- Open Access
Mapping of the complement C1q binding site on Trichinella spiralis paramyosin
Parasites & Vectorsvolume 11, Article number: 666 (2018)
Trichinella spiralis is a tissue-dwelling parasite has developed the ability to evade the host immune attack to establish parasitism in a host. One of the strategies evolved by the nematode is to produce proteins that immunomodulate the host immune system. TsPmy is a paramyosin secreted by T. spiralis on the surface of larvae and adult worms that can interact with complement components C1q and C8/C9 to compromise their activation and functions. To better understand the mechanism of TsPmy involved in the C1q inactivation and immune evasion, the C1q-binding site on TsPmy was investigated.
The TsPmy C1q-binding site was investigated by sequential narrow-down fragment expression in bacteria and peptide binding screening. C1q binding activity was identified by Far-Western blotting and ELISA assays.
After several runs of sequential fragment expression, the C1q binding site was narrowed down to fragments of N-terminal TsPmy226-280aa and TsPmy231-315aa, suggesting the final C1q binding site is probably located to TsPmy231-280aa. A total of nine peptides covering different amino acid sequences within TsPmy231-280aa were synthesized. The binding assay to C1q determined that only P2 peptide covering TsPmy241-280aa binds to C1q, indicating that the C1q binding domain may need both the linearized sequence and conformational structure required for binding to C1q. The binding of peptide P2 to C1q significantly inhibited both C1q-initiated complement classical activation and C1q-induced macrophage chemotaxis.
This study identifies the C1q binding site within TsPmy which provides helpful information for developing a vaccine against trichinellosis by targeting the C1q-binding activity of TsPmy.
Trichinella spiralis is a parasitic nematode that has a broad host range, including humans and more than 150 mammalian species . Trichinellosis is a worldwide foodborne zoonotic disease caused by the ingestion of undercooked or raw meat infected with infective larvae of T. spiralis . This tissue-dwelling parasite has developed the ability to evade the host immune attack in order to become established in the host [3,4,5]. The mechanism by which Trichinella evades the host immune response and survives in a hostile environment is not well understood [6, 7].
The complement has been identified as a first line immune defense system against invading pathogens. C1q is the first subcomponent of the C1 complex of classical complement pathway activation and plays a vital role in linking innate and acquired immunity. More evidence has suggested that C1q is an important target for immune evasion of some pathogens [8,9,10,11,12,13,14,15].
Paramyosin is a fibrous protein in invertebrates which is not only a structural component of muscle, but also an important immunomodulatory protein expressed on the surface of parasitic helminths [5, 16, 17]. TsPmy, the paramyosin expressed by the larval and adult T. spiralis , plays an important role in the process of the nematode’s immune evasion from host complement attack by binding to complement component C1q  and C8/C9 [5, 18]. It has therefore become the leading candidate for a vaccine against T. spiralis infection, particularly since mice immunized with different forms of TsPmy (recombinant protein, DNA, multiepitope) have shown to develop significant protection against a challenge of T. spiralis infective larvae [19,20,21,22,23,24,25]. The binding domain to C9 has been pinned down to the 14 amino acid residues within the TsPmy C-terminal region (866Val-879Met) . Further studies have revealed that TsPmy’s binding to C1q inhibited not only the classical complement activation, but also C1q-induced macrophage migration and functions . However, the C1q binding site on TsPmy has not been determined. Identification of the C1q binding site on TsPmy would further facilitate our understanding of the complement binding and evasion activity of TsPmy. This information will assist the design of drugs and vaccines based on its complement regulatory functions.
To ascertain the C1q binding site of TsPmy, sequential fragment expressions were performed in this study and their binding activities to C1q were investigated. As a result, the C1q binding site was narrowed down to the region between241Val and 280Ile at the N-terminus of TsPmy. The synthesized peptide based on this region was able to bind to C1q and subsequently inhibit the activation of the C1q-initiated classical pathway and C1q-induced macrophage migration. Our results provide important information to further understand the interaction between TsPmy and C1q and complement evasion mechanisms of T. spiralis, therefore facilitating the design of novel drugs and vaccines to control trichinellosis.
