Identification of the C-terminal region of 70 kDa heat shock protein from Leishmania (Viannia) braziliensis as a target for the humoral immune response

A Leishmania (Viannia) braziliensis (LB) promastigote cDNA library in λgt11 was screened with patients' sera with the aim of identifying antigens specifically related to mucocutaneous leishmaniasis (MCL). One of the clones isolated, 133P, consistently reacted with MCL sera; it was sequenced and found to encode the C-terminal three-quarters of a protein belonging to the highly conserved Hsp70 family. Since Hsp70 proteins from different species tend to be less conserved through the C-terminal end, it was predicted that this region would be more antigenic and was likely to bear the discriminatory epitopes. In order to test this hypothesis, the N- and C-terminal halves of the polypeptide encoded by 133P, 133P-N and 133P-C, respectively, were expressed in Esherichia coli. Immunoblotting analysis demonstrated that 133P-C reacted more strongly with a pool of MCL sera than 133P-N, and both recombinant proteins reacted faintly with pools of cutaneous (CL) and visceral (VL) leishmaniasis sera. These results confirmed the predicted epitope location in the C-terminal region. The 133P-C fragment was also expressed as a fusion protein with glutathione-S-transferase (GST-133P-C), affinity-purified with glutathione-agarose and tested by ELISA with individual sera. From 46 Lb-infected patients, 41 sera (89%) were positive, no cross-reactivity was observed with healthy, Trypanosoma cruzi- or L. amazonensis-infected individuals. Despite a relatively high cross-reactivity with VL sera, the enhanced humoral response of MCL as compared with CL patients might be interesting for studies on disease aggravation.     

The sera from individuals suffering from cutaneous leishmaniasis due to Leishmania brazilensis present antibodies against parasitic conserved proteins, but not their human counterparts

Sera from individuals suffering from leishmaniasis have been shown to strongly react against conserved proteins from the parasite, such as ribosomal, histones and heat-shock proteins. Some of these proteins have also been described as immunogenic in several auto-immune syndromes, and the detection of antibodies against them is considered to be indicative of disorder of the immune system. In this paper, we investigate whether there is any relationship between the recognition of some conserved proteins from leishmania braziliensis by individuals suffering from cutaneous (CL) and mucocutaneous (MCL) leishmaniasis, and the recognition of the human homologues of these antigens found in sufferers from autoimmune diseases. Our findings reveal that the immune response generated during CL and MCL is elicited specifically by the parasitic histone H1 and Hsp70, since the CL and MCL sera do not react against their human counterparts. In addition, evidence is presented showing the specific recognition of human proteins by the autoimmune sera, showing only a weak cross-reaction with the most divergent regions of the parasitic proteins.     

Antigenic differences among Leishmania amazonensis isolates and their relationship with distinct clinical forms of the disease.

Immunoblot analysis was used to investigate antigenic differences among clinical isolates of Leishmania amazonensis and their role in the etiology of the disease. Western blots of promastigote homogenates were analyzed with either monoclonal antibodies (MAbs) specific for the L. mexicana complex (M-4, M-6, M-9, and M-11) or polyclonal sera from L. amazonensis infected patients with the various forms of clinical disease. In the case of the MAbs, no significant variation was observed among the strains of L. amazonensis, isolated from cases of cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL), diffuse cutaneous leishmaniasis (DCL), visceral leishmaniasis (VL) or post kala-azar dermal leishmaniasis (PKDL), in either the relative mobility (Mr) or the quantitative amount (intensity) of the antigenic determinants. In the case of the sera of the infected patients, the patterns of antigenic reactivity of these strains revealed that, despite showing the presence of shared antigens, differences were observed between some of the antigenic components of the various isolates of L. amazonensis that were recognized by a single serum. Differences were also demonstrated between the antigenic determinants of a single isolate of L. amazonensis that were recognized by the different patients' sera. No apparent association was consistently found, however, between the Mr components identified in these isolates and the clinical form of the disease or the geographical area of isolation. In addition, the spectrum of antigens recognized by the sera from patients with the same clinical form were not identical; although in some instances, similar Mr antigens were shared.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro and in vivo behaviour of sympatric Leishmania (V.) braziliensis, L. (V.) peruviana and their hybrids

