The crystal structure of the Leishmania major surface proteinase leishmanolysin (gp63).

BACKGROUND: Despite their medical importance, there is little available structural information for the surface antigens of infectious protozoa. Diseases caused by the protozoan parasite Leishmania are common in many developing countries. Human infection occurs during the bite of infected sandfilies, when Leishmania promastigote cells from the insect gut enter the bloodstream. Promastigotes in the blood parasitize macrophages, often causing serious disease. Leishmanolysin is the predominant protein surface antigen of promastigotes, and is assumed to have a key role during infection. Leishmanolysin is a membrane-bound zinc proteinase, active in situ. Similar molecules exist in other trypanomastid protozoa. RESULTS: Two crystal forms of leishmanolysin were obtained from protein purified from promastigote membranes. A single lead derivative in both crystal forms was used to solve the structure. The structure reveals three domains, two of which have novel folds. The N-terminal domain has a similar structure to the catalytic modules of zinc proteinases. The structure clearly shows that leishmanolysin is a member of the metzincin class of zinc proteinases. CONCLUSIONS: The unexpected metzincin features of the leishmanolysin structure suggest that the metzincin fold may be more widespread than indicated by sequence homologies amongst existing metzincin zinc proteinases. The similarity of the active-site structure to previously well characterized metzincin class zinc proteinases should aid the development of specific inhibitors. These inhibitors might be used to determine the function of leishmanolysin in the insect and during mammalian infection, and may aid the development of drugs for human leishmaniasis.

Progressive disease or protective immunity to Leishmania major infection: the result of a network of stimulatory and inhibitory interactions.

The study of experimental infection of inbred strains of mice with the intracellular protozoan parasite Leishmania major has contributed significantly not only to our understanding of this fascinating host/parasite relationship but also to that of many basic immunological phenomena. Much has been learned about the cognate interaction of antigen-specific T cells and antigen-presenting cells, about cytokine and T cell subset regulation, and the requirements for costimulation. Specifically, the immune response to experimental L. major infection is the paradigm for polarized T helper cell (Th) 1 and Th2 differentiation. In this model system a Th1 response characterized by interleukin (IL)-2 and interferon (IFN)-gamma secretion leads to self-curing disease, whereas a Th2 response (IL-4, IL-10) leads to nonhealing disease. Numerous manipulations, including the injection of cytokines and of neutralizing anti-cytokine antibodies, cytokine transgene expression, and more recently cytokine and cytokine receptor gene knockout studies, have all provided intriguing new pieces to the still incomplete mosaic of our understanding of the immune response. Some of these findings were clearly unexpected and are still incompletely understood. For instance, based on earlier neutralizing anti-IL-4 monoclonal antibody injection studies, IL-4 gene-disrupted BALB/c mice were expected to be unable to mount the biased Th2 response typical of the IL-4+/+ wild-type mice and to be able to control their lesions; quite unexpectedly, the BALB/c IL-4 knockout mice remain unable to heal their L. major infection. Based on these unexpected findings, we reexamine the literature in an attempt to resolve this apparent paradox and to relate the large body of experimental findings in the mouse system to that which is known about natural and experimental infections in the human.

Characterization of T cell-mediated responses in nonhealing and healing Leishmania major infections in the absence of endogenous IL-4.

IL-4 drives polarized Th2 responses, and differentiating Th2 cells down-regulate their sensitivity to IL-12. Therefore, the failure of BALB/c mice to heal Leishmania major infection could be due to an IL-4-dependent biased Th2 response or to a reduced capacity of Leishmania-specific Th cells to respond to IL-12. We examined the ability of CD4+ Th cells from L. major-infected wild-type and IL-4-deficient BALB/c mice to respond to IL-12. We show that the inability of normal and IL-4-deficient BALB/c mice to heal L. major infections is due to their inability to generate effective Th1 responses and not to persistent IL-4-dominated Th2 responses. Redirection of immune responses in vivo by administration of IL-12 or anti-CD4 mAb treatment in the early phase of infection (+/-12 days) allows both normal and IL-4-deficient BALB/c mice to heal their lesions by allowing them to develop an efficient Th1 response regardless of the presence or the absence of IL-4. Finally, on a population level, Ag-specific Th cells from infected animals induced to heal display a strongly elevated response to IL-12.

Crystallization and preliminary X-ray diffraction studies of leishmanolysin, the major surface metalloproteinase from Leishmania major.