Normal human serum (NHS) was selected from healthy human volunteers. Human C1q-deficient serum (C1q D) was purchased from Merck (Merck Darmstadt, Germany).
Expression of TsPmy and its fragments
To identify and locate the complement C1q binding site on TsPmy, the full-length T. spiralis paramyosin (TsPmy1-885aa, NCBI accession numbers for nucleotides sequence: EF429310.1 and protein sequence: ABO09862.1) was expressed as recombinant protein using a baculovirus insect expression system (Invitrogen, Carlsbad, CA, USA), and the subsequent fragments of TsPmy, with 30 amino acids overlapped (TsPmy1-315aa, TsPmy286-600aa, TsPmy571-885aa), were sub-cloned and expressed in an E. coli expression system (Novagen, Merck, Darmstadt, Germany) as described in . Further fragmenting expression on the C1q-binding fragments of TsPmy1-315aa (TsPmy1-125aa, 96-220aa, 191-315aa) and TsPmy191-315aa (TsPmy191-245aa, 226-280aa, 261-315aa) was performed in E. coli. All recombinant proteins were expressed with 6-histidine tag and purified by nickel affinity chromatograph (GE Healthcare, Boston, MA, USA).
Peptide design and synthesis
To finally determine the amino acid sequence of the C1q binding site on TsPmy, the peptides with different amino acid sequences were synthesized by solid-phase peptide synthesis (China Peptides, Shanghai, China). The acquired 9 peptides were designed (P1-P9, TsPmy231-280aa, TsPmy241-280aa, TsPmy251-280aa, TsPmy261-280aa, TsPmy231-275aa, TsPmy231-276aa, TsPmy231-277aa, TsPmy231-278aa, TsPmy231-279aa), purified up to 95% by preparative RP-HPLC and verified by mass spectrometry.
C1q binding assay of TsPmy and its fragments
To detect whether the expressed recombinant TsPmy and its fragment proteins bind to human C1q, Far-Western blotting was used based on the method as previously described . Briefly, 1 μg of each protein was subject to 12 or 15% SDS-PAGE under reducing conditions, and then transferred onto a nitrocellulose membrane (GE Healthcare). After being blocked with 5% (w/v) dry milk in PBS, the membrane was incubated with human C1q (5 μg/ml in PBS, pH 7.4, 0.5 mM Ca2+, 0.5 mM Mg2+, 1% dry milk) at 37 °C for 2 h, then probed with anti-C1q antibody (Abnova, Taipei City, Taiwan, China) at a 1:10,000 dilution in the same dilution buffer for 1 h at room temperature. IRDye 800CW-labled goat anti-mouse IgG (LI-COR, Lincoln, NE, USA) at 1:10,000 was used as the secondary antibody. Bovine serum albumin (BSA) (Thermo Fisher, Life Technologies, Carlsbad, CA, USA) and Ts87, a T. spiralis expressed protein , were used as negative and non-relevant controls. The membranes were visualized and imaged with an Odyssey CLx Infrared Imaging System (LI-COR).
An ELISA-based assay was also used to determine the C1q binding activity of TsPmy and its fragments according to the method as previously described . Briefly, a 96-well ELISA plate was coated with human C1q at 5.0 ug/ml in carbonate buffer (100 mM Na2CO3/NaHCO3, pH 9.6), blocked with 5.0% (w/v) BSA in PBS at 37 °C for 2 h, then incubated with different concentrations of recombinant TsPmy fragments at 25 °C for 1 h. Anti-His mAb at 1:1000 in PBS/1% BSA was used to probe recombinant proteins, and HRP-conjugated goat anti-mouse IgG (BD Biosciences, Franklin Lakes, NJ, USA; 1:5000 in PBS/1% BSA) was used as secondary antibody. For the peptide binding assay, synthesized peptide P2 was labeled with biotin and the C1q coated plate was incubated with different concentrations of biotinylated peptide P2 (B-P2). HRP-conjugated streptavidin (Abcam, Cambridge, UK; 1:10,000 in PBS/1% BSA) served as the detection antibody. After adding the substrate o-phenylendiamine dihydrochloride (OPD, Sigma-Aldrich, St. Louis, MO, USA), absorbance was measured at 450 nm with a plate reader (Thermo Fisher, Life Technologies). BSA coated on the same plate was used as a negative control.
