Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Prostaglandin production from arachidonic acid and evidence for a 9,11-endoperoxide prostaglandin H2 reductase in Leishmania

Lysates of Leishmania promastigotes can metabolise arachidonic acid to prostaglandins. Prostaglandin production was heat sensitive and not inhibited by aspirin or indomethacin. We cloned and sequenced the cDNA of Leishmania major, Leishmania donovani, and Leishmania tropica prostaglandin F(2alpha) synthase, and overexpressed their respective 34-kDa recombinant proteins that catalyse the reduction of 9,11-endoperoxide PGH(2) to PGF(2alpha). Database search and sequence alignment showed that L. major prostaglandin F(2alpha) synthase exhibits 61, 99.3, and 99.3% identity with Trypanosoma brucei, L. donovani, and L. tropica prostaglandin F(2alpha) synthase, respectively. Using polymerase chain reaction amplification, Western blotting, and immunofluorescence, we have demonstrated that prostaglandin F(2alpha) synthase protein and gene are present in Old World and absent in New World Leishmania, and that this protein is localised to the promastigote cytosol.     

Evidence of transferrin binding sites on the surface of Leishmania promastigotes.

A glycoprotein of 78,000 molecular mass (78 kDa), associated with the membrane of Leishmania infantum promastigotes, was identified and immunopurified by monoclonal antibody (mAb) LD9 produced against isolated membrane preparations. mAb LD9 was subsequently found to bind to human transferrin, also of 78 kDa. Binding of LD9 to transferrin was completely abolished when the mAb was preabsorbed by Leishmania membranes, thereby indicating that the 78-kDa Leishmania membrane-associated glycoprotein and transferrin have common antigenic epitope(s). The 78-kDa Leishmania membrane-associated protein was released in soluble nonaggregated form by mild treatment with acetic acid saline. Anti-transferrin polyclonal antibodies, recognized both the membrane-associated and the soluble form of the 78-kDa glycoprotein. The 78-kDa soluble form was characterized further as an iron-containing protein. The above data combined with iron uptake by promastigotes as demonstrated by the Prussian blue reaction indicate that the 78-kDa Leishmania membrane-associated glycoprotein is transferrin. The binding of 125I-human transferrin to Leishmania-purified membrane preparations was then investigated. The results indicate the presence of a high affinity saturable binding site (Kd = 2.2 10(-8) M) that is specific for transferrin. We suggest that the 78-kDa glycoprotein recognized by mAb LD9 is transferrin that binds to the surface of Leishmania promastigotes via a transferrin receptor.     

C-reactive protein binds to phosphorylated carbohydrates

C-reactive protein (CRP) is a major acute phase protein in man. In order to more fully understand the physiological role of this serum protein, we have demonstrated high avidity binding for a defined chemically synthesized carbo-hydrate ligand which represents the repeating disaccharide of lipophosphoglycan, the major surface glycoconjugate of the unicellular parasite Leishmania donovani. Increasing the number of phosphorylated disaccharides in a molecule from one up to seven did not increase the avidity for CRP, however increasing this to 10 potential CRP binding sites did. In order to define the important features of this complex and variable structure for CRP binding we competed CRP binding to whole Leishmania parasites with amino, sulfated, phosphorylated, and unsubstituted monosaccharides, of which only phosphorylated monosaccharides were able to inhibit. Both the carbohydrate and the position of phosphorylation influenced the avidity for CRP. Synthetic oligosaccharides and phospho-oligosaccharides of various lengths and conformations were used to define the structural requirements for CRP recognition. The optimum structure for recognition of a single phosphate group was between two monosaccharide pyranose rings, and within a linear rather than a cyclic molecule. This stresses the importance of the interaction of the CRP binding site with both the carbohydrate and the phosphate group. CRP function may be mediated via the recognition of large arrays of phosphorylated carbohydrates as are characteristic of the surface of microorganisms.     

