From the W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 21205; and
Division of Parasitology, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom
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ABSTRACT |
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One of the first events in T. gondii invasion, coincident with apical attachment of the parasite to the host cell, is the release of numerous "soluble" and transmembrane (TM)1-anchored proteins from secretory vesicles called micronemes (6, 7). These micronemal proteins (MICs) contain a variety of conserved adhesive domains, and several have been demonstrated to bind to receptors on mammalian cells (8). MIC2 (9) is a micronemal TM protein consisting of six thrombospondin-like adhesive repeats (M-domain), an integrin-like adhesive sequence (A-domain), and a short cytoplasmic tail indirectly connected to the actin-myosin system beneath the plasma membrane of the parasite (10). Within 15 min of synthesis, MIC2 is assembled into a complex with another micronemal protein, M2AP (11). M2AP is composed of a short pro-domain, a ß-sheet domain, and a C-terminal coil domain. The complex remains associated during transit through the Golgi complex and storage in the micronemes. Upon parasite attachment to a host cell, the MIC2/M2AP complex is rapidly secreted onto the apical surface of the parasite. As T. gondii uses its actin-myosin system to actively penetrate into a target cell (12, 13), the MIC2/M2AP complex is translocated over the parasite body to its posterior end (14), where the ectodomain of MIC2 (A-domain and M-domain) is released through C-terminal proteolysis of MIC2, with M2AP still bound in the complex (15). The observation that discharge and translocation of MIC2/M2AP is tightly correlated with invasion has strongly implied an important role for the complex during invasion (11, 14). This is further supported by recent evidence showing that blocking C-terminal proteolysis of MIC2 can impair host cell entry (16). Together with the observation that M2AP-null mutant parasites are markedly defective in host-cell attachment and invasion (17), the collective evidence strongly suggests that MIC2/M2AP is a key protein complex that is required for efficient invasion. Despite this progress, however, the precise molecular details of how MIC2/M2AP facilitates invasion remain poorly understood.
MIC2 and M2AP undergo extensive proteolytic processing during maturation and invasion (11, 15). Indeed, proteolytic processing is a common post-translational modification for Toxoplasma MICs including, for example, AMA1 (18), MIC3 (19), and MIC6 (20). Processing occurs either within the parasite secretory pathway during protein maturation or on the parasite surface during invasion. In the case of M2AP, the pro-domain of M2AP is removed in the Golgi or nascent micronemes. When M2AP is discharged onto the surface of the parasite, it is further truncated into four smaller polypeptides (11). However, the precise cleavage sites and the proteases responsible for the processing remain unknown. Extensive processing of MIC2 also occurs on the parasite surface where this adhesin is multiply cleaved at its N terminus by a parasite-derived protease MPP2 (15). In addition, another parasite-derived protease MPP1 processes MIC2 at an undefined site in its C terminus, releasing the ectodomain into the medium. Recently, the Soldati group (20) demonstrated that a MIC2 hybrid protein containing multiple mutations within its TM anchor was resistant to C-terminal cleavage and shedding. This genetic evidence suggested that C-terminal cleavage might occur within the membrane. However, another recent genetic study by the Sibley group (16) demonstrated that a lysine-containing motif outside the TM segment is necessary for C-terminal processing. Because this C-terminal proteolysis is required for invasion, it is important to address this apparent discrepancy with compelling structural evidence. Also, because MPP1 has not been molecularly cloned or characterized, analysis of its cleavage site specificity could reveal important clues to its enzymatic properties.
Herein we unequivocally demonstrate that MIC2 is liberated from the parasite surface by intramembrane cleavage, based on precise identification of the cleavage site using complementary mass spectrometric approaches. Furthermore, we used proteomic analysis of cleavage sites and protease inhibitor profiling to show that M2AP is multiply processed by MPP2 and at least one additional novel parasite-derived protease we term MPP3. Based on these new findings, we propose a detailed two-step model for the dynamic processing of MIC2/M2AP, which includes primary and secondary processing steps that culminate in shedding of the adhesive complex from the parasite surface.