Leishmania (Viannia) braziliensis is the main cause of highly disfiguring mucocutaneous leishmaniasis (MCL) in South America. The related species L. (V.) peruviana has only been identified in simple cutaneous lesions (CL). Hybrids between L. braziliensis and L. peruviana have been reported although genetic exchange in Leishmania is considered to be rare. Here we compared growth in vitro, adaptive capacity under thermal and oxidative stress and behaviour in a hamster model, of L. braziliensis, L. peruviana, and their putative hybrids. At 24°C, the optimal temperature for in vitro growth, L. braziliensis had the highest growth rate. In in vitro studies hybrid clones presented heterogeneous phenotypes, from slower growth rates, similar to L. peruviana, to higher growth rates, as observed in L. braziliensis. Hamsters infected with hybrid strains, presented the highest parasite densities and aggressive relapses at a later stage of infection. Hybrids generally presented higher plasticity and phenotypic diversity than the putative parental species, with potential eco-epidemiological implications, including an impact on the success of disease control.     

Epitopes of the p70 and p80 (Ku) lupus autoantigens.

High titer autoantibodies to the Ku Ag, a DNA-protein complex containing 70- and approximately 80-kDa protein subunits (p70 and p80, respectively), are found in sera of certain patients with systemic lupus erythematosus and related disorders. Autoepitopes of the Ku Ag were identified and partially characterized by expressing fragments of the p70 and p80 cDNA as fusion proteins in bacteria. Systemic lupus erythematosus sera reacted on immunoblots with at least three epitopes of p70 (amino acids 560-609, 506-535, and 115-467), and three epitopes of p80 (amino acids 682-732, 558-681, and 1-374). These six antigenic regions had distinct amino acid sequences, and were also immunologically distinct, as determined by using immunoaffinity-purified auto-antibodies to particular epitopes. Detailed mapping of the strongly antigenic region near the C terminus of p70 revealed a complex conformational or discontinuous epitope, the antigenicity of which was abolished by deleting either amino acids 560-571 or 601-609. The C terminus of p80 may also contain a discontinuous or conformational epitope(s). Although only some sera reacted with p70 or p80 on immunoblots, all sera that immunoprecipitated the native Ku complex reacted with native Ku by ELISA, and inhibited the binding of mAb directed at epitopes of native Ku. Taken together, these studies indicate that anti-Ku autoantibodies target a diversity of independent epitopes located on p70, p80, and the intact Ku complex, and that a significant portion of the autoantibodies in most patients' sera is directed against conformational/discontinuous epitopes.     

The Molecular Chaperone Hsp70 Activates Protein Phosphatase 5 (PP5) by Binding the Tetratricopeptide Repeat (TPR) Domain

Protein phosphatase 5 (PP5) is auto-inhibited by intramolecular interactions with its tetratricopeptide repeat (TPR) domain. Hsp90 has been shown to bind PP5 to activate its phosphatase activity. However, the functional implications of binding Hsp70 to PP5 are not yet clear. In this study, we find that both Hsp90 and Hsp70 bind to PP5 using a luciferase fragment complementation assay. A fluorescence polarization assay shows that Hsp90 (MEEVD motif) binds to the TPR domain of PP5 almost 3-fold higher affinity than Hsp70 (IEEVD motif). However, Hsp70 binding to PP5 stimulates higher phosphatase activity of PP5 than the binding of Hsp90. We find that PP5 forms a stable 1:1 complex with Hsp70, but the interaction appears asymmetric with Hsp90, with one PP5 binding the dimer. Solution NMR studies reveal that Hsc70 and PP5 proteins are dynamically independent in complex, tethered by a disordered region that connects the Hsc70 core and the IEEVD-TPR contact area. This tethered binding is expected to allow PP5 to carry out multi-site dephosphorylation of Hsp70-bound clients with a range of sizes and shapes. Together, these results demonstrate that Hsp70 recruits PP5 and activates its phosphatase activity which suggests dual roles for PP5 that might link chaperone systems with signaling pathways in cancer and development.     

