INFECTION AND IMMUNITY, Nov. 2003, p. 6402–6410 Vol. 71, No.

11

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0019-9567/03/$08.00⫹0 DOI: 10.1128/IAI.71.11.6402–6410.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Inhibition of the Complement Membrane Attack Complex by

Schistosoma mansoni Paramyosin
Jiusheng Deng,1 Daniel Gold,1 Philip T. LoVerde,2 and Zvi Fishelson3*
Departments of Human Microbiology1 and Cell and Developmental Biology,3 Sackler School of Medicine,
Tel Aviv University, Tel Aviv, Israel, and Department of Microbiology and Immunology, School of Medicine
and Biomedical Sciences, State University of New York, Buffalo, New York2
Received 28 April 2003/Returned for modification 18 June 2003/Accepted 18 August 2003

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 deter-
mined SCIP-1 to be a surface-exposed form of the muscle protein paramyosin. As shown by immunofluores-
cence, 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 paramyo-
sin. 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 Zn2ⴙ-
induced C9 polymerization and poly-C9 deposition onto rabbit erythrocytes (ER). In addition, paramyosin
inhibited lysis of ER 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.

Schistosomiasis is one of the most prevalent parasitic dis-
eases, affecting over 200 million people who live in areas of
endemicity in Africa, South America, and Asia (12). Schisto-
soma mansoni, one of the causative agents of this disease,
resides within patients’ mesenteric and portal blood systems
and successfully evades host immune responses (43). During
skin penetration into the mammalian host and shortly after-
wards, the larvae convert from exhibiting sensitivity to comple-
ment-mediated killing to bearing resistance to complement-
mediated killing (32, 49). The first step in that conversion is the
removal of the glycocalyx coat that contains strong comple-
ment activators. Subsequently, the transformed schistosomula
and the adult worms employ several strategies to evade the
host’s complement system (14), thus enabling the parasite to
reside for years within the host’s circulatory system in direct
contact with complement proteins. This parasite contains pro-
teins that bind in vitro to the complement proteins C1 (25) and
C2 (22) and C8 and C9 (40) and may thus block the comple-
ment cascade at multiple steps. Paramyosin, one of the inhib-
itory proteins, was shown to bind in vitro to C1q and inhibit C1
and the classical pathway of complement activation (25).
Paramyosin is in essence an invertebrate muscle protein (11)
that serves as a major component of the thick filament and is
thought to play an important role in certain specialized con-
tractile states (7, 23). It has also been found to be an immuno-
gen during infection of mice and humans with helminth para-
sites (27, 37, 47). A nonfilamentous membrane-bound form of
* Corresponding author. Mailing address: Department of Cell and
Developmental Biology, Sackler School of Medicine, Tel Aviv Univer-
sity, P.O. Box 39040, Tel Aviv 69978, Israel. Phone: 972 3 6409620.
Fax: 972 3 6407432. E-mail: lifish@post.tau.ac.il.
paramyosin was also found in the tegumental outer layer (16,
33) and on the surface (18, 31) of S. mansoni schistosomes.
Analyzed on the basis of its capacity to bind to Fc (31), it has
been suggested to have immunomodulatory activities.
Paramyosin served in experimental systems as a vaccine mol-
ecule potent against infection with S. mansoni and Schistosoma
japonicum (27, 46) and is currently under investigation as a
candidate vaccine against schistosomiasis in humans (2).
Parizade et al. (40) reported that schistosomula, lung stage
worms, and adult worms of S. mansoni express on their surface
a 94-kDa complement inhibitor (SCIP-1) that has characteris-
tics similar to those of human CD59. CD59 is an 18-kDa
membrane glycoprotein expressed in most vertebrate tissues
(9, 10) that protects homologous cells from damage by com-
plement. It binds to C8 and C9 (30, 39) and inhibits assembly
of the C5b-9n membrane attack complex (MAC) (36, 48). As
described here, sequencing and specific antibody binding data
indicate that SCIP-1 is a tegumental form of paramyosin. Our
data further prove that paramyosin binds in vitro to C8 and C9
and inhibits MAC formation. This finding extends the range of
potential immunomodulatory activities of paramyosin.
MATERIALS AND METHODS
Parasites and parasite extracts. An Egyptian strain of S. mansoni was used in
this work. The life cycle was maintained in Puerto Rican Biomphalaria glabrata
snails and outbred ICR mice (Charles River Laboratories, Wilmington, Mass.).