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EXPERIMENTAL PROCEDURES |
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Parasite Culture and Large-scale Excretory/Secretory Antigen (ESA) Preparation
Parasite culture and ESA preparation was performed according to the protocol described previously (15). Briefly, T. gondii strain 2F was propagated in human foreskin fibroblast cells. Freshly egressed tachyzoites, a rapidly dividing parasite stage responsible for acute infection, were harvested by passage twice through a 20-gauge needle followed by filtration through a 3-µm pore size membrane to remove host cell debris. Parasites were washed twice by centrifugation in invasion medium prior to use in experiments. Large-scale preparation of ESA proteins was performed by incubating 4x109 2F strain tachyzoites in 15 ml of Dulbeccos modified Eagles medium (DMEM) containing 2 mM glutamine, 10 mM HEPES, and 1% (v/v) ethanol at 37 °C for 20 min followed by cooling on ice 5 min. Parasites were removed by centrifugation (1000 x g, 10 min, 4 °C), and the supernatant was concentrated to
400 µl using C-20 concentrators according to the manufacturers instructions (Millipore, Bedford, MA).
Secretion Experiment and Protease Inhibitor Assay
Tachyzoites (4 x 107/ml) in modified invasion medium (DMEM + 10 mM HEPES) were incubated for 3 min at 37 °C with 1% (v/v) ethanol to stimulate microneme secretion. Tachyzoites were removed by centrifugation (twice at 1000 x g, 10 min, 4 °C), and supernatants were mixed with SDS-PAGE sample buffer containing 2.0% (v/v) 2-mercaptoethanol for SDS-PAGE and Western blotting (see below). Before being assayed for secretion as described above, tachyzoites were preincubated for 10 min at room temperature with 1% DMSO (solvent control); 1 µM cytochalasin D (CytD); 5 µM pepstatin; 10 mM EDTA (sodium salt); 10 mM EGTA (sodium salt); 5 mM 1,10-phenanthroline; 50 µg ml1 matrix metalloprotease-1 inhibitor; 10 µM 3,4-dichloroisocoumarin; 100 µM L-1-chloro-3-[4-tosylamido]-7-amino-2-heptanone HCl; 100 µM 4-(2-aminoethyl)benzene-sulfonyl)fluoride hydrochloride; 1 mM phenylmethanesulfonyl fluoride (PMSF); 40 µg ml1 leupeptin; 10 µM E-64; and 40 µg ml1 calpain inhibitor I (N-acetyl-Leu-Leu-norleucinal (ALLN)) or 40 µg ml1 calpain inhibitor II (N-acetyl-Leu-Leu-methioninal (ALLM)).
SDS-PAGE and Western Blotting
Proteins were resolved on 12.5% or 10% reducing SDS-polyacrylamide gels. For Western blotting, PAGE gels were semidry-transferred to nitrocellulose membranes. Blotting was performed as described previously (9) using RrM2AP (whole serum, 1:5000).
Two-dimensional Gel Electrophoresis (2DE) and Two-dimensional Differential Gel Electrophoresis (2D-DIGE)
ESA proteins were separated in the first dimension using the Ettan IPGphor isoelectric focusing system (Amersham Biosciences). Then 120 µg of ESA protein was mixed with rehydration buffer containing 8 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), 0.5% carrier ampholytes buffer, and 65 mM dithiothreitol (DTT). Rehydration and isoelectric focusing were performed according to the manufacturers instructions using pH 47 IPG strips (13 cm for 2DE, 18 cm for 2D-DIGE). Following isoelectric focusing, proteins were reduced and alkylated by successive 15-min treatments with equilibration buffer containing 2% dithiothreitol followed by 2.5% iodoacetamide. Then, proteins were resolved in the second dimension on a 12.5% SDS-PAGE gel. Resolved proteins were then either transferred directly to PVDF membrane for N-terminal sequencing or stained with colloidal Coomassie Blue stain (21) for MS analysis. 2D-DIGE experiments (22) were carried out as follows: 50 µg lyophilized ALLN-treated and solvent (DMSO) control-treated ESA were each dissolved in 10 µl of labeling buffer (30 mM Tris, 8 M urea, 2% CHAPS, pH 8.5). Then 400 pmol each of Cy3 and Cy5 was added to the ALLN and control samples, respectively, and samples were incubated on ice for 30 min in the dark. After labeling, 1 µl of 10 mM lysine was added to stop the reaction. Cy3- and Cy5-labeled samples were mixed together and resolved by 2DE as described above. Fluorescence scanning was performed using a Typhoon 9200 variable mode imager (Molecular Dynamics, Sunnyvale, CA)
N-terminal Sequencing
Proteins on a 2DE gel were transferred to Immobilon® PSQ membranes (Millipore) and stained with Coomassie Brilliant Blue R-250. Bands were excised and subjected to Edman degradation on a Perkin-Elmer Biosystems Model 477A Gas-Phase Protein Sequencer (Wellesley, MA).
