C-terminal Processing of the Toxoplasma Protein MIC2 Is Essential for Invasion into Host Cells*

Fabien Brossier, Travis J. JewettDagger, Jennie L. LovettDagger, and L. David Sibley§

From the Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri 63110

Received for publication, September 25, 2002, and in revised form, December 3, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Host cell invasion by apicomplexan parasites is accompanied by the rapid, polarized secretion of parasite proteins that are involved in cell attachment. The Toxoplasma gondii micronemal protein MIC2 contains several extracellular adhesive domains, a transmembrane domain, and a short cytoplasmic tail. Following apical secretion, MIC2 is transiently present on the parasite surface before being translocated backward and released by proteolytic cleavage. Mutations in the extracellular domain of MIC2, directly upstream of the transmembrane domain, prevented processing and release of the soluble protein into the supernatant. A conserved basic residue in MIC2 was essential for cleavage, and basic residues are similarly positioned in other microneme proteins. Following the induction of secretion, MIC2 processing mutants were stably expressed on the surface of the parasite. Surface MIC2-expressing mutants showed increased adhesion to host cells, yet were impaired in their capacity to invade. These data demonstrate that proteolysis is essential for releasing cell surface adhesins prior to cell entry by apicomplexan parasites.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Toxoplasma gondii is member of the phylum Apicomplexa, a group of medically and economically important obligate intracellular parasites that includes Plasmodium, the causative agent of malaria. One of the first events in host cell recognition by T. gondii is the polarized release of parasite adhesins from secretory vesicles called micronemes (1). Micronemal proteins contain a variety of conserved adhesive domains, and several have been demonstrated to bind to host cells (2). Despite their important role in cell attachment, microneme proteins only transiently occupy the parasite cell surface during the brief interval of cell invasion before being released into the extracellular medium. MIC2 is composed of a short cytoplasmic tail, a transmembrane (TM)1 domain, and an extracellular region composed of an integrin A-domain and six type 1 thrombospondin (TSP-1)-like repeats (3). The T. gondii microneme protein MIC2 belongs to the family of thrombospondin-related anonymous proteins (TRAPs) previously shown to be essential for host cell invasion by Plasmodium sporozoites and ookinetes (4-6). MIC2 is normally stored in the micronemes and is rapidly discharged at the apical tip of the parasite when it contacts the host cell (7). Following secretion, MIC2 is translocated backward as the parasite enters the cell, occupying the junction that forms between the host and parasite cell membranes. MIC2 is released into the medium by C-terminal processing that is carried out by a parasite protease called MPP1 (8). The cleavage specificity of this protease is unknown, and it is insensitive to a variety of commonly used protease inhibitors. Throughout the processes of secretion, translocation, and release, MIC2 is complexed with another protein called MIC2-associated protein (M2AP), which is comprised of two extracellular domains but is not predicted to be anchored in the membrane (9).

The cleavage of micronemal proteins upon secretion is a common feature of this protein family that occurs in MIC2, MIC6, MIC8, MIC12 (10), and AMA-1 (11). It was recently reported that extensive mutations within the conserved TM domains of MIC2, MIC6, and MIC12 prevented their release into the medium, suggesting that processing of micronemal proteins may occur within the membrane (10). Intramembrane processing has been reported for proteins produced by a broad range of organisms from bacteria to humans (12). However, in other examples of intramembrane processing, cleavage within the membrane-spanning domain is always preceded by a separate proteolytic processing step that releases the extracellular domain (12).

We show here that mutations in the exodomain of MIC2, directly upstream of the TM domain, prevent the cleavage and release of the protein from the cell surface, revealing that microneme proteins are likely processed in multiple steps. Parasites expressing uncleaved MIC2 on their surface showed increased adherence to host cells but impaired invasion, demonstrating an important role for the processing of MIC2 during cell entry.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Antibodies-- MIC2 was detected with monoclonal antibody 6D10 as described previously (3). The c-Myc epitope (EQKLISEEDL) was detected with mouse monoclonal antibody 9E10 (Zymed Laboratories Inc.), and the HA9 epitope was detected using rabbit antisera (Zymed Laboratories Inc.). Rabbit anti-SAG1 was provided by Lloyd Kasper (Dartmouth Medical School). M2AP and MIC5 were detected using rabbit antisera provided by Vern Carruthers (Johns Hopkins University). Chemicals were obtained from Sigma.

