Identification of factors involved in ribosome assembly in the protozoan parasite Leishmania major

The immunosuppressive effects of the fungal metabolite cyclosporin A (CsA) are mediated primarily by binding to cyclophilins (Cyps). The resulting CsA–Cyp complex inhibits the Ca2+-regulated protein phosphatase calcineurin and down-regulates signal transduction events. Previously we reported that CsA is a potent inhibitor of infections transmitted by the human pathogenic protozoan parasite Leishmania major in vitro and in vivo, but does not effect the extracellular growth of L. major itself. It is unknown how L. major exerts this resistance to CsA. Here we report that a major Cyp, besides additional isoforms with the same N-terminal amino acid sequence, was expressed in L. major. The cloned and sequenced gene encodes a putative 174-residue protein called L. major Cyp 19 (LmCyp19). The recombinant LmCyp19 exhibits peptidyl-prolyl cis/trans isomerase activity with a substrate specificity and an inhibition by CsA that are characteristic of other eukaryotic Cyps. To determine whether calcineurin is involved in the discrimination of the effects of CsA we also examined the presence of a parasitic calcineurin and tested the interaction with Cyps. Despite the expression of functionally active calcineurin by L. major, neither LmCyp19 nor other L. major Cyps bound to its own or mammalian calcineurin. The amino acid sequence of most Cyps includes an essential arginine residue around the calcineurin-docking side. In LmCyp19 this is replaced by an asparagine residue. This exchange and additional charged residues are apparently responsible for the lack of LmCyp19 interaction with calcineurin. These observations indicate that resistance of L. major to CsA in vitro is mediated by the lack of complex formation with calcineurin despite CsA binding by parasitic Cyp.

Download Full-text

Ribosomal RNA Genes in the Protozoan Parasite Leishmania major Possess a Nucleosomal Structure

Protist ◽

10.1016/j.protis.2016.02.001 ◽

2016 ◽

Vol 167 (2) ◽

pp. 121-135 ◽

Cited By ~ 8

Author(s):

Juan C. Vizuet-de-Rueda ◽

Luis E. Florencio-Martínez ◽

Norma E. Padilla-Mejía ◽

Rebeca Manning-Cela ◽

Rosaura Hernández-Rivas ◽

...

Keyword(s):

Ribosomal Rna ◽

Leishmania Major ◽

Protozoan Parasite ◽

Ribosomal Rna Genes ◽

Nucleosomal Structure ◽

Rna Genes ◽

Parasite Leishmania

Download Full-text

Biosynthesis of the glycolipid anchor of lipophosphoglycan and the structurally related glycoinositolphospholipids from Leishmania major

Biochemical Journal ◽

10.1042/bj3080045 ◽

1995 ◽

Vol 308 (1) ◽

pp. 45-55 ◽

Cited By ~ 27

Author(s):

L Proudfoot ◽

P Schneider ◽

M A J Ferguson ◽

M J McConville

Keyword(s):

Cell Surface ◽

Leishmania Major ◽

Significant Proportion ◽

Specific Radioactivity ◽

Protozoan Parasite ◽

Before And After ◽

Surface Labelling ◽

Glycolipid Anchor ◽

Kinetics Of ◽

Parasite Leishmania

The major macromolecule on the surface of the protozoan parasite Leishmania major is a lipophosphoglycan (LPG) which contains a glycosylphosphatidylinositol glycolipid anchor. This parasite also synthesizes a complex family of abundant low-molecular-mass glycoinositolphospholipids (GIPLs) which are structurally related to the LPG anchor. In this study, L. major promastigotes were metabolically labelled with [3H]GlcN, and the kinetics of incorporation into free glycolipids and the LPG anchor followed to elucidate the pathway of GIPL biosynthesis and possible precursor-product relationships between the GIPLs and LPG. Labelled GIPLs were identified by TLC and by liquid chromatography of the released headgroups, before and after enzymic and chemical cleavage. On the basis of the measured specific radioactivities of the GIPLs, and their kinetics of radiolabelling, we suggest the pathway GlcN-PI-->Man1GlcN-PI (M1)-->Man2GlcN-PI (iM2)-->GalfMan2GlcN-PI (GIPL-1)-->Gal1GalfMan2GlcN-PI (GIPL-2)-->Gal2GalfMan2GlcN-PI (GIPL-3). All of the GIPLs were shown to contain alkylacylglycerol or lyso-alkylglycerol lipid moieties with the exception of the earliest intermediate, glucosaminylphosphatidylinositol (GlcN-PI), which contained both alkylacylglycerol and diacylglycerol. A significant proportion (approx. 50%) of GIPL-3 appeared to be selectively modified by the addition of a Glc-1-PO4 residue to one of the mannose residues (P-GIPL-3). On the basis of the specific radioactivity and kinetics of labelling of GIPL-3 and P-GIPL-3 we suggest that both of these low-abundance species are rapidly utilized as LPG precursors. The turnover of LPG and the GIPLs was also studied by [3H]Gal pulse-chase labelling and cell-surface labelling experiments. Whereas LPG was rapidly shed from the cell surface, consistent with previous studies, the GIPLs (both the total cellular and cell-surface pools) had a much slower turnover. These results suggest that the majority of the GIPLs do not act as LPG precursors and indicate that the cellular levels of these molecules is determined, at least in part, by the rate at which they are shed from the cell surface.

