The Toxoplasma gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesin
2002; Springer Nature; Volume: 21; Issue: 11 Linguagem: Inglês
10.1093/emboj/21.11.2526
ISSN1460-2075
Autores Tópico(s)Cytomegalovirus and herpesvirus research
ResumoArticle3 June 2002free access The Toxoplasma gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesin Odile Cérède Odile Cérède UMR Université-INRA d'Immunologie Parasitaire, Faculté des Sciences Pharmaceutiques et Biologiques, 31 Avenue Monge, F-37200 Tours, France Search for more papers by this author Jean François Dubremetz Jean François Dubremetz UMR 5539 CNRS, Université de Montpellier 2, CP 107, Place Eugène Bataillon, F-34090 Montpellier, France Search for more papers by this author Daniel Bout Daniel Bout UMR Université-INRA d'Immunologie Parasitaire, Faculté des Sciences Pharmaceutiques et Biologiques, 31 Avenue Monge, F-37200 Tours, France Search for more papers by this author Maryse Lebrun Corresponding Author Maryse Lebrun UMR Université-INRA d'Immunologie Parasitaire, Faculté des Sciences Pharmaceutiques et Biologiques, 31 Avenue Monge, F-37200 Tours, France Search for more papers by this author Odile Cérède Odile Cérède UMR Université-INRA d'Immunologie Parasitaire, Faculté des Sciences Pharmaceutiques et Biologiques, 31 Avenue Monge, F-37200 Tours, France Search for more papers by this author Jean François Dubremetz Jean François Dubremetz UMR 5539 CNRS, Université de Montpellier 2, CP 107, Place Eugène Bataillon, F-34090 Montpellier, France Search for more papers by this author Daniel Bout Daniel Bout UMR Université-INRA d'Immunologie Parasitaire, Faculté des Sciences Pharmaceutiques et Biologiques, 31 Avenue Monge, F-37200 Tours, France Search for more papers by this author Maryse Lebrun Corresponding Author Maryse Lebrun UMR Université-INRA d'Immunologie Parasitaire, Faculté des Sciences Pharmaceutiques et Biologiques, 31 Avenue Monge, F-37200 Tours, France Search for more papers by this author Author Information Odile Cérède1, Jean François Dubremetz2, Daniel Bout1 and Maryse Lebrun 1 1UMR Université-INRA d'Immunologie Parasitaire, Faculté des Sciences Pharmaceutiques et Biologiques, 31 Avenue Monge, F-37200 Tours, France 2UMR 5539 CNRS, Université de Montpellier 2, CP 107, Place Eugène Bataillon, F-34090 Montpellier, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:2526-2536https://doi.org/10.1093/emboj/21.11.2526 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The phylum Apicomplexa is a large group of mostly intracellular protozoan parasites of considerable medical and veterinary importance, responsible for diseases such as malaria, toxoplasmosis, neosporosis, coccidiosis and cryptosporidiosis. A key step in infection is host cell invasion. The sophisticated invasion apparatus of the Apicomplexa involves the sequential exocytosis of two types of apical secretory organelles, i.e. micronemes and rhoptries. Recent investigations have highlighted the central role of micronemes in the recognition–adhesion to host cells (for reviews see Menard, 2001; Soldati et al., 2001). Immunofluorescence observations of invading Apicomplexa parasites have shown that micronemal proteins (MICs) are rapidly redistributed onto the apical end of the parasite at the attachment site and then progressively capped, in an actin-dependent manner, toward the posterior end of the parasite, where they are finally released. This surface capping and posterior relocalization during penetration superimposes exactly with the timing of invasion. Moreover, in being able to bind to host cell surface receptors, MICs function as adhesins (Soldati et al., 2001). The recruitment of a high density of secretory adhesins on the surface of parasites at the time of invasion and their ability to interact with host cell receptors are likely to induce a tight attachment of the parasites to the host cell surface, which could then initiate invasion. Many MICs are processed extensively either before storage in the microneme compartment or after release from micronemes (Soldati et al., 2001). This latter event may be responsible for disengaging receptors prior to completing invasion. The biological significance of the processing event occurring before sorting in micronemes and the proteases involved are still unknown. MICs contain structural domains that are morphologically and functionally conserved at the molecular level (Kappe et al., 1999; Di Cristina et al., 2000). Some of these domains present homology with known adhesive domains from