Cercariae were collected from infected snails, mechanically transformed into
schistosomula by repeated transfers through a syringe needle (5, 32), and frac-
tionated over a Percoll gradient to separate the bodies from the tails (28). The
schistosomula were then incubated in defined synthetic medium (DSM) com-
posed of RPMI 1640 and nutrient F12 (GIBCO, Paisley, United Kingdom) (1:1)
for 3 h or overnight at 37°C in a 6% CO2 incubator. Outbred ICR mice were
infected by subcutaneous infection with 400 to 450 cercariae. Adult worms were

6402
VOL. 71, 2003
collected by perfusion of the hepatic portal system of infected mice at 6 to 8
weeks postinfection (52). Nonidet P-40 (NP-40)-released material (NPRM) was
extracted from 24-h-old schistosomula by treatment with 1% NP-40 in phos-
phate-buffered saline (PBS) for 2 h on ice, and particulate debris was removed by
centrifugation for 1 min at 20,000 ⫻ g.
SCIP-1 purification and sequencing. NPRM from schistosomula was fraction-
ated over a DEAE-cellulose (DE-52; Whatman, Maidstone, Kent, United King-
dom) column. Bound proteins were eluted with a salt concentration gradient (0
to 0.6 M NaCl in 0.01 M sodium acetate buffer, pH 6.0) in the presence of
protease inhibitors (Inhibitor Cocktail [catalog no. P8340]; Sigma, St. Louis,
Mo.). Protein concentrations were determined using a protein assay kit (Bio-
Rad, Richmond, Va.). Fractions containing SCIP-1 were identified in Western
blot assays with rabbit anti-human CD59 antibodies (received from Peter Lach-
mann, Cambridge, United Kingdom). Partially purified SCIP-1 was analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 8%
acrylamide gels under nonreducing conditions. The SCIP-1 band was excised
from the gel and submitted to the Protein Center of Technion (Haifa, Israel) for
mass spectrometry analysis.
Purification of native and recombinant paramyosin. Mouse monoclonal anti-
paramyosin antibodies (4B1 and immunoglobulin G2a [IgG2a] [obtained from
Edward Pearce]) were coupled to cyanogen bromide-activated Sepharose
(Sigma) according to Sigma’s recommended protocol. NPRM from schistoso-
mula (50,000 total) was passed through a column of packed anti-paramyosin–
Sepharose (0.5 ml). The bound protein was eluted with glycine-HCl (pH 2.3),
immediately titrated to pH 7.0 with carbonate buffer (pH 8.6), and dialyzed with
PBS. Recombinant paramyosin was expressed as inclusion bodies in Escherichia
coli transformed with an expression vector, BL-21-DE3 pLys (obtained from
Alan Sher and John Anderson). The recombinant paramyosin was purified in
inclusion bodies, renatured by dialysis with a high salt buffer, and purified over
a W-pore C4 high-pressure liquid chromatography column (Phenomenex, Tor-
rance, Calif.).
Western blot analysis. NPRM or purified protein was subjected to SDS-PAGE
on an 8% acrylamide gel and transferred onto a nitrocellulose membrane
(Schleicher & Schuell, Dassel, Germany). The membrane was blocked with a 5%
skim milk solution (Tnuva, Rehovot, Israel) in Tris-buffered saline containing
0.05% Tween 20 (Sigma) (TBST) (pH 8.0) for 1 h at room temperature. Next,
the membrane was treated with rabbit anti-CD59 or anti-paramyosin antibody
(1:300), washed, and treated with peroxidase-conjugated goat anti-rabbit IgG
(Sigma) (1:5,000) as a second antibody. Bands were developed with an enhanced
chemiluminescence reagent (Pierce, Rockford, Ill.) and exposed to a BioMax
film (Kodak, Rochester, N.Y.).
Immunofluorescence of schistosomula and adult worms. Schistosomula (100
total, 24 h old) or fresh adult worms (20 mixed male and female worms) were
treated with a blocking solution containing 0.5% bovine serum albumin (BSA),
0.1% ␥-globulin, and 10% heat-inactivated goat serum (Sigma) in DSM (50 ␮l)
for 30 min at room temperature. They were then incubated with polyclonal rabbit
anti-paramyosin antibodies or normal rabbit serum (diluted 1:50 in 50 ␮l of
DSM) for 30 min at 37°C. After three washes with DSM on ice, they were treated
with fluorescein isothiocyanate-conjugated goat anti-rabbit IgG (Sigma) (1:100
in DSM) for 30 min on ice. The schistosomes were washed with DSM and
examined under a fluorescence microscope (Olympus, Hamburg, Germany).