Growth of Host Cells and Toxoplasma Strains-- T. gondii tachyzoites of the RH hxgprt- strain (obtained form David Roos, University of Pennsylvania) were maintained by growth in monolayers of human foreskin fibroblast (HFFs), propagated in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 2 mM glutamine, 20 mM HEPES, pH 7.5, and 20 µg ml-1 gentamicin (referred to as D10). Transformed strains were cultivated in D10 supplemented with 25 µg ml-1 of mycophenolic acid and 50 µg ml-1 of xanthine.

Construction of an Expression Cassette for the MIC2 Gene-- Constructs for expression of MIC2 in T. gondii were based on the HXGPRT selection plasmids described previously (13). The full-length MIC2 cDNA encoding the complete open reading frame and ~ 1 kb of 5'- and 3'-flanking genomic regions was inserted into this vector to create MIC2 expression plasmids for T. gondii. PCR-based cloning was used to add an HA9 tag (consisting of the residues YPYDVPDYAL) after amino acid 769, followed by a stop codon. The c-Myc epitope was inserted in-frame, replacing blocks of 10 amino acids at a time upstream of the TM domain of MIC2 from Ser-659 to Gly-698. PCR amplification was used to create point mutations in a plasmid bearing the MIC2 gene harboring the c-Myc tag from position Glu-679 to Gly-688 and the HA9 tag at position 770. Gene-specific primers were designed to change the residues 692SKKE695 to SAAE, SAKE, SKAE, SRAE, or SKER. The correct sequence of constructs was confirmed by cycle sequencing, and expression of the epitope-tagged forms of MIC2 was verified by transient expression in T. gondii as described previously (13).

Microneme Secretion Assay-- Freshly harvested parasites were purified and resuspended in 100 µl of PBS-Ca2+ containing 1% ethanol and incubated for 15 min at 37 °C. After centrifugation (400 × g, 10 min), the supernatant containing secreted microneme proteins was used for Western blot experiments and the parasite pellet was used for Western blot and immunofluorescence experiments. Samples were separated by 10% SDS-PAGE, and proteins were transferred semi-dry to nitrocellulose membranes. Western blots were performed as described previously using ECL (Pierce) (8).

Immunoprecipitation-- Polyclonal anti-M2AP or pre-immune rabbit sera were incubated separately with 100 µl of rec-protein A-coated Sepharose beads (Zymed Laboratories Inc.) in 300 µl of PBS overnight at 4 °C. Antibody coated beads were washed three times in 500 µl of RIPA buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 0.5% sodium deoxycholate, 0.2% SDS, 100 mM NaCl2, 5 mM EDTA). Purified parasites (5 × 107) were resuspended in 100 µl of RIPA buffer and incubated for 15 min at room temperature. Insoluble material was removed by centrifugation at 14,000 rpm for 10 min at room temperature. The lysates were added to the beads and incubated at 4 °C for 4 h. Beads were washed five times with RIPA buffer, and proteins bound to the beads were dissolved in 40 µl of SDS-PAGE sample buffer. The amount of proteins loaded onto SDS-PAGE gels was normalized to the percentage of parasites expressing each epitope-tagged MIC2 protein. Proteins were transferred to nitrocellulose membranes for immunoblotting with the mouse monoclonal antibody anti-c-Myc.

Indirect Immunofluorescence (IF) Microscopy-- To detect microneme proteins within the parasite, HFF cell monolayers were infected with parasites and incubated overnight prior to being fixed and processed for IF (14). Permeabilized cells were incubated with anti-c-Myc, anti-MIC2, or anti-MIC5 antibodies and then washed and incubated with secondary antibodies conjugated to Alexa 494 or Alexa 488 (Molecular Probes, Eugene, OR). Coverslips were washed and mounted in Vectashield containing DAPI (Vector Laboratories, Burlingame, CA). To detect microneme proteins at the cell surface, parasites were stimulated to secrete (see above) and then fixed and processed for IF as described previously (15). Non-permeabilized parasites were incubated with anti-c-Myc, rabbit anti-SAG1, or rabbit anti-M2AP primary antibodies followed by the appropriate secondary antibodies, and the cells were mounted in Vectashield. Slides were examined using a Zeiss Axioplan (Carl Zeiss, Inc.) microscope equipped with phase contrast and epifluorescence optics. Images were obtained using a Zeiss AxioCam cooled charge-coupled device camera directed by Zeiss Axiovision software and processed using Photoshop 4.0 (Adobe Systems, San Jose, CA).

