Evidence for a recent mutation giving rise to a truncated copy of a cysteine proteinase gene in Leishmania pifanoi

We have previously identified and characterized two amastigote-specific cysteine proteinases of Leishmania pifanoi. The slightly different isoforms of the more abundant proteinase are coded by a gene family of approximately 20 gene copies, that contain a C-terminal extension characteristic of cysteine proteinases of trypanosomatids. In this gene family, we have detected a copy that codes for a truncated form of this proteinase, lacking the C-terminal extension. Interestingly, when the deletion of a nucleotide that creates a stop codon causing this truncation is disregarded, the translated sequence gives rise to a divergent C-terminal extension that has many conserved amino acids when compared to Leishmania and Trypanosome, suggesting that a recent mutation led to the truncation.     

Leishmania amazonensis: early proteinase activities during promastigote-amastigote differentiation in vitro

Leishmania proteinase activity is known as parasite differentiation marker, and has been considered relevant for leishmanial survival and virulence. These properties suggest that Leishmania proteinases can be promising targets for development of anti-leishmania drugs. Here, we analyze the activities of four proteinases during the early phase of the Leishmania amazonensis promastigotes differentiation into amastigotes induced by heat shock. We have examined activities of cysteine-, metallo-, serine-, and aspartic-proteinase by hydrolysis of specific chromogenic substrates at pH 5.0 and at the optimal pH for each enzyme. Our results show that metallo-, serine-, and aspartic-proteinases activities were down-regulated during the shock-induced transformation of promastigotes into amastigotes. In contrast, cysteine-proteinase activity increased concomitantly with the promastigote differentiation. Immunocytochemical localization using two anti-cysteine-proteinase monospecific rabbit antibodies detected the enzyme in several cell compartments of both parasite stages. Our results show different proteinase activity modulation and expression during the early phases of the shock-induced parasite transformation.     

The iron-induced cysteine proteinase TvCP4 plays a key role in Trichomonas vaginalis haemolysis

Trichomonas vaginalis has multiple proteinases, mainly of the cysteine type (CPs), including a 34 kDa precursor cathepsin L-like CP dubbed TvCP4. TvCP4 is an iron-up-regulated CP. The goal of this work was to identify the role of TvCP4 in the virulence of T. vaginalis. We cloned, expressed, and purified the recombinant mature enzyme region of TvCP4 (TvCP4r) to produce a rabbit polyclonal antibody (α-TvCP4r). This antibody reacted with a ∼24 kDa protein band in total protein extracts that could correspond to the mature enzyme. By two-dimensional western blot assays TvCP4 corresponded to three protein spots of ∼24 kDa with pI values of ∼6.7, 6.9, and 7.0 and two spots of ∼22 and ∼21 kDa with a pI of 6.9, as confirmed by mass spectrometry. As expected, a higher amount of TvCP4 was detected in cytoplasmic vesicles, lysosomes, and on the surface of iron-rich parasites when compared with normal and iron-depleted parasites. The α-TvCP4r antibody protected human erythrocytes from trichomonal lysis. Additionally, TvCP4 is expressed during infection and is part of the released products detected in vaginal fluids of patients with trichomonosis. Thus, data show that TvCP4 is an iron-induced CP that participates in T. vaginalis haemolysis.     

A major cysteine proteinase is developmentally regulated in Trypanosoma cruzi

Epimastigotes of different stocks of Trypanosoma cruzi contain similar levels of proteinase activity on azocasein; amastigotes and trypomastigotes contain 10-fold lower levels of this proteolytic activity, which seems, therefore, to be developmentally regulated. The proteinase could be detected as a broad band, centered at about 60 kDa, which in some cases resolved into two close bands, in (a) SDS-polyacrylamide gels containing fibrinogen, and (b) Western blots probed with a polyclonal rabbit antiserum prepared against purified cysteine proteinase. No proteinase activity was observed at molecular weights lower than 55 kDa. The results show that the enzyme previously purified is the major cysteine proteinase present in epimastigotes of all stocks of T. cruzi tested.     

Dynamic identification of H2 epitopes from Leishmania (Leishmania) amazonensis cysteine proteinase B with potential immune activity during murine infection

Peptides from the COOH‐terminal extension of cysteine proteinase B from Leishmania (Leishmania) amazonensis (cyspep) can modulate immune responses in vertebrate hosts. With this hypothesis as base, we used the online analysis tool SYFPEITHI to predict seven epitopes from this region with potential to bind H2 proteins. We performed proliferation tests and quantified reactive T lymphocytes applying a cytometry analysis, using samples from draining lymph node of lesions from L. (L.) amazonensis‐infected mice. To define reactivity of T cells, we used complexes of DimerX (H2 D^b:Ig and H2 L^d:Ig) and the putative epitopes. Additionally, we applied surface plasmon resonance to verify real time interactions between the putative epitopes and DimerX proteins. Five peptides induced blastogenesis in BALB/c cells, while only two presented the same property in C57BL/6 mouse cells. In addition, our data indicate the existence of CD8⁺ T lymphocyte populations able to recognize each tested peptide in both murine strains. We observed an overlapping of results between the peptides that induced lymphocyte proliferation and those capable of binding to the DimerX in the surface plasmon resonance assays thus indicating that using these recombinant proteins in biosensing analyses is a promising tool to study real time molecular interactions in the context of major histocompatibility complex epitopes. The data gathered in this study reinforce the hypothesis that cyspep‐derived peptides are important factors in the murine host infection by L. (L.) amazonensis. Copyright © 2014 John Wiley & Sons, Ltd.     