Paramyosin binding to C8 and C9. Purified human C8 and C9 (Advanced
Research Technologies, San Diego, Calif.) and BSA (Sigma) (1 ␮g each) were
subjected to SDS-PAGE (8% acrylamide gel) and transferred onto a nitrocellu-
lose membrane. After blocking with 5% milk in TBST for 1 h at room temper-
ature, the membrane was incubated with recombinant paramyosin (4 ␮g/ml) or
with paramyosin purified from NPRM of schistosomula over a DEAE-cellulose
column in 5% milk in TBST for 2 h at 37°C. After two washes with TBST, the
membrane was reacted with monoclonal anti-paramyosin antibody (1:4 in TBST)
for 1 h at room temperature, washed with TBST, and incubated with peroxidase-
conjugated goat anti-mouse IgG (Sigma) (1:5,000) for 1 h at room temperature.
Bands were visualized with an enhanced chemiluminescence reagent.
To show paramyosin binding competition between native C9 and blotted C9,
recombinant paramyosin (1 ␮g) was premixed with native C9 (1:5 [wt/wt]) for 1 h
at 37°C before it was added to the membrane. For determination of the binding
site of paramyosin within C9, purified human C9 was cleaved into C9a and C9b
by treatment with thrombin (Sigma) for 4 h at 37°C or into C9a⬘ and C9b⬘ by
treatment with trypsin (Sigma) for 35 min at 37°C (3). The resultant fragments
were separated by SDS–12% PAGE, blotted onto a nitrocellulose membrane,
and reacted with recombinant paramyosin (data shown) or purified paramyosin
from NPRM of schistosomula (data not shown) for 2 h at 37°C and then reacted
with monoclonal anti-paramyosin antibodies and goat anti-mouse IgG (1:5,000)
as described above.
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In competition assays between paramyosin and human CD59 for binding to
human C9, C9 (1 ␮g) was blotted onto a nitrocellulose membrane that was then
blocked with 5% milk–TBST. The membrane was pretreated with purified hu-
man CD59 (kindly provided by Paul Morgan, Cardiff, United Kingdom) (0, 0.33,
or 1 ␮g) for 1.5 h and then with recombinant paramyosin (2 ␮g) for 1.5 h at 37°C.
Next, the membrane was incubated with monoclonal anti-paramyosin antibodies
overnight at 4°C and then reacted with goat anti-mouse IgG as described above.
C9 polymerization assays. Purified human C9 (2 ␮g) was incubated with 42
␮M ZnCl2 in 20 mM Tris (pH 7.2) for 2 h at 37°C (54). To test the effect of
paramyosin on C9 polymerization, C9 was pretreated with various amounts of
recombinant paramyosin at 37°C for 30 min. The samples were subjected to
SDS-PAGE on a 3 to 10% acrylamide gradient gel under reducing conditions
and stained with Coomassie blue.
C9 polymerization on rabbit erythrocytes (ER) was performed as previously
described (55). Briefly, erythrocytes (2 ⫻ 108/ml) were mixed with 100 ␮l of
normal human serum (NHS) supplemented with 6 ␮g of C9 and were incubated
for 1 h at 37°C. To examine the effect of paramyosin on C9 polymerization, the
NHS supplemented with 6 ␮g of C9 was premixed for 30 min at 37°C with 40 ␮g
of recombinant paramyosin. The lysed cells were washed twice with 3 ml of TBS
and sedimented for 20 min at 4,800 ⫻ g at 4°C, once with 3 ml of 5 mM EDTA
(pH 8.0), and once with 3 ml of 0.5 mM phosphate buffer (pH 8.0). The lysed cell
pellet was dissolved in 30 ␮l of reducing sample buffer and heated for 5 min at
95°C. The samples were analyzed by SDS-PAGE on a 3 to 10% gradient acryl-
amide gel under reducing conditions and stained with Coomassie blue.
Hemolytic assays. Lysis of ER by the alternative complement pathway (15).
