Limits...
Architecture of a Host-Parasite Interface: Complex Targeting Mechanisms Revealed Through Proteomics.

Gadelha C, Zhang W, Chamberlain JW, Chait BT, Wickstead B, Field MC - Mol. Cell Proteomics (2015)

Bottom Line: This surface proteome contains previously known flagellar pocket proteins as well as multiple novel components, and is significantly enriched in proteins that are essential for parasite survival.Validation shows that the majority of surface proteome constituents are bona fide surface-associated proteins and, as expected, most present at the flagellar pocket.This work provides a paradigm for the compartmentalization of a cell surface and a resource for its analysis.

View Article: PubMed Central - PubMed

Affiliation: From the ‡School of Life Sciences, University of Nottingham, Nottingham, UK, NG2 7UH; §Department of Pathology, University of Cambridge, Cambridge, UK, CB2 1QP; catarina.gadelha@nottingham.ac.uk.

No MeSH data available.


Related in: MedlinePlus

Workflow of biochemical, semi-quantitative mass spectrometry and bioinformatic methods used to identify putative cell surface proteins.A, Scheme illustrating key steps in purification. B, Micrograph of cells following chemical modification with fluorescein (live at 0 °C). Native fluorescence at plasma membrane is predominantly derived from fluoresceinated VSG (which makes up ∼90% of proteins at the parasite surface). DNA has been counter-stained with DAPI (magenta); the FP is indicated by yellow arrowhead. C, Immunoblots showing isolation of known surface proteins (ISG65, found on the cell surface and TfR, found in the FP) in the final purified eluate. Note faster migration of deglycosylated ISG and TfR in eluate. Common contaminants from the ER (BiP) and lysosome (p67) are highly depleted in final eluate. D, Schematic showing enrichment analysis (for exclusion of contaminants by comparison of labeled samples with controls) and bioinformatic filters (for prediction of membrane proteins features) applied to protein identification to produce “high-confidence” sets. The numbers of unique proteins present in each set are shown in red. The high-confidence set of 175 putative surface membrane proteins enriched 5x in labeled samples is herein referred to as the T. brucei bloodstream surface proteome (TbBSP). Experimental replicates of protein isolation from fluorescein-labeled live cells (“Labeled”), unlabeled cells (“Unlabeled”), and fluorescein-labeled material from lysed cells (“Dead”) are indicated between brackets. See Experimental Procedures for details of protein feature prediction, and supplemental Table S2 for bioinformatics filter abbreviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4587319&req=5

Figure 1: Workflow of biochemical, semi-quantitative mass spectrometry and bioinformatic methods used to identify putative cell surface proteins.A, Scheme illustrating key steps in purification. B, Micrograph of cells following chemical modification with fluorescein (live at 0 °C). Native fluorescence at plasma membrane is predominantly derived from fluoresceinated VSG (which makes up ∼90% of proteins at the parasite surface). DNA has been counter-stained with DAPI (magenta); the FP is indicated by yellow arrowhead. C, Immunoblots showing isolation of known surface proteins (ISG65, found on the cell surface and TfR, found in the FP) in the final purified eluate. Note faster migration of deglycosylated ISG and TfR in eluate. Common contaminants from the ER (BiP) and lysosome (p67) are highly depleted in final eluate. D, Schematic showing enrichment analysis (for exclusion of contaminants by comparison of labeled samples with controls) and bioinformatic filters (for prediction of membrane proteins features) applied to protein identification to produce “high-confidence” sets. The numbers of unique proteins present in each set are shown in red. The high-confidence set of 175 putative surface membrane proteins enriched 5x in labeled samples is herein referred to as the T. brucei bloodstream surface proteome (TbBSP). Experimental replicates of protein isolation from fluorescein-labeled live cells (“Labeled”), unlabeled cells (“Unlabeled”), and fluorescein-labeled material from lysed cells (“Dead”) are indicated between brackets. See Experimental Procedures for details of protein feature prediction, and supplemental Table S2 for bioinformatics filter abbreviations.