Sequence Analysis
Protein sequence alignments, pI, and molecular mass predictions were performed using Lasergene DNAStar, version 5.00. The web-based programs, ExPASy PeptideMass (ca.expasy.org/tools/peptide-mass.html) and MS-Digest (prospector.ucsf.edu), were also used to predict theoretical molecular mass of protein and peptide digests, respectively.
MALDI Mass Spectrometry
MALDI time-of-flight (TOF) MS was performed using a Voyager DE-STR from PerSeptive Biosystem (Framingham, MA), equipped with a nitrogen laser (337 nm). In all cases unless otherwise specified, spectra were obtained in positive acquisition mode, and external peptides or proteins adjacent to the sample spots were used for MS calibration. Reported peptide masses are monoisotopic, while intact protein masses are average masses.
MALDI-MS Analysis of Intact Protein
Protein bands from 2DE were cut out and solvent-extracted as described (23) with minor modifications. Briefly, excised bands in microtubes were washed with 50 mM ammonium bicarbonate for 10 min, 50% acetonitrile/200 mM NH4HCO3 for another 30 min (twice), and finally washed thoroughly with HPLC-grade water. Gel pieces were crushed with a sharp-pointed dental tool. Then 20 µl of 10 mg ml1 sinapinic acid (matrix) in 0.5% TFA/acetonitrile (1:1) was added to cover each gel piece. Samples were extracted by vortexing (moderate speed) at room temperature for 3 h. Following extraction, tubes were opened to evaporate for 20 min with vortexing until the matrix solution became slightly cloudy, and 1 µl of supernatants were drawn out and deposited onto the MALDI probe for MS analysis. MS spectra were acquired in the linear mode with aldolase as external calibration standard. The mass error of aldolase was less than 10 atomic mass units (amu) after calibration. Acquisition parameters were as follows: acceleration voltage, 25 k; grid voltage, 91%; extraction delay time, 1000 ns; laser shots, 100 per spectrum. The average mass was assigned to each acquired peak in the spectra according to the centroid at 50% height. For assessing experimental deviation, three spectra were acquired for each sample deposited on the plate.
MALDI-MS Analysis of Digested Peptides
Protein spots from PAGE gels were excised, in-gel digested with endoproteinases (12.5 ng µl1 trypsin or 10 ng µl1 Asp-N in 50 mM NH4HCO3), and solvent-extracted as described previously (24). Proteins excised from one-dimensional (1D) gels were reduced with DTT (50 mM DTT in 100 mM NH4CO3, 56 °C, 1 h) and alkylated with iodoacetamide (275 mM iodoacetamide in 100 mM NH4CO3 in the dark at room temperature 45 min) prior to digestion. After extraction, peptide mixtures were dried down and redissolved in 2 µl of 50% acetonitrile and 0.3% TFA, mixed with saturated -cyano-4-hydroxycinnamic acid (matrix) solution, and deposited on the MALDI-MS target plate according to a general two-layer method (25). The MS spectra were acquired in reflection mode with delayed extraction and were calibrated using calibration peptide mixture II. Peptide mass fingerprints (PMF; a list of highly accurate peptide monoisotopic masses) were used to search against the Toxoplasma NCBI database via the MS-Fit algorithm (prospector.ucsf.edu) or searching the FindPept tool (ca.expasy.org/tools/findpept.html). A threshold mass deviation of 50 ppm was used to judge true positive matches in all cases unless otherwise specified.
For in-gel digestion with 18O labeling, digests were performed in 5 mM NH4HCO3 with or without the presence of 50% 18O water, using the protease Asp-N at a final concentration of 2 µg ml1. Digests were acidified and analyzed by MALDI-TOF as previously described (26).