higher eukaryote proteins (for a review see Tomley and Soldati, 2001). They include: (i) the type I repeat of thrombospondin; (ii) the I-domains (an integrin-like adhesive domain); (iii) epidermal growth factor (EGF)-like domains; (iv) the Apple domains; and (v) chitin-binding domains. These motifs are present in one or multiple copies and a growing number of possible combinations of these modules has been identified, so that every MIC is structurally unique. Among Apicomplexa parasites, Toxoplasma gondii has the broadest host range. It is able to invade and infect virtually any nucleated cell from warm-blooded vertebrates. The molecular basis controlling host cell-type specificity has not yet been elucidated, but might involve receptor-mediated adhesive interactions and therefore the repertoire of MICs. This repertoire is currently expanding. It contains membrane-spanning or soluble MICs (Soldati et al., 2001). Recent studies have indicated that soluble and transmembrane MICs assemble into multimolecular complexes, and that transmembrane proteins function as escorts, targeting soluble adhesins to the micronemes (Meissner et al., 2002; Rabenau et al., 2001; Reiss et al., 2001). We have characterized, in T.gondii, a soluble micronemal protein, MIC3 (Achbarou et al., 1991). MIC3 is a dimeric 90 kDa protein, synthesized as 40 kDa precursors that are proteolytically processed to 38 kDa final products during their trafficking through the secretory pathway and before storage in mature organelles (J.F.Dubremetz, unpublished). It is escorted to micronemes by MIC8 (Meissner et al., 2002) and binds to all nucleated cells tested to date, and does so only in non-reducing conditions, indicating that an appropriate conformation of the protein is required for binding (Garcia-Reguet et al., 2000). MIC3 contains five partially overlapping EGF-like domains and a chitin-binding-like domain found in lectins and plant chitinases, which can be involved in protein–protein or protein–carbohydrate interactions, respectively. In this study we have carried out a structure–function analysis of MIC3, which has demonstrated that the lectin-like domain is responsible for the adhesion and that N-terminal pro-peptide cleavage and C-terminal dimer formation are needed to allow the expression of this binding property. Results In mammalian cells, MIC3 is synthesized as a dimeric pro-protein In order to determine the functional domain(s) of MIC3, which promotes adhesion to cell surfaces, we have produced recombinant MIC3 proteins in eukaryotic cells and studied their interaction with host cells. The entire MIC3 nucleotide-coding sequence was therefore cloned in the mammalian expression vector pcDNA3 and transiently expressed in baby hamster kidney (BHK)-21 cells. Lysates of transfected cells were then analyzed on immunoblots. As shown in Figure 1A, anti-MIC3 antibodies reacted specifically against cells transfected with the MIC3 gene. Recombinant MIC3 migrated at the size expected for a dimer (100 kDa) under unreducing conditions and for a monomer (42 kDa) in reducing conditions. This result showed that dimerization of recombinant MIC3 was obtained in mammalian cells. However, recombinant MIC3 migrated at a higher molecular weight than native MIC3 found in T.gondii tachyzoites. The difference was consistent with a failure to cleave the MIC3 pro-peptide in BHK-21 cells. This was confirmed by probing the immunoblots with a specific anti-pro-peptide serum. This serum strongly labeled recombinant MIC3 in BHK-21 cells and the small amount of immature MIC3 (proMIC3) found in tachyzoite lysate, providing direct evidence that the pro-peptide was not cleaved (Figure 1B). Identical results were obtained with 293 T or Vero cells transfected with the MIC3 gene (data not shown). This recombinant MIC3 protein was thereafter named R-proMIC3. Taken together, these results indicate that the natural processing of MIC3 involves a protease that does not traffic with MIC3 in mammalian cells, or is specific to T.gondii. Figure 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. Download figure Download PowerPoint Processing of the MIC3 pro-peptide is a prerequisite to the expression of the binding function of the protein To determine whether R-proMIC3 possessed adhesin function, its interaction with putative host cells was tested in 'cell blot' experiments. Extracts of cells transfected with the MIC3 gene (p-SS-PRO-MIC3 plasmid) were separated by SDS–PAGE and transferred onto a nitrocellulose sheet, which