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doi: 10.1089/omi.2014.0163.

Comprehensive proteomics analysis of glycosomes from Leishmania donovani

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Comprehensive proteomics analysis of glycosomes from Leishmania donovani

Mahendra D Jamdhade et al. OMICS. 2015 Mar.

Abstract

Leishmania donovani is a kinetoplastid protozoan that causes a severe and fatal disease kala-azar, or visceral leishmaniasis. L. donovani infects human host after the phlebotomine sandfly takes a blood meal and resides within the phagolysosome of infected macrophages. Previous studies on host-parasite interactions have not focused on Leishmania organelles and the role that they play in the survival of this parasite within macrophages. Leishmania possess glycosomes that are unique and specialized subcellular microbody organelles. Glycosomes are known to harbor most peroxisomal enzymes and, in addition, they also possess nine glycolytic enzymes. In the present study, we have carried out proteomic profiling using high resolution mass spectrometry of a sucrose density gradient-enriched glycosomal fraction isolated from L. donovani promastigotes. This study resulted in the identification of 4022 unique peptides, leading to the identification of 1355 unique proteins from a preparation enriched in L. donovani glycosomes. Based on protein annotation, 566 (41.8%) were identified as hypothetical proteins with no known function. A majority of the identified proteins are involved in metabolic processes such as carbohydrate, lipid, and nucleic acid metabolism. Our present proteomic analysis is the most comprehensive study to date to map the proteome of L. donovani glycosomes.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Workflow for glycosome purification and enrichment from L. donovani promastigotes. Promastigotes of L. donovani were cultured and used for the isolation of glycosomes. The cells were resuspended in hypotonic buffer, followed by lysis of the promastigotes by passing the suspension through a narrow gauge syringe. This was followed by brief centrifugation at 3000 g, followed by sucrose density gradient centrifugation at 39,000 rpm for 6 h in an ultracentrifuge. The glycosomes sedimented at the interphase of 2 M and 1.75 M sucrose on the sucrose density gradient.
<b>FIG. 2.</b>
FIG. 2.
Workflow for sample processing, fractionation, and proteomic analysis of L. donovani glycosomes. L. donovani promastigotes were used for the isolation and purification of glycosomes for proteomic analysis as indicated. Proteins from the glycosome were extracted using SDS and subjected to SDS-PAGE or digested with trypsin and subjected to strong cation exchange (SCX) chromatography. LC-MS/MS analysis of the digested gel bands or SCX fractions was carried out on a high resolution mass spectrometer. The mass spectrometry data were analyzed against a protein database of L. donovani.
<b>FIG. 3.</b>
FIG. 3.
Identification of PTS-1 containing protein ATP-dependent phosphofructokinase. (A) ATP-dependent phosphofructokinase (blue bar) in L. donovani is encoded by the gene LDBPK_292620 encoding a 486 amino acid long protein. The red bars indicate the peptides identified in our study mapping to ATP-dependent phosphofructokinase. (B) Sequence of ATP-dependent phosphofructokinase (LDBPK_292620) with its C-terminal PTS-1 sequence "SKV" marked in gray, and the 21 unique peptides that were identified marked in red. (C) A representative MS/MS spectrum of peptide TFGFQTAVEQAVNAVR identified from ATP-dependent phosphofructokinase.
<b>FIG. 4.</b>
FIG. 4.
Identification of PTS-2 containing protein hexokinase. (A) Hexokinase (blue bar) in L. donovani is encoded by the gene LDBPK_210300 encoding a 471 amino acid long protein The red bars indicate the peptides identified in our study. (B) Sequence of hexokinase (LDBPK_210300) with its N-terminal PTS-2 sequence "RVNNLLSHI" marked in gray, along with 22 unique peptides that were identified and marked in red. (C) A representative MS/MS spectrum of peptide ATGVYYALDLGGTNFR identified from hexokinase.
<b>FIG. 5.</b>
FIG. 5.
Comparison of glycosomal proteomic data from L. donovani, L. tarentolae, and T. brucei. The comparison of proteins detected in proteomic study of three kintoplastid glycosomal preparation shown by Venn diagram indicates number of orthologues found in all three preparations.
<b>FIG. 6.</b>
FIG. 6.
Gene ontology based bioinformatics analysis of proteins identified in L. donovani glycosome. (A) Distribution of L. donovani proteins based on biological processes. (B) Distribution of L. donovani proteins based on molecular function. (C) Distribution of secreted and transmembrane proteins identified in L. donovani. A great percentage of proteins remain unclassified.

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