Pentameric Two-Dimensional Crystallization of Rabbit C-Reactive Protein on Lipid Monolayers

As a member of the pentraxin family, C-reactive protein plays various roles in the nonspecific immunity of animals. Though soluble, C-reactive protein always functions on membranes. In order to study the structure of the membrane-bound protein and the reaction between protein and membranes, two-dimensional (2D) crystallization of rabbit C-reactive protein on lipid monolayers was performed. The 2D crystals composed of pentameric proteins were obtained on lipid monolayers by specific adsorption for the first time. The projection map at 26-A resolution is presented, which exhibits P2 symmetry with lattice parameters a = 158(+/-3) A, b = 92(+/-1) A, and gamma = 107(+/-1) degrees. The current work may give a basis for the further study on the structure of complexes made up of C-reactive protein with its functional binding molecules on membranes.     

Hemoglobin receptor in Leishmania is a hexokinase located in the flagellar pocket

Hb endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket. To understand the nature of the Hb receptor (HbR), we have purified the 46-kDa protein to homogeneity from Leishmania promastigote membrane. Purified HbR specifically binds Hb. The gene for HbR was cloned, and sequence analysis of the full-length HbR gene indicates the presence of hexokinase (HK) signature sequences, ATP-binding domain, and PTS-II motif. Four lines of evidence indicate that HbR in Leishmania is a hexokinase: 1) the recombinant HbR binds Hb, and the Hb-binding domain resides in the N terminus of the protein; 2) recombinant proteins and cell lysate prepared from HbR-overexpressing Leishmania promastigotes show enhanced HK activity in comparison with untransfected cells; 3) immunolocalization studies using antibodies against the N-terminal fragment (Ld-HbR-DeltaC) of Ld-HbR indicate that this protein is located in the flagellar pocket of Leishmania; and 4) binding and uptake of (125)I-Hb by Leishmania is significantly inhibited by anti-Ld-HbR-DeltaC antibody and Ld-HbR-DeltaC, respectively. Taken together, these results indicate that HK present in the flagellar pocket of Leishmania is involved in Hb endocytosis.     

Effect of pH and temperature on protein kinase release by Leishmania donovani

During their life cycle Leishmania are exposed to environments that differ markedly in pH and temperature. The effect of these factors on protein kinase release into the surrounding environment by Leishmania donovani promastigotes was examined. Promastigotes release protein kinase activity both constitutively and following induction by incubation with an exogenous substrate, phosvitin. The substrate specificity of the constitutive and induced activities was similar, unlike that previously described for Leishmania major promastigotes. The Leishmania donovani enzymes phosphorylate phosvitin, but not casein, mixed histones or protamine sulphate, and both activities are shed over a wide pH range from 6 to 9. Transfer of promastigotes from pH 7.4/30 degrees C to pH 5.0-5.5/37 degrees C, conditions that mimic those encountered by parasites following transmission from sandflies to a mammalian host and uptake by macrophages, inhibited release of the constitutive activity. Identical conditions had only a minor effect on induced protein kinase release. Both types of protein kinase activities released at pH 7.4 were still active when assayed at pH 5.0. Characterisation of the constitutive and induced promastigote protein kinases showed that casein kinase 1- and casein kinase 2-like activities are released by Leishmania donovani. Constitutive enzyme release decreased over time, however, the addition of phosvitin to these "casein kinase-depleted" promastigotes induced elevated casein kinase 1 and casein kinase 2 shedding. These results suggest that shed protein kinase might play a role in parasite survival and adaptation to host environments.     