Electrospray Ionization (ESI) Tandem Mass Spectrometry
Peptide sequencing was performed by liquid chromatography tandem MS analysis of peptide fragments from collision-induced dissociation (CID). Peptides were fractionated by reverse-phase HPLC in a 060% methanol gradient with 0.5% formic acid over 30 min at 200 nl min1 on a 75 µm x 100 mm C18 PepMap column using an UltiMate HPLC system, equipped with a FAMOS autoinjector (LC Packings, San Francisco, CA). Eluting peptides were sprayed through a 10-µm picotip (New Objectives, Worburn, MA) directly into a QSTAR Pulsar quadrupole orthogonal TOF tandem mass spectrometer (Applied Biosystems/MDX Sciex, Foster City, CA), equipped with a Protana XYZ manipulator source (MDS Protana A/S, Odense, Denmark). Using the software of Analyst QS (Applied Biosystems), spray, declustering, and focusing potentials were set at 1500, 60, and 220 V, respectively. Peptides were selected for fragmentation in information-dependent acquisition experiments monitoring charge states 24 of the two most intense ions. Parent ions were surveyed for 0.5 s, and selected ions were fragmented by CID at 25, 30, and 35 V collision energies. Fragmentation spectra were collected in enhance-all mode over 1.5-s durations. Specific ions of interest were placed in the included list for fragmentation. Errors on fragmentation ions were less than 0.02 amu. The acquired MS data were analyzed with the programs of Mascot (Matrix Science, London, United Kingdom) or BioAnalyst (Applied Biosystems).
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RESULTS |
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M2AP Is Processed by Two Distinct Proteolytic Activities
To examine the protease(s) responsible for the surface proteolysis of the M2AP coiled domain, we performed protease inhibitor profiling experiments. As shown in Fig. 4A, proteolytic processing of M2AP was affected by a subset of protease inhibitors. These included chymostatin and PMSF, which showed partial inhibition, and the calpain inhibitors ALLN and ALLM, which strongly blocked processing. These inhibitors blocked the formation of the three smallest fragments of M2AP (M2AP24), without impairing the two largest fragments (mM2AP and M2AP-1). It should be noted that because the C-terminal coiled domain is highly antigenic, the smaller, more heavily processed species are less well recognized by the antiserum. Therefore, inhibition of M2AP24 formation by ALLN and ALLM is more pronounced than indicated by Western blotting. ALLN and ALLM are tripeptide aldehydes previously shown to block removal of an N-terminal extension from MIC2 by MPP2 (15). The following additional evidence further supports that MPP2 is responsible for generating M2AP24. First, similar to the observed processing of M2AP, trimming of the MIC2 N terminus occurs in multiple steps resulting in removal of a loosely structured region adjacent to a globular domain. Second, M2AP processing is dramatically enhanced by parasite treatment with CytD (Fig. 4A), which was previously shown to promote processing by MPP2 (15). Finally, based on processing of MIC2 at V50, G66, and S53, MPP2 prefers to cleave at a small amino acid in the P1 position, which is consistent with the cleavage specificity for M2AP24 (L222, A207, and S203, respectively). Although formation of M2AP24 appeared to be somewhat sensitive to EGTA, because levels of mM2AP and M2AP-1 were also lower, at least some of this effect is due to inhibition of microneme secretion, which is calcium dependent.
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In addition to the current findings for M2AP, MPP2 has been previously reported to cleave MIC2 (15) and MIC4 (29). However, it is not known whether MPP2 is exclusively dedicated to these substrates or if it is responsible for processing additional parasite proteins. To globally examine the MPP2 substrate repertoire, we compared ESA proteins from control and ALLN-treated parasites by 2D-DIGE. Control ESA proteins were covalently labeled with Cy3 (pseudocolored green) while ALLN ESA proteins were labeled with Cy5 (pseudocolored red), and the samples were mixed and separated on a single 2D gel. Protein spots that are diminished or absent in the ALLN sample are green, whereas proteins that increase in abundance are red. Spots that do not change significantly are yellow or shades thereof. As expected (Fig. 4B), ALLN treatment inhibited the formation of M2AP24, MIC295 (product of MPP2s removal of the N-terminal extension), and the MIC4 cleavage products including the 50-kDa N-terminal product and the "15"-kDa C-terminal product (29). However, ALLN also disrupted processing of SUB1, a micronemal subtilase of unknown function, by blocking the formation of several isoforms in the 70-kDa cluster (SUB170) and the 40-kDa product (SUB140). Although this implies that MPP2 is responsible for the processing of SUB1, we cannot rule out the possibility that ALLN inhibits other proteases in addition to MPP2. Verification of the complete MPP2 substrate repertoire will require its molecular cloning and the identification of additional selective inhibitors.