Parasite Adherence and Invasion Assays-- HFF monolayers were infected with parasites during a 5-min pulse, washed, fixed gently in 3.4% paraformaldehyde, and incubated with anti-SAG1 antibodies to reveal extracellular parasites. Following detergent permeabilization, cells were incubated with an anti c-Myc antibody to reveal transfected parasites expressing epitope-tagged forms of MIC2. Monolayers were washed and incubated with appropriate secondary antibodies and mounted in Vectashield containing DAPI.

To determine the percentage of transfected parasites that were adherent, the proportion of c-Myc-positive parasites in the starting inoculum (c-Myc positive) was compared with the proportion of parasites that were attached to host cells following a brief invasion pulse and staining for the surface protein SAG1 (c-Myc positive and SAG positive). Data are presented as fold increase that was calculated as the percentage of c-Myc-positive parasites bound to host cells per the percentage of c-Myc-positive parasites in the inoculum. Values represent the mean ± S.E. from three experiments.

To determine the percentage of transformed parasites that successfully entered the host cells, the number of c-Myc positive parasites that was attached to host cells (SAG1 positive and DAPI positive) was compared with the number that was intracellular (SAG1 negative but DAPI positive). Values are presented as the percentage of invasion based on three similar experiments (mean ± S.E.).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Mutations in the Extracellular Domain of MIC2 Block Processing-- The c-Myc epitope was inserted to generate in-frame mutations upstream of the predicted TM domain of MIC2 (Fig. 1A). To detect processing, the proteins were also tagged with the HA9 epitope at the C terminus (Fig. 1A). Immunofluorescence staining with antibodies to c-Myc and another microneme protein, MIC5, revealed that all the epitope-tagged proteins were faithfully targeted to micronemes in the parasite (Fig. 1B). Epitope-tagged genes were transiently transfected into T. gondii, and secretion of the expressed proteins was evaluated by Western blot analysis of proteins released into the supernatant. Insertion of the c-Myc epitope at residues 689-698 in the construct referred to as mut4 completely prevented the release of the protein into the supernatant following stimulation of secretion (Fig. 2A). The region of MIC2 that was removed in creating mut4 contains two lysines located at positions 692 and 693, and this site resembles the substrates for trypsin-like enzymes, including subtilisins. To test the involvement of these residues, the two lysines were replaced by alanines to create mutAA (Fig. 1A). The mutAA form of MIC2 was not released into the supernatant after induction of secretion (Fig. 2A). This lack of processing was not a consequence of the c-Myc epitope, as placement of the tag at residues 679-688 in the construct referred to as MIC2-TAG, or at other sites upstream of this, did not prevent secretion (Fig. 2A, and data not shown). Transient expression of epitope-tagged forms of MIC2 did not discernibly affect secretion of the endogenous wild type MIC2 protein (Fig. 2A, and data not shown). The HA9 tag was only detected in the full-length form of the protein found in the cell pellet (Fig. 2A), indicating that, when secreted, the C terminus was removed by proteolytic processing as described previously (8).


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Fig. 1.   Epitope-tagged and mutant constructs of MIC2 are normally packaged in the micronemes. A, schematic representation of MIC2 constructs. The wild type amino acid sequence from position 689 to 696 is shown beneath the wild type protein (top) and in the epitope-tagged form MIC-TAG (second from top). The location of the c-Myc tag (gray box) is indicated in parentheses where the amino acids (single letter code) and position within the protein are given in superscript. Point mutations introduced in the mutant mutAA are shown beneath the lower construct. The HA9 epitope is located between residues 770 and 780 in each construct (white box). Black bars represent the transmembrane domain. B, MIC2-TAG and mutant forms of MIC2 were correctly localized to micronemes. Intracellular parasites were permeabilized, incubated with anti-c-Myc and anti-MIC5 antibodies, and the proteins were revealed using secondary antibodies coupled to Alexa 594 (red) and Alexa 488 (green), respectively. Scale bar = 2 µm.


