Global Quantitative SILAC Phosphoproteomics Reveals Differential Phosphorylation Is Widespread between the Procyclic and Bloodstream Form Lifecycle Stages of <i>Trypanosoma brucei</i>

We report a global quantitative phosphoproteomic study of bloodstream and procyclic form <i>Trypanosoma brucei</i> using SILAC labeling of each lifecycle stage. Phosphopeptide enrichment by SCX and TiO₂ led to the identification of a total of 10096 phosphorylation sites on 2551 protein groups and quantified the ratios of 8275 phosphorylation sites between the two lifecycle stages. More than 9300 of these sites (92%) have not previously been reported. Model-based gene enrichment analysis identified over representation of Gene Ontology terms relating to the flagella, protein kinase activity, and the regulation of gene expression. The quantitative data reveal that differential protein phosphorylation is widespread between bloodstream and procyclic form trypanosomes, with significant intraprotein differential phosphorylation. Despite a lack of dedicated tyrosine kinases, 234 phosphotyrosine residues were identified, and these were 3–4 fold over-represented among site changing >10-fold between the two lifecycle stages. A significant proportion of the <i>T. brucei</i> kinome was phosphorylated, with evidence that MAPK pathways are functional in both lifecycle stages. Regulation of gene expression in <i>T. brucei</i> is exclusively post-transcriptional, and the extensive phosphorylation of RNA binding proteins observed may be relevant to the control of mRNA stability in this organism

Global Quantitative SILAC Phosphoproteomics Reveals Differential Phosphorylation Is Widespread between the Procyclic and Bloodstream Form Lifecycle Stages of <i>Trypanosoma brucei</i>

We report a global quantitative phosphoproteomic study of bloodstream and procyclic form <i>Trypanosoma brucei</i> using SILAC labeling of each lifecycle stage. Phosphopeptide enrichment by SCX and TiO₂ led to the identification of a total of 10096 phosphorylation sites on 2551 protein groups and quantified the ratios of 8275 phosphorylation sites between the two lifecycle stages. More than 9300 of these sites (92%) have not previously been reported. Model-based gene enrichment analysis identified over representation of Gene Ontology terms relating to the flagella, protein kinase activity, and the regulation of gene expression. The quantitative data reveal that differential protein phosphorylation is widespread between bloodstream and procyclic form trypanosomes, with significant intraprotein differential phosphorylation. Despite a lack of dedicated tyrosine kinases, 234 phosphotyrosine residues were identified, and these were 3–4 fold over-represented among site changing >10-fold between the two lifecycle stages. A significant proportion of the <i>T. brucei</i> kinome was phosphorylated, with evidence that MAPK pathways are functional in both lifecycle stages. Regulation of gene expression in <i>T. brucei</i> is exclusively post-transcriptional, and the extensive phosphorylation of RNA binding proteins observed may be relevant to the control of mRNA stability in this organism

Molecular modeling of TfR in the VSG coat of <i>T. brucei</i>.

<p>(A) A single glycosylated ESAG6/ESAG7 TfR heterodimer is shown flanked by two glycosylated VSG homodimers. The molecular models suggest that TfR is likely to be recessed into the VSG surface coat. (B) Top and side views of a glycosylated TfR molecule surrounded by VSG molecules. (C) The same views as (B) but with a glycosylated transferrin molecule approaching the TfR. TfR: peptide chains – green; N-glycans – yellow; GPI anchor – orange. VSG: peptide chains – blue; N-glycans – yellow; GPI anchor – orange. Transferrin: peptide chains – red; N- and O-glycans – purple.</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

Experimental and theoretical occupancy of TfR N-glycosylation sites.

1<p>Based on the predicted amino acid sequences for ESAG6 (GenBank: CAQ57442.1) and ESAG7 (GenBank: CAQ57441.1) minus their predicted 17-residue N-terminal signal peptides.</p>2<p>Predicted isoelectric point of the glycosylation sequon ±5 amino acid residues, as shown, calculated using ExPASy Compute pI/MW.</p>3<p>Experimentally determined (E) or theoretically predicted (P) sensitivity (+) or resistance (−) of the N-glycan to Endo H at the glycosylation site.</p>4<p>The major triantennary Endo H sensitive oligomannose Man₅GlcNac₂ structure is Manα1-3(Manα1-3(Manα1-6-Manα1-6)Manβ1-4GlcNAcβ1-4GlcNAc and the major biantennary Endo H resistant paucimannose Man₄GlcNac₂ structure is Manα1-2Manα1-3(Manα1-6Manβ1-4GlcNAcβ1-4GlcNAc.</p

SDS-PAGE analysis of purified <i>T. brucei</i> TfR and endoglycosidase digestions.

<p>(A) An aliquot of <i>T. brucei</i> TfR, affinity purified on transferrin-Sepharose, was analyzed by SDS-PAGE and silver staining. (B) Aliquots of purified TfR were incubated with Endo H (lane 1), PNGase F (lane 3) or mock treated (lane 2) and analyzed by SDS-PAGE and Western blot with an antibody that reacts with the ESAG6 and ESAG7 subunits of TfR.</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

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

TfR does not bind directly to tomato lectin but binds to other glycoproteins.

<p>A ricin-binding glycoprotein fraction (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002618#ppat.1002618-Atrih1" target="_blank">[27]</a> was purified from <i>T. brucei</i>, separated by SDS-PAGE and transferred to nitrocellulose. Identical lanes were incubated without (lane 1) or with (lane 2) purified TfR, followed by anti-TfR antibody, or with tomato lectin (TL) in the absence (lane 3) or presence (lane 4) of competing chitin hydrolysate. The positions of molecular weight markers are shown on the left.</p

Fluorographs of HPTLC analyses of released and radiolabeled N-glycans from purified TfR.

<p>(A) Total N-glycan fraction of TfR released by PNGase F and radiolabeled by reduction with NaB[³H]₄. The positions of oligomannose N-linked glycan standards reduced with NaB[³H]₄ are shown on the left. The proposed structures of the principal TfR glycans are shown on the right. The top three biantennary structures (Man₃GlcNAc₂ to Man₅GlcNAc₂) are of the paucimannose series and the bottom three triantennary structures (Man₅GlcNAc₂ to Man₇GlcNAc₂) are of the oligomannose series. (B) The three major components of the radiolabeled TfR N-glycan fraction isolated by Dionex HPAEC (<i>peaks a, b and c</i>; lanes 1, 2 and 3, respectively). The positions of NaB[³H]₄ reduced oligomannose N-linked glycan and dextran oligomer standards are shown on the left and right, respectively. (C) The paucimannose Man₄GlcNAc₂ structure (Dionex <i>peak a</i>) before (lane 1) and after (lane 2) digestion with Manα1-2Man specific α-mannosidase (ASαM) and the triantennary oligomannose Man₅GlcNAc₂structure Dionex <i>peak b</i> before (lane 3) and after (lane 4) digestion with ASαM. The positions of NaB[³H]₄-reduced dextran oligomers are shown on right.</p