Location of the cell-binding domain of CP65, a 65kDa cysteine proteinase involved in Trichomonas vaginalis cytotoxicity

The cysteine proteinase (CP) of 65 kDa, CP65, binds to the surface of HeLa cells and is involved in Trichomonas vaginalis cellular damage. To identify and locate the CP65 cellular-binding domain, we enriched the CP65 protein band by ammonium sulfate fractionation and ion-exchange chromatography and the N-terminal sequence was obtained. A 618 bp gene fragment was obtained by PCR using genomic DNA as template and primers derived from the N-terminal sequence of CP65 and the Asn papain-catalytic conserved region. This gene fragment encodes for 206 amino acid (aa) residues corresponding to the N-terminal region of a mature CP with 67–76% identity to the reported trichomonad cathepsin-L-like CPs. This gene fragment was expressed in a bacterial system for antibody production and functional analysis. Antibodies against the native trichomonad CP65 recognized the recombinant protein, referred to as rCP65, confirming its relationship with the CP65 gene. The rCP65 protein was bound to the surface of HeLa cells and competed with the native CP65 for binding. Antibodies to the rCP65 (-rCP65) reacted with the trichomonad CP65 located on the parasite surface, and inhibited trichomonal cytotoxicity in a concentration-dependent manner. These data strongly suggest that this gene fragment encodes for the putative cell-binding domain (CBD) of CP65 located at its N-terminal region.     

Analysis of the cysteine proteinases of Leishmania mexicana and Trypanosoma cruzi using specific antisera

Abstract Antisera raised against papain and cysteine proteinases (CPs) purified from Leishmania mexicana and Trypanosoma cruzi have been used to study the proteins in the two parasites. The antisera against the major CP of T. cruzi (cruzipain) not only cross-reacted with known CPs of L. mexicana but also detected stage-specific molecules that may represent previously unrecognised CPs. The binding of the same abtisera to extracts of different life cycle stages of T. cruzi suggested that the stages possess different isoforms of cruzipain. The lack of cross-reactivity of anti-papain antiserum against cruzipain suggests that the major immunogenic epitopes of these CPs are different, whereas the detection of the major CPs of L. mexicana with both heterologous antisera shows that the parasite's enzymes share epitopes with the other CPs.     

Characterization of a multi-copy gene for a major stage-specific cysteine proteinase of Leishmania mexicana.

lmcpb, a gene from Leishmania mexicana that encodes a major cysteine proteinase in the parasite, has been cloned and sequenced. LmCPb is related more to cysteine proteinases from Trypanosoma brucei and Trypanosoma cruzi than to a previously characterized cysteine proteinase, LmCPa, of L. mexicana. It contains a long C-terminal extension characteristic of similar enzymes of T. brucei and T. cruzi. The gene is multi-copy and tandemly arranged. lmcpb RNA levels are developmentally regulated with steady state levels being high in amastigotes, low in metacyclic promastigotes and undetectable in multiplicative promastigotes. This variation correlates with and may account for the stage-specific expression of LmCPb enzyme activity.     

Leishmania aethiopica: identification and characterization of cathepsin L-like cysteine protease genes

There is limited information on the biology and pathogenesis of Leishmania aethiopica, causative agent of cutaneous leishmaniasis (CL) in Ethiopia. In this study we have identified and characterized two cathepsin L-like cysteine protease genes, Laecpa and Laecpb, from L. aethiopica. The predicted amino acid sequence of Laecpa and Laecpb is more than 75% identical with homologous cathepsin L-like cysteine protease genes of other Leishmania species and less than 50% identical with human cathepsin L. Laecpa is expressed predominantly in the stationary, and to a lower level, during the amastigote stage while Laecpb is specifically expressed in the stationary stage of L. aethiopica development. Phylogenetic analysis showed that the two genes are grouped into separate clades which are the result of gene duplication. The isolation of these genes will be useful in developing Leishmania species specific diagnostics for molecular epidemiological studies and serves as a first step to study the role of cysteine proteases in L. aethiopica pathogenesis.     

Purification and partial characterization of a cysteine proteinase from Trypanosoma rangeli

Abstract Epimastigotes of the American Trypanosome Trypanosoma rangeli contain a very low cysteine proteinase (CP) activity. The enzyme was purified to homogeneity by affinity chromatography on ConA-Sepharose and Cystatin-Sepharose. This CP had a similar apparent molecular mass and an identical N-terminal sequence (15 amino acids) as compared with cruzipain from Trypanosoma cruzi ; cross-reacted immunologically with the latter enzyme, it was inhibited by E-64 and TLCK, but not by PMSF, o-phenanthroline or Pepstatin, and was able to use the same substrates, although with different order of effectiveness and optimum pH.     