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Fig. 2.   MIC2 processing mutants are stably retained on the cell surface following secretion. A, Western blot analysis of secreted microneme proteins by transiently transfected parasites. Although epitope-tagged MIC2 (MIC2-TAG) was secreted normally and appeared in the supernatant (S), insertion of the c-Myc tag within mut4 or the point mutations in mutAA disrupted secretion and resulted in retention in the pellet (P). The tagged proteins were detected using antibodies against c-Myc (middle panel) or HA9 (bottom panel). As an internal control for secretion of the endogenous form of MIC2, the monoclonal antibody against MIC2 (6D10) was used in the top panel. All three panels contain proteins from the MIC2-TAG, mut4, and mutAA parasites, respectively. B, immunolocalization of epitope-tagged MIC2 proteins at the surface of the parasites following secretion. Although the wild type (MIC2-TAG) protein was turned over rapidly, both mut4 and mutAA forms accumulated on the surface, primarily at the posterior end of the cell. Following stimulation of secretion, cells were fixed and then incubated with anti-c-Myc and anti-SAG1 antibodies in the absence of permeabilization. Epitope-tagged MIC2 proteins and SAG1 were detected using secondary antibodies coupled to Alexa 494 (red) and Alexa 488 (green), respectively. The arrows represent the extruded conoid at the apical end of the parasites. Scale bar = 2 µm.

MIC2 Processing Mutants Remain Associated with the Parasite Surface after Secretion-- MPP1 is a parasite protease that acts on MIC2 only once it has been released onto the parasite surface (8). Thus, the lack of secretion of mut4 and mutAA could be due to their failure to be released onto the cell surface following stimulation of secretion. The localization of epitope-tagged forms of MIC2 was examined after stimulation of microneme discharge. Parasites were stained with antibodies in the absence of permeabilization to reveal proteins on the cell surface (Fig. 2B). Epitope-tagged MIC2 (MIC2-TAG) was not detected on the parasite cell surface (Fig. 2B), consistent with the fact that this protein is rapidly secreted, processed, and released into the supernatant as described previously (8) (see also Fig. 2A). In contrast, parasites expressing mut4 or mutAA were strongly labeled with anti c-Myc antibodies on their surface as shown by colocalization with staining for the cell surface protein SAG1 (Fig. 2B). Moreover, mut4 and mutAA were often concentrated at the posterior end of the parasites, whereas the extruded conoid, located at the apical end, was weakly labeled (Fig. 2B). Absence of staining with antibodies to the HA9 epitope, which is attached to the cytoplasmic domain (8), was used to verify that the parasites that stained positively for epitope-tagged MIC2 were not permeabilized during the IF procedure (data not shown).

During secretion and surface translocation, MIC2 remains associated with the protein M2AP (9). Epitope-tagged MIC2 proteins co-immunoprecipitated with M2AP (Fig. 3A), showing that the mutations and/or the insertion of the c-Myc tag do not alter the interaction with M2AP. In parasites expressing the non-processed forms of MIC2, M2AP was retained on the cell surface (Fig. 3B). In contrast, in parasites expressing wild type epitope-tagged MIC2 (MIC2-TAG), M2AP was not retained on the surface of the cell (Fig. 3B). To examine whether the surface localization of other MIC proteins was affected by the expression of non-processed forms of MIC2, we performed surface IF assay staining as described above. No accumulation of MIC4, MIC5, or MIC6 was observed in parasites expressing non-processed forms of MIC2 (data not shown). Collectively, these data show that, upon secretion, mutAA and mut4 are transferred to the surface of the parasite where they are complexed with M2AP; however, they are not released into the supernatant because of the failure of MIC2 to be proteolytically processed.


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Fig. 3.   M2AP interacts with epitope-tagged MIC2 proteins. A, immunoprecipitation of epitope-tagged MIC2 using M2AP-specific antibodies. Lysates of strains producing MIC2-TAG, mut4, and mutAA were incubated with beads coated with rabbit anti-M2AP (M2AP) or prebleed sera (prebleed). Proteins bound to the beads were resolved on 10% SDS-PAGE gels and Western blotted using anti-c-Myc antibodies. B, immunolocalization of endogenous M2AP and epitope-tagged MIC2 at the surface of parasites following secretion. Although wild type parasites (MIC2-TAG) rapidly shed MIC2/M2AP from the surface, this complex accumulated on the cell surface in parasites expressing mut4 and mutAA mutants. Following stimulation of secretion, parasites were fixed and then incubated with anti c-Myc and anti M2AP antibodies in the absence of permeabilization. The presence of the proteins at the surface of the parasites was detected using a secondary antibody coupled to Alexa 494 (red) and Alexa 488 (green), respectively. Unlabeled parasites correspond to untransfected parasites. Scale bar = 2 µm.

