A novel PAN/apple domain-containing protein from Toxoplasma gondii: characterization and receptor identification

Abstract

Toxoplasma gondii is an intracellular parasite that invades nucleated cells, causing toxoplasmosis in humans and animals worldwide. The extremely wide range of hosts susceptible to T. gondii is thought to be the result of interactions between T. gondii ligands and receptors on its target cells. In this study, a host cell-binding protein from T. gondii was characterized, and one of its receptors was identified. P104 (GenBank Access. No. CAJ20677) is 991 amino acids in length, containing a putative 26 amino acid signal peptide and 10 PAN/apple domains, and shows low homology to other identified PAN/apple domain-containing molecules. A 104-kDa host cell-binding protein was detected in the T. gondii lysate. Immunofluorescence assays detected P104 at the apical end of extracellular T. gondii. An Fc-fusion protein of the P104 N-terminus, which contains two PAN/apple domains, showed strong affinity for the mammalian and insect cells evaluated. This binding was not related to protein-protein or protein-lipid interactions, but to a protein-glycosaminoglycan (GAG) interaction. Chondroitin sulfate (CS), a kind of GAG, was shown to be involved in adhesion of the Fc-P104 N-terminus fusion protein to host cells. These results suggest that P104, expressed at the apical end of the extracellular parasite, may function as a ligand in the attachment of T. gondii to CS or other receptors on the host cell, facilitating invasion by the parasite.

Figure 1. Analysis of the P104 protein sequence.

A. The signal peptide is indicated by black rectangle. A1, A3, A5, and A7 are indicated by a dark gray rectangle. A2, A4, A6, and A8 are indicated by a light gray rectangle. A9 and A10 are shown in light black and shiny black rectangles. The arrows at the N- and C-termini indicate the designated primers; the corresponding PCR products are indicated by black bands. B. The putative apple-like structure of a PAN/apple domain. C1–C6 (shown in bold) indicate the six cysteine residues that form three disulfide bridges.

Figure 2. Recombinant proteins synthesized by eukaryotic and prokaryotic expression systems.

A. Left panel, silver-stained gel showing the recombinant proteins with their expected molecular weights purified from the Tn5 (insect) cell culture medium. Right panel, purified recombinant proteins were transferred onto a PVDF membrane and anti-mouse Fc antibody was employed to detect the Fc-recombinant protein bands. B. CBB-stained gel showing the fusion protein rP104-1-S/GST purified from E. coli. The purified proteins are shown by arrows. The molecular masses in kDa are shown on the left.

Figure 3. Localization of P104 in intracellular and extracellular T. gondii.

A. Western blot analysis of P104 in the T. gondii lysate. Lane 1 and 4, Vero cell lysate; lane 2 and 5, lysate of T. gondii-infected Vero cells; lane 3 and 6, lysate of purified T. gondii. Lane 1–3, CBB-staining of Vero cell and T. gondii lysate; lane 4–6, Western blotting of the lysates. Mouse anti-rP104-1-S/GST was used as the primary antibody. The molecular masses in kDa are shown on the left. B. Co-localization assays of P104 with other proteins in extracellular (upper 3 panels) and intracellular (lower 3 panels) T. gondii. Green, anti-rP104-1-S/GST antibodies; red, anti-M2AP, anti-ROP1 and anti-GRA6 antibodies; blue, nuclei (with TO-PRO-3 staining). In intracellular T. gondii (lower 3 panels), mouse anti-FLAG antibody, instead of anti-rP104-1-S/GST, was used to stain the recombinant protein of P104 with a C-terminal FLAG tag (green stained, arrows). Wild-type parasites that failed to be stained with anti-FLAG antibody were indicated with stars.

Figure 4. FACS analysis of the cellular binding activity of rP104-1-S/Fc and rP104-1-B/Fc.

A. Different types of cells were incubated with rP104-1-S/Fc before being stained with anti-Fc-FITC antibodies and subjected to FACS analysis with Cell Quest software. B. FACS analysis of cells incubated with rP104-1-B/Fc. C. FACS analysis of cells incubated with rP104-2/Fc. In all panels, cells (solid line) and cells incubated with Fc (dotted line) were employed as negative controls. Gray-filled histograms indicate the cells incubated with recombinant proteins.

Figure 5. The attachment of rP104-1-S/Fc to host cells was not affected by protease digestion of cell surface proteins, and the receptor was not a major lipid on the cell surface.

Vero, K562, and P3U1 cells were treated with trypsin (A) or chymotrypsin (B) to remove surface proteins, and then incubated with rP104-1-S/Fc (gray-filled histograms) or Fc (dotted line). The bound signals were detected by FACS analysis. The solid line indicates the cell-only control. C. rP104-1-S/Fc was incubated with a membrane spotted with 15 major lipids from the cell surface (right panel). The membrane was then immersed in HRP-conjugated anti-mouse antibodies, and reaction spots were revealed using the ECL system. Fc was used as a negative control (central panel). The names of lipids were shown on the left panel.

Figure 6. CS is necessary for the adhesion of rP104-1-S/Fc to cells.

A. Inhibitory effect of CS on the binding activity of rP104-1-S/Fc to CHO-K1 cells. The inhibitory effect of CSA and CSC on the activity of rP104-1-S/Fc was calculated as the ratio of the geometric mean fluorescence intensity value (GMean): (1-CSA-treated cells/no treated cells) ×100. The experiment was performed in triplicate. B. Binding activity of rP104-1-S/Fc to CHO-K1 mutant cells. The upper panel shows the ratio of the GMean as (rP104-1-S/Fc-mutant cells/rP104-1-S/Fc-wild cells-1) ×100; the experiment was performed in triplicate. The lower panel shows the change in fluorescence as evaluated by FACS assay. Different colors indicate the different types of cells and treatments: CHO-K1 (black), Fc-treated CHO-K1 (light green), rP104-1-S/Fc-treated CHO-K1 (red filled histogram), pgsA-745 (dark blue), Fc-treated pgsA-745 (purple), rP104-1-S/Fc-treated pgsA-745 (light blue), pgsD-677 (yellow), Fc-treated pgsD-677 (dark red), and rP104-1-S/Fc-treated pgsD-677 (dark green). C. Binding of CSA-conjugated beads to rP104-1-S/Fc. CSA-conjugated beads were incubated with rP104-1-S/Fc and then stained with HRP-conjugated anti-mouse antibodies. The bands indicate that rP104-1-S/Fc was co-purified with the CSA-coupled beads. Lane 1, input rP104-1-S/Fc; 2, mock beads incubated with rP104-1-S/Fc; 3, CSA-treated beads incubated with rP104-1-S/Fc. The molecular masses in kDa are shown on the left.

Figure 7. In vitro inhibitory effect of rP104-1-S/Fc or CSA on the invasion of T. gondii.

Monolayers of Vero cells in an 8-well chamber slide were infected with 2×10⁵ GFP-expressing T. gondii after incubation with different concentrations of rP104-1-S/Fc (A) or CSA (B). Extracellular and intracellular parasites were stained differentially and enumerated as described in method section. The invasion rate was calculated as the ratio of parasites/cells in the tested group compared to that in the mock group (Vero cells treated with no protein prior to infection with T. gondii). Fc was used as a negative control. Student's t-test, asterisk, P<0.05; mean ± standard deviation (n = 3 experiments).