infection. From a number of recent observations with parasitic protists and helminths, emerges evidence that also in these organisms the interaction with plasminogen may be important for infection and virulence. A group of molecules that act as plasminogen receptors have been identified in parasites. This group comprises the glycolytic enzymes enolase, glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-biphosphate aldolase, in common with the plasminogen receptors known in prokaryotic pathogens. The interaction with the fibrinolytic system may arm the parasites with the host protease plasmin, thus helping them to migrate and cross barriers, infect cells and avoid clot formation. In this context, plasminogen receptors on the parasite surface or as secreted molecules, may be considered virulence factors. A possible evolutionary scenario for the recruitment of glycolytic enzymes as plasminogen receptors by widely different pathogens is discussed.

Characteristics of plasminogen binding to Trypanosoma cruzi epimastigotes.

The binding constants of the interaction between plasminogen and Trypanosoma cruzi epimastigotes were determined. An indirect method in which the bound plasminogen is detached from the cell by epsilon-aminocaproic acid and a direct method through biotinylated plasminogen were used. The analyses revealed a dissociation constant (Kd) from 0.4 to 1.2microM, these values being compatible with recognition in vivo. Moreover, epimastigotes from the gut of Rhodnius prolixus were able to bind plasminogen from the blood meal. Fragments derived from elastase digestion of plasminogen were tested for their ability to bind T. cruzi cells. The fragment with highest ability to interact with the parasite was miniplasminogen that bound in a concentration-dependent and saturable manner with a Kd similar to that for plasminogen. This binding was inhibited by epsilon-aminocaproic acid indicating that the lysine-binding site of kringle 5 may be responsible for the interaction of plasminogen with T. cruzi.

Plasminogen Interaction with Trypanosoma cruzi

The ability of Trypanosoma cruzi to interact with plasminogen, the zimogenic form of the blood serin protease plasmin, was examined. Immunohistochemistry studies revealed that both forms, epimastigotes and metacyclic trypomastigotes, were able to fix plasminogen in a lysine dependant manner. This interaction was corroborated by plasminogen activation studies. Both forms of the parasite enhanced the plasminogen activation by tissue-type plasminogen activator. The maximal enhancements obtained were 15-fold and 3.4-fold with epimastigotes and metacyclic trypomastigotes, respectively, as compared to plasminogen activation in absence of cells. Ligand-blotting analysis of proteins extracted with Triton X-114 from a microsomal fraction of epimastigotes revealed at least five soluble proteins and one hydrophobic protein able to bind plasminogen.

Leishmania mexicana: LACK (Leishmania homolog of receptors for activated C-kinase) is a plasminogen binding protein

Leishmania mexicana is able to interact with the fibrinolytic system through its component plasminogen, the zymogenic form of the protease plasmin. In this study a new plasminogen binding protein of this parasite was identified: LACK, the Leishmania homolog of receptors for activated C-kinase. Plasminogen binds recombinant LACK with a K(d) value of 1.6±0.4 μM, and binding is lysine-dependent since it is inhibited by the lysine analog ε-aminocaproic acid. Inhibition studies with specific peptides and plasminogen binding activity of a mutated recombinant LACK have highlighted the internal motif (260)VYDLESKAV(268), similar to those found in several enolases, as involved in plasminogen binding. Recombinant LACK and secreted proteins, in medium conditioned by parasites, enhance plasminogen activation to plasmin by the tissue plasminogen activator (t-PA). In addition to its localization in the cytosol, in the microsomal fraction and as secreted protein in conditioned medium, LACK was also localized on the external surface of the membrane. The results presented here suggest that LACK might bind and enhance plasminogen activation in vivo promoting the formation of plasmin. Plasminogen binding of LACK represents a new function for this protein and might contribute to the invasiveness of the parasite.