Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin

Abstract

Larvae and adults of the parasitic blood fluke Schistosoma mansoni are resistant to killing by human complement. An earlier search by Parizade et al. for a schistosome complement inhibitor identified a 94-kDa surface protein which was named SCIP-1 (M. Parizade, R. Arnon, P. J. Lachmann, and Z. Fishelson, J. Exp. Med. 179:1625-1636, 1994). Following partial purification and analysis by mass spectrometry, we have determined SCIP-1 to be a surface-exposed form of the muscle protein paramyosin. As shown by immunofluorescence, anti-paramyosin antibodies label the surface of live schistosomula and adult worms. Like SCIP-1, purified native paramyosin reacts with a polyclonal rabbit anti-human CD59 antiserum, as shown by Western blot analysis. Also, the human complement components C8 and C9 bind to recombinant and native paramyosin. Analysis of paramyosin binding to fragments of C9 generated by thrombin or trypsin has demonstrated that paramyosin binds to C9 at a position located between Gly245 and Arg391. Paramyosin inhibited Zn(2+)-induced C9 polymerization and poly-C9 deposition onto rabbit erythrocytes (E(R)). In addition, paramyosin inhibited lysis of E(R) and of sensitized sheep erythrocytes by human complement. Finally, anti-paramyosin antibodies enhanced in vitro killing of schistosomula by normal and C4-depleted human complement. Taken together, these findings suggest that an exogenous form of S. mansoni paramyosin inhibits activation of the terminal pathway of complement and thus has an important immunomodulatory role in schistosomiasis.

FIG. 1.

Binding of anti-CD59 antibody (1:300) to paramyosin (Pmy). (Lanes a to c) NPRM from schistosomula or affinity purified protein were subjected to SDS-8% PAGE and transferred onto a nitrocellulose membrane. After blocking with a 5% milk solution in TBST for 1 h at room temperature, the membrane was treated with rabbit anti-CD59 antibody or normal rabbit serum (NRS) (1:300) and then washed and treated with peroxidase-conjugated goat anti-rabbit IgG as a second antibody (1:5,000). Bands were developed by enhanced chemiluminescence. Lane a, NPRM of schistosomula; lane b, effluent of affinity column; lane c, eluate of affinity column; lane d, SCIP-1 (arrowhead) eluted from DEAE-cellulose (peak tube) was subjected to SDS-PAGE and the gel was stained with Coomassie blue; lane m, stained molecular mass markers (200, 120, 97, and 67 kDa).

FIG. 2.

Immunofluorescence analysis showing the presence of paramyosin on the surface of schistosomula and adult worms. After treatment with blocking solution, 100 24-h-old schistosomula (A and B) or 20 fresh adult worms (C and D) were incubated with polyclonal rabbit anti-paramyosin antibodies (A and C) or normal rabbit serum (B and D) for 30 min at 37°C. After three washes with DSM on ice, they were treated with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (1:100 in DSM) for 30 min on ice. The schistosomes were washed with DSM and examined under a fluorescence microscope.

FIG. 3.

Binding of paramyosin to C8 and C9. Purified human C8 and C9 and BSA (1 μg each) were subjected to SDS-8% PAGE and transferred onto a nitrocellulose membrane. After blocking with 5% milk in TBST, the membrane was incubated with recombinant paramyosin (4 μg/ml) in blocking solution for 2 h at 37°C. After two washes with TBST, the membrane was reacted with monoclonal anti-paramyosin antibody (1:4) (Anti-Pmy) or an isotype-matched, unrelated monoclonal antibody (Control) and then incubated with peroxidase-conjugated goat anti-mouse IgG (1:5,000) for 1 h at room temperature. Bands were visualized by enhanced chemiluminescence. Lane a, C8α and C8γ (upper band) and C8β (lower band); lane b, C9; lane c, BSA.

FIG. 4.

Binding of paramyosin to blotted C9: competition with soluble C9 or CD59. (Lanes a and b) Purified human C9 (lanes a) or BSA (lanes b) (1 μg each) was blotted onto nitrocellulose membrane. The membrane was reacted with paramyosin (Pmy) or paramyosin preincubated with C9 (1:5 [wt/wt]) (Pmy+C9) or buffer (control) and then with monoclonal anti-paramyosin antibody, peroxidase-conjugated goat anti-mouse IgG, and enhanced chemiluminescence reagent. (Lanes c to e) Purified human C9 was blotted onto nitrocellulose membrane and reacted with recombinant paramyosin preincubated with or without purified human CD59. Further details are provided in Materials and Methods. Bound paramyosin was detected with monoclonal anti-paramyosin antibodies, goat anti-mouse IgG, and enhanced chemiluminescence reagent. Lane c, 2 μg of paramyosin without CD59; lane d, 2 μg of paramyosin plus 0.33 μg of CD59; lane e, 2 μg paramyosin plus 1 μg of CD59.