Toll-like receptor 4 (TLR4) is essential for Hsp70-like protein 1 (HSP70L1) to activate dendritic cells and induce Th1 response

Toll-like receptors (TLRs) play important roles in initiation of innate and adaptive immune responses. Emerging evidence suggests that TLR agonists can serve as potential adjuvant for vaccination. Heat shock proteins (HSPs), functionally serving as TLR4 agonists, have been proposed to act as Th1 adjuvant. We have identified a novel Hsp70 family member, termed Hsp70-like protein 1 (Hsp70L1), shown that Hsp70L1 is a potent T helper cell (Th1) polarizing adjuvant that contributes to antitumor immune responses. However, the underlying mechanism for how Hsp70L1 exerts its Th1 adjuvant activity remains to be elucidated. In this study, we found that Hsp70L1 binds directly to TLR4 on the surface of DCs, activates MAPK and NF-κB pathways, up-regulates I-a(b), CD40, CD80, and CD86 expression and promotes production of TNF-α, IL-1β, and IL-12p70. Hsp70L1 failed to induce such phenotypic maturation and cytokine production in TLR4-deficient DCs, indicating a role for TLR4 in mediating Hsp70L1-induced DC activation. Furthermore, more efficient induction of carcinoembryonic antigen (CEA)-specific Th1 immune response was observed in mice immunized by wild-type DCs pulsed with Hsp70L1-CEA(576-669) fusion protein as compared with TLR4-deficient DCs pulsed with same fusion protein. In addition, TLR4 antagonist impaired induction of CEA-specific human Th1 immune response in a co-culture system of peripheral blood lymphocytes (PBLs) from HLA-A2.1(+) healthy donors and autologous DCs pulsed with Hsp70L1-CEA(576-669) in vitro. Taken together, these results demonstrate that TLR4 is a key receptor mediating the interaction of Hsp70L1 with DCs and subsequently enhancing the induction of Th1 immune response by Hsp70L1/antigen fusion protein.     

Trypanosoma cruzi ubiquitin as an antigen in the differential diagnosis of Chagas disease and leishmaniasis

In the present report we describe Trypanosoma cruzi ubiquitin as an antigen to be utilized in the differential diagnosis of Chagas disease and leishmaniasis. Initially, recombinant T. cruzi ubiquitin was evaluated against a panel of sera by phage dot immunoassay, showing a good performance against chagasic sera. However, the presence of a carboxy-terminal tail region encoding a ribosomal protein homologous to a related protein present in the genome of Leishmania sp. gave significant cross-reactivity with leishmanial sera. Therefore, ubiquitin was purified by a simple biochemical protocol and its immunoreactivity was studied by enzyme-linked immunosorbent assay. Analysis of 104 sera indicates that the response to ubiquitin is very sensitive towards chronic chagasic sera (98%) and, more important, highly species-specific, presenting better performance compared to the use of the recombinant protein or the total epimastigote extracts when tested against a panel of leishmanial sera, where out of a total of 70 sera tested, only five sera from the mucocutaneous form of the disease reacted with T. cruzi ubiquitin. On the other hand, Leishmania ubiquitin was not recognized by chagasic sera, but was recognized by sera from different forms of leishmaniasis. These results make ubiquitin an excellent candidate to be used in the differential diagnosis of these two parasitic diseases. The molecular basis for this highly species-specific response is discussed.     

Antiphospholipid reactivity against cardiolipin metabolites occurring during endothelial cell apoptosis

We have recently shown that cardiolipin (CL) and its metabolites move from mitochondria to other cellular membranes during death receptor-mediated apoptosis. In this study, we investigate the immunoreactivity to CL derivatives occurring during endothelial apoptosis in patients with antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). We compared the serum immunoreactivity to CL with that of its derivatives monolysocardiolipin (MCL), dilysocardiolipin (DCL), and hydrocardiolipin (HCL) by means of both enzyme-linked immunosorbent assay and thin-layer chromatography (TLC) immunostaining. In addition, we investigated the composition of phospholipid extracts from the plasma membrane of apoptotic endothelial cells and the binding of patients' sera to the surface of the same cells by using high-performance TLC and immunofluorescence analysis. The average reactivity to MCL was comparable with that of CL and significantly higher than that for DCL and HCL in patients studied, both in the presence or in the absence of beta2-glycoprotein I. Of relevance for the pathogenic role of these autoantibodies, immunoglobulin G from patients' sera showed an increased focal reactivity with the plasma membrane of endothelial cells undergoing apoptosis. Interestingly, the phospholipid analysis of these light membrane fractions showed an accumulation of both CL and MCL. Our results demonstrated that a critical number of acyl chains in CL derivatives is important for the binding of antiphospholipid antibodies and that MCL is an antigenic target with immunoreactivity comparable with CL in APS and SLE. Our finding also suggests a link between apoptotic perturbation of CL metabolism and the production of these antibodies.     

