The Toxoplasma gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesin

Abstract

Attachment and invasion of host cells by apicomplexan parasites involve the exocytosis of the micronemal proteins (MICs). Most MICs are adhesins, which show homology with adhesive domains from higher eukaryote proteins and undergo proteolytic processing of unknown biological significance during their transport to micronemes. In Toxoplasma gondii, the micronemal homodimeric protein MIC3 is a potent adhesin that displays features shared by most Apicomplexa MICs. We have developed an original MIC3-binding assay by transfection of mammalian cells with complete or truncated MIC3 gene sequences and demonstrated that the receptor binding site of MIC3 is located in the N-terminal chitin-binding-like domain, which remains poorly accessible until the adjacent pro-peptide has been cleaved, and that binding requires dimerization. We have localized the dimerization domain in the C-terminal end of the protein and shown that it is able to convert MIC8, a monomeric micronemal protein sharing the MIC3 lectin-like domain, into a dimer able to interact with host cell receptors. These findings shed new light on molecular mechanisms that control functional maturation of MICs.

Fig. 1. In mammalian cells, the complete ORF of MIC3 is expressed as a dimeric pro-protein (R-proMIC3). Western blot analysis of cells transfected with MIC3 gene. Lane 1, T.gondii lysate; lane 2, lysate of BHK-21 cells transfected with plasmid p-SS-PRO-MIC3; lane 3, control (BHK-21 cells transfected with empty plasmid pcDNA3). Molecular weight standards are indicated. Anti-MIC3 mAb (A) and anti-pro-peptide serum (B) labeled the same band in immunoblot of cells transfected with the entire MIC3 gene. In contrast, anti- pro-peptide labeled only faintly the small amount of the proMIC3 in tachyzoite lysate, which co-migrated with recombinant MIC3.

Fig. 2. Recombinant R-MIC3 has a strong affinity to host cell surfaces. Western blot and cell blot analysis of cells transfected with MIC3 constructs. Lane 1, T.gondii lysate; lane 2, control (BHK-21 cells transfected with empty plasmid pcDNA3); lane 3, lysate of BHK-21 cells transfected with plasmid p-SS-PRO-MIC3; lane 4, lysate of BHK-21 cells transfected with plasmid p-SS-MIC3. Molecular weight standards are indicated. (A) The nitrocellulose membrane was probed with anti-MIC3 mAb. (B) A duplicate nitrocellulose membrane was incubated with BHK-21 cells, washed, and bound cells were stained with amidoblack (cell blot). Cells bind to native MIC3 (lane 1) and R-MIC3 (lane 4), but not to R-proMIC3 (lane 3).

Fig. 3. Immunofluorescence assay (IFA) and biotinylation demonstrate the difference in affinity to host cell surface between R-MIC3 and R-proMIC3. (A) Localization of recombinant MIC3 proteins in transfected cells by IFA. BHK-21 cells were transfected with pOC1 or pOC2 (see Figure 4A), which encode, respectively, R-proMIC3 and R-MIC3. Intracellular staining was performed on permeabilized cells with mAb anti-V5 followed by an anti-mouse antibody conjugated to TRITC (left panel). Surface expression of recombinant proteins was performed on unpermeabilized cells (right panel). GFP fluorescence (green) monitored transfected cells. In contrast to R-proMIC3, which is barely detectable in the absence of cell permeabilization, mature R-MIC3 covers the entire cell surface of transfected cells. Scale bar = 10 µm. (B) Comparative distribution of R-MIC3 and R-proMIC3 on cell surface and in supernatants. Transfected cells expressing R-proMIC3 or R-MIC3 were surface biotinylated and recombinant MIC3 proteins were immunopurified from cell lysates with anti-MIC3 agarose. The western blot was revealed by anti-V5–alkaline phosphatase (IP Total) and streptavidin–alkaline phosphatase conjugates (IP Surface). Note that equal amounts of R-proMIC3 and R-MIC3 were immunoprecipitated, in contrast to unequal amounts that were biotinylated, R-MIC3 being strongly biotinylated. Analysis of supernatants obtained from the same number of cells shows that R-proMIC3 is highly enriched, whereas R-MIC3 is almost absent. (C) Three-color overlay (green, GFP; red, anti-V5; blue, Hoechst DNA stain) of a cell monolayer transfected with pOC2, showing surface binding of R-MIC3 on neighboring untransfected BHK-21 cells (arrows). Scale bar = 10 µm. (D) Single confocal section through a BHK-21 cell expressing R-MIC3 (on the surface) and GFP (in cytoplasm). Scale bar = 10 µm.