MIC2 Is Shed from the Parasite Surface by Intramembrane Cleavage
As mentioned above, recent studies have implied that MIC2 is released from the parasite surface by cleavage within the membrane (20) or outside the membrane (16). To resolve this issue, we purified the secreted form of MIC2 collected from parasite cultures and subjected it to MS analysis by several approaches including PMF analysis, 18O labeling, and tandem MS sequencing.
ESA proteins were resolved by 1D SDS-PAGE, and the band corresponding to MIC2 (indicated in Fig. 5A) was excised and subjected to tryptic or Asp-N in-gel digestion. Digests were analyzed by MALDI-TOF, and PMF data were acquired. Analysis of tryptic PMF data confirmed that the excised band was MIC2; however, this data was not suitable for defining the C terminus because trypsin recognizes lysine residues, which potentially constitute the juxtamembrane cleavage site (16). Analysis of the Asp-N spectrum revealed an ion at m/z 2748.262 (Fig. 5B, lower spectrum) corresponding to the peptide 678DENETPTNEEGEQSKKESGSGIAGAIA704 (predicted m/z 2748.245). This peptide does not have an Asp-N cleavage site at its C terminus, suggesting that it represents the terminal peptide for the shed form of MIC2. Also, this peptide terminates precisely at the intramembrane cleavage site corresponding to that of MIC6 (20) (Fig. 6). In contrast, despite extensive efforts, we did not observe by MALDI-TOF the C-terminal peptide (678DENETPTNEEGEQSK692, theoretical m/z 1706.700) representing a juxtamembrane cleavage event at the lysine-containing motif identified by the Sibley group (16).
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To unambiguously verify the identity of the C-terminal peptide, we analyzed the Asp-N digests of the shed form of MIC2 with an ESI quadrupole-TOF (QTOF) tandem mass spectrometer. As expected, a triply charged ion ([M+3H]3+) with m/z 916.7 whose singly charged ion ([M+H]+) corresponds to the MALDI ion m/z 2748.262 was observed in the ESI-MS spectra. Subsequently, this peptide was selected and fragmented by CID. In the acquired fragmentation spectrum (Fig. 5C), the major fragmentation peaks were assigned to "y" and "b" series ions representing truncation from the C and N terminus of the peptide, respectively (fragmentation ions are labeled according to Biemanns nomenclature (31)). Largely based on the assigned "y" series ions (y1, y2, y3, y4, y5, y7, y8, and y9; although the y6 ion was missing, the corresponding b22 ion was observed), the partial sequence ... GSGIAGAIA704 was specified, which precisely terminates at the cleavage site identified above. Similarly, the N-terminal sequence 678DENETPTN ... was largely specified by the "b" series ions in the spectrum (b2, b3, b4, b5, b7, b8; the b1 and b6 ions were missing, but the corresponding y27 and y22 ions were observed). Similar to MALDI-TOF analysis, ESI-QTOF also failed to detect a peptide corresponding to juxtamembrane cleavage at the conserved lysine motif that is necessary for processing.
Based on the above MS evidence, we conclude that MIC2 is shed from the parasite surface by intramembrane proteolytic cleavage at a site near the N-terminal side of its TM anchor. As shown in Fig. 6, alignment of the TM sequences of MIC2 and related proteins reveals that the cleavage site is strikingly well conserved, strongly suggesting that intramembrane cleavage is a general mechanism for shedding adhesive proteins from the surface of apicomplexan parasites.