Structural Analysis of the Ribosome-associated Complex (RAC) Reveals an Unusual Hsp70/Hsp40 Interaction

Yeast Zuotin and Ssz are members of the conserved Hsp40 and Hsp70 chaperone families, respectively, but compared with canonical homologs, they atypically form a stable heterodimer termed ribosome-associated complex (RAC). RAC acts as co-chaperone for another Hsp70 to assist de novo protein folding. In this study, we identified the molecular basis for the unusual Hsp70/Hsp40 pairing using amide hydrogen exchange (HX) coupled with mass spectrometry and mutational analysis. Association of Ssz with Zuotin strongly decreased the conformational dynamics mainly in the C-terminal domain of Ssz, whereas Zuotin acquired strong conformational stabilization in its N-terminal segment. Deletion of the highly flexible N terminus of Zuotin abolished stable association with Ssz in vitro and caused a phenotype resembling the loss of Ssz function in vivo. Thus, the C-terminal domain of Ssz, the N-terminal extension of Zuotin, and their mutual stabilization are the major structural determinants for RAC assembly. We furthermore found dynamic changes in the J-domain of Zuotin upon complex formation that might be crucial for RAC co-chaperone function. Taken together, we present a novel mechanism for converting Zuotin and Ssz chaperones into a functionally active dimer.     

Preparation of P52 recombinant antigenic protein from human cytomegalovirus (HCMV)

Analysis of published reports helped us single out the most potent antigens among HCMV proteins: phosphoproteins pp150(UL32) and p52(UL44). Theoretical computer analysis of p52 epitopes showed the main antigenic determinants not cross-reacting with antigens of other viruses. Virus-containing (strain AD169) material was obtained and genome DNA was isolated. Amplification of a site of gene UL44 coding for unique determinants detected a PCR fragment of required electrophoretic mobility. The fragment was cloned in vector pLBE. The specificity of cloning was confirmed by restriction analysis of theoretical sites. Nucleotide sequence of cloned fragment of UL44 gene was studied by Maxam-Gilbert's method. Cloning in expressing bacterial vectors helped obtain HCMV recombinant protein p52 in the pure form and fused with beta-galactosidase. Enzyme immunoassay with HCMV-positive and negative donor sera and ABBOTT HCMV sera showed that recombinant p52 increased the sensitivity and specificity of a previously obtained recombinant pp150 as an antigen to HCMV-IgG and HCMV-IgM. The sensitivity and specificity is 100% with 98-99% reliability.     

Characterization of a receptor for heat shock protein 70 on macrophages and monocytes

Heat shock proteins (Hsps) and molecular chaperones isolated from tumors or virally infected cells elicit an efficient CD8+ T cell response against bound antigenic peptides. This immune response is mediated by presentation of the peptides on MHC class I complexes of antigen-presenting cells (APCs), but the cellular mechanism of this presentation process is not yet understood. Here we provide evidence for the existence of a proteinaceous receptor on the surface of APCs that is specific for mammalian Hsp70. Using a flow cytometry-based assay, saturable binding of Hsp70 to the cell surface of macrophages and peripheral blood monocytes, but not of lymphocytes, can be demonstrated. The affinity of the receptor is in the sub-micromolar range (Kd < 100 nM). Only mammalian Hsc70/Hsp70, but not bacterial Hsp70, is bound with high affinity. Subsequent to binding, Hsp70 is taken up by endocytosis, resulting in an intracellular localization. Our results suggest that receptor-mediated endocytosis forms the basis for the demonstrated efficacy of Hsp70-peptide complexes as anti-tumor vaccines.     

Calcium signaling in dendritic cells by human or mycobacterial Hsp70 is caused by contamination and is not required for Hsp70-mediated enhancement of cross-presentation

Extracellular heat shock proteins (HSPs) can stimulate antigen-specific immune responses. Using recombinant human (rhu)Hsp70, we previously demonstrated that through complex formation with exogenous antigenic peptides, rhuHsp70 can enhance cross-presentation by antigen-presenting cells (APCs) resulting in stronger T cell stimulation. T cell stimulatory activity has also been described for mycobacterial (myc)Hsp70. MycHsp70-assisted T cell activation has been reported to act through the binding of mycHsp70 to chemokine receptor 5 (CCR5), calcium signaling, phenotypic maturation, and cytokine secretion by dendritic cells (DCs). We report that highly purified rhuHsp70 and mycHsp70 proteins both strongly enhance cross-presentation of exogenous antigens. Augmentation of cross-presentation was seen for different APCs, irrespective of CCR5 expression. Moreover, neither of the purified Hsp70 proteins induced calcium signals in APCs. Instead, calcium signaling activity was found to be caused by contaminating nucleotides present in Hsp70 protein preparations. These results refute the hypothesis that mycHsp70 proteins require CCR5 expression and calcium signaling by APCs for enhanced antigen cross-presentation for T cell stimulation.     

