A Toxoplasma gondii phosphoinositide phospholipase C (TgPI-PLC) with high affinity for phosphatidylinositol

Abstract

The Toxoplasma gondii phosphoinositide-specific phospholipase C gene (TgPI-PLC) was cloned, sequenced and expressed in Escherichia coli and its enzymatic characteristics were investigated. TgPI-PLC is present in the genome as a single-copy gene consisting of 22 exons interrupted by 21 introns, and encodes a polypeptide of 1097 amino acids with a predicted molecular mass of 121 kDa. In addition to the conserved catalytic X and Y domains, TgPI-PLC contains an apparent N-terminal PH domain, an EF hand motif and a C-terminal C2 domain. When compared with mammalian delta-type PI-PLC, TgPI-PLC has an additional extended N-terminus and two insertions in the region between the X and Y domains, with a 31-35% identity over the whole sequence. Recombinant TgPI-PLC, as well as the native enzyme obtained from crude membrane extracts of the parasite, was more active with phosphatidylinositol than with phosphatidylinositol 4,5-bisphosphate as substrate. Indirect immunofluorescence analysis using an affinity-purified antibody against TgPI-PLC revealed that this enzyme localizes in the plasma membrane of the parasites.

Figure 1. Organization and predicted protein sequence of TgPI-PLC

(A) Schematic map of the TgPI-PLC gene locus. The relative position of exons in the genomic DNA is indicated by black boxes with numbers. The grey boxes show the 5′ end flanking region, the introns and the 3′ end flanking region. A Kozak translation initiation site (tccgatATGG, where the lower-case letters indicate untranslated sequence) is indicated above with a bent arrow. The isolated TgPI-PLC cDNA clone (5.5 kb) contains a 3.3 kb ORF. (B) Deduced amino acid sequence of the TgPI-PLC cDNA clone. The unique N-terminal extension and the catalytic X and Y domains are highlighted in grey. The additional two inserts between the X and Y domains are underlined. The consensus NES of TgPI-PLC (H²⁷⁰LHGLLRKLNIQAD²⁸³) within the EF hand is underlined in white. An additional putative leucine-rich NES site (L⁵⁶⁸EELELRM⁵⁷⁵) predicted using NetNES 1.1 (http://www.cbs.dtu.dk/services/NetNES/) is indicated by downward-pointing triangles downstream of the X domain. The dileucine (L¹⁰⁶⁰L¹⁰⁶¹) and a mammalian sorting YXXΦ motif (Y¹⁰⁶³SRI¹⁰⁶⁶) is indicated with closed circles and diamonds respectively under the sequence at the C-terminal tail. (C) The numbers show the starting and finishing amino acid for each domain. The bold line indicates the N-terminal extension and two triangles represent the inserts in the X-Y domain linker. The peptide used to generate polyclonal antibody against TgPI-PLC (Antigen) is indicated above the Y domain. The protein size is not to scale. The consensus NES of the EF hand domain is compared with those of other δ-type PLCs. The important hydrophobic residues are in black boxes. Ψ represents a hydrophobic residue (isoleucine, valine or methionine); X represents any amino acid.

Figure 2. Expression and affinity purification of recombinant TgPI-PLC from E. coli

(A) Protein samples were separated by SDS/10% PAGE and visualized by Coomassie Brilliant Blue staining. Lane 1, standard protein size markers (MagicMark™ XP standards) with sizes given in kDa. Lane 2, nickel column-purified fraction (2.5 μg of protein/lane). The position of TgPI-PLC (120 kDa) is shown by the arrow. (B) Detection of TgPI-PLC by immunoblot using an affinity-purified polyclonal antibody against the peptide shown in Figure 1(C). Proteins were separated by SDS/10% PAGE, transferred on to nitrocellulose membranes and probed with affinity-purified anti-TgPI-PLC antibody (1:2000). Lane 1, protein standards (sizes are given in kDa); lane 2, recombinant TgPI-PLC (0.5 μg/lane) is shown by the arrow; lane 3, crude total cell lysate of E. coli BL21 CodonPlus(DE3)-RIPL transformed with TgPI-PLC/pET28a recombinant plasmid; lane 4, crude total cell lysate of non-transformed E. coli BL21 CodonPlus(DE3)-RIPL.

Figure 3. PI-PLC activity of recombinant TgPI-PLC as a function of temperature (A), medium pH (B), Mg²⁺ concentration (C), substrate concentration (D, E) and free Ca²⁺ concentration (F, G)

Experimental conditions were as described under the Materials and methods section with 250 μM free Ca²⁺ and 40 μM PIP₂ or 40 μM PI, adjusted to different temperatures (A), pH values (B), MgCl₂ concentration (C), different concentrations of PIP₂ (D) or PI (E) and 250 μM Ca²⁺, or in the same buffer with different free Ca²⁺ concentrations and 40 μM PIP₂ (F) or 40 μM PI (G), with (open symbols) or without (closed symbols) 3 mM MgCl₂. Insets in (D) and (E) represent the linear transformation, by double-reciprocal plot, of the curve. The results are representative of three experiments, each one carried out in duplicate.

Figure 4. Effect of PI-PLC inhibitors on the activity of recombinant TgPI-PLC

TgPI-PLC activity was measured as described in the Materials and methods section in the presence of 40 μM substrate (PI, grey bars; PIP₂, black bars) and 250 μM Ca²⁺, pH 7.5, and different concentrations of U-73122 (A), neomycin (B), and compound 48/80 (C).

Figure 5. PI-PLC activity of a T. gondii membrane fraction as a function of free Ca²⁺ concentration

Freshly isolated parasites were ruptured by freezing and thawing at low osmolarity, and the homogenate was centrifuged at 15000 g. The pellet fraction was used to measure PI-PLC activity using 40 μM PI (A) or 40 μM PIP₂ (B) as substrate, with different free Ca²⁺ concentrations. Other experimental details were as described in the Materials and methods section. The results are representative of three experiments, each one performed in duplicate.

Figure 6. Distribution of TgPI-PLC in intracellular and extracellular tachyzoites

Cells were permeabilized with 0.05% saponin for 30 min. The Figure shows the co-localization of TgPI-PLC (A, E) with SAG1 (B, F) in the plasma membrane. (C) and (G) show the overlap of (A) and (B), and (E) and (F) respectively. (D) and (H) are phase images. DAPI (4,6-diamidino-2-phenylindole) staining is shown in (H). Scale bar, 5 μm.

Figure 7. Biotinylation of cell-surface proteins in tachyzoites

Upper panel, tachyzoites of the 2F1 strain were incubated with 2 mM sulpho-NHS-biotin for 30 min. After lysis of the cells with RIPA buffer, the lysates were immunoprecipitated by the affinity-purified anti-TgPI-PLC polyclonal antibody, and the immunoprecipitates were subjected to Western blot analysis. Detection of biotinylation was carried out using streptavidin–peroxidase conjugate and ECL®. No band was detected in the immunoprecipitates with anti-PI-PLC (lane 1). Lane 2 shows a positive control with anti-SAG1 antibody instead of anti-TgPI-PLC antibody for immunoprecipitation. Lane 3 shows a negative control with anti-β-galactosidase antibody for immunoprecipitation. Migration of molecular-mass standards (in kDa) is shown to the left of the gels. Lower panel, the positive control experiment shows that the anti-TgPI-PLC antibody can immunoprecipitate both the native PLC from T. gondii (left-hand lane) and the recombinant TgPI-PLC (right-hand lane), as probed with the guinea-pig anti-TgPI-PLC antibody. The position of a 120 kDa protein is indicated.