Fresh, washed erythrocytes (2 ⫻ 107) were incubated with NHS (6%) as a source
of complement in the presence of Mg-EGTA (2.5 mM MgCl2, 10 mM EGTA)
and various amounts of recombinant paramyosin (0, 2, 10, or 20 ␮g [premixed
with NHS for 15 min at 37°C]) in 0.1 ml of GVB (Veronal-buffered saline [pH
7.4] containing 0.1% gelatin and 0.02% NaN3) for 30 min at 37°C. A total of 1
ml of cold GVB containing 10 mM EDTA was added to stop lysis. Following
centrifugation at 4,400 ⫻ g for 10 min at 4°C, light absorption of the supernatant
at 412 nm was measured and percent lysis (relative to cells completely lysed by
water) was calculated. Percent inhibition of lysis by paramyosin was calculated
relative to samples containing no paramyosin.
Lysis of antibody-sensitized sheep erythrocytes by the classical complement
pathway. To produce antibody-sensitized erythrocytes (EAs), fresh washed
sheep erythrocytes (109 cells/ml) were incubated with rabbit hemolysin (DIFCO)
(1:2,000 in GVB) for 30 min at 37°C and washed with GVB containing 0.5 mM
MgCl2 and 0.15 mM CaCl2 (34). A total of 15 ␮l of EAs (1.5 ⫻ 107) was
incubated with 15 ␮l of C8-deficient human serum (1:10 in GVB) (41) for 15 min
at 37°C, and the resultant EAC5b-7 cells were washed with GVB. Concomitantly,
various amounts of recombinant paramyosin (0, 1, 2, and 4 ␮g) were added to 30
␮l of C7-deficient human serum (diluted 1:4,000 in GVB containing 10 mM
EDTA) for 20 min at 37°C. The treated C7-deficient serum was then added to
EAC5b-7 cells for 30 min at 37°C. A total of 1 ml of cold GVB containing 10 mM
EDTA was added to stop lysis, the cells were subjected to centrifugation at 4,400
⫻ g for 10 min at 4°C, and light absorption of the supernatants at 412 nm was
measured. Percent lysis (relative to cells completely lysed by water) was calcu-
lated. Percent inhibition of lysis by paramyosin was calculated relative to samples
containing no paramyosin.
Complement-mediated killing of schistosomula in vitro. Schistosomula (200
total [3 h old] in 15 ␮l of DSM) were pretreated with heat-inactivated polyclonal
rabbit anti-CD59 or anti-paramyosin antiserum or normal rabbit serum (2, 10, or
40 ␮l) in wells of a 96-well microtiter plate (Corning Incorporated, New York,
N.Y.) for 30 min at room temperature. Then, 100 ␮l of normal or C4-depleted
human serum (32) was added into each well and the plates were incubated
overnight in a 6% CO2 incubator at 37°C. Heat-inactivated serum served as a
control. The mortality of the schistosomula was assessed under an inverted
microscope on the basis of motility and granularity data (19). Experiments were
run in triplicate. Percent mortality values for control cultures were subtracted
from those for experimental cultures, and percent net mortality was calculated
according to the following formula: percent mortality ⫽ [(E ⫺ C) ⫻ 100]/(100 ⫺
C), where C represents the percentage of dead schistosomula in control cultures
and E represents the percentage of dead schistosomula in experimental cultures.
Statistical analysis. Student’s unpaired and paired t tests were used to deter-
mine the statistical significance of differences between various data sets. Results
are expressed as arithmetic means ⫾ standard deviations (SDs). Statistical sig-
nificance was assumed when P ⬍ 0.05.
6404 DENG ET AL.
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 en-
hanced 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).
RESULTS
Identification of SCIP-1 as paramyosin. As explained in
Materials and Methods, a DEAE-cellulose column was used to
partially purify SCIP-1 from NPRM of S. mansoni schistoso-
mula. Bound proteins were eluted from the DEAE-cellulose
by a salt concentration gradient (0 to 0.6 M NaCl) in a 0.01 M
NaAc buffer (pH 6.0). A large protein peak eluted between 0.1
and 0.2 M NaCl. Fractions containing SCIP-1 were identified
by Western blot analysis with polyclonal rabbit anti-CD59 an-
tibodies, pooled, and concentrated. The SCIP-1 pool was sub-
jected to SDS-PAGE under nonreducing conditions on two
lanes of an 8% gel (0.1 mg per lane). The 94- to 97-kDa SCIP-1
band was identified by Western blotting with polyclonal rabbit
anti-CD59 antibodies in one lane, while the second lane was
stained with Coomassie blue. The SCIP-1 band was cut from
the stained gel and subjected to trypsin digestion and mass
spectrometry analysis of the peptides. A total of 15 peptides
were identified; 10 of them were identical to sequences from S.
mansoni paramyosin (SwissProt accession no. P06198). A sim-
ilar analysis performed with adult worm NPRM also identified
paramyosin in the SCIP-1 band. Frequently, SCIP-1 ran on
SDS-PAGE gels as a doublet. Analysis of the upper and lower
bands demonstrated that both contained paramyosin se-
quences.