The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals. Protein microheterogeneity in molecular size and charge was incorporated into both crystal forms. The tetragonal crystal form belongs to the space group P4(1)2(1)2 or the enantiomorph P4(3)2(1)2, has unit cell parameters of a = b = 63.6 A, c = 251.4 A, and contains one molecule per asymmetric unit. The second crystal form is monoclinic, space group C2, with unit cell dimensions a = 107.2 A, b = 90.6 A, c = 70.6 A, beta = 110.6 degrees, and also contains one molecule per asymmetric unit. Both crystal forms diffract X-rays beyond 2.6 A resolution and are suitable for X-ray analysis. Native diffraction data sets have been collected and the structure determination of leishmanolysin using a combination of the isomorphous replacement and the molecular replacement methods is in progress.

Gamma interferon response in secondary Leishmania major infection: role of CD8+ T cells.

CD8+ T cells have been shown to contribute to the rapid resolution of secondary lesions developing in immune mice challenged with Leishmania major. In the present study, we assessed directly the participation of specific CD8+ T cells in the memory response induced in immune mice by reinfection. Lymphocyte populations from reinfected immune mice exhibit marked secondary gamma interferon (IFN-gamma) responses. The participation of IFN-gamma-producing CD8+ T cells in the memory response elicited by secondary infectious challenge was demonstrated in both genetically resistant immune CBA mice and genetically susceptible immune BALB/c mice that were rendered resistant by administration of anti-CD4 monoclonal antibody in the early phase of the primary infection. The protective function of CD8+ T cells in experimental murine cutaneous leishmaniasis might thus be explained in part by their ability to secrete IFN-gamma. In this context, the neutralization of IFN-gamma at the time of reinfection reduced the Leishmania-specific delayed-type hypersensitivity response, showing that this cytokine is involved in the recall of immunological memory to L. major in vivo.

Structure of Leishmania mexicana lipophosphoglycan.

Lipophosphoglycan (LPG) was isolated from the culture supernatant of Leishmania mexicana promastigotes and its structure elucidated by a combination of 1H NMR, fast atom bombardment mass spectrometry, methylation analysis, and chemical and enzymatic modifications. It consists of the repeating phosphorylated oligosaccharides PO4-6Gal beta 1-4Man alpha 1- and PO4-6[Glc beta 1-3]Gal beta 1-4Man alpha 1-, which are linked together in linear chains by phosphodiester linkages. Each chain of repeat units is linked to a phosphosaccharide core with the structure PO4-6Gal alpha 1-6Gal alpha 1-3Galf beta 1- 3[Glc alpha 1-PO4-6]Man alpha 1-3Man alpha 1-4GlcNH2 alpha 1-6 myo-inositol, where the myo-inositol residue forms the head group of a lyso-alkylphosphatidylinositol moiety. The nonreducing terminus of the repeat chains appear to be capped with the neutral oligosaccharides Man alpha 1-2Man, Man alpha 1-2Man alpha 1-2Man, or Man alpha 1-2[Gal beta 1-4]Man. Cellular LPG, isolated from promastigotes, has a very similar structure to the culture supernatant LPG. However, it differs from culture supernatant LPG in the average number of phosphorylated oligosaccharide repeat units (20 versus 28) and in alkyl chain composition. Although culture supernatant LPG contained predominantly C24:0 alkyl chains, cellular LPG contained approximately equal amounts of C24:0 and C26:0 alkyl chains. It is suggested that culture supernatant LPG is passively shed from promastigotes and that it may contribute significantly, but not exclusively, to the "excreted factor" used for serotyping Leishmania spp. Comparison of L. mexicana LPG with the LPGs of Leishmania major and Leishmania donovani indicate that these molecules are highly conserved but that species-specific differences occur in the phosphorylated oligosaccharide repeat branches and in the relative abundance of the neutral cap structures.

Identification of a surface metalloproteinase on 13 species of Leishmania isolated from humans, Crithidia fasciculata, and Herpetomonas samuelpessoai.

Promastigotes of thirteen species of Leishmania isolated from human patients, as well as L. enriettii, Crithidia fasciculata and Herpetomonas samuelpessoai, were examined for the expression of an amphiphilic, surface-oriented metalloproteinase by surface radioiodination of living cells, fractionation by Triton X-114 extraction and phase separation, and zymogram analysis by fibrinogen-SDS-PAGE. In all species of Leishmania, and the two monoxenous trypanosomatid parasites of insects, an ectoproteinase similar to the Promastigote Surface Protease, or PSP, was observed. In contrast, neither Phytomonas sp. nor 'Leishmania tarentolae' express a detectable surface metalloproteinase. The presence of the functionally conserved metalloproteinase at the surface of Crithidia and Herpetomonas suggest the enzyme may not be involved in the infection of the mammalian host by Leishmania, but rather contributes to the survival of the protozoan in the environment of the insect midgut.

