Biochemistry and Pharmacology - Research Publications
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ItemSTAGE-SPECIFIC BINDING OF LEISHMANIA-DONOVANI TO THE SAND FLY VECTOR MIDGUT IS REGULATED BY CONFORMATIONAL-CHANGES IN THE ABUNDANT SURFACE LIPOPHOSPHOGLYCAN(ROCKEFELLER UNIV PRESS, 1995-02-01)The life cycle of Leishmania parasites within the sand fly vector includes the development of extracellular promastigotes from a noninfective, procyclic stage into an infective, metacyclic stage that is uniquely adapted for transmission by the fly and survival in the vertebrate host. These adaptations were explored in the context of the structure and function of the abundant surface lipophosphoglycan (LPG) on Leishmania donovani promastigotes. During metacyclogenesis, the salient structural feature of L. donovani LPG is conserved, involving expression of a phosphoglycan chain made up of unsubstituted disaccharide-phosphate repeats. Two important developmental modifications were also observed. First, the size of the molecule is substantially increased because of a twofold increase in the number of phosphorylated disaccharide repeat units expressed. Second, there is a concomitant decrease in the presentation of terminally exposed sugars. This later property was indicated by the reduced accessibility of terminal galactose residues to galactose oxidase and the loss of binding by the lectins, peanut agglutinin, and concanavalin A, to metacyclic LPG in vivo and in vitro. The loss of lectin binding was not due to downregulation of the capping oligosaccharides as the same beta-linked galactose or alpha-linked mannose-terminating oligosaccharides were present in both procyclic and metacyclic promastigotes. The capping sugars on procyclic LPG were found to mediate procyclic attachment to the sand fly midgut, whereas these same sugars on metacyclic LPG failed to mediate metacyclic binding. And whereas intact metacyclic LPG did not inhibit procyclic attachment, depolymerized LPG inhibited as well as procyclic LPG, demonstrating that the ligands are normally buried. The masking of the terminal sugars is attributed to folding and clustering of the extended phosphoglycan chains, which form densely distributed particulate structures visible on fracture-flip preparations of the metacyclic surface. The exposure and subsequent masking of the terminal capping sugars explains the stage specificity of promastigote attachment to and release from the vector midgut, which are key events in the development of transmissible infections in the fly.
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ItemChewing the fat on natural killer T cell development(ROCKEFELLER UNIV PRESS, 2006-10-02)Natural killer T cells (NKT cells) are selected in the thymus by self-glycolipid antigens presented by CD1d molecules. It is currently thought that one specific component of the lysosomal processing pathway, which leads to the production of isoglobotrihexosylceramide (iGb3), is essential for normal NKT cell development. New evidence now shows that NKT cell development can be disrupted by a diverse range of mutations that interfere with different elements of the lysosomal processing and degradation of glycolipids. This suggests that lysosomal storage diseases (LSDs) in general, rather than one specific defect, can disrupt CD1d antigen presentation, leading to impaired development of NKT cells.
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ItemEvidence that intracellular β1-2 mannan is a virulence factor in Leishmania parasites(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2003-10-17)The protozoan parasite Leishmania mexicana proliferates within macrophage phagolysosomes in the mammalian host. In this study we provide evidence that a novel class of intracellular beta1-2 mannan oligosaccharides is important for parasite survival in host macrophages. Mannan (degree of polymerization 4-40) is expressed at low levels in non-pathogenic promastigote stages but constitutes 80 and 90% of the cellular carbohydrate in the two developmental stages that infect macrophages, non-dividing promastigotes, and lesion-derived amastigotes, respectively. Mannan is catabolized when parasites are starved of glucose, suggesting a reserve function, and developmental stages having low mannan levels or L. mexicana GDPMP mutants lacking all mannose molecules are highly sensitive to glucose starvation. Environmental stresses, such as mild heat shock or the heat shock protein-90 inhibitor, geldanamycin, that trigger the differentiation of promastigotes to amastigotes, result in a 10-25-fold increase in mannan levels. Developmental stages with low mannan levels or L. mexicana mutants lacking mannan do not survive heat shock and are unable to differentiate to amastigotes or infect macrophages in vitro. In contrast, a L. mexicana mutant deficient only in components of the mannose-rich surface glycocalyx differentiates normally and infects macrophages in vitro. Collectively, these data provide strong evidence that mannan accumulation is important for parasite differentiation and survival in macrophages.
