High-confidence glycosome proteome for procyclic form <em>Trypanosoma brucei</em> by epitope-tag organelle enrichment and SILAC proteomics

The glycosome of the pathogenic African trypanosome Trypanosoma brucei is a specialized peroxisome that contains most of the enzymes of glycolysis and several other metabolic and catabolic pathways. The contents and transporters of this membrane-bounded organelle are of considerable interest as potential drug targets. Here we use epitope tagging, magnetic bead enrichment, and SILAC quantitative proteomics to determine a high-confidence glycosome proteome for the procyclic life cycle stage of the parasite using isotope ratios to discriminate glycosomal from mitochondrial and other contaminating proteins. The data confirm the presence of several previously demonstrated and suggested pathways in the organelle and identify previously unanticipated activities, such as protein phosphatases. The implications of the findings are discussed

Proteomics workflow.

<p>Procyclic cells were cultured in SDM-79+R₆K₆ then mixed 1∶1 with either unlabeled procyclic or bloodstream form cells. Sample complexity was reduced prior to LC-MS/MS analysis by either fractionation at the protein level by SDS-PAGE or at the peptide level by SCX chromatography.</p

Histograms of Log₂ fold change.

<p><b>A.</b> Procyclic form labeled with heavy isotopes (R₆K₆) mixed 1∶1 with unlabeled procyclic form (R₀K₀). <b>B.</b> Procyclic form labeled with heavy isotopes (R₆K₆) mixed 1∶1 with unlabeled bloodstream form (R₀K₀).</p

Agreement of comparative proteomic data with known biology.

<p>Heatmap showing the Log₂ FC (procyclic to bloodstream) derived from comparative proteomic data (this study) and previous transcriptomic studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036619#pone.0036619-Jensen1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036619#pone.0036619-Kabini1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036619#pone.0036619-Queiroz1" target="_blank">[12]</a>. Grey – not observed. Heatmap generated with GENEE (<a href="http://www.broadinstitute.org/cancer/software/GENE-E/" target="_blank">http://www.broadinstitute.org/cancer/software/GENE-E/</a>).</p

GO term enrichment.

<p><b>A.</b> Proteins with greater than ten-fold up- or down regulation, with enrichment P<0.01. <b>B.</b> Constitutively expressed proteins, with enrichment P<0.01.</p

Comparison of Proteomic and transcriptomic data.

<p>Scatterplot of the Log₂ FC (procyclic to bloodstream) derived from comparative proteomic data (this study) and previous transcriptomic studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036619#pone.0036619-Jensen1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036619#pone.0036619-Kabini1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036619#pone.0036619-Queiroz1" target="_blank">[12]</a>.</p

Growth of <i>T. brucei</i> procyclic form cells in original SDM-79 and SILAC labelling media.

<p><b>A.</b> Cumulative growth curve. Growth in original SDM-79 containing non-dialysed FBS (open squares) is shown in parallel to SDM-79+R₀K₀ (open circles) and SDM-79+R₆K₆ (closed circles), both containing dialysed FBS. <b>B.</b> DIC light microscopy. <i>T. brucei</i> procyclic cells grown in original SDM-79, SDM-79+R₀K₀ or SDM-79+R₆K₆ for ten days were fixed in 4% paraformaldehyde and DIC images acquired on a Zeiss confocal microscope.</p

A gene of the beta3-glycosyltransferase family encodes N-acetylglucosaminyltransferase II function in Trypanosoma brucei

The bloodstream form of the human pathogen Trypanosoma brucei expresses oligomannose, paucimannose and complex N-linked glycans, including some exceptionally large poly-N-acetyllactosamine-containing structures. Despite the presence of complex N-glycans in this organism, no homologues of the canonical N-acetylglucosaminyltransferase I or II genes can be found in the T. brucei genome. These genes encode the activities that initiate the elaboration of the Manalpha1-3 and Manalpha1-6 arms, respectively, of the conserved trimannosyl-N-acetylchitobiosyl core of N-linked glycans. Previously, we identified a highly divergent T. brucei N-acetylglucosaminyltransferase I (TbGnTI) among a set of putative T. brucei glycosyltransferase genes belonging to the beta3-glycosyltransferase superfamily (1). Here, we demonstrate that TbGT15, another member of the same beta3-glycosyltransferase family, encodes an equally divergent N-acetylglucosaminyltransferase II (TbGnTII) activity. In contrast to multicellular organisms, where GnTII activity is essential, TbGnTII null mutants of T. brucei grow in culture and are still infectious to animals. Characterization of the large poly-N-acetyllactosamine containing N-glycans of the TbGnTII null mutants by methylation linkage analysis suggests that, in wild-type parasites, the Manalpha1-6 arm of the conserved trimannosyl core may carry predominantly linear poly-N-acetyllactosamine chains whereas the Manalpha1-3 arm may carry predominantly branched poly-N-acetyllactosamine chains. These results provide further detail on the structure and biosynthesis of complex N-glycans in an important human pathogen and provide a second example of the adaptation by trypanosomes of beta3-glycosyltransferase family members to catalyze beta1-2 glycosidic linkages

Comparative SILAC proteomic analysis of Trypanosoma brucei bloodstream and procyclic lifecycle stages

The protozoan parasite Trypanosoma brucei has a complex digenetic lifecycle between a mammalian host and an insect vector, and adaption of its proteome between lifecycle stages is essential to its survival and virulence. We have optimized a procedure for growing Trypanosoma brucei procyclic form cells in conditions suitable for stable isotope labeling by amino acids in culture (SILAC) and report a comparative proteomic analysis of cultured procyclic form and bloodstream form T. brucei cells. In total we were able to identify 3959 proteins and quantify SILAC ratios for 3553 proteins with a false discovery rate of 0.01. A large number of proteins (10.6%) are differentially regulated by more the 5-fold between lifecycle stages, including those involved in the parasite surface coat, and in mitochondrial and glycosomal energy metabolism. Our proteomic data is broadly in agreement with transcriptomic studies, but with significantly larger fold changes observed at the protein level than at the mRNA level