Biochemistry and Pharmacology - Research Publications
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ItemLeishCyc: a biochemical pathways database for Leishmania major(BMC, 2009-06-05)BACKGROUND: Leishmania spp. are sandfly transmitted protozoan parasites that cause a spectrum of diseases in more than 12 million people worldwide. Much research is now focusing on how these parasites adapt to the distinct nutrient environments they encounter in the digestive tract of the sandfly vector and the phagolysosome compartment of mammalian macrophages. While data mining and annotation of the genomes of three Leishmania species has provided an initial inventory of predicted metabolic components and associated pathways, resources for integrating this information into metabolic networks and incorporating data from transcript, protein, and metabolite profiling studies is currently lacking. The development of a reliable, expertly curated, and widely available model of Leishmania metabolic networks is required to facilitate systems analysis, as well as discovery and prioritization of new drug targets for this important human pathogen. DESCRIPTION: The LeishCyc database was initially built from the genome sequence of Leishmania major (v5.2), based on the annotation published by the Wellcome Trust Sanger Institute. LeishCyc was manually curated to remove errors, correct automated predictions, and add information from the literature. The ongoing curation is based on public sources, literature searches, and our own experimental and bioinformatics studies. In a number of instances we have improved on the original genome annotation, and, in some ambiguous cases, collected relevant information from the literature in order to help clarify gene or protein annotation in the future. All genes in LeishCyc are linked to the corresponding entry in GeneDB (Wellcome Trust Sanger Institute). CONCLUSION: The LeishCyc database describes Leishmania major genes, gene products, metabolites, their relationships and biochemical organization into metabolic pathways. LeishCyc provides a systematic approach to organizing the evolving information about Leishmania biochemical networks and is a tool for analysis, interpretation, and visualization of Leishmania Omics data (transcriptomics, proteomics, metabolomics) in the context of metabolic pathways. LeishCyc is the first such database for the Trypanosomatidae family, which includes a number of other important human parasites. Flexible query/visualization capabilities are provided by the Pathway Tools software and its Web interface. The LeishCyc database is made freely available over the Internet http://www.leishcyc.org.
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ItemHumans lack iGb3 due to the absence of functional iGb3-synthase: Implications for NKT cell development and transplantation(PUBLIC LIBRARY SCIENCE, 2008-07)The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galalpha(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galalpha(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation.
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ItemA dynamic programming approach for the alignment of signal peaks in multiple gas chromatography-mass spectrometry experiments(BMC, 2007-10-29)BACKGROUND: Gas chromatography-mass spectrometry (GC-MS) is a robust platform for the profiling of certain classes of small molecules in biological samples. When multiple samples are profiled, including replicates of the same sample and/or different sample states, one needs to account for retention time drifts between experiments. This can be achieved either by the alignment of chromatographic profiles prior to peak detection, or by matching signal peaks after they have been extracted from chromatogram data matrices. Automated retention time correction is particularly important in non-targeted profiling studies. RESULTS: A new approach for matching signal peaks based on dynamic programming is presented. The proposed approach relies on both peak retention times and mass spectra. The alignment of more than two peak lists involves three steps: (1) all possible pairs of peak lists are aligned, and similarity of each pair of peak lists is estimated; (2) the guide tree is built based on the similarity between the peak lists; (3) peak lists are progressively aligned starting with the two most similar peak lists, following the guide tree until all peak lists are exhausted. When two or more experiments are performed on different sample states and each consisting of multiple replicates, peak lists within each set of replicate experiments are aligned first (within-state alignment), and subsequently the resulting alignments are aligned themselves (between-state alignment). When more than two sets of replicate experiments are present, the between-state alignment also employs the guide tree. We demonstrate the usefulness of this approach on GC-MS metabolic profiling experiments acquired on wild-type and mutant Leishmania mexicana parasites. CONCLUSION: We propose a progressive method to match signal peaks across multiple GC-MS experiments based on dynamic programming. A sensitive peak similarity function is proposed to balance peak retention time and peak mass spectra similarities. This approach can produce the optimal alignment between an arbitrary number of peak lists, and models explicitly within-state and between-state peak alignment. The accuracy of the proposed method was close to the accuracy of manually-curated peak matching, which required tens of man-hours for the analyzed data sets. The proposed approach may offer significant advantages for processing of high-throughput metabolomics data, especially when large numbers of experimental replicates and multiple sample states are analyzed.
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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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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.