Mentions: We used bloodstream-form Trypanosoma brucei Lister 427 expressing VSG221 (BES1/MITat 1.2/VSG427–2/TAR 40), as monitored by immunofluorescence microscopy using an affinity-purified polyclonal antibody anti-VSG221. 5 × 108 mid-log phase cells were harvested by centrifugation and resuspended at 2 × 108 cells ml−1 in PBS (10 mm PO4, 137 mm NaCl, 2.7 mm KCl, pH 7.5) plus 20 mm glucose. Cells were held on ice while pulsed with 500 μm fluorescein-hexanoate-NHS (referred hereafter to as fluorescein) dissolved in DMSO and HPG buffer (20 mm HEPES pH 7.5, 140 mm NaCl, 20 mm glucose). Pulse duration was 15 min on ice, during which time cells remained actively motile and morphologically normal (as assessed by light microscopy). Fluorescence microscopy showed fluorescein to be exclusively associated to the parasite cell surface (Fig. 1B). At the end of this period, unreacted fluorescein was blocked by the addition of TBS (25 mm Tris-HCl pH 7.5, 150 mm NaCl) plus 0.25% w/v glycine, and removed by washing cells in TBS plus 20 mm glucose. Fluorescein-labeled cells were lysed with 2% v/v Igepal CA-630 and 2% w/v CHAPS in the presence of protease inhibitors (5 μm E-64d, 2 mm 1,10-phenanthroline, 50 μm leupeptin, 7.5 μm pepstatin A, 500 μm phenylmethylsulfonyl fluoride, 1 mm EDTA, 1 mm DTT) and 200 μg ml−1 DNase I, and centrifuged at 20,000 × g for 30 min to separate soluble labeled proteins from the insoluble fraction. To increase identification sensitivity toward less abundant surface membrane proteins, we included a VSG-depletion step by affinity chromatography, for which a polyclonal antibody anti-VSG221 was generated (please see below). The soluble fraction was allowed to bind to 8 mg polyclonal antibody anti-VSG221 conjugated to protein G-Sepharose 4 fast flow (GE Healthcare). Then the soluble fraction partially depleted of VSG was allowed to bind to 30 mg protein G-Dynabeads (Invitrogen) cross-linked to 400 μg polyclonal antibody anti-fluorescein for 1 h, after which period unbound material was collected as flow-through and beads were washed several times in the presence of high salt and detergent (500 mm NaCl, 0.02% v/v Tween-20). Bound proteins were deglycosylated native on column with 1000U of PNGase F for 1 h before acid then basic elutions in 0.2 m glycine pH 2.5 and 0.2 m triethanolamine pH 11 respectively. To control for nonspecific binding to anti-fluorescein column, a parallel isolation was carried out with unlabeled cells. To account for possible cell lysis during the surface labeling step, 1 × 108 cells were subjected to hypotonic lysis by resuspension in 20 mm Hepes pH 7.5 in the presence of the protease inhibitors aforementioned for 30 min at room temperature, and then pulsed with fluorescein as above.


Architecture of a Host-Parasite Interface: Complex Targeting Mechanisms Revealed Through Proteomics.

Gadelha C, Zhang W, Chamberlain JW, Chait BT, Wickstead B, Field MC - Mol. Cell Proteomics (2015)

Workflow of biochemical, semi-quantitative mass spectrometry and bioinformatic methods used to identify putative cell surface proteins.A, Scheme illustrating key steps in purification. B, Micrograph of cells following chemical modification with fluorescein (live at 0 °C). Native fluorescence at plasma membrane is predominantly derived from fluoresceinated VSG (which makes up ∼90% of proteins at the parasite surface). DNA has been counter-stained with DAPI (magenta); the FP is indicated by yellow arrowhead. C, Immunoblots showing isolation of known surface proteins (ISG65, found on the cell surface and TfR, found in the FP) in the final purified eluate. Note faster migration of deglycosylated ISG and TfR in eluate. Common contaminants from the ER (BiP) and lysosome (p67) are highly depleted in final eluate. D, Schematic showing enrichment analysis (for exclusion of contaminants by comparison of labeled samples with controls) and bioinformatic filters (for prediction of membrane proteins features) applied to protein identification to produce “high-confidence” sets. The numbers of unique proteins present in each set are shown in red. The high-confidence set of 175 putative surface membrane proteins enriched 5x in labeled samples is herein referred to as the T. brucei bloodstream surface proteome (TbBSP). Experimental replicates of protein isolation from fluorescein-labeled live cells (“Labeled”), unlabeled cells (“Unlabeled”), and fluorescein-labeled material from lysed cells (“Dead”) are indicated between brackets. See Experimental Procedures for details of protein feature prediction, and supplemental Table S2 for bioinformatics filter abbreviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587319&req=5