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ItemCharacterization of a Leishmania mexicana mutant defective in synthesis of free and protein-linked GPI glycolipids(ELSEVIER, 2002)The cell surface of the promastigote stage of the protozoan parasite, Leishmania mexicana is coated by a number of glycosylphosphatidylinositol (GPI)-anchored proteins, a GPI-anchored lipophosphoglycan (LPG) and an abundant class of free GPIs, termed glycoinositolphospholipids (GIPLs). We have developed a new screen for isolating L. mexicana mutants that are defective in GPI biosynthesis, involving concanavalin A selection of a parental strain with a modified surface coat. One mutant was isolated that lacked the major GIPL species and mature GPI-protein anchor precursors, but synthesized normal levels of LPG anchor precursors. Based on analysis of apolar GIPLs that accumulate in this mutant and in vivo and in vitro synthesized GPIs, this mutant was found to have a defect in the addition of an alpha1-6 linked mannose to the common precursor, Man(1)GlcN-PI. The apolar GIPLs were transported to the cell surface with the same kinetics as mature GIPLs. However, non-anchored isoforms of the major GPI-anchored protein, gp63, were either slowly secreted (with a t(1/2) of 2 h) or retained within the endoplasmic reticulum, respectively. These findings suggest that common enzymes are involved in the synthesis of GIPLs and protein anchors and have implications for understanding how the biosynthesis of the major surface components of these parasites is regulated.
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ItemRegulated degradation of an endoplasmic reticulum membrane protein in a tubular lysosome in Leishmania mexicana(AMER SOC CELL BIOLOGY, 2001-08)The cell surface of the human parasite Leishmania mexicana is coated with glycosylphosphatidylinositol (GPI)-anchored macromolecules and free GPI glycolipids. We have investigated the intracellular trafficking of green fluorescent protein- and hemagglutinin-tagged forms of dolichol-phosphate-mannose synthase (DPMS), a key enzyme in GPI biosynthesis in L. mexicana promastigotes. These functionally active chimeras are found in the same subcompartment of the endoplasmic reticulum (ER) as endogenous DPMS but are degraded as logarithmically growing promastigotes reach stationary phase, coincident with the down-regulation of endogenous DPMS activity and GPI biosynthesis in these cells. We provide evidence that these chimeras are constitutively transported to and degraded in a novel multivesicular tubule (MVT) lysosome. This organelle is a terminal lysosome, which is labeled with the endocytic marker FM 4-64, contains lysosomal cysteine and serine proteases and is disrupted by lysomorphotropic agents. Electron microscopy and subcellular fractionation studies suggest that the DPMS chimeras are transported from the ER to the lumen of the MVT via the Golgi apparatus and a population of 200-nm multivesicular bodies. In contrast, soluble ER proteins are not detectably transported to the MVT lysosome in either log or stationary phase promastigotes. Finally, the increased degradation of the DPMS chimeras in stationary phase promastigotes coincides with an increase in the lytic capacity of the MVT lysosome and changes in the morphology of this organelle. We conclude that lysosomal degradation of DPMS may be important in regulating the cellular levels of this enzyme and the stage-dependent biosynthesis of the major surface glycolipids of these parasites.