Download Full-text

PTR1: a reductase mediating salvage of oxidized pteridines and methotrexate resistance in the protozoan parasite Leishmania major.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.24.11442 ◽

1994 ◽

Vol 91 (24) ◽

pp. 11442-11446 ◽

Cited By ~ 113

Author(s):

A. R. Bello ◽

B. Nare ◽

D. Freedman ◽

L. Hardy ◽

S. M. Beverley

Keyword(s):

Leishmania Major ◽

Protozoan Parasite ◽

Methotrexate Resistance ◽

Parasite Leishmania

Download Full-text

Expression profiling using random genomic DNA microarrays identifies differentially expressed genes associated with three major developmental stages of the protozoan parasite Leishmania major

Molecular and Biochemical Parasitology ◽

10.1016/j.molbiopara.2004.03.002 ◽

2004 ◽

Vol 136 (1) ◽

pp. 71-86 ◽

Cited By ~ 82

Author(s):

Natalia S Akopyants ◽

Robin S Matlib ◽

Elena N Bukanova ◽

Matthew R Smeds ◽

Bernard H Brownstein ◽

...

Keyword(s):

Differentially Expressed Genes ◽

Expression Profiling ◽

Genomic Dna ◽

Dna Microarrays ◽

Leishmania Major ◽

Developmental Stages ◽

Protozoan Parasite ◽

Differentially Expressed ◽

Parasite Leishmania

Download Full-text

A glycosylphosphatidylinositol (GPI)-negative phenotype produced in Leishmania major by GPI phospholipase C from Trypanosoma brucei: topography of two GPI pathways

The Journal of Cell Biology ◽

10.1083/jcb.124.6.935 ◽

1994 ◽

Vol 124 (6) ◽

pp. 935-947 ◽

Cited By ~ 45

Author(s):

K Mensa-Wilmot ◽

JH LeBowitz ◽

KP Chang ◽

A al-Qahtani ◽

BS McGwire ◽

...

Keyword(s):

Phospholipase C ◽

Mammalian Cells ◽

Leishmania Major ◽

Protozoan Parasite ◽

T Lymphoma ◽

Major Surface ◽

Parasite Leishmania ◽

Negative Phenotype

The major surface macromolecules of the protozoan parasite Leishmania major, gp63 (a metalloprotease), and lipophosphoglycan (a polysaccharide), are glycosylphosphatidylinositol (GPI) anchored. We expressed a cytoplasmic glycosylphosphatidylinositol phospholipase C (GPI-PLC) in L. major in order to examine the topography of the protein-GPI and polysaccharide-GPI pathways. In L. major cells expressing GPI-PLC, cell-associated gp63 could not be detected in immunoblots. Pulse-chase analysis revealed that gp63 was secreted into the culture medium with a half-time of 5.5 h. Secreted gp63 lacked anti-cross reacting determinant epitopes, and was not metabolically labeled with [3H]ethanolamine, indicating that it never received a GPI anchor. Further, the quantity of putative protein-GPI intermediates decreased approximately 10-fold. In striking contrast, lipophosphoglycan levels were unaltered. However, GPI-PLC cleaved polysaccharide-GPI intermediates (glycoinositol phospholipids) in vitro. Thus, reactions specific to the polysaccharide-GPI pathway are compartmentalized in vivo within the endoplasmic reticulum, thereby sequestering polysaccharide-GPI intermediates from GPI-PLC cleavage. On the contrary, protein-GPI synthesis at least up to production of Man(1 alpha 6)Man(1 alpha 4)GlcN-(1 alpha 6)-myo-inositol-1-phospholipid is cytosolic. To our knowledge this represents the first use of a catabolic enzyme in vivo to elucidate the topography of biosynthetic pathways. GPI-PLC causes a protein-GPI-negative phenotype in L. major, even when genes for GPI biosynthesis are functional. This phenotype is remarkably similar to that of some GPI mutants of mammalian cells: implications for paroxysmal nocturnal hemoglobinuria and Thy-1-negative T-lymphoma are discussed.

Download Full-text