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DISCUSSION |
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C-terminal processing of MIC2 by MPP1 is probably a crucial step in invasion based on the finding that parasites expressing a noncleavable copy of MIC2 were significantly impaired in cell entry (16). However, as mentioned above, conflicting evidence regarding the precise cleavage site was reported in two recent studies. Based on biochemical analysis of MIC6 and genetic analysis of MIC2 and MIC12, Soldati and coworkers (20) proposed that MIC2 is cleaved within its TM anchor by regulated intramembrane proteolysis (RIP), a recently identified mechanism of liberating membrane proteins. On the other hand, data reported by Sibley and coworkers (16) suggested that MIC2 is cleaved by juxtamembrane proteolysis (JP) at a dibasic motif consisting of K692/K693, i.e. several residues outside the TM anchor. In the present article, we resolve this issue by unequivocally demonstrating that MIC2 is cleaved within its TM anchor at A704/G705. While we cannot completely rule out the possibility of a separate JP cleavage, especially because it has been reported (32, 33) that JP often precedes RIP, we prefer the model that cleavage occurs exclusively by RIP for the following reasons. First, when ALLN or CytD treatment is used to eliminate MIC2 size heterogeneity due to N-terminal processing, MIC2 migrates as a well-defined band, suggesting it is generated by a single cleavage event (15). Second, as exemplified by its similarity to the Rhomboid substrate Spitz (Fig. 6), the MIC2 TM cleavage site closely resembles the recognition sequence for a Rhomboid-like protease (34), which is one of several enzymes capable of mediating RIP. Third, several genes encoding Rhomboid-like proteases are evident in the Toxoplasma genome database (D. Soldati, personal communication). Fourth, a recent study demonstrated that Drosophila Rhomboid-1 is capable of cleaving a hybrid protein containing the TM region of MIC2 (34). Finally, Rhomboid proteases typically do not require primary processing by JP (32). If indeed MIC2 processing occurs exclusively by RIP, it is possible that the membrane-proximal dibasic motif necessary for MIC2 C-terminal processing is either a distal recognition site for MPP1 (a putative Rhomboid-like protease) or is necessary to maintain the structural integrity of MIC2 for proper intramembrane cleavage.
RIP is generally carried out by multipass membrane proteases and occurs in a wide variety of organisms ranging from bacteria to humans (33). Recently, RIP has gained much attention as a recognized mechanism for intercellular and intracellular signaling (32). It was revealed that RIP mediates important signal transduction events associated with sterol regulation (e.g. SREBP cleaved by S2P (35)), cell fate selection (e.g. surface receptor Notch cleaved by -secretase (36)), and developmental growth regulation (e.g. Rhomboid cleavage of the Drosophila epidermal growth factor-like membrane proteins Spitz, Keren, and Gurken (37)). Also, the recently identified mammalian signal peptide peptidase is a multipass membrane aspartyl protease responsible for secondary processing of secretory signal peptides (38). In addition, RIP of amyloid precursor protein to produce the amyloid ß peptide plays a key role in Alzheimers disease (39). Despite these established instances, the mechanism of RIP is only beginning to be unveiled, largely because the lipid bilayer presents a challenging experimental environment. Nonetheless, it was recently shown that RIP proteases probably use catalytic mechanisms similar to known proteases that require water to hydrolyze the peptide (32). Given this and considering that TM segments generally adopt a difficult-to-cleave helical conformation, the TM domain of the substrate protein must be at least partially unwound and exposed to the aqueous environment before or during the cleavage to allow water access to the cleavage site buried in the membrane. Accordingly, Urban and Freeman (34) recently suggested that Rhomboid substrates are susceptible to RIP cleavage because their TM domain contains potential helix-breaking residues (e.g. G and A), which tend to form a partially disordered conformation. This presumably contributes to the specificity of Rhomboid-like RIP proteases. Our defined RIP cleavage site at A704/G705 in the outer portion of the MIC2 membrane segment appears to be consistent with this mechanism for specificity.
Based on the analysis of MIC2/M2AP proteolysis presented herein, we propose a two-step processing model for MIC2/M2AP during invasion (Fig. 7). Once the parasites apical end attaches to a host cell, the MIC2/M2AP complex is mobilized from the micronemes onto the parasite surface where it undergoes primary processing by MPP2 and MPP3 at multiple sites (N terminus of MIC2 by MPP2, C terminus of M2AP by MPP2 and MPP3). The role of these primary processing events in invasion remains unknown because the most effective inhibitor of MPP2, ALLN, adversely affects host cells in addition to the parasite (B. F. C. Kafsack and V. B. Carruthers, unpublished observations). Nonetheless, we speculate that primary processing of MIC2/M2AP may facilitate its binding to host receptors or it may activate clustering of the complex with other adhesive proteins at the attachment junction. After primary processing, the receptor-bound complex translocates over the parasite body to the posterior end as the parasite penetrates into the host cell (other MIC adhesins also work in concert here to aid penetration). Upon reaching the extreme posterior end, our data suggests that the complex is disengaged from the parasite surface by MPP1-mediated RIP cleavage within the TM domain of MIC2. If MPP1 is confined to the posterior end of the parasite, this step would be spatially regulated by movement of the substrate into the vicinity of the protease. This type of spatial regulation is commonly associated with RIP (35, 37). However, because the MIC2/M2AP complex is efficiently cleaved by MPP1 in the presence of CytD, at least some MPP1 is likely present on the apical surface. Definitive identification, molecular cloning, and analysis of the MPP1 should help clarify the regulatory mechanism governing the release of adhesive complexes.