was then incubated with Vero cell suspension. As expected, the cells bound strongly to native MIC3 (Figure 2B, lane 1). In contrast, cells were unable to bind to R-proMIC3 (lane 3). The possibility that the presence of the pro-sequence was inhibiting the adhesive function of the protein was then considered. We thus constructed a plasmid encoding the MIC3 signal sequence fused directly to the mature MIC3 coding sequence, and the resulting p-SS-MIC3 plasmid was transfected into BHK-21 cells. After 24 h of expression, cells were analyzed by western and cell blots. Anti-MIC3 antibodies reacted with protein bands migrating with molecular weights expected for MIC3 dimers in unreducing conditions and monomers in reducing conditions, indicating that the pro-sequence is not essential for dimerization of MIC3 (Figure 2A). This recombinant protein was thereafter named R-MIC3. It appeared as two closely migrating bands co-migrating with MIC3 from the T.gondii lysate. These two bands showed affinity to host cell surfaces in cell blot experiments (Figure 2B, lane 4), contrasting with the absence of binding of R-proMIC3 (lane 3) and showing that processing of the pro-peptide is a prerequisite for the expression of MIC3 binding properties. Figure 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). Download figure Download PowerPoint R-MIC3 is strongly expressed at the cell surface of transfected mammalian cells, although it contains no transmembrane sequence The presence of the recombinant proteins in the supernatant of transfected cells and their localization in cells were then investigated using western blotting and immunofluorescence microscopy. Sequences allowing expression of R-proMIC3 and R-MIC3 were first subcloned in-frame with a sequence coding for the V5 epitope, in pTRACER-A vector, which allows co-expression of the sequence of interest and of green fluorescent protein (GFP). GFP is expressed in the cytoplasm and its intrinsic fluorescence allows the direct visualization of transfected cells. In transfected cells expressing R-proMIC3 and permeabilized after fixation, anti-MIC3 (data not shown) and anti-V5 antibodies (Figure 3A, left panel) gave a perinuclear and vesicular labeling pattern, suggesting that R-proMIC3 was present in the secretory pathway. Western blots of supernatant showed abundant secretion in the cell culture media (Figure 3B). GFP was not detected in supernatants (data not shown), confirming that the presence of R-proMIC3 in supernatants was not due to cell lysis. When cells were not permeabilized, a few fluorescent dots were found on some transfected cells (Figure 3A, right panel). In contrast, in cells expressing R-MIC3, strong anti-MIC3 (data not shown) or anti-V5 labeling was detected on transfected cells, permeabilized (Figure 3A, left panel) or not (right panel), indicating that R-MIC3 was bound to the transfected cell surface. The surface expression of R-MIC3 was essentially observed in transfected cells (GFP fluorescence), but also in some cases in non-transfected adjacent cells (absence of GFP; Figure 3C). Apart from a putative signal peptide, MIC3 has no other hydrophobic region suggestive of a transmembrane domain. Therefore, these results indicate that surface localization of mature MIC3 occurs by direct binding with its cell surface receptor. Single optical sections in confocal analysis (Figure 3D) confirmed that, in contrast to GFP, which was homogeneously distributed in the cytosol, R-MIC3 was found essentially associated with the plasma membrane, with barely detectable cytosol staining. Remarkably, the labeling extended beyond the edge of the cell surface. To confirm the difference between R-proMIC3 and R-MIC3 in their affinity for the host cell surface, we biotinylated the surface proteins of transfected cells, immunopurified recombinant MIC3 proteins from the cells and compared the amount of R-proMIC3 and R-MIC3 at the cell surface (streptavidin probing), and analyzed protein secretion in supernatants. When comparing similar amounts of immunopurified proteins, more R-MIC3 than R-proMIC3 was found to be biotinylated, showing a higher expression on the surface, whereas comparison of similar volumes of supernatant showed a large amount of R-proMIC3 and little R-MIC3, demonstrating that the absence of R-proMIC3 on the surface was not due to a defect in secretion (Figure 3B). Taken together, these results showed that the signal peptide of MIC3 allowed correct sorting of recombinant proteins into the secretory pathway of