A developmentally regulated cysteine proteinase gene of Leishmania mexicana

We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single-copy gene (lmcpa) [corrected]. has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C-terminal extension of only 10 amino acids and a three-amino-acid insertion, GlyValMet, close to the predicted N-terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life-cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective metacyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro-region, but was barely detectable in early log-phase promastigotes. Anti-central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPheValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life-cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.     

A developmentally regulated cysteine proteinase in Dictyostelium discoideum.

We have determined the sequence of a Dictyostelium mRNA encoding a protein with a high degree of homology to plant and animal cysteine proteinases. The degree of homology is highest in the region of the cysteine residue which is transiently acylated during peptide hydrolysis but all other residues known to be important in catalysis are also conserved. We have named this protein cysteine proteinase 1. There is a hydrophobic signal peptide of 18 amino acids and an additional 99 amino acids at the N terminus, which are not present in other cysteine proteases and which may be cleaved off during processing of the enzyme. There is a single copy of the gene in the Dictyostelium genome. The cysteine proteinase 1 mRNA is absent from growing cells and from cells isolated during the first 6 h of development but it constitutes approximately 1% of cellular mRNA by 10-12 h of development. During the development of Dictyostelium a major fraction of cellular protein is degraded to provide amino acids and a source of energy. Cysteine proteinase 1 may play a role in this auto-digestion.     

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.     

Identification and cloning of an aspartyl proteinase from Coccidioides immitis

A 45 kDa protein was isolated from a soluble vaccine prepared from formaldehyde-killed spherules of Coccidioides immitis. From the N-terminal amino acid sequence, the protein yielded a 17-amino-acid peptide that was homologous to sequences of other fungal aspartyl proteinases. The coccidioidal cDNA encoding the proteinase was amplified using oligonucleotide primers designed from the 45 kDa N-terminal amino acid sequence and a fungal aspartyl proteinase consensus amino acid sequence. The PCR product was cloned and sequenced, and the remaining 5' upstream and 3' downstream cDNA was amplified, cloned, and sequenced. The cDNA encoding the coccidioidal aspartyl proteinase open reading frame was cloned and the fusion protein containing a C-terminal His-tag expressed in E. coli. The recombinant aspartyl proteinase was purified by immobilized metal affinity chromatography. This recombinant protein will be used for further studies to evaluate its antigenicity, including protective immunogenicity.     

Leishmania mexicana: identification and characterization of an aspartyl proteinase activity

An aspartyl proteinase activity was detected in the soluble fraction (SF) of Leishmania mexicana promastigotes by the use of the synthetic substrate benzoyl-Arg-Gly-Phe-Phe-Leu-4-methoxy-beta-naphthylamide selective for Cathepsin D like aspartyl-proteinases. This peptide was hydrolyzed with an apparent K(m) of 2.3+/-0.3 microM. The classic inhibitor of aspartyl-proteinases, diazo-acetyl-norleucinemethylester (DAN) inhibited the proteolytic activity with an IC(50) of 400 microM. The soluble fraction degraded (in absence of thiol groups) human fibrinogen with a specific activity of 533 U/mg protein. When tested for the ability to inhibit the "in vitro" proliferation of L. mexicana promastigotes, DAN showed concentration dependent anti-proliferative effects with a LD(50) of 466 microM at 48 h, with a significant fall in this value to 22 microM after 72 h. This is the first characterization of an aspartyl-proteinase activity in Leishmania, calling for further studies directed towards the physiologic role of these enzymes in the parasite. The anti-proliferative effect of its inhibition makes this enzyme a putative new target for the development of leishmanicidal drugs.     

Genomic organization, chromosomal localization, and developmentally regulated expression of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei.