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Interestingly, proteins on the surface of the malaria merozoite are also subjected to proteolytic processing and shedding during invasion (41). Merozoite surface protein-1 (MSP-1) is initially present as a high-molecular-mass protein expressed on the surface of malaria merozoites and noncovalently complexed with other parasite proteins (41). The 200-kDa MSP-1 precursor is processed into four smaller fragments coincident with merozoite release from an infected cell. During erythrocyte invasion, one of these primary processing products (MSP-142) is further subjected to a secondary processing, thereby releasing the fragmented MSP-1 ectodomain and its associated partners into the surrounding medium. Only the 19-kDa processed product, MSP-119, remains on the merozoite surface. The Plasmodium falciparum ortholog of AMA1 (PfAMA1) is also shed as a result of juxtamembrane cleavage following its translocation from micronemes onto the merozoite surface (26). Similar to MPP1, the protease responsible for shedding of both these malaria proteins is a membrane-bound enzyme (26, 41). MSP-1 secondary processing has been demonstrated to be essential for successful erythrocyte invasion, and accordingly MSP-119 is emerging as a promising target for drug or vaccine development designed to block invasion (42). However, despite the striking similarities between processing of these malaria proteins and MIC2, several important differences exist in their features. First, MSP-1 circumferentially decorates the free merozoite surface, whereas MIC2 is a transient surface protein that is initially deployed only to the parasite apical surface. Second, in contrast to MSP-1, which is "shaved" off the merozoite surface as the moving junction slides over the parasite, MIC2 is excluded by the moving junction, resulting in it being swept toward the posterior end of the parasite during penetration. While some shedding of MIC2 may occur throughout invasion, it appears that much of the surface MIC2 is released from the posterior end of the parasite just prior to completion of invasion (14). Third, and most importantly, shedding of both malaria proteins occurs at JP cleavage sites, whereas secondary processing of MIC2 by MPP1 occurs within the TM anchor by RIP.
In summary, the present study suggests that T. gondii uses a dynamic and complex system for processing of MIC proteins during invasion, culminating in the intramembrane cleavage of adhesive complexes from the parasite surface. Given that these proteolytic events are likely crucial for Toxoplasma invasion, these findings shed new light on the invasive mechanism of T. gondii and provide new insight regarding the properties of parasite proteases that could be attractive therapeutic targets. In addition, these studies provide a novel example of sequential processing associated with RIP-mediated liberation of membrane proteins.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, February 24, 2004, DOI 10.1074/mcp.M300123-MCP200
1 The abbreviations used are: TM, transmembrane; MICs, micronemal proteins; M2AP, MIC2-associated protein; mM2AP, mature M2AP; MPP1, Microneme protein protease 1; MPP2, microneme protein protease 2; MPP3, microneme protein protease 3; amu, atomic mass units; ESA, excretory/secretory antigen; DMEM, Dulbeccos modified Eagles medium; 2DE, two-dimensional gel electrophoresis; MS, mass spectrometry; MALDI, matrix-assisted laser desorption/ionization; ESI, electrospray ionization; TOF, time-of-flight; CID, collision-induced dissociation; CytD, cytochalasin D; ALLN, N-acetyl-Leu-Leu-norleucinal; ALLM, N-acetyl-Leu-Leu-methioninal; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; 2D-DIGE, two-dimensional differential gel electrophoresis; Cy3, 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide N-hydroxy-succinimidyl ester; Cy5, 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide N-hydroxysuccinimidyl ester; TFA, trifluoroacetic acid; IPG, immobilized pH gradient; HPLC, high-pressure liquid chromatography; PMSF, phenylmethanesulfonyl fluoride; 1D, one dimensional; DTT, dithiothreitol; PMF, peptide mass fingerprinting; QTOF, quadrupole TOF; RIP, regulated intramembrane proteolysis; JP, juxtamembrane proteolysis; MSP, merozoite surface protein.
* This work is supported by the National Institutes of Health (AI46675; to V. B. C.), the Burroughs Wellcome Fund (to V. B. C.), and the Medical Research Council (to M. J. B.).
¶ To whom correspondence should be addressed: The W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD 21205. Tel.: 410-614-5592; Fax: 410-955-0105; E-mail address: vcarruth{at}jhsph.edu
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REFERENCES |
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