Secreted acid phosphatase of Leishmania mexicana: a filamentous phosphoglycoprotein polymer.

In the promastigote, or insect stage, most species of the parasitic protozoan Leishmania secrete an acid phosphatase. The enzyme purified from the culture medium of Leishmania mexicana is shown to be a complex [13.3% (wt/wt) protein, 74.4% (wt/wt) carbohydrate, and 12.3% (wt/wt) phosphate] composed of a predominant phosphorylated glycoprotein with a relative molecular mass of 100 kDa and noncovalently associated high molecular mass (proteo)phosphoglycans. Electron microscopy discloses long filaments composed of a central chain of protein subunits surrounded by a diffuse glycocalix that can be decorated by monoclonal antibodies or concanavalin A. In contrast to the polymeric structure of the L. mexicana enzyme, the acid phosphatase secreted by Leishmania donovani is mono- or oligomeric but not filamentous.

Monoclonal antibodies to Leishmania mexicana promastigote antigens. I. Secreted acid phosphatase and other proteins share epitopes with lipophosphoglycan.

The abundant surface glycolipid, lipophosphoglycan (LPG), of Leishmania promastigotes is composed of phosphosaccharide repeating units linked via a phosphosaccharide core to a conserved lyso alkylphosphatidylinositol membrane anchor. It is shown in this paper that monoclonal antibodies (mAbs) directed against LPG also react with an acid phosphatase secreted by L. mexicana promastigotes. Acid phosphatase purified by column chromatography (apparent Mr = 100,000) reacts in immunoblots with the anti-LPG mAb AP3 and another mAb, L3.13, which does not recognize LPG. mAb L3.13 was used to purify the enzyme by affinity chromatography. The resulting glycoprotein has the same molecular weight and binds AP3 on immunoblots. The secreted phosphatase is non-covalently associated with a high molecular weight, galactose-containing glycan or proteoglycan that is recognized by both AP3 and L3.13. In addition to acid phosphatase, other parasite proteins appear to be modified by LPG epitopes.

Genetic control of the immune response in mice to Leishmania mexicana surface protease.

Congenic mouse strains were tested in the lymphocyte proliferation assay for their response to the purified surface protease of Leishmania mexicana (gp63). The data obtained allow us to distinguish three different patterns of response, influenced both by H-2 (class II) and non-H-2 genes. Mice of the C57BL/10 (B10) background carrying H-2 haplotypes b,q, and r were found to be high responders; those carrying H-2 haplotypes d, j, v, and z were low responders; and those with H-2a, H-2f, H-2k, H-2p, and H-2u haplotypes were intermediate responders. Studies with H-2 recombinant strains indicated that the high responsiveness on the B10 background was determined by the Ab allele and the low responsiveness influenced by the Ad allele. Other genes besides H-2 appear to have a role in the immune response as shown by the fact that some strains with BALB, DBA, or C3H background differed in their pattern of responsiveness from B10 background strains carrying the corresponding H-2 haplotypes. By using recombinant protein, the influence of the leishmanial surface lipophosphoglycan that might co-purify with gp63, on the MHC restriction of the response to gp63 was excluded. The immune response to gp63 did not correlate with susceptibility of mouse strains to cutaneous infection with L. mexicana promastigotes.

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania.

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.

Characterization of the promastigote surface protease of Leishmania as a membrane-bound zinc endopeptidase.

The effects of a variety of inhibitors suggested that the promastigote surface protease (PSP) of Leishmania might be a zinc metalloprotease. To investigate this possibility, we conducted atomic emission and absorption spectroscopic analyses, which show that PSP contains 1 atom of zinc per 63-kDa monomer. Further studies showed that the enzyme can be biosynthetically labeled with 65ZnCl2. The comparison of the amino acid sequence of Leishmania major PSP with nine other zinc metalloproteinases revealed significant similarity in the area of their zinc-binding sites. These data show clearly that the promastigote surface protease of Leishmania is a zinc metalloproteinase. Secondary structure analysis by circular dichroism spectroscopy indicates that PSP contains over 40% beta-strand and less than 20% alpha-helical structure. The molecular masses of amphiphilic PSP (152 kDa) and of hydrophilic PSP (142 kDa), determined by quantitative electron scattering, suggest that the purified enzyme occurs in solution, and presumably at the cell surface, as a non-covalent homodimer.

Secondary structure of the promastigote surface protease of Leishmania.