To further demonstrate that paramyosin, like SCIP-1, can
bind to anti-CD59 antibodies, paramyosin purified from
NPRM over an affinity column of monoclonal anti-paramyosin
antibody was analyzed by SDS-PAGE and Western blotting
with polyclonal rabbit anti-CD59 antibodies. As shown in Fig.
1, affinity-purified native paramyosin (running here as a dou-
blet) bound the polyclonal anti-human CD59 antibodies. The
two bands perhaps represented isoforms generated by phos-
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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.
phorylation of paramyosin (50) or products of an alternative
splicing or of proteolysis.
Immunofluorescence of schistosomula and adult worms
with anti-paramyosin antibody. To examine whether paramyo-
sin is located on the surface of the parasite, live, intact schis-
tosomula and adult worms were analyzed by immunofluores-
cence with anti-paramyosin antibodies. Labeling was performed
with rabbit anti-paramyosin antibodies as explained in Mate-
rials and Methods. Examination under a fluorescence micro-
scope showed that paramyosin is present on the surface of
schistosomula and adult worms (males and females) (Fig. 2).
Paramyosin was distributed over the tegument with patches of
darker and brighter regions.
Paramyosin binds to C8 and C9. Purified human C8 and C9
and BSA were subjected to SDS-PAGE and transferred onto a
nitrocellulose membrane. After blocking, the membrane was
reacted with recombinant paramyosin (4 ␮g/ml) or paramyosin
purified from NPRM of schistosomula over a DEAE-cellulose
column for 2 h at 37°C. Western blot analysis with monoclonal
anti-paramyosin antibody showed that both recombinant
paramyosin (Fig. 3) and purified paramyosin (data not shown)
bound to C8 and C9 on the membrane but not to BSA. When
identical amounts of native and recombinant paramyosin (as
determined by immunoblotting with anti-paramyosin antibod-
ies) were reacted with C9, recombinant paramyosin produced
a stronger band than native paramyosin (data not shown). This
suggested that recombinant paramyosin binds to C9 with a
higher affinity or that the native paramyosin preparation con-
tains an inhibitor of C9 binding. Paramyosin bound to both ␣-␥
and ␤ chains of C8. To test whether native C9 can compete
with blotted C9 in binding to paramyosin, recombinant
paramyosin was premixed with native C9 (1:5) for 1 h at 37°C
before it was added to the membrane containing C9 and BSA.
VOL. 71, 2003
FIG. 3. Binding of paramyosin to C8 and C9. Purified human C8
and C9 and BSA (1 ␮g 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 ␮g/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, unre-
lated monoclonal antibody (Control) and then incubated with perox-
idase-conjugated goat anti-mouse IgG (1:5,000) for 1 h at room
temperature. Bands were visualized by enhanced chemiluminescence.
Lane a, C8␣ and C8␥ (upper band) and C8␤ (lower band); lane b, C9;
lane c, BSA.
As shown in Fig. 4 lanes a, soluble C9 almost completely
blocked paramyosin binding to blotted C9, suggesting the in-
volvement of native epitopes on C9 in the binding. Further-
more, soluble CD59 blocked paramyosin binding to blotted C9
(Fig. 4, lanes c to e), implying that paramyosin binds to C9
roughly to the same site where CD59 binds to it (21), i.e.,
between Cys359 and Cys384.
To further characterize the region in C9 involved in binding
to paramyosin, purified human C9 was cleaved into C9a and
C9b with human thrombin or into C9a⬘ and C9b⬘ with trypsin
(3). The resultant fragments were subjected to SDS-PAGE and
transferred to a nitrocellulose membrane. After blocking with
milk, the membrane was incubated with recombinant paramyo-
sin and then with monoclonal anti-paramyosin antibody as
described above. Paramyosin bound to the C9b fragment of
thrombin cleavage and to the C9a⬘ fragment of trypsin cleav-
age (Fig. 5A). The C9 cleavage sites are shown schematically in
Fig. 5B. The data indicate that the paramyosin binding site in
C9 is located between Gly245 and Arg391, the sequence over-
lapping C9b and C9a⬘.