MIC2 Processing Mutants Show Enhanced Adherence to Host Cells but Are Impaired in Invasion-- We analyzed whether the enhanced retention of MIC2 on the surface of parasites expressing processing mutants would result in alterations in binding to host cells. Because the percentage of cells expressing the epitope-tagged MIC2 proteins (c-Myc positive parasites) was different between constructs and in separate experiments (a natural feature of transient expression assays), binding was expressed as a fold change in the proportion of positively expressing cells that were attached to host cells versus the proportion that was present in the challenge inoculum (varied from 5-35%). Parasites expressing the processing mutant mut4 or mutAA strains showed enhanced binding to HFF cells by 3.8- and 2.8-fold, respectively (Fig. 4A). In contrast, the ratio observed for parasites expressing MIC2-TAG was 0.9, indicating that their capacity to adhere to the cells was not significantly different to that observed in the untransfected population (Fig. 4A). These data indicate that parasites expressing non-processed forms of MIC2 on their cell surface have an enhanced capacity to adhere to host cells.


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Fig. 4.   MIC2 processing mutants show enhanced cell binding but decreased entry. Analysis of cell attachment and invasion by parasites expressing epitope-tagged MIC2. MIC2 processing mutants showed enhanced attachment but are inhibited in cell entry. A, capacity of the mutants to adhere to the surface of the cells. The proportion of adherent parasites was compared with the frequency of positively expressing cells in the inoculum (i.e. percentage of positively expressing cells bound per the percentage of positively expressing cells in the inoculum) to determine a fold difference in attachment. An asterisk (*) indicates a statistically significant difference versus MIC2-TAG, p < 0.05 (Student's t test). B, capacity of the mutants to invade host cells. The percentage of the parasites able to enter host cells is shown for each construct. CTL denotes control or non-transfected parasites. An asterisk (*) indicates a statistically significant difference versus CTL or MIC2-TAG-expressing parasites, p < 0.05 (Student's t test).

To determine whether the enhanced attachment to host cells by surface MIC2-expressing parasites also affected invasion, the efficiency of cell entry was evaluated. When parasites transfected with the MIC2-TAG form were used to challenge host cell monolayers, between 50-70% of the parasites that were associated with the monolayer were also able to enter the host cell (Fig. 4B). Although the efficiency of invasion was reduced in parasites expressing MIC2-TAG, it was not significantly different from that of non-transfected parasites (Fig. 4B). In contrast, only 10% of adherent parasites expressing mut4 or mutAA were able to enter the cells, a result that was significantly different from that observed with MIC2-TAG (p < 0.05, Student's t test). These data indicate that the unusual persistence of MIC2 at the surface of the parasite enhances binding but does not lead to productive cell entry.

A Conserved, Basic Residue Is Required for the Cleavage of MIC2-- To analyze the contribution of Lys-692 and Lys-693 to the cleavage of MIC2, we substituted each amino acid independently with an alanine leading to the construction of mutAK and mutKA constructs (Fig. 5A). Additionally, we generated a mutant, mutRA, in which Lys-692 was substituted by arginine (Fig. 5A). These constructs were transiently expressed in T. gondii, and the capacity of the parasite to secrete the epitope-tagged proteins was analyzed by Western blotting as described above. The supernatants from parasites expressing MIC2-TAG, mutKA, and mutRA contained the secreted form of MIC2, indicating that these forms were properly cleaved by MPP1 (Fig. 5B). In contrast, epitope-tagged MIC2 was not found in the supernatants of mutAA- and mutAK-expressing parasites (Fig. 5B). These data demonstrate that Lys-693 is not essential for the cleavage of MIC2, whereas Lys-692, which can be replaced by arginine, is required for processing. The sequence KER, which is found upstream of the TM domain of MIC6, was also inserted in place of the 691SKK693 of MIC2 to create mutKER. When transfected into T. gondii, the mutKER form of MIC2 was properly secreted, processed, and released into the supernatant (Fig. 5B). The ability of this sequence to be recognized in the context of MIC2 suggests that it may fulfill a similar role in MIC6. Furthermore, analysis of other microneme proteins from T. gondii (TgMIC6, TgMIC12, and TgMIC8), Plasmodium falciparum (PfTRAP) or Neospora caninum (NcMIC2) revealed that a conserved lysine is located within residues -11 and -13 from the putative beginning of their transmembrane domains (Fig. 5C).