The surface of the bloodstream form of the African trypanosome, Trypansoma brucei, is covered with about 10(7) molecules of the variant surface glycoprotein (VSG), a protein tethered to the plasma membrane by a glycosyl-phosphatidylinositol (GPI) membrane anchor. This anchor is cleavable by an endogenous GPI-specific phospholipase C (GPI-PLC). GPI-PLC activity is down regulated when trypanosomes differentiate from the bloodstream form to the procyclic form found in the tsetse fly vector. We have mapped the GPI-PLC locus in the trypanosome genome and have examined the mechanism for this developmental regulation in T. brucei. Southern blot analysis indicates a single-copy gene for GPI-PLC, with two allelic variants distinguishable by two NcoI restriction fragment length polymorphisms. The gene was localized solely to a chromosome in the two-megabase compression region by contour-clamped homogeneous electric field gel electrophoresis. No rearrangement of the GPI-PLC gene occurs during differentiation to procyclic forms, which could potentially silence GPI-PLC gene expression. Enzymological studies give no indication of a diffusible inhibitor of GPI-PLC activity in procyclic forms, and Western immunoblot analysis reveals no detectable GPI-PLC polypeptide in these forms. Therefore, it is highly unlikely that the absence of GPI-PLC activity in procyclic forms is due to posttranslational control. Northern (RNA) blot analysis reveals barely detectable levels of GPI-PLC mRNA in procyclic forms; therefore, regulation of GPI-PLC activity in these forms correlates with the steady-state mRNA level.     

VEGF expression is developmentally regulated during human brain angiogenesis

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor working as an endothelial cell-specific mitogen and exerting a trophic effect on neurons and glial cells, both these activities being essential during central nervous system vascularisation, development and repair. The vascularisation of human telencephalon takes place by means of an angiogenic mechanism, which starts at the beginning of corticogenesis and actively proceeds up to the last neuronal migration, when the basic scheme of the vascular network has been drawn. Our study focused on VEGF during this critical developmental period with the aim of identifying the cells that express VEGF and of correlating the events of angiogenesis with the main events of cerebral cortex formation. The results show that in fetal human brain VEGF protein is located on multiple cell types, cells proper to the nervous tissue, neuroepithelial cells, neuroblasts and radial glia cells, and non-neuronal cells, endothelial and periendothelial cells. In these cells VEGF expression appears developmentally regulated and is correlated with angiogenesis, which in turn responds to the high metabolic demands of the differentiating neocortex.     

Genomic organisation and nervous system expression of radial spoke protein 3

Following their generation in the germinal zones, young neurons of the neocortex, hippocampus and cerebellum undergo long-distance migration to reach their final destinations. This locomotive activity depends on active deployment of cytoskeletal elements including the microtubule apparatus. In this study, we report the identification and expression of radial spoke protein 3 (RSP3), a member of a protein cluster responsible for anchoring and modifying dynein motor activity known to be crucial to microtubule sliding. The mouse RSP3 gene consists of eight exons and seven introns and spans over 230 kb. The genomic organisations of the human and rat RSP3 genes are similar although they span approximately 23 and 53 kb, respectively. This is in contrast to the Chlamydomonas RSP3 gene, where RSP3 was first isolated, which consists of four exons and three introns and spans approximately 2.7 kb. Despite these differences, the nucleotide and amino acid sequences upstream of, and throughout the RPII-binding domain of RSP3 are highly conserved between all the above-mentioned species. Mouse RSP3 mRNA was restricted to the developing neocortex, hippocampus and cerebellum during the stages when these structures are known to contain large numbers of migratory neurons. Gene expression studies suggest that RSP3 function is consistent with a locomotory role for this protein in migrating young neurons. In addition, expression of RSP3 mRNA in adult neurons point to additional, though still unknown functions. Our data provides the first evidence for the expression of radial spoke proteins in higher eukaryotes, and provides a biological framework for how these proteins may participate in microtubule sliding and neuronal migration in the embryonic brain.     