Structure and interactions of the helical and U-box domains of CHIP, the C terminus of HSP70 interacting protein

The heat-shock proteins Hsp70 and Hsp90 play a crucial role in regulating protein quality control both by refolding and by preventing the aggregation of misfolded proteins. It has recently been shown that Hsp70 and Hsp90 act not only in protein refolding but also cooperate with the C terminus of Hsp70 interacting protein (CHIP), a multidomain ubiquitin ligase, to mediate the degradation of unfolded proteins. We present the crystal structure of the helical linker domain and U-box domain of zebrafish CHIP (DrCHIP-HU). The structure of DrCHIP-HU shows a symmetric homodimer. The conformation of the helical linker domains and the relative positions of the helical and U-box domains differ substantially in DrCHIP-HU from those in a recently published structure of an asymmetric dimer of mammalian (mouse) CHIP. We used an in vitro ubiquitination assay to identify residues, located on two long loops and a central alpha helix of the CHIP U-box domain, that are important for interacting with the ubiquitin-conjugating enzyme UbcH5b. In addition, we used NMR spectroscopy to define a complementary interaction surface located on the N-terminal alpha helix and the L4 and L7 loops of UbcH5b. Our results provide insights into conformational variability in the domain arrangement of CHIP and into U-box-mediated recruitment of UbcH5b for the ubiquitination of Hsp70 and Hsp90 substrates.     

Antibodies to murine leukemia virus gp70 and p15(E) in sera of BALB/c mice immunized with syngeneic chemically induced sarcomas

The sera of BALB/c mice immunized with syngeneic methylcholanthrene-induced sarcomas commonly contain antibodies that react with the immunizing tumor line and also with other chemically induced sarcomas. Three lines of evidence suggest that the cross-reactive antibodies are directed against antigenic determinants of gp70 and p15(E), envelope proteins of endogenous murine leukemia virus (MuLV). 1) The antibodies bind only to sarcomas expressing MuLV antigens, 2) they are removed by absorption with purified MuLV antigens, and 3) they precipitate proteins identified as gp70 and p15(E) from 125I-labeled sarcoma cell lysates.     

Extracellular amastigote-like forms of Leishmania panamensis and L. braziliensis. II. Stage- and species-specific monoclonal antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania. Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to greater than 200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.     

Extracellular Amastigote-Like Forms of Leishmania panamensis and L. braziliensis. II. Stage- and Species-Specific Monoclonal Antibodies

Immunochemical evidence, employing monoclonal antibodies, shows that the forms of L. braziliensis complex axenically grown at elevated temperature are amastigote-like. The monoclonal antibodies were raised against membrane proteins of amastigote-like forms, strains of both L. panamensis (WR442) and L. braziliensis (M5052), which were grown axenically. The specificities of these antibodies were examined by indirect radioimmune binding assay, indirect immunofluorescent assay and Western blot analyses. Two distinct groups of monoclonal antibodies were obtained and their specificities were consistent with the 3 methods used. Four antibodies are specific for the species L. panamensis and react with both developmental stages. Six antibodies specifically recognize amastigote-like forms grown at elevated temperature and intracellular amastigotes of both L. panamensis (WR442) and L. braziliensis (M5052). These monoclonal antibodies do not bind to promastigotes of these species, nor to promastigotes of any other species of Leishmania . Therefore these antibodies are specific for amastigotes of L. panamensis (WR442) and L. braziliensis (M5052), and suggest that immunochemically both amastigote forms (culture and macrophage) are developmentally very close, if not identical. The molecules associated with the amastigote-specific antigenic determinants consist of a Mr 12-kD component and a heterogeneous component (Mr from 50 kD to >200 kD); these molecules appear to be identical for both amastigote-like forms and amastigotes isolated from macrophages.