Dot blot was used to detect whether the synthesized peptides bind to human C1q. The peptides (5 μg in total volume of 2.5 ul) were spotted onto a nitrocellulose membrane. After blocking with 5% (w/v) dry milk in PBS, the membrane was incubated with human C1q (5 μg/ml) at 37 °C for 2 h, washed with PBS, then probed with anti-C1q antibody (1:10,000) at room temperature for 1 h. The same amount of C1q and BSA were used as controls.
Detection of peptide P2-mediated inhibition of complement C4 deposition
To evaluate whether the binding of peptide P2 to C1q inhibits the C1q-initiated complement activation, the intermediate complement C4 deposition following complement activation was analyzed . To initiate the classical activation, purified human IgM (Calbiochem, Merck, Darmstadt, Germany) was coated on plates (0.2 μg/well). The plates were blocked with 3% (w/v) BSA, then incubated at 37 °C for 1 h with NHS which was pre-incubated with various doses of peptide P2 (0, 2, or 6 μg) in 1× Veronal buffer (VB; Lonza, Basel, Switzerland) containing 0.05% (v/v) Tween-20 and 0.1% (w/v) gelatin. Subsequently, goat anti-human C4 mAb (1:10,000, Abcam) was added and incubated for 1 h. HRP-conjugated rabbit anti-goat IgG (1:10,000; BD Biosciences) was used as the secondary antibody. OPD was used as substrate and absorbance at 450 nm was measured.
To determine whether the binding of P2 peptide to C1q inhibits the classical complement activation-mediated hemolysis in the same way as the full-length TsPmy, 100 μl of fresh sheep red blood cells (SRBC 5 × 108 cells/ml) were sensitized with rabbit anti-SRBC antibody (Sigma-Aldrich) at a dilution 1:200 in HBSS++ buffer (Hank’s Balanced Salt Solution containing 0.6 mM MgSO4·7H2O, 0.5 mM MgCl2·6H2O, 1.3 mM CaCl2) at 37 °C for 30 min; 2 μg C1q was pre-incubated with various amounts of peptide P2 (0, 0.5 and 1.0 μg) at 37 °C for 90 min before the addition of 100 μl of 1:50 diluted human C1q-deficient serum (C1q D). Subsequently, sensitized SRBCs were mixed with the treated C1q D at 37 °C for 30 min. Cold HBSS++ containing 10 mM EDTA was added to stop the reaction and the solution was centrifuged at 1000× g for 10 min. The absorbance of the supernatants was measured at 412 nm to calculate the reduced hemolysis compared to the total lysis in water.
Transwell migration assay
To evaluate the effect of the C1q-binding peptide P2 on the C1q-induced migration of THP-1-derived macrophages, a cell migration assay was performed using a 24-well-transwell permeable support system with 8 μm pores (Corning, New York, NY, USA). Briefly, M2 macrophages were induced from human leukemia monocytic THP-1 cells (an acute monocytic leukemia cell line, Infrastructure of Cell Line Resource, http://cellresource.cn/contact.aspx, China) by adding phorbol-12-myristate-13-acetate (PMA) (100 ng/ml, Sigma-Aldrich) for 48 h and then human IL-4 (Prepro Tech, Rocky Hill, NJ, USA) for 24 h as previously described . The THP-1-derived M2 macrophages (2 × 105) were added to the upper chamber. RPMI 1640 containing human C1q (10 nM) mixed with different amounts of peptide P2 (0, 2, 4 and 6 μg) was placed in lower chamber. After incubation at 5% CO2, 37 °C for 24 h, the cells that migrated to the bottom surface of the membrane were stained with Giemsa and counted in 10 randomly chosen fields under a microscope as described previously . TsPmy (6 μg) was added as a positive control and BSA (6 μg) as a negative control. LPS (100 ng/ml) was used as positive control to induce the migration of macrophages.