A proatherogenic role for C-reactive protein in vivo

PURPOSE OF REVIEW: We have selectively reviewed some of the latest papers on the mechanistic role of C-reactive protein in atherosclerotic cardiovascular disease. RECENT DEVELOPMENTS: C-reactive protein is known to activate the classic pathway of the complement system. One paper examined the role of C-reactive protein in complement activation by enzymatically remodeled LDL proteins. Enzymatically remodeled LDL was found to induce complement activation with or without C-reactive protein, but in the presence of C-reactive protein the activation of complement halted before its terminal sequence. Complement activation by C-reactive protein in atherogenesis remains controversial. Different laboratories have reported the multi-organ origin of C-reactive protein. The atherosclerotic lesion itself is another place where C-reactive protein could be produced. Numerous studies have continued to dissect the potential diverse proatherogenic actions of C-reactive protein on cultured vascular cells. Caution must be exercised in inadequately controlled studies that have unwittingly used commercial C-reactive protein preparations contaminated by other bioactive components. In contrast to in-vitro experiments, in-vivo studies that support a proatherogenic role of C-reactive protein are less likely to be subject to misinterpretation. SUMMARY: Evidence suggests that C-reactive protein is a proatherogenic molecule that plays an active role. The amount of C-reactive protein in lesions is determined by its plasma levels and its local production. The biological effect of C-reactive protein on atherosclerosis development seems to encompass a complex network of interactions with other players in immunity and inflammation, such as the complement system, as well as a direct effect of C-reactive protein on the cells involved in lesion growth and development.     

Synthesis of viruslike particles by expression of the putative capsid protein of Leishmania RNA virus in a recombinant baculovirus expression system.

The putative capsid open reading frame (ORF2) of the Leishmania RNA virus LRV1-4 was expressed in a baculovirus expression system. The expressed protein was identified by Western immunoblot analysis with polyclonal antiserum raised to purified LRV1-4 virus. Electron microscopy and sedimentation analysis indicated that the expressed protein self-assembles into empty viruslike particles of similar size and shape to authentic virus particles, thus confirming that ORF2 encodes the viral capsid. The expressed particles are present exclusively in the cytoplasm of infected SF9 cells and are able to assemble in the absence of LRV1-4 RNA, viral polymerase, or any Leishmania host factors.     

The toxic oil syndrome: An example of an exogenously induced autoimmune reaction

The toxic oil syndrome (TOS) is a chemically induced autoimmune-like reaction in humans. The etiologic agent(s) have so far not clearly been identified. A short overview is given on this disease which is associated with a unique antibody specificity to cryptic epitopes of C-reactive protein in affected patients. In addition a murine model for TOS is described which suggests that genetic suscepbility might play a role in inducing a similar syndrome in mice. These differences are reflected in the immunological alterations and cytokine production caused by the toxicant.Abbreviations CRP C-reactive protein - OAA oleic acid anilide - PAP 3-phenylamino-1,2-propanediol - TOS toxic oil syndrome     

Molecular and cellular characterization of an AT-hook protein from Leishmania

AT-rich DNA, and the proteins that bind it (AT-hook proteins), modulate chromosome structure and function in most eukaryotes. Unlike other trypanosomatids, the genome of Leishmania species is unusually GC-rich, and the regulation of Leishmania chromosome structure, replication, partitioning is not fully understood. Because AT-hook proteins modulate these functions in other eukaryotes, we examined whether AT-hook proteins are encoded in the Leishmania genome, to test their potential functions. Several Leishmania ORFs predicted to be AT-hook proteins were identified using in silico approaches based on sequences shared between eukaryotic AT-hook proteins. We have used biochemical, molecular and cellular techniques to characterize the L. amazonensis ortholog of the L. major protein LmjF06.0720, a potential AT-hook protein that is highly conserved in Leishmania species. Using a novel fusion between the AT-hook domain encoded by LmjF06.0720 and a herpesviral protein, we have demonstrated that LmjF06.0720 functions as an AT-hook protein in mammalian cells. Further, as observed for mammalian and viral AT-hook proteins, the AT-hook domains of LmjF06.0720 bind specific regions of condensed mammalian metaphase chromosomes, and support the licensed replication of DNA in mammalian cells. LmjF06.0720 is nuclear in Leishmania, and this localization is disrupted upon exposure to drugs that displace AT-hook proteins from AT-rich DNA. Coincidentally, these drugs dramatically alter the cellular physiology of Leishmania promastigotes. Finally, we have devised a novel peptido-mimetic agent derived from the sequence of LmjF06.0720 that blocks the proliferation of Leishmania promastigotes, and lowers amastigote parasitic burden in infected macrophages. Our results indicate that AT-hook proteins are critical for the normal biology of Leishmania. In addition, we have described a simple technique to examine the function of Leishmania chromatin-binding proteins in a eukaryotic context amenable to studying chromosome structure and function. Lastly, we demonstrate the therapeutic potential of compounds directed against AT-hook proteins in Leishmania.     