Inhibition of Zn2ⴙ-induced C9 polymerization by paramyo-
sin. Human C9 has a tendency to polymerize on prolonged
incubation (for 3 days) at 37°C to a circular polymer of C9
(polyC9), a process that is accelerated by the presence of Zn2⫹
ions and that occurs in their presence within 2 h (54). PolyC9
has a tubular structure resistant to dissociation by boiling in
SDS (44). To examine whether paramyosin can inhibit Zn2⫹-
induced C9 polymerization, C9 was mixed with various
amounts of paramyosin for 30 min at 37°C and then incubated
with ZnCl2 (at a final concentration of 42 ␮M) for 2 h at 37°C.
The samples were analyzed by SDS-PAGE on a 3 to 10%
acrylamide gradient gel under reducing conditions, and the gel
was stained with Coomassie blue. As shown in Fig. 6, paramyo-
sin inhibited the Zn2⫹-induced C9 polymerization in a dose-
dependent manner. At about threefold molar excess (8 ␮g),
paramyosin completely inhibited polymerization of C9.
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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 ␮g 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-conju-
gated 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 mono-
clonal anti-paramyosin antibodies, goat anti-mouse IgG, and enhanced
chemiluminescence reagent. Lane c, 2 ␮g of paramyosin without
CD59; lane d, 2 ␮g of paramyosin plus 0.33 ␮g of CD59; lane e, 2 ␮g
paramyosin plus 1 ␮g of CD59.
Inhibition of polyC9 formation on ER by paramyosin. Com-
plement activation by cells like ER culminates in formation of
the complement MAC composed of C5b, C6, C7, C8, and C9
and cell lysis (4). The C9 within the MAC expresses a hydro-
phobic domain and polymerizes and inserts into the plasma
membrane of the target cell (45). To further test whether
paramyosin can inhibit polyC9 formation on ER, recombinant
paramyosin was added to 100 ␮l of NHS supplemented with 6
␮g of C9, incubated for 30 min at 37°C, and then incubated
with ER for 1 h at 37°C. The ER were washed, dissolved in
reducing sample buffer, and analyzed by SDS-PAGE on a 3 to
10% gradient gel. As shown in Fig. 7, lane a, paramyosin
inhibited polyC9 deposition on ER.
Inhibition of complement-mediated hemolysis by paramyo-
sin. That paramyosin inhibits C9 polymerization suggested that
paramyosin might block complement-mediated lysis of target
cells. The alternative and classical pathways of human comple-
ment are activated by ER and EAs (antibody-coated sheep
erythrocytes), respectively. This leads to lysis of the red blood
cells by the complement MAC. To test whether paramyosin
can inhibit the lysis of ER by the alternative pathway, NHS was
incubated with recombinant paramyosin (0, 2, 10, or 20 ␮g) for
15 min at 37°C and then with ER in GVB containing 2.5 mM
MgCl2 and 10 mM EGTA for 30 min at 37°C. As shown in Fig.
8, paramyosin significantly (P ⬍ 0.05) inhibited lysis of ER by
the complement in a dose-dependent manner. Lysis of ER was
reduced to 30% of that of the control by 10 ␮g of paramyosin
and completely blocked by 20 ␮g of paramyosin.
Next, we tested whether paramyosin can inhibit lysis of EAs
by binding to C8 and C9. EAs were treated with C8-deficient
human serum for 15 min at 37°C to generate EAs with bound
C5b-7 complexes (EAC5b-7 cells) and washed. Next, C7-defi-
cient human serum (as a source for C8 and C9) preincubated
with or without recombinant paramyosin for 20 min at 37°C
was added to EAC5b-7 cells in GVB containing 10 mM EDTA
and incubated for 30 min at 37°C. EDTA was added to block
further classical pathway activation. As shown in Fig. 9,
paramyosin significantly (P ⬍ 0.005) inhibited lysis of EAC5b-7
by C8 and C9 in a dose-dependent manner. This clearly dem-
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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
␣-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.

onstrates that paramyosin can block cell lysis at the stage of
MAC formation.
Paramyosin on schistosomula is protective from comple-
ment-mediated killing. To block the postulated complement
inhibitory activity of paramyosin, schistosomula were treated
with heat-inactivated rabbit anti-paramyosin or anti-CD59 an-
tibodies for 30 min at room temperature. Normal rabbit serum
served as a control. Next, 100 ␮l of normal or C4-depleted
human serum was added as a source of complement for an
overnight incubation at 37°C. As shown in Fig. 10, preincuba-
tion with anti-paramyosin or with anti-CD59 antibodies signif-
icantly (P ⬍ 0.05) enhanced the in vitro killing of schistoso-
mula by human complement in a dose-dependent manner even
in the absence of an active classical complement pathway. In
contrast, normal rabbit serum produced only a small enhance-
ment of schistosomula killing. No significant difference was
observed between the results obtained with anti-paramyosin
and anti-CD59 antibodies.