mammalian cells and that recombinant R-MIC3 bound to the surface of transfected cells, whereas R-proMIC3 had a much lower affinity for the host cell surface. Figure 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. Download figure Download PowerPoint Dimerization plays a crucial role in the expression of the binding properties of the lectin-like motif The deduced protein sequence of MIC3 contains a lectin-like domain (Wright et al., 1991) and five EGF-like domains, of which three are found in tandem (EGF234) whereas two other less conserved domains overlap with the others (EGF1 and EGF5; Figure 4A). Figure 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–AA294–359, 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. Download figure Download PowerPoint In order to identify domains of MIC3 essential for the binding process, we cloned the corresponding sequences in pTRACER-A, transfected mammalian cells, and analyzed the recombinant proteins by immunofluorescence, taking advantage of the capacity of recombinant mature MIC3 to relocalize at the surface of transfected cells and using this parameter as an indicator of the binding properties. All constructs contained the MIC3 signal peptide, were deleted of the pro-peptide and fused at their C-terminal end to the V5 epitope. The efficiency of secretion of the different constructs was analyzed by western blotting of equivalent amounts of cells and supernatants after 18 h of expression. Dimerization status was analyzed by western blotting in reduced and unreduced conditions. The nomenclature of recombinant proteins, their dimerization, their binding properties and their secretion are summarized in Figure 4. Consistent with the strong binding properties of R-MIC3, a large amount of this protein was present in pellets compared with the supernatant (Figure 4A). Conversely, in the absence of binding, R-proMIC3 was essentially secreted in the supernatant. The mobility shift observed for R-proMIC3 and R-MIC3 compared with that in Figure 2B is consistent with the addition of V5 epitope followed by His6 residues. Two recombinant proteins were then generated that comprised either the EGF-like motifs or the lectin-like motif. The recombinant EGF2345 protein was expressed as variable amounts of protein bands migrating with molecular weights expected for dimers in non-reducing conditions and monomers in reducing conditions (Figure 4B). They were all recognized by anti-MIC3 antibodies (data not shown). The recombinant EGF2345 protein was dimeric and abundantly secreted, but did not bind to the surface of transfected cells (Figure 4A) and failed to bind cells in cell blot experiments (data not shown). When expressed alone, the lectin-like domain was expressed as a monomer (Figure 4B). This construct was always weakly expressed and secreted, and no transfected cell expressed the protein at the surface. These results indicate that the lectin-like domain is probably essential for binding, and has no potential for dimerization. New constructs were generated in which EGF-like motifs were successively added to the lectin-like domain. In summary, R-lectin–EGF12, R-lectin–EGF123 and R-lectin–EGF1234 were all monomerics, secreted, and not adhesins (Figure 4). These results suggest that the C-terminal part of MIC3 is involved in dimerization and that the dimerization may be essential for the adhesive function. Finally, the last amino acids (294–359) of MIC3 were fused to R-lectin, R-lectin–EGF12 or R-lectin–EGF123 to create R-lectin–AA294–359, R-lectin–EGF12–AA294–359 and R-lectin–EGF123–AA294–359 chimeras. The three chimeras were dimerics (Figure 4B), and R-lectin–EGF12–AA294–359 and R-lectin–EGF123–AA294–359 chimeras exhibited surface relocalization in transfected cells (Figure 4A). R-lectin–AA294–359 was poorly secreted and accumulated in wide perinuclear vesicules. The binding properties of this construct were, therefore, difficult to analyze. A gradual increase in the intensity of sur face detection was observed (R-lectin–EGF12–AA294–359 < R-lectin–EGF123–AA294–359 < R-MIC3), parallel to an increasing retention of the proteins in pellets. Taken together, these results showed that the amino acid stretch 294–359 is sufficient for dimerization and that dimerization plays a crucial role in the expression of the binding properties of the lectin-like motif, which may itself be modulated by the addition of the EGF motifs. Dimerization of another lectin-like-containing protein, MIC8, leads to acquisition of adhesive function