Leishmania EF-1alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation

The human leishmaniasis are persistent infections of macrophages caused by protozoa of the genus Leishmania. The chronic nature of these infections is in part related to induction of macrophage deactivation, linked to activation of the Src homology 2 domain containing tyrosine phosphatase-1 (SHP-1) in infected cells. To investigate the mechanism of SHP-1 activation, lysates of Leishmania donovani promastigotes were subjected to SHP-1 affinity chromatography and proteins bound to the matrix were sequenced by mass spectrometry. This resulted in the identification of Leishmania elongation factor-1alpha (EF-1alpha) as a SHP-1-binding protein. Purified Leishmania EF-1alpha, but not host cell EF-1alpha, bound directly to SHP-1 in vitro leading to its activation. Three independent lines of evidence indicated that Leishmania EF-1alpha may be exported from the phagosome thereby enabling targeting of host SHP-1. First, cytosolic fractions prepared from macrophages infected with [(35)S]methionine-labeled organisms contained Leishmania EF-1alpha. Second, confocal, fluorescence microscopy using Leishmania-specific antisera detected Leishmania EF-1alpha in the cytosol of infected cells. Third, co-immunoprecipitation showed that Leishmania EF-1alpha was associated with SHP-1 in vivo in infected cells. Finally, introduction of purified Leishmania EF-1alpha, but not the corresponding host protein into macrophages activated SHP-1 and blocked the induction of inducible nitric-oxide synthase expression in response to interferon-gamma. Thus, Leishmania EF-1alpha is identified as a novel SHP-1-binding and activating protein that recapitulates the deactivated phenotype of infected macrophages.     

Binding of C-reactive protein to chromatin and nucleosome core particles. A possible physiological role of C-reactive protein

By using a variety of biochemical techniques, chromatin and chromatin fragments have been identified as probable physiological ligands for C-reactive protein. Studies using 14C-labeled C-reactive protein show that binding to chromatin is saturable with a Kd = 8 X 10(-7) M, a value indicating that the affinity of C-reactive protein for chromatin is at least four times its affinity for phosphorylcholine. At saturation, there is approximately one C-reactive protein-binding site for every 160 base pairs of DNA in chromatin. The interaction of C-reactive protein with chicken erythrocyte nucleosome core particle has been studied. Fifty per cent inhibition of the binding of C-reactive protein to phosphorylcholine is obtained at a core particle concentration of 1.25 X 10(-9) M, indicating that the affinity of C-reactive protein for one of the sites on core particles is at least 2400 times greater than the affinity of C-reactive protein for phosphorylcholine. The possibility that C-reactive protein may act as a scavenger for chromatin fragments released from damaged cells is discussed.     

Phosphoproteomic analysis of Leishmania donovani pro- and amastigote stages

Following transmission to the vertebrate host, the protozoan parasite Leishmania donovani differentiates into the pathogenic amastigote stage that is adapted for intracellular survival. This developmental transition is induced by environmental factors including elevated temperature and acidic pH and is likely transduced by signaling cascades involving protein kinases and their downstream phosphoprotein substrates. These signaling networks are highly adapted to the specific nutritional and physiological requirements of the organism and thus studying Leishmania phosphorylation may allow important insight into the parasite-specific biology. We used a gel-based approach to investigate qualitative and quantitative changes of the phosphoproteome of the major L. donovani life cycle stages. Phosphoproteins were purified by immobilized metal affinity chromatography (IMAC), separated by IEF and SDS-PAGE using pH 4-7 IPG immobiline strips, revealed by fluorescent multiplex staining, and identified by MALDI-MS and MS/MS. Our analysis allowed us to establish a first repertoire of the Leishmania phosphoproteome and to identify phosphoproteins implicated in stress- and heat shock response, RNA/protein turnover, metabolism, and signaling.