DISCUSSION
Results shown here demonstrate that paramyosin of the
blood fluke S. mansoni is the inhibitor of the MAC C5b-9 of

FIG. 6. Inhibition of Zn2⫹-induced C9 polymerization by recombi-

nant paramyosin (Pmy). C9 (2 ␮g) was mixed with various amounts of
paramyosin (0, 2, 4, or 8 ␮g) for 30 min at 37°C and then incubated
with 42 ␮M 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 ␮g
of paramyosin; lane b, C9 plus 4 ␮g of paramyosin; lane c, C9 plus 2 ␮g
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 prob-
ably represent fragments of paramyosin (lanes a to c) or C9 (lanes d
and e).
FIG. 7. Inhibition of polyC9 deposition on ER by paramyosin.
Fresh ER were treated with 100 ␮l of NHS supplemented with 6 ␮g of
C9 preincubated for 30 min at 37°C with (lane a) or without (lane b)
40 ␮g of recombinant paramyosin. The lysed ER 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 Coomas-
sie 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.
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FIG. 8. Inhibition of complement-mediated lysis of ER by recombinant paramyosin (Pmy). Fresh ER (2 ⫻ 107 cells) were incubated with NHS
(6%) in the presence of Mg-EGTA and various amounts (0, 2, 10, or 20 ␮g) 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.

human complement, previously named SCIP-1 (40). SCIP-1
was originally referred to as a CD59-like protein on the basis of
two criteria: (i) binding to anti-CD59 antibodies and (ii) bind-
ing to and inhibition of complement C9. By using column
chromatography and acrylamide gel fractionation, SCIP-1 has
been partially isolated. This permitted its identification as
paramyosin by mass spectrometry. Purified, native paramyosin
fulfills the two above-mentioned criteria. In addition, recom-
binant paramyosin is shown to bind C8 and C9, inhibit forma-
tion of the MAC, and block complement-mediated cell lysis.
That anti-paramyosin antibodies sensitize schistosomula to
killing by human complement strongly supports the claim that
paramyosin acts as a complement inhibitor in its native form
on the surface of schistosomes.
Paramyosin, a muscle protein of invertebrates, assembles
with myosin to form the thick muscle filaments (7, 11). Our
data confirm previous reports (18, 31, 33) that paramyosin can
be found in schistosomes of S. japonicum and S. mansoni not
only within muscles but also within the tegument and on the
surface. Antibodies directed to paramyosin label schistoso-
mula, as well as adult worms, by immunofluorescence (31)
(Fig. 2). External paramyosin can be removed from schistoso-
mula by detergent solubilization of the tegument (Fig. 1) and,
at least partly, by treatment with a phosphatidylinositol-specific
phospholipase C (unpublished data). In addition, paramyosin
can be exogenously biotinylated on the surface of live S. man-
soni adult worms (31). Adults of the Asian schistosome S.
japonicum also contain a surface form of paramyosin (31).
Paramyosin was also detected in the tegument of other para-
sitic worms such as Echinococcus granulosus (37) and Taenia
solium (26). It is likely that the external and intrategumental
paramyosin is produced by the subtegumental cells within cell

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 ␮g) 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.
6408 DENG ET AL.
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.
bodies situated beneath the outer muscle fibers (20). Alterna-
tively, the paramyosin may be produced within the muscle
fibers and secreted into the tegument coating it and may reach
the external surface through it. Another source for surface
paramyosin, at least in larvae, could be secretions of the posta-
cetabular glands, as shown for S. japonicum cercariae (18).