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Fig. 5.   A similar basic residue is found upstream of the TM domain in a variety of MIC proteins. A, schematic representation of MIC2 containing point mutations between residues 691 and 693. The position of the c-Myc tag is indicated in parentheses. The HA9 is located between positions 770 and 780 in each mutant. Black bars represent the transmembrane domain, white bars the HA9 tag, and gray bars the c-Myc tag. B, Western blot analysis of microneme proteins secreted by parasites transiently expressing mutant proteins. Following stimulation of secretion, proteins associated with the parasites (pellet, P) or released into the medium (supernatant, S) were electrophoresed on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. The mutant proteins were detected using antibodies against c-Myc. C, comparison of the region upstream of the transmembrane domains of microneme proteins from apicomplexans. The beginning regions of the predicted transmembrane domains are underlined. Boldfaced lysines represent amino acids thought to be important for the cleavage of the proteins. Their localization upstream the transmembrane domain is numbered. Accession numbers are as follows: TgMIC2, AAB63303; TgMIC6, AAD28185; TgMIC12, AAK58479; TgMIC8, AF353165; NcMIC2, AF061273; and PfTRAP, AAC18657.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Proteolytic processing plays an important role in the maturation and activation of microneme protein apicomplexans (2). Processing of the micronemal protein MIC3 has been shown to be essential to activate its adhesive properties (16). Similarly, processing of the malarial protein MSP1 on the surface of merozoites is important for exposing an epidermal growth factor (EGF)-like domain that is involved in cell recognition (17). Following the release of full-length MIC2 from the anterior end of the parasite, it is translocated to the posterior end of the cell before being released into the medium (8, 14). MIC2 occurs as a complex with M2AP that is released from the cell surface following cleavage of MIC2 by a surface or secreted protease called MPP1, which is insensitive to a variety of inhibitors (8).

We demonstrate here that mutations in a sequence just upstream of the predicted transmembrane domain of MIC2 disrupt the processing and prevent release of the protein complex into the medium. Importantly, these mutant forms of MIC2 are still normally packaged in the micronemes, secreted upon stimulation, and efficiently delivered to the cell surface. Processing of cell surface MIC2 was critically dependent on a basic amino acid positioned -11 residues upstream of the predicted TM domain, and a mutation of this residue abolished processing. A similar basic residue is found 11-13 residues upstream of the TM domains of a variety of microneme proteins, suggesting a similar requirement in proteolytic processing. Collectively, these results suggest that MPP1 is responsible for cleavage of a variety of MIC proteins from the cell surface and that it may recognize a sequence that includes a basic residue. A more complete characterization of the processing site of MPP1 awaits additional mutational analysis and determination of the specific cleavage site by peptide sequencing or mass spectrometry.

Our results lead to a different model from that recently proposed for the release of microneme proteins by intramembrane processing (10). Mutations in the conserved TM domains of MIC2 and MIC6 blocked release of chimeric, heterologous proteins into the supernatant (10). This result is somewhat unexpected, because in other well studied examples of intramembrane processing there is little sequence specificity to the cleavage site (18). Rather, the major determinant of which TM domains are susceptible to intramembrane proteolysis is the lack of a significantly sized lumenal or extracellular domain (18). Furthermore, in other examples of intramembrane protein processing, cleavage within the membrane is secondary and is always preceded by a primary cleavage that removes the extracellular or lumenal domain of the protein (12, 19). Our findings clearly establish that the extracellular domain of MIC2 is essential for proteolytic processing that releases MIC2 into the supernatant. Interestingly, Opitz et al., (10) also reported that insertion of c-Myc outside the TM domain disrupted processing of MIC2; however, they did not follow up on the precise residues involved in this effect. Our studies implicate a basic residue just outside the TM domain as being critical for processing of MIC2. Whether this basic residue is involved in the cleavage site per se or is simply part of an extended recognition domain will require further studies to determine.

