The merozoite surface protein 1 complex is a platform for binding to human erythrocytes by Plasmodium falciparum

Abstract

Plasmodium falciparum is the causative agent of the most severe form of malaria in humans. The merozoite, an extracellular stage of the parasite lifecycle, invades erythrocytes in which they develop. The most abundant protein on the surface of merozoites is merozoite surface protein 1 (MSP1), which consists of four processed fragments. Studies indicate that MSP1 interacts with other peripheral merozoite surface proteins to form a large complex. Successful invasion of merozoites into host erythrocytes is dependent on this protein complex; however, the identity of all components and its function remain largely unknown. We have shown that the peripheral merozoite surface proteins MSPDBL1 and MSPDBL2 are part of the large MSP1 complex. Using surface plasmon resonance, we determined the binding affinities of MSPDBL1 and MSPDBL2 to MSP1 to be in the range of 2-4 × 10(-7) m. Both proteins bound to three of the four proteolytically cleaved fragments of MSP1 (p42, p38, and p83). In addition, MSPDBL1 and MSPDBL2, but not MSP1, bound directly to human erythrocytes. This demonstrates that the MSP1 complex acts as a platform for display of MSPDBL1 and MSPDBL2 on the merozoite surface for binding to receptors on the erythrocyte and invasion.

Keywords: Cell Invasion; Complex; Infection; Malaria; Merozoite; Parasite; Plasmodium; Protein Complex; falciparum.

FIGURE 1.

MSPDBL1 and -2 are components of the MSP1 complex. Invasion supernatant harvested postinvasion from 3D7/DBL1HA, 3D7/DBL2HA, and 3D7 parasite lines was used for immunoprecipitation experiments with either anti-HA monoclonal or anti-MSP1 polyclonal antibodies, and bound proteins were detected by immunoblotting with rat anti-HA monoclonal antibodies (Roche Applied Science) under reducing conditions or mouse anti-MSP1₃₀ monoclonal m195 under non-reducing conditions (A and B). Anti-HA antibodies pulled down both MSPDBL1 and MSP1 (A) from 3D7/DBL1HA parasites and MSPDBL2 and MSP1 from 3D7/DBL2HA parasites (B). In the control lane, anti-HA antibodies did not pull down either MSPDBL or MSP1 from 3D7 parasites. In reciprocal experiments, anti-MSP1 antibodies pulled down both MSPDBL1 and MSP1 from 3D7/DBL1HA parasites and MSPDBL2 and MSP2 from 3D7/DBL2HA parasites, indicating the interaction of MSP1 with MSPDBL1 and MSPDBL2. In the control assay, anti-MSP1 antibodies were able to pull down native MSP1 material from 3D7 invasion supernatant.

FIGURE 2.

Recombinant MSPDBL1, MSPDBL2, MSP1, and MSP6 proteins expressed in E. coli. A, schematic representation of MSPDBL1 and -2 and the four recombinant proteins encompassing different domains: FL, DBL, SL, and SO. B, Coomassie Brilliant Blue-stained gels of final protein products of MSPDBL1 domains: FL, 71.7 kDa; DBL, 38.9 kDa; SL, 22.7 kDa; and SO, 16.6 kDa. C, Coomassie Brilliant Blue-stained gels of final protein products of MSPDBL2 domains: FL, 76.4 kDa; DBL, 38.9 kDa; SL, 24.6 kDa; and SO, 18.7 kDa with an N-terminal His₆ tag under non-reducing (NR) and reducing (Rd) conditions with molecular mass standards in kDa denoted (left). D, schematic representation of MSP1 and the six recombinant proteins expressed in E. coli: MSP1-D (heterodimer of MSP1_83/30 and MSP1_38/42), p83, p30, p38, and p42. E, Coomassie Brilliant Blue-stained gels of final protein products of MSP1-D (102 + 89.7 kDa) and the N-terminally His₆ tagged subunits p83 (83.5 kDa), p30 (25.0 kDa), p38 (49.1 kDa), and p42 (46.7 kDa) with an N-terminal His₆ tag under non-reducing (NR) and reducing (Rd) conditions with molecular mass standards in kDa denoted. F, schematic representation of MSP6 with a putative PfSub1 cleavage site where MSP6₃₆ was designed to include the C-terminal region of this site. G, final purified MSP6₃₆ (28.1 kDa) on SDS-PAGE under reducing (Rd) and non-reducing conditions (NR).

FIGURE 3.

Size exclusion chromatography of the MSPDBL1 and MSPDBL2 constructs. The chromatograms for MSPDBL1 and MSPDBL2 FL, DBL, SL, and SO are plotted. The linear regression was derived from molecular standards: thyroglobulin (670 kDa), bovine γ-globulin (158 kDa), chicken ovalbumin (44 kDa), equine myoglobulin (17 kDa), and vitamin B₁₂ (1.4 kDa) with elution volumes of 8.97, 12.77, 14.17, 17.69, and 20.77 ml, respectively. Experimentally derived results were fitted to y = −0.11x + 4.881 where r² = 0.9701 and are reported in Table 1.