Human CD59 is an 18-kDa glycosylphosphatidylinositol-
linked membrane glycoprotein, widely expressed in all circu-
lating cells and most tissues, that serves as an inhibitor of the
C5b-9 MAC of human complement (9, 17, 39, 48). The com-
plement inhibitory activity of CD59 depends on its ability to
bind to C8 in the C5b-8 complex (30) and to C9 between
Cys359 and Cys384 (21), thereby inhibiting ion channel forma-
tion by C5b-8 (13) and generation of the polymerized C9
responsible for MAC cytolytic activity (36, 39, 48). In contrast
to CD59 that binds to C8␣ (30), paramyosin appears to bind
both C8␣ and C8␤ chains (Fig. 3). As shown here, paramyosin
binds to C9 at a site located between Gly245 and Arg391 that
overlaps with the postulated CD59 binding site. It is suggested
that CD59 and paramyosin bind to the same location in C9 or
in close proximity. This is supported by the competition be-
tween purified human CD59 and paramyosin for binding to C9
blotted onto nitrocellulose membrane (Fig. 4, right panel). The
fact that paramyosin shares no continuous sequence homology
with CD59, as concluded from alignment trials, is perhaps
surprising in light of the fact that rabbit anti-CD59 antibodies
bind specifically to paramyosin. However, monoclonal antibod-
ies directed to CD59 do not bind to paramyosin and rabbit
anti-paramyosin polyclonal antibodies do not bind to human
CD59 (unpublished data). Therefore, it is suggested that the
rabbit antiserum raised against human CD59 contains antibod-
ies directed to a specific conformational peptide epitope or to
a modifying nonpeptide residue of CD59 (e.g., carbohydrate or
lipid) present also on paramyosin.
Paramyosin is considered a vaccine candidate against schis-
tosomiasis mansoni (42), schistosomiasis japonicum (24, 46),
filariasis (29, 38), and cysticercosis (57). It acts as one of the
major schistosome immunogens during infection with S. man-
INFECT. IMMUN.
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soni in mice (27) and humans (47). Vaccination trials per-
formed with S. mansoni (16, 42) or S. japonicum (6, 35, 53)
infections in mice, pigs, or water buffaloes demonstrated that
immunization with native or recombinant paramyosin can re-
duce the worm burden and liver or fecal egg counts in infected
animals. It will soon become clear whether or not paramyosin
exerts a similar vaccination activity in humans (2).
The relationship between the paramyosin B cell epitopes
contributing to protection from infection and the complement
modulatory sites in paramyosin is unclear. It is conceivable that
protective antibodies targeted at the paramyosin active sites
would be more effective. Such antibodies would mediate acti-
vation of humoral and cellular immune responses, and at the
same time inhibit the capacity of paramyosin to serve as an
immune evasion molecule. Remarkably, paramyosin is capable
of inhibiting complement activation by binding to at least three
complement components: C1q (25), C8, and C9 (Fig. 3 to 5).
By binding to complement C1 it may inhibit initial activation of
the classical pathway, and by binding to C8 and C9 it may
inhibit generation of the membranolytic terminal complement
complexes. In addition, by binding to Fc of IgG (31) it may
mask the surface and block binding of immune antibodies.
Paramyosin is a multifunctional alpha-helical coiled-coil mus-
cle protein (8, 11) and has various zones of positive and neg-
ative charges on the outer surface of the alpha-helices (23).
These highly charged zones have been suggested to account for
the ability of paramyosin to bind to ligands (31) such as Fc,
collagen, and C1. Possibly, they are also involved in the binding
of paramyosin to C8 and C9.
In summary, these results suggest a novel immunomodula-
tory role for paramyosin in inhibition of the terminal pathway
of complement. It is anticipated that paramyosin of other par-
asitic worms, e.g., S. japonicum (1), T. solium (56) and Opis-
thorchis felineus (51), which share a high degree of homology
with S. mansoni paramyosin, also have a C9 inhibitory activity.
To date, the complement system has not been counted among
the major immune effector mechanisms involved in limiting
infection by parasitic worms. This is largely because of the
VOL. 71, 2003
most efficient control of complement activation by these par-
asites. Yet, as can be shown in vitro, the complement system
has all of the arsenal required to execute killing of schisto-
somes. Future attempts to establish passive or active parasite
humoral immunotherapy will have to consider and cope with
the multitude of complement evasion molecules, such as the
surface paramyosin.
ACKNOWLEDGMENTS
This work was supported in part by the U.S.-Israel Binational Sci-
ence Foundation, the Cooperation Program of the Deutsches
Krebsforschungszentrum (DKFZ) and the Israeli Ministry of Science
(MOS), and the National Institutes of Health (AI 18867 and
TW006280).
We are grateful to Sir Peter Lachmann for the anti-CD59 antibodies
and for his continuous interest and support, to Edward Pearce for the
anti-paramyosin monoclonal antibodies, and to Alan Sher and John
Anderson for the plasmid expressing paramyosin.
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