Mechanism of aspartyl proteinase action. VII. Noncovalent complexes of HIV-1 aspartyl proteinase with substrate and substrate-like inhibitors

A computer model of a noncovalent complex of HIV-1 aspartyl protease with substrate-like inhibitor JG-365 was a priori constructed by using the approaches of theoretical conformational analysis and molecular mechanics. The root mean square deviation of the calculated conformation of the inhibitor from the X-ray diffraction analysis data was 0.87 A. These results enabled the a priori calculation of the structure of noncovalent complex of HIV-1 protease with a hexapeptide fragment of its native specific substrate Ser-Gln-Asn-Tyr-Pro-Ile-Val. The only possible orientation of the cleavable peptide bond in this and the nucleophilic water molecule relative to the catalytically active Asp residues of the enzyme (Asp25 and Asp125) was found that provides for the chemical transformation of the substrate to a tetrahedral intermediate. An action mechanism of enzymes of this class was proposed on the basis of the analysis of calculated distances. We showed that neither steric distortion of the cleavable bond nor the formation of unfavorable contacts in molecules of the enzymes and their substrates accompany the optimum orientation of substrate molecules at the active sites of HIV-1 aspartyl proteases and rhizopuspepsin.     

Differential localisation of an Eimeria tenella aspartyl proteinase during the infection process

Aspartyl proteinases are essential for the survival of many pathogens. A single copy gene in species of Eimeria encodes an aspartyl proteinase, which we propose should be called eimepsin to conform to the commonly used names of this family of proteinases. An epitope map, constructed using BIAcore technology, confirmed the specificity of 14 mAbs for eimepsin and defined four antigenic domains, which were conserved between native and recombinant forms of eimepsin. In resting sporozoites, mAb defining antigenic domains I and II stained the refractile body organelles, whereas those defining antigenic domains III and IV stained cytoplasmic granules. During host cell invasion, the staining patterns of mAb defining antigenic domains I, III and IV changed dramatically with the apical tips of invading sporozoites becoming strongly stained. In contrast, mAb defining antigenic domain II continued to stain only the refractile bodies. During early schizogony, mAb to all four domains stained the single fused refractile body, but when schizonts matured, mAb to antigenic domains I, III and IV stained the apical tip of merozoites whereas those to antigenic domain II continued to follow the developmental redistribution of the refractile body. Irrespective of localisation, mAb to three antigenic domains recognised a polypeptide of 49 kDa, which from N-terminal sequencing corresponds to a mature form of eimepsin. Staining with fluorescent pepstatin localised a mature, active form of eimepsin to the refractile bodies of the sporozoite, schizont and first generation merozoite. It remains to be determined whether eimepsin has a catalytic function within the refractile body or whether the activated enzyme is stored in the refractile body so that it can be rapidly redistributed to the apical tip during parasite invasion.     

Ddi1-like protein from Leishmania major is an active aspartyl proteinase

Eukaryotic cells respond to DNA damage by activating damage checkpoint pathways, which arrest cell cycle progression and induce gene expression. We isolated a full-length cDNA encoding a 49-kDa protein from Leishmania major, which exhibited significant deduced amino acid sequence homology with the annotated Leishmania sp. DNA damage-inducible (Ddi1-like) protein, as well as with the Ddi1 protein from Saccharomyces cerevisiae. In contrast to the previously described Ddi1 protein, the protein from L. major displays three domains: (1) an NH2-terminal ubiquitin like; (2) a COOH terminal ubiquitin-associated; (3) a retroviral aspartyl proteinase, containing the typical D[S/T]G signature. The function of the L. major Ddi1-like recombinant protein was investigated after expression in baculovirus/insect cells and biochemical analysis, revealing preferential substrate selectivity for aspartyl proteinase A₂ family substrates, with optimal activity in acidic conditions. The proteolytic activity was inhibited by aspartyl proteinase inhibitors. Molecular modeling of the retroviral domain of the Ddi1-like Leishmania protein revealed a dimer structure that contained a double Asp-Ser-Gly-Ala amino acid sequence motif, in an almost identical geometry to the exhibited by the homologous retroviral aspartyl protease domain of yeast Ddi1 protein. Our results indicate that the isolated Ddi1-like protein is a functional aspartyl proteinase in L. major, opening possibility to be considered as a potential target for novel antiparasitic drugs.