Figure 1: Workflow of biochemical, semi-quantitative mass spectrometry and bioinformatic methods used to identify putative cell surface proteins.A, Scheme illustrating key steps in purification. B, Micrograph of cells following chemical modification with fluorescein (live at 0 °C). Native fluorescence at plasma membrane is predominantly derived from fluoresceinated VSG (which makes up ∼90% of proteins at the parasite surface). DNA has been counter-stained with DAPI (magenta); the FP is indicated by yellow arrowhead. C, Immunoblots showing isolation of known surface proteins (ISG65, found on the cell surface and TfR, found in the FP) in the final purified eluate. Note faster migration of deglycosylated ISG and TfR in eluate. Common contaminants from the ER (BiP) and lysosome (p67) are highly depleted in final eluate. D, Schematic showing enrichment analysis (for exclusion of contaminants by comparison of labeled samples with controls) and bioinformatic filters (for prediction of membrane proteins features) applied to protein identification to produce “high-confidence” sets. The numbers of unique proteins present in each set are shown in red. The high-confidence set of 175 putative surface membrane proteins enriched 5x in labeled samples is herein referred to as the T. brucei bloodstream surface proteome (TbBSP). Experimental replicates of protein isolation from fluorescein-labeled live cells (“Labeled”), unlabeled cells (“Unlabeled”), and fluorescein-labeled material from lysed cells (“Dead”) are indicated between brackets. See Experimental Procedures for details of protein feature prediction, and supplemental Table S2 for bioinformatics filter abbreviations.
Mentions: We used bloodstream-form Trypanosoma brucei Lister 427 expressing VSG221 (BES1/MITat 1.2/VSG427–2/TAR 40), as monitored by immunofluorescence microscopy using an affinity-purified polyclonal antibody anti-VSG221. 5 × 108 mid-log phase cells were harvested by centrifugation and resuspended at 2 × 108 cells ml−1 in PBS (10 mm PO4, 137 mm NaCl, 2.7 mm KCl, pH 7.5) plus 20 mm glucose. Cells were held on ice while pulsed with 500 μm fluorescein-hexanoate-NHS (referred hereafter to as fluorescein) dissolved in DMSO and HPG buffer (20 mm HEPES pH 7.5, 140 mm NaCl, 20 mm glucose). Pulse duration was 15 min on ice, during which time cells remained actively motile and morphologically normal (as assessed by light microscopy). Fluorescence microscopy showed fluorescein to be exclusively associated to the parasite cell surface (Fig. 1B). At the end of this period, unreacted fluorescein was blocked by the addition of TBS (25 mm Tris-HCl pH 7.5, 150 mm NaCl) plus 0.25% w/v glycine, and removed by washing cells in TBS plus 20 mm glucose. Fluorescein-labeled cells were lysed with 2% v/v Igepal CA-630 and 2% w/v CHAPS in the presence of protease inhibitors (5 μm E-64d, 2 mm 1,10-phenanthroline, 50 μm leupeptin, 7.5 μm pepstatin A, 500 μm phenylmethylsulfonyl fluoride, 1 mm EDTA, 1 mm DTT) and 200 μg ml−1 DNase I, and centrifuged at 20,000 × g for 30 min to separate soluble labeled proteins from the insoluble fraction. To increase identification sensitivity toward less abundant surface membrane proteins, we included a VSG-depletion step by affinity chromatography, for which a polyclonal antibody anti-VSG221 was generated (please see below). The soluble fraction was allowed to bind to 8 mg polyclonal antibody anti-VSG221 conjugated to protein G-Sepharose 4 fast flow (GE Healthcare). Then the soluble fraction partially depleted of VSG was allowed to bind to 30 mg protein G-Dynabeads (Invitrogen) cross-linked to 400 μg polyclonal antibody anti-fluorescein for 1 h, after which period unbound material was collected as flow-through and beads were washed several times in the presence of high salt and detergent (500 mm NaCl, 0.02% v/v Tween-20). Bound proteins were deglycosylated native on column with 1000U of PNGase F for 1 h before acid then basic elutions in 0.2 m glycine pH 2.5 and 0.2 m triethanolamine pH 11 respectively. To control for nonspecific binding to anti-fluorescein column, a parallel isolation was carried out with unlabeled cells. To account for possible cell lysis during the surface labeling step, 1 × 108 cells were subjected to hypotonic lysis by resuspension in 20 mm Hepes pH 7.5 in the presence of the protease inhibitors aforementioned for 30 min at room temperature, and then pulsed with fluorescein as above.

Bottom Line: This surface proteome contains previously known flagellar pocket proteins as well as multiple novel components, and is significantly enriched in proteins that are essential for parasite survival.Validation shows that the majority of surface proteome constituents are bona fide surface-associated proteins and, as expected, most present at the flagellar pocket.This work provides a paradigm for the compartmentalization of a cell surface and a resource for its analysis.

View Article: PubMed Central - PubMed

Affiliation: From the ‡School of Life Sciences, University of Nottingham, Nottingham, UK, NG2 7UH; §Department of Pathology, University of Cambridge, Cambridge, UK, CB2 1QP; catarina.gadelha@nottingham.ac.uk.

No MeSH data available.


Related in: MedlinePlus