MIC3 shares a significant degree of homology with the recently described T.gondii microneme protein MIC8 (Meissner et al., 2002). The two proteins contain a peptide signal, a chitin-binding-like domain and several EGF-like domains (Figure 5A). In MIC8, the last EGF motif is followed by a hydrophobic region, likely to promote transmembrane insertion, and a short cytoplasmic domain. MIC8 lacks the dimerization domain of MIC3, and is detectable in immunoblots of tachyzoites as a monomer of uncharacterized adhesive properties. Homologous to what we had observed with MIC3, we suspected that dimerization of MIC8 could reveal adhesive properties. Figure 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–AA294–359-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. Download figure Download PowerPoint New constructs were generated in pTRACER-A to transiently express: (i) R-MIC8; (ii) R-MIC8ΔTMCD (deletion of transmembrane and cytoplasmic domains); and (iii) the chimera R-MIC8ΔTMCD–AA294–359MIC3 in which the dimerization stretch of MIC3 was fused in-frame with the C-terminal end of MIC8ΔTMCD (Figure 5A). A V5 epitope sequence was fused at the end of R-MIC8 and of the chimera. Dimerization and binding properties were analyzed as described above, using anti-V5 or anti-MIC8 serum (this serum was raised against EGF domains and does not recognize the last 66 amino acids of MIC3). R-MIC8 was strongly expressed at the plasma membrane of transfected cells (Figure 5C). Under permeabilization, both polyclonal anti-MIC8 and anti-V5 (data not shown) strongly labeled R-MIC8, while in the absence of permeabilization, only anti-MIC8 serum revealed R-MIC8 at the surface of transfected cells. The inaccessibility of the C-terminal V5 epitope suggested that the C-terminal hydrophobic region of MIC8 promoted efficient type 1 transmembrane insertion in mammalian cells. Consistently, R-MIC8 was not found in culture media (Figure 5B, lane 2). As expected, deletion of the transmembrane domain (R-MIC8ΔTMCD) led to secretion of the protein in supernatants (Figure 5B, lane 3). R-MIC8ΔTMCD was not found on the surface of transfected cells (Figure 5C). While R-MIC8 and R-MIC8ΔTMCD were exclusively detected as monomers in immunoblots, R-MIC8ΔTMCD–AA294–359MIC3 was detected as a monomer and a dimer with both anti-MIC8 (Figure 5B, lane 4) and anti-V5 (data not shown) antibodies. The recombinant chimera was detected in supernatant essentially as a dimer. It was detected by IFA at the surface of transfected cells, with both anti-MIC8 (Figure 5C) and anti-V5 (data not shown) antibodies, showing that unlike R-MIC8, the surface localization of R-MIC8ΔTMCD–AA294–359MIC3 was not due to transmembrane insertion and, like R-MIC3, probably resulted from direct binding of the chimera with cell surface molecules. Taken together, these results confirmed that amino acids 294–359 of MIC3 allow dimerization, and that such dimerization could induce binding of a MIC3 homolog. Dimerization of MIC3 occurs through protein–protein interaction Dimerization of proteins may occur through inter-chain disulfide bonds or through protein–protein dimerization motifs like leucine zipper or helix–loop–helix motifs. No consensus for such motifs was found in the dimerization stretch of MIC3. However, four cysteines were present. All four cysteine residues were mutagenized individually to glycine, and the dimerization of recombinant mutated proteins was analyzed (Figure 6). No single cysteine mutation (Cys302, Cys304, Cys339 or Cys354) abolished dimerization (Figure 6B). We then performed double mutations (Cys302,304 and Cys339,354) and a quadruple mutation (Cys302,304,339,354), but all mutants dimerized (Figure 6B). These data clearly indicated that dimerization through the amino acid stretch 294–359 does not involve inter-chain disulfide links and is probably due to protein–protein interaction. Figure 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. Download figure Download PowerPoint Discussion During their transport to micronemes, some of the MICs undergo proteolytic processing of unknown biological significance. The data reported here stress the importance of two post-translational events, N-terminal pro-peptide cleavage and C-terminal dimer formation, in the expression of the binding properties of the micronemal protein MIC3. We also show that EGF-like domains of MIC3 are not able to promote recognition of host cell receptor, a function that can be attributed
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