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ItemIntracellular trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins and free GPIs in Leishmania mexicana(PORTLAND PRESS, 2002-04-15)Free glycosylphosphatidylinositols (GPIs) are an important class of membrane lipids in many pathogenic protozoa. In this study, we have investigated the subcellular distribution and intracellular trafficking of an abundant class of free GPIs [termed glycosylinositolphospholipids (GIPLs)] in Leishmania mexicana promastigotes. The intracellular transport of the GIPLs and the major GPI-anchored glycoprotein gp63 was measured by following the incorporation of these molecules into sphingolipid-rich, detergent-resistant membranes (DRMs) in the plasma membrane. In metabolic-labelling experiments, mature GIPLs and gp63 were transported to DRMs in the plasma membrane with a t(1/2) of 70 and 40 min, respectively. Probably, GIPL transport to the DRMs involves a vesicular mechanism, as transport of both the GIPLs and gp63 was inhibited similarly at 10 degrees C. All GIPL intermediates were quantitatively recovered in Triton X-100-soluble membranes and were largely orientated on the cytoplasmic face of the endoplasmic reticulum, as shown by their sensitivity to exogenous phosphatidylinositol-specific phospho-lipase C. On the contrary, a significant proportion of the mature GIPLs ( approximately 50% of iM4) were accessible to membrane-impermeable probes on the surface of live promastigotes. These results suggest that the GIPLs are flipped across intracellular or plasma membranes during surface transport and that a significant fraction may populate the cytoplasmic leaflet of the plasma membrane. Finally, treatment of L. mexicana promastigotes with myriocin, an inhibitor of sphingolipid biosynthesis, demonstrated that ongoing sphingolipid biosynthesis is not required for the plasma-membrane transport of either gp63 or the GIPLs and that DRMs persist even when cellular levels of the major sphingolipid are depleted by 70%.
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ItemSMP-1, a member of a new family of small myristoylated proteins in kinetoplastid parasites, is targeted to the flagellum membrane in Leishmania(AMER SOC CELL BIOLOGY, 2004-11)The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum. SMP-1 is myristoylated and palmitoylated in vivo, and mutation of Gly-2 and Cys-3 residues showed that both fatty acids are required for flagellar localization. SMP-1 is associated with detergent-resistant membranes based on its recovery in the buoyant fraction after Triton X-100 extraction and sucrose density centrifugation and coextraction with the major surface glycolipids in Triton X-114. However, the flagellar localization of SMP-1 was not affected when sterol biosynthesis and the properties of detergent-resistant membranes were perturbed with ketoconazole. Remarkably, treatment of Leishmania with ketoconazole and myriocin (an inhibitor of sphingolipid biosynthesis) also had no affect on SMP-1 localization, despite causing the massive distension of the flagellum membrane and the partial or complete loss of internal axoneme and paraflagellar rod structures, respectively. These data suggest that flagellar membrane targeting of SMP-1 is not dependent on axonemal structures and that alterations in flagellar membrane lipid composition disrupt axoneme extension.
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ItemPimE is a polyprenol-phosphate-mannose-dependent mannosyltransferase that transfers the fifth mannose of phosphatidylinositol mannoside in mycobacteria(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2006-09-01)Phosphatidylinositol mannosides (PIMs) are a major class of glycolipids in all mycobacteria. AcPIM2, a dimannosyl PIM, is both an end product and a precursor for polar PIMs, such as hexamannosyl PIM (AcPIM6) and the major cell wall lipoglycan, lipoarabinomannan (LAM). The mannosyltransferases that convert AcPIM2 to AcPIM6 or LAM are dependent on polyprenol-phosphate-mannose (PPM), but have not yet been characterized. Here, we identified a gene, termed pimE that is present in all mycobacteria, and is required for AcPIM6 biosynthesis. PimE was initially identified based on homology with eukaryotic PIG-M mannosyltransferases. PimE-deleted Mycobacterium smegmatis was defective in AcPIM6 synthesis, and accumulated the tetramannosyl PIM, AcPIM4. Loss of PimE had no affect on cell growth or viability, or the biosynthesis of other intracellular and cell wall glycans. However, changes in cell wall hydrophobicity and plasma membrane organization were detected, suggesting a role for AcPIM6 in the structural integrity of the cell wall and plasma membrane. These defects were corrected by ectopic expression of the pimE gene. Metabolic pulse-chase radiolabeling and cell-free PIM biosynthesis assays indicated that PimE catalyzes the alpha1,2-mannosyl transfer for the AcPIM5 synthesis. Mutation of an Asp residue in PimE that is conserved in and required for the activity of human PIG-M resulted in loss of PIM-biosynthetic activity, indicating that PimE is the catalytic component. Finally, PimE was localized to a distinct membrane fraction enriched in AcPIM4-6 biosynthesis. Taken together, PimE represents the first PPM-dependent mannosyl-transferase shown to be involved in PIM biosynthesis, where it mediates the fifth mannose transfer.