The results are presented as the mean ± standard error of the mean (SEM) or the mean ± standard deviation (SD). The data were statistically analyzed using SPSS v.17.0 software. One-way ANOVA with LSD-t multiple comparison test analysis was utilized to analyze the differences between groups. P < 0.05 was considered statistically significant.
Binding of TsPmy to human C1q
The binding of full-length TsPmy1-885aa to human complement C1q was confirmed by Far-Western blotting analysis. The results demonstrated that C1q was able to bind onto full-length TsPmy on the membrane (Fig. 1). As a control, C1q could not bind to the same amount BSA at the same condition.
Mapping of C1q binding fragment of TsPmy
To identify the C1q binding domain(s) within TsPmy, three different fragments covering the whole molecule with 30 amino acids overlapped were expressed as recombinant proteins (Fig. 2a). The binding of recombinant fragments to human complement C1q was determined by ELISA with C1q coated on a plate. Only fragment TsPmy1-315aa bound to C1q in a dose-dependent manner, detected by anti-His Ab; the rest of the fragments did not bind (Fig. 2b). All fragments did not bind to BSA coated on a plate with the same conditions (Fig. 2c). Since TsPmy 1-315aa is a fragment that binds to C1q, further fragment expressions were performed within amino acid 1-315aa (Fig. 3a). The C1q binding site was narrowed down to TsPmy191-315aa detected by Far-Western blotting with fragments transferred on a membrane which was incubated with C1q and probed with anti-C1q Ab (Fig. 3b, c). A further narrow-down fragment assay identified the final C1q-binding site was located at TsPmy226-280aa (Fig. 3d, e). ELISA with C1q coated on a plate also concluded similar results (data not shown). Another similar fragment expression approach showed that the fragment TsPmy231-315aa bound to C1q at a similar level to fragment TsPmy226-280aa (data not shown), indicating that the C1q binding site should fall into the fragment between 231-280aa. The TsPmy fragments binding to C1q was TsPmy-specific since the same amount of BSA or Ts87 did not show any binding activity.
Determination of the C1q binding peptide
The fragment expression assay determined that the C1q binding domain should be within TsPmy231-280aa. To further pinpoint the C1q binding site within this fragment, 9 peptides with different amino acids (20–50) covering TsPmy231-280aa (P1-P9) were synthesized for evaluating their binding activity to human C1q (Fig. 4a). After being spotted onto a nitrocellulose membrane, the peptides (P1-P9) were probed with human C1q and detected with anti-C1q antibody. The dot-blot analysis demonstrated that only peptide P2 (TsPmy241-280aa) was able to bind C1q (Fig. 4b), narrowing down the final C1q binding site to TsPmy241-280aa. To further confirm whether the 40 amino acid peptide P2 can bind to C1q or not, an ELISA was performed with C1q coated on a plate, then incubated with biotinylated P2 (B-P2) and detected with streptavidin. Results showed that peptide B-P2 was capable of binding C1q in a dose-dependent manner (Fig. 4c). The same amount of B-P2 did not bind to BSA control (Fig. 4d). These findings confirm that P2 is able to bind to C1q and the 40 amino acid re sidues between 241Val and 280Ile of TsPmy (VKTQLAQQLEEARRRLEDAERERSQMQTQLHQMQLELDSI) contains the C1q- binding site.
Inhibition of classical complement activation and complement-mediate hemolysis by peptide P2
To assess whether the C1q-binding peptide P2 has the ability to interfere with the human C1q complement function, IgM-C1q-initiated C4 deposition and complement-mediated hemolysis were tested. The results showed that the addition of P2 (0, 2, 6 μg) to NHS inhibited C4 deposition in a dose-dependent manner (P2 0 μg vs P2 2 μg, t = 2.566, df = 