FIGURE 4.

Circular dichroism spectra of MSPDBL1 and MSPDBL2 FL. The change in extinction coefficient is plotted as a function of wavelength for MSPDBL1 FL (A) and MSPDBL2 FL (B) at an initial concentration of 0.5 mg/ml. The data (open circles) were collected within a 4-s averaging time and at 20 °C, and the solid lines represent the nonlinear least square regression analysis best fits using the CONTINLL algorithm from the CDPro software package using the SP29 protein database. All results resulted in a root mean square deviation ≤0.081, and the secondary structure proportions are reported in Table 2.

FIGURE 5.

Probing the formation of a complex between MSP1-D and MSPDBL1 or MSPDBL2. A, co-immunoprecipitation of MSP1-D with MSPDBL1 (top) or MSP1-D with MSPDBL2 (bottom) with either anti-MSP1 monoclonal (5.2) or anti-MSPDBL monoclonal antibodies (MSPDBL1, 2A7; MSPDBL2, 7D11) isolated a complex containing both recombinant MSP1 and MSPDBL. Samples were analyzed by SDS-PAGE and stained with Coomassie Blue with molecular weights indicated on the left. B, the same samples were subjected to immunoblot analysis with either anti-MSP1 or MSPDBL monoclonal antibodies as described above, confirming that MSP1 interacts with MSPDBL1 and MSPDBL2. In the bottom panel, MSP1 and MSPDBL were allowed to interact with His₆-tagged AMA1 prior to immunoprecipitation with either anti-AMA1 monoclonal 3F9 or anti-MSP1 monoclonal 5.2 and MSPDBL monoclonal antibodies (MSPDBL1, 2A7; MSPDBL2, 7D11). Immunoblots show that anti-MSP1 monoclonal and anti-MSPDBL antibodies immunoprecipitate MSP1-D and MSPDBL but not AMA1. C, MSPDBL1 and MSPDBL2 bind to MSP1. ELISA plates were coated with MSPDBL1, MSPDBL2, or AMA1 at 2 μg/ml. A 2-fold dilution of MSP1 was added at 0–2 μg/ml. Binding was detected using anti-MSP1 monoclonal 5.2 and quantified at A₄₅₀. Binding of AMA1 is a negative control. The binding was fitted to a one-site-specific binding curve where y = A_450max(x)/(K_d + x). D and E, SPR sensorgrams of MSPDBL1 FL (D) and MSPDBL2 FL (E) binding to MSP1-D between concentrations of 0.03125 and 2 μm. The maximum responses were plotted to a one-site total binding model to determine the steady-state affinity (K_d). R.U., resonance units; IP, immunoprecipitation. Error bars represent standard deviation (S.D.).

FIGURE 6.

Dissecting the interactions of MSP1-D with MSPDBL FL, DBL, SL, and SO. SPR sensorgrams (left panels) show binding of MSP1-D to MSPDBL1 DBL (A), SL (B), and SO (C) and MSPDBL2 DBL (D), SL (E), and SO (F) between concentrations of 0.03125 and 10 μm. The resulting maximum responses were plotted to a one-site total binding model to determine the steady-state affinity (K_d), where error bars represent S.D. (right panels). The binding affinity of MSP1-D to MSPDBL1 DBL and SL was determined to be 390 nm and 1.3 μm, respectively. No binding was observed for both MSPDBL1 SO and MSPDBL2 SO to MSP1-D. MSPDBL2 DBL and SL constructs bound to MSP1 at 350 nm and 1.0 μm, respectively. R.U., resonance units.

FIGURE 7.

Dissecting the interactions of MSPDBL1/2 with MSP1-D fragments. Shown is the analysis of the interaction of MSPDBL1 with p42 (A), p38 (B), p30 (C), and p83 (D) and MSPDBL2 with p42 (E), p38 (F), p30 (G), and p38 (H). SPR assays determined that both MSPDBL1 and MSPDBL2 bound to three of the four MSP1 fragments, p42, p38, and p83. Sensorgrams (left panels) are shown at 2-fold increases in concentration between 0.03125 and 4 μm. The resulting maximum responses were plotted to a one-site total binding model where the steady-state affinity (K_d) was determined (right panels). MSPDBL1 bound to p42 at 1.1 μm, p38 at 860 nm, and p83 at 1.4 μm, whereas MSPDBL2 bound to p42 at 840 nm, p38 at 2.5 μm, and p83 at 1.9 μm. R.U., resonance units. Error bars represent S.D.

FIGURE 8.