An unusual intronic mutation in the CYBB gene giving rise to chronic granulomatous disease

The most common, X-linked, form of chronic granulomatous disease (CGD) is caused by mutations in the CYBB gene located at Xp21.1. The product of this gene is the large subunit of flavocytochrome b558, gp91phox, which forms the catalytic core of the antimicrobial superoxide-generating enzyme, NADPH oxidase. In the overwhelming majority of cases, mutations are family-specific and occur in the exonic regions of the gene, or more rarely at the intron/exon borders. Alternatively, they are large (often multi-gene) deletions. In addition, four mutations have been found in the promoter region. In contrast, very few intronic mutations have been reported. Here we describe an intronic mutation that causes X-linked CGD. A single nucleotide substitution in the middle of intron V creates a novel 5' splice site and results in multiple abnormal mRNA products.     

Leishmania major metacaspase can replace yeast metacaspase in programmed cell death and has arginine-specific cysteine peptidase activity

The human protozoan parasite Leishmania major has been shown to exhibit several morphological and biochemical features characteristic of a cell death program when differentiating into infectious stages and under a variety of stress conditions. Although some caspase-like peptidase activity has been reported in dying parasites, no caspase gene is present in the genome. However, a single metacaspase gene is present in L. major whose encoded protein harbors the predicted secondary structure and the catalytic dyad histidine/cysteine described for caspases and other metacaspases identified in plants and yeast. The Saccharomyces cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In this study, we describe the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA) in cell death induced by oxidative stress. We show that LmjMCA is involved in yeast cell death, similar to YCA1, and that this function depends on its catalytic activity. LmjMCA was found to be auto-processed as occurs for caspases, however LmjMCA did not exhibit any activity with caspase substrates. In contrast and similarly to Arabidopsis thaliana metacaspases, LmjMCA was active towards substrates with arginine in the P1 position, with the activity being abolished following H147A and C202A catalytic site mutations. These results suggest that metacaspases are members of a family of peptidases with a role in cell death conserved in evolution notwithstanding possible differences in their catalytic activity.     

Evidence for expression of a single distinct form of mammalian cysteine dioxygenase

Summary. Cysteine dioxygenase (CDO) plays a critical role in the regulation of cellular cysteine concentration. Because multiple forms of CDO (23kDa, 25kDa, and 68kDa) have been claimed based upon separation and detection using SDS-PAGE/western blotting (with antibodies demonstrated to immunoprecipitate CDO), we further investigated the possibility of more than one CDO isoform. Using either rabbit antibody raised against purified rat liver CDO or against purified recombinant his₆-tagged CDO (r-his₆-CDO) and using 15% (wt/vol) polyacrylamide for the SDS-PAGE, we consistently detected the 25kDa band, but never detected a 68kDa band, in rat liver, kidney, lung and brain. Nondenatured gel electrophoresis of r-his₆-CDO yielded a molecular mass estimate of 25.7kDa and no evidence of dimerization. Mass spectrometry of r-his₆-CDO yielded two peaks with molecular masses of 24.1kDa and 24.3kDa. Anion-exchange FPLC of r-his₆-CDO also gave two peaks, with the first containing CDO that was 7.5-times as active as the more anionic form that eluted second. When the two peaks recovered from FPLC were run on SDS/PAGE, the first (more active) CDO fraction yielded two bands (perhaps as an artifact of SDS/PAGE), whereas the second (less active) CDO fraction yielded only the 23kDa band. We conclude that the physiologically active form of CDO is the 25kDa (i.e., 23.5kDa based on mass spectrometry) monomer and that this active form is probably derived by post-translational modification of the 23kDa gene product.     

Purification and characterization of a low molecular weight cysteine proteinase inhibitor from bovine muscle

A low-M_r tight binding proteinase inhibitor was purified from bovine muscle by alkaline denaturation of cysteine proteinases, gel filtration on Sexphadex G-75 and affinity chromatography on carboxymethyl-papain-Sepharose. Chromatofocusing separated three isoforms which are similar in their M_r of about 14 000, their stability with heating at 80°C and their inhibitory activity towards cathepsin H, cathepsin B and papain. The equilibrium constants (K_i) were determined for these three cysteine proteinases but for cathepsin H, association (k_ass) and dissociation (k_diss) rate constants were also evaluated. K_i values of 56 nM and 8.4 nM were found for cathepsin B and cathepsin H, respectively. For papain, K_i was in the range of 0.1–1 nM. The kinetic features of enzyme-inhibitor binding suggest a possible role for this low-M_r protein inhibitor in controlling ‘in vivo’ cathepsin H proteolytic activity. With regard to cathepsin B, such a physiological role was less evident.