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ItemIdentification of a novel protein with a role in lipoarabinomannan biosynthesis in mycobacteria(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2006-04-07)All species of Mycobacteria synthesize distinctive cell walls that are rich in phosphatidylinositol mannosides (PIMs), lipomannan (LM), and lipoarabinomannan (LAM). PIM glycolipids, having 2-4 mannose residues, can either be channeled into polar PIM species (with 6 Man residues) or hypermannosylated to form LM and LAM. In this study, we have identified a Mycobacterium smegmatis gene, termed lpqW, that is required for the conversion of PIMs to LAM and is highly conserved in all mycobacteria. A transposon mutant, Myco481, containing an insertion near the 3' end of lpqW exhibited altered colony morphology on complex agar medium. This mutant was unstable and was consistently overgrown by a second mutant, represented by Myco481.1, that had normal growth and colony characteristics. Biochemical analysis and metabolic labeling studies showed that Myco481 synthesized the complete spectrum of apolar and polar PIMs but was unable to make LAM. LAM biosynthesis was restored to near wild type levels in Myco481.1. However, this mutant was unable to synthesize the major polar PIM (AcPIM6) and accumulated a smaller intermediate, AcPIM4. Targeted disruption of the lpqW gene and complementation of the initial Myco481 mutant with the wild type gene confirmed that the phenotype of this mutant was due to loss of LpqW. These studies suggest that LpqW has a role in regulating the flux of early PIM intermediates into polar PIM or LAM biosynthesis. They also suggest that AcPIM4 is the likely branch point intermediate in polar PIM and LAM biosynthesis.
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ItemCompartmentalization of lipid biosynthesis in mycobacteria(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2005-06-03)The plasma membrane of Mycobacterium sp. is the site of synthesis of several distinct classes of lipids that are either retained in the membrane or exported to the overlying cell envelope. Here, we provide evidence that enzymes involved in the biosynthesis of two major lipid classes, the phosphatidylinositol mannosides (PIMs) and aminophospholipids, are compartmentalized within the plasma membrane. Enzymes involved in the synthesis of early PIM intermediates were localized to a membrane subdomain termed PMf, that was clearly resolved from the cell wall by isopyknic density centrifugation and amplified in rapidly dividing Mycobacterium smegmatis. In contrast, the major pool of apolar PIMs and enzymes involved in polar PIM biosynthesis were localized to a denser fraction that contained both plasma membrane and cell wall markers (PM-CW). Based on the resistance of the PIMs to solvent extraction in live but not lysed cells, we propose that polar PIM biosynthesis occurs in the plasma membrane rather than the cell wall component of the PM-CW. Enzymes involved in phosphatidylethanolamine biosynthesis also displayed a highly polarized distribution between the PMf and PM-CW fractions. The PMf was greatly reduced in non-dividing cells, concomitant with a reduction in the synthesis and steady-state levels of PIMs and amino-phospholipids and the redistribution of PMf marker enzymes to non-PM-CW fractions. The formation of the PMf and recruitment of enzymes to this domain may thus play a role in regulating growth-specific changes in the biosynthesis of membrane and cell wall lipids.