MSP1, MSPDBL, and MSP6 form a three-component complex. The interaction of MSP6 with either MSP1-D, MSPDBL1, or MSPDBL2 was monitored with SPR. The SPR sensorgrams show the binding responses of MSP6 to immobilized MSP1-D (A), MSPDBL1 (B), and MSPDBL2 (C) between 0.03125 and 4 μm. Where binding was observed in A, the maximum response at each concentration was plotted to a one-site total binding model, and the steady-state affinity (K_d) of the MSP1-MSP6 interaction was determined to be 79 nm. D, immunoprecipitation assays using MSP1-MSPDBL1-MSP6 or MSP1-MSPDBL2-MSP6 showed that all three components were pulled down using anti-MSP1 and anti-MSPDBL antibodies but not with anti-AMA1 antibodies. E, anti-MSP1 5.2 monoclonal antibodies only detect MSP1. ELISA plates were coated with a 2 μg/ml concentration of either MSP1, MSP6, MSPDBL1, or MSPDBL2, and anti-MSP1 5.2 monoclonal antibodies were used to detect binding. Measurements were taken at A₄₅₀. F, competition ELISA. Plates were coated with 2 μg/ml MSP6 to assay the effect of site-specific saturation of MSP1 in the presence of either MSP6, MSPDBL1, or MSPDBL2 at 10 μg/ml and MSP1-D at 2 μg/ml. Upon incubation of MSP1-MSP6, MSP1-MSPDBL1, or MSP1-MSPDBL2 to the MSP6-coated plate, binding was detected with anti-MSP1 mAb5.2 and secondary anti-mouse antibodies conjugated to HRP. Absorbance values at A₄₅₀ were measured. G, ELISA plates were coated with 2 μg/ml FL MSPDBL1 or MSPDBL2. A 2-fold titration of MSP1-D across the concentration range of 0–6.4 μg/ml was allowed to incubate with 10 μg/ml MSP6, and binding was detected as described above. All ELISA experiments (F and G) were repeated, and similar results were obtained. The error bars represent the S.E. of four independent assays. R.U., resonance units; IP, immunoprecipitation.

FIGURE 9.

MSPDBL1 and MSPDBL2, but not MSP1 or MSP6, bind to human erythrocytes. A, 3 μg of recombinant FL, DBL, SL, and SO proteins of MSPDBL1 and MSPDBL2 together with MSP1, MSP6, and Rh4.9 (control) were subjected to either binding in the presence of red blood cells (+) or not subjected to binding as a molecular weight reference (−). Bound proteins were eluted in the presence of 1 m NaCl and separated by SDS-PAGE before analysis by immunoblotting with anti-MSPDBL1 polyclonal R1277, anti-MSPDBL2 polyclonal R1296, hyperimmune sera for MSP6 and MSP1, or anti-Rh4.9 monoclonal 10C9 as indicated. B, MSPDBL1 and MSPDBL2 erythrocyte binding activity is not affected by enzyme treatments. 3 μg each of MSPDBL1 FL, MSPDBL2 FL, and Rh4.9 were incubated with red blood cells untreated (U) or treated with neuraminidase (N), trypsin (T), or chymotrypsin (C), and binding was detected in immunoblots. Rh4.9 was used as a control for successful enzyme treatments. C, full-length MSPDBL1 and MSPDBL2 recombinant proteins incubated with anti-MSPDBL1, anti-MSPDBL2, or preimmune sera at final concentrations between 0 and 4 mg/ml were added to red blood cells (RBCs). The bound proteins were eluted and analyzed on immunoblots with anti-MSPDBL-specific antibodies as described above. D, MSPDBL1 and MSPDBL2 proteins are not produced in 3D7ΔMSPDBL1 and 3D7ΔMSPDBL2, respectively. Late stage schizonts were harvested and extracted using Tris/NaCl/EDTA/Triton X-100, and protein expression was tested in each of 3D7, 3D7ΔMSPDBL1, and 3D7ΔMSPDBL2 using anti-MSPDBL1 polyclonal R1277, anti-MSPDBL2 polyclonal R1296, anti-SERA5 polyclonal, and anti-AMA1 polyclonal sera. E, parasite growth was reduced in 3D7 wild type and 3D7/DBL2KO lines in the presence of anti-MSPDBL1 antigen-specific antibodies. Final concentrations of 0–1 mg/ml anti-MSPDBL1 affinity-purified antibodies were added to the culture. The observed inhibition was concentration-dependent. In the presence of anti-MSPDBL1 antibodies at 1 mg/ml, growth decreased by 33.66 ± 4.82% (***, p = 0.0001) in 3D7 and 41.95 ± 3.454% (***, p = 0.0067) in 3D7ΔMSPDBL2 using Fisher's exact test. Growth was measured as a percentage of non-inhibitory growth in PBS, and error bars represent the S.E. of three separate experiments in duplicate.

FIGURE 10.

MSPDBL1 and MSPDBL2 form a complex with MSP1 to mediate interaction with erythrocytes during invasion. A schematic representation of MSPDBL in complex with MSP1 based on our data and published reports shows that MSPDBL interacts with p42, p38, and p83 of MSP1, whereas MSP6 interacts exclusively with MSP1. This MSP1-MSPDBL complex interacts with an unknown receptor on the surface of erythrocytes to mediate invasion. We have represented MSPDBL1 and -2 proteins in the complex as a single unit; however, it is likely that they are present as oligomers as suggested by our data (Fig. 3 and Table 1).