By Raman spectroscopic analysis we have determined the secondary structure of the promastigote surface protease, named PSP or gp63, of Leishmania major. It consist of nearly 50% antiparallel beta-strand, and less than 20% alpha-helix. These results are contrasted with the predominantly alpha-helical VSGs of the African trypanosomes and the alpha-helical metalloprotease thermolysin. The PSP of Leishmania thus represents a novel kind of membrane-anchored protease.

Purification and characterization of a metabolite-regulated pyruvate kinase from Leishmania major promastigotes.

The pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) of Leishmania major promastigotes is a multimer of 59 kDa subunits having an Mr 181000. It is activated by its substrate phosphoenolpyruvate (PEP) in a positively cooperative manner, and heterotropically by fructose 1,6-bisphosphate (FBP). Kinetics with regard to the phosphate acceptor adenosine 5'-diphosphate (ADP), MgCl2, and KCl are hyperbolic and unaffected by FBP. The enzyme is strongly inhibited by the reaction product ATP, as well as GTP and ITP, and to a lesser degree by citrate. Of seven amino acids reported to inhibit the pyruvate kinases of other organisms, none have any effect on the L. major pyruvate kinase in vitro. The enzyme shows its maximum activity at pH 7.0 in the absence of FBP, and at pH 7.6 in its presence. Contrary to previous suggestions, the enzyme appears to be well-suited for a regulatory role in the metabolism of an aerobic organism capable of net glucose synthesis.

Identification of the promastigote surface protease in seven species of Leishmania.

Twelve different strains of Leishmania, including L. major, L. donovani, L. infantum, L. tropica, L. mexicana, L. amazonensis, L. braziliensis, and L. enriettii were examined for the presence of an ectoenzyme structurally and functionally related to the promastigote surface protease found in L. major LEM 513. All strains examined possess a protease that is labelled by surface iodination of living promastigotes. The electrophoretic migrations of the labelled proteases are similar in all species showing distinct ectoprotease activity. In addition, proteases that cross-react immunologically with the polypeptide moiety of the surface protease of L. major LEM 513 were found in 10 strains. These proteases were in all cases labelled by surface radioiodination. Two of the strains, L. amazonensis and L. braziliensis, do not show a strict correlation between protease activity, surface iodination, and immunological cross-reactivity with the promastigote surface protease of L. major LEM 513, although both strains possess distinct neutral proteases with electrophoretic behavior similar to that of the enzyme of L. major. The amount of proteolytic activity detected at the surface of living cells depends on the strain tested, and correlates qualitatively with the amount of promastigote surface protease detected on zymograms. We conclude that the proteolytic activity found at the surface of Leishmania promastigotes is a common feature of the species infective for humans and that the promastigote surface protease described in this article is structurally and functionally conserved in Old and New World Leishmania.

An assay of membrane-bound Trypanosoma brucei phospholipase using an integral membrane protein substrate and detergent phase separation.

The technique of phase separation in a solution of the non-ionic detergent Triton X-114 was used to measure the enzymatic conversion of a membrane protein to a soluble product via removal of a hydrophobic moiety. The substrate was the major surface protein (p63), of Leishmania promastigotes and the enzyme was a phospholipase C purified from Trypanosoma brucei. This membrane-bound enzyme is responsible for the cleavage of the hydrophobic lipid membrane anchor of the variant surface glycoprotein (VSG), of T. brucei. The assay is fast, simple and uses small amounts of reagents. It has been used to determine the pH optimum, thermal resistance, and the sensitivity to inhibitors of the trypanosomal phospholipase.

Leishmania and Trypanosoma surface glycoproteins have a common glycophospholipid membrane anchor.

The variant surface glycoprotein (VSG) of the African trypanosomes is the major membrane protein of the plasma membrane of the bloodstream stage of the parasite. It is anchored in the plasma membrane by a glycolipid covalently bound to the C-terminal amino acid of the protein. The VSG is released through the action of a phosphatidylinositol-specific phospholipase C that removes dimyristoylglycerol and exposes the carbohydrate antigenic determinant common to all VSGs. Promastigotes of Leishmania have a predominant surface glycoprotein, termed p63, that is anchored in the plasma membrane in a similar way. A water-soluble form of p63 can be generated through the action of phosphatidylinositol-specific phospholipase C from trypanosomes or from Bacillus cereus. Either treatment exposes on the Leishmania p63 an antigenic determinant recognized by antibody prepared against the trypanosomal crossreacting determinant. These findings indicate that p63 and VSG have a common membrane anchor and are structurally related.