Fig. 4. Analysis of the binding domain of MIC3. (A) Schematic drawings of the MIC3 constructs produced. The color code is yellow for pro-peptide, blue for lectin and green for tandemly repeated EGF domains. EGF domains are numbered as indicated. The two overlapping EGF domains are I and V. The C-terminal stretch (amino acids 294–359) is represented by a checkered green area. The V5 epitope tag is indicated by a red circle. BHK-21 cells were transfected with plasmids pOC1–pOC10. Intracellular or surface detection were performed with mAb anti-V5 (red) on cells permeabilized or not before incubation, respectively. GFP fluorescence monitored transfected cells. Surface staining was considered as an indicator of the binding property of the recombinant protein. Scale bar = 10 µm. Secretion was monitored by western blotting: identical fractions of cell monolayers and corresponding supernatants after 18 h of expression were analyzed in reduced conditions and probed with anti-V5. Note that, except for the chimera R-lectin–AA_294–359, all non-adhesive recombinant proteins were well secreted (amount in supernatant greater than amount in pellet), in contrast to adhesive proteins, which were present in supernatant and pellet, most likely resulting from their surface association. (B) Dimerization status of MIC3 constructs expressed in BHK-21 cells. Cell lysates (pellet) of BHK-21 cells expressing the different constructs were separated by SDS–PAGE in reduced or unreduced conditions, western blotted and probed with anti-V5 antibodies. Molecular weight standards are indicated.

Fig. 4. Analysis of the binding domain of MIC3. (A) Schematic drawings of the MIC3 constructs produced. The color code is yellow for pro-peptide, blue for lectin and green for tandemly repeated EGF domains. EGF domains are numbered as indicated. The two overlapping EGF domains are I and V. The C-terminal stretch (amino acids 294–359) is represented by a checkered green area. The V5 epitope tag is indicated by a red circle. BHK-21 cells were transfected with plasmids pOC1–pOC10. Intracellular or surface detection were performed with mAb anti-V5 (red) on cells permeabilized or not before incubation, respectively. GFP fluorescence monitored transfected cells. Surface staining was considered as an indicator of the binding property of the recombinant protein. Scale bar = 10 µm. Secretion was monitored by western blotting: identical fractions of cell monolayers and corresponding supernatants after 18 h of expression were analyzed in reduced conditions and probed with anti-V5. Note that, except for the chimera R-lectin–AA_294–359, all non-adhesive recombinant proteins were well secreted (amount in supernatant greater than amount in pellet), in contrast to adhesive proteins, which were present in supernatant and pellet, most likely resulting from their surface association. (B) Dimerization status of MIC3 constructs expressed in BHK-21 cells. Cell lysates (pellet) of BHK-21 cells expressing the different constructs were separated by SDS–PAGE in reduced or unreduced conditions, western blotted and probed with anti-V5 antibodies. Molecular weight standards are indicated.

Fig. 5. Replacement of the transmembrane insertion domain of MIC8 by the C-terminal end of MIC3 led to dimerization of MIC8 and acquisition of adhesive function. (A) Schematic drawing of the domains of MIC8 and recombinant MIC8 constructs produced. (B) Western blot analysis of MIC8 constructs. Supernatant and corresponding cell lysates (pellet) of BHK-21 cells expressing full-length MIC8 (lane 2), MIC8ΔTMCD (lane 3) and the chimera MIC8ΔTMCD–AA_294–359-MIC3 (lane 4) were western blotted and probed with rabbit anti-MIC8 serum. Lane 1 shows cells transfected by empty vector. Molecular weight standards are indicated. The chimera is secreted as a dimer also present in the pellet. The asterisk indicates the band corresponding to the dimer. (C) Intracellular detection and surface expression of MIC8 constructs were performed on cells permeabilized (P) or not (NP) by incubation with rabbit anti-MIC8 serum. The chimera is detected on the cell surface. Scale bar = 10 µm.

Fig. 6. Expression of site-directed mutagenized R-MIC3 proteins in BHK-21 cells. (A) Schematic drawing of the domains of MIC3 and position of substituted amino acids. The sequence of the dimerization stretch is given and the cysteine residues are indicated in bold letters. (B) BHK-21 cells were transfected with plasmids pOC302, pOC304, pOC339, pOC354, pOC302-304, pOC339-354 and pOC302-304-339-354 coding for R-MIC3 with cysteine mutations in the dimerization stretch. Cell lysates were separated by SDS–PAGE in unreduced conditions, western blotted and probed with anti-V5 antibody. Molecular weight standards are indicated. Dimerization occurs for all constructs.