FIG. 5.

Location of the paramyosin binding site in C9. C9 was cleaved into C9a (molecular weight, 34,000) and C9b (37,000) by incubation with α-thrombin for 4 h at 37°C or into C9a′ (47,000) and C9b′ (24,000) by incubation with trypsin for 35 min at 37°C. The fragments were separated by SDS-12% PAGE and stained with Coomassie blue or blotted onto nitrocellulose membrane. The membrane was incubated with recombinant paramyosin for 2 h at 37°C and then reacted with monoclonal anti-paramyosin and goat anti-mouse IgG (1:5,000) as described above. (A) Profile of the C9 and C9 fragments bands after staining (left panel) or Western blotting (right panel) with paramyosin and anti-paramyosin. Lanes a, C9 plus PBS; lanes b, C9 plus thrombin; lanes c, C9 plus trypsin. (B) Schematic presentation of cleavage sites in C9. The C9 fragments that bind paramyosin are shown in boxes shaded in gray.

FIG. 6.

Inhibition of Zn²⁺-induced C9 polymerization by recombinant paramyosin (Pmy). C9 (2 μg) was mixed with various amounts of paramyosin (0, 2, 4, or 8 μg) for 30 min at 37°C and then incubated with 42 μM ZnCl₂ for 2 h at 37°C. The samples were subjected to a SDS-PAGE gradient gel (3 to 10% acrylamide). Lane a, C9 plus 8 μg of paramyosin; lane b, C9 plus 4 μg of paramyosin; lane c, C9 plus 2 μg of paramyosin; lane d, C9 without paramyosin; lane e, C9 without ZnCl₂; lane f, Western blot of C9 without paramyosin (from lane d) with anti-C9 antibody (1:300). The 97-kDa bands seen in lanes a to c represent paramyosin. The 67-kDa bands in lanes d and e represent monomeric C9 and in lanes a to c represent a mixture of C9 with a 67-kDa fragment of recombinant paramyosin. Lower weak bands probably represent fragments of paramyosin (lanes a to c) or C9 (lanes d and e).

FIG. 7.

Inhibition of polyC9 deposition on E_R by paramyosin. Fresh E_R were treated with 100 μl of NHS supplemented with 6 μg of C9 preincubated for 30 min at 37°C with (lane a) or without (lane b) 40 μg of recombinant paramyosin. The lysed E_R were sedimented, and the pellet was dissolved in reducing sample buffer and analyzed on a SDS-PAGE gradient gel (3 to 10% acrylamide) stained with Coomassie blue. Lane m, molecular mass markers (200, 120, 97, and 67 kDa). The arrow indicates a band of polyC9. Other bands represent proteins originated from the red blood cells and serum proteins adhering to blood cells.

FIG. 8.

Inhibition of complement-mediated lysis of E_R by recombinant paramyosin (Pmy). Fresh E_R (2 × 10⁷ cells) were incubated with NHS (6%) in the presence of Mg-EGTA and various amounts (0, 2, 10, or 20 μg) of recombinant paramyosin for 30 min at 37°C. Cold GVB containing 10 mM EDTA was added to stop lysis. Following centrifugation, light absorption of the supernatant was measured at 412 nm and percent lysis was calculated. Results shown are means ± SD representative of three independent experiments.

FIG. 9.

Inhibition of complement-mediated lysis of EAs by paramyosin (Pmy). EAs (1.5 × 10⁷ cells) were incubated with C8-deficient human serum (1:10) for 15 min at 37°C. After washes with GVB containing EDTA, C7-deficient human serum (1:4,000) premixed with recombinant paramyosin (0, 1, 2, or 4 μg) was added to EAC5b-7 in the presence of 10 mM EDTA. After 30 min of incubation at 37°C, the cells were sedimented and the light absorption of the supernatant was measured at 412 nm. Percent hemolysis was calculated. Results shown are means ± SD representative of three independent experiments.

FIG. 10.

Enhanced complement-mediated killing of schistosomula by anti-CD59 and anti-paramyosin (anti-Pmy) antibodies. Schistosomula (3 h old) were treated with rabbit anti-CD59 or anti-paramyosin antisera or normal rabbit serum (NRS) for 30 min at room temperature and then with NHS or C4-depleted human serum (C4D) overnight at 37°C. Percent mortality of schistosomula was determined under the microscope on the basis of motility and granularity data. Results shown are means ± SD representative of three independent experiments.