MIC2 is a homologue of the malaria protein called TRAP, which is essential for gliding motility and cell invasion by malaria sporozoites (5, 20). TRAP mediates entry into mammalian and insect cells, indicating that it contains recognition motifs for highly conserved receptor molecules (21, 22). Recognition of host cell receptors by TRAP is likely mediated by an integrin A domain and a single type 1 thrombospondin domain that have been shown to bind to sulfated glycans in vitro (23-25). MIC2 contains similar adhesive domains for the recognition of glycosaminoglycans and binds to host cells in vitro, suggesting that it may participate in the recognition of host cell glycosaminoglycans by the parasite (14, 26, 27).

Parasites expressing mutant forms of MIC2 on the cell surface exhibited enhanced binding to host cells, suggesting that MIC2 plays a role in cell attachment. Recently, it was shown that disruption of the M2AP gene results in reduced targeting of MIC2 to the micronemes and decreased attachment and entry of host cells.2 Collectively, these findings indicate that the MIC2/M2AP complex plays an important role in cell recognition. Although the stable expression of MIC2 on the parasite surface led to enhance binding to host cells, it had the opposite effect on entry. These findings indicate that the expression of MIC2/M2AP on the cell surface must be carefully regulated and that turnover of the complex is essential for productive entry. Precedent for such a dual role in binding and regulated release has been reported for the vertebrate cell surface hyaluronic acid receptor CD44, which participates in cell-cell and cell-matrix interactions. Proteolytic cleavage of CD44 from the surface of vertebrate cells decreases attachment with the substratum, thus facilitating cell migration, a process important for metastasis of some tumor cells (28).

Typically, adhesins are stably expressed on the surface of microorganisms, where their ability to bind host cell receptors often facilitates entry by activating host cell endocytosis (29). Apicomplexan parasites accomplish this process in a fundamentally different manner. Adhesins are stored in apical secretory organelles and discharged at the time of cell contact. The directional translocation of adhesin-receptor complexes from the anterior to the posterior end of the parasite accompanies host cell penetration. TRAP family adhesins participate in this directional attachment and likely facilitate cell entry by interaction with the parasite's actin-myosin cytoskeleton, which provides the essential propulsive force for cell entry (30-32). Importantly, uncoupling of this process by the prevention of proteolytic processing of MIC2 enhanced binding to host cells but did not lead to productive entry. Our findings underscore the fact that polarized secretion, translocation, and proteolytic processing must be tightly coupled for efficient invasion.

Proteolysis is important in controlling a variety of complex processes in parasites, including cell entry and egress by Plasmodium (33), tissue migration by Schistosoma (34), and development in Giardia (35). Proteolysis is known to activate parasite proteins, enabling them to recognize host cell receptors in a variety of adhesins including MIC3 (16), MSP1 (36), and AMA-1 (37). We demonstrate here that proteolysis is also critically important for releasing parasite adhesins from the cell surface, thus breaking the attachment to host cell receptors prior to the completion of cell invasion by apicomplexan parasites.

    ACKNOWLEDGEMENTS

We thank Antonio Barragan and Dan Goldberg for fruitful discussions and Vern Carruthers for critical review of the manuscript.

    FOOTNOTES

* This work was supported in part by National Institutes of Health Grant AI34036.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Partially supported by National Institutes of Health Institutional Training Grant AI010172 (to Washington University).

§ Recipient of a Scholar Award in Molecular Parasitology from the Burroughs Wellcome Fund and to whom correspondence should be addressed. Tel.: 314-362-8873; Fax: 314-362-3203; E-mail: sibley@borcim.wustl.edu.

Published, JBC Papers in Press, December 5, 2002, DOI 10.1074/jbc. M209837200

2 V. B. Carruthers, personal communication.

    ABBREVIATIONS

The abbreviations used are: TM, transmembrane; TRAP, thrombospondin-related anonymous protein; MPP1, microneme protein protease 1; M2AP, MIC2-associated protein; HFF, human foreskin fibroblast; HA, hemagglutinin; RIPA, radioimmune precipitation assay buffer; IF, immunofluorescence; DAPI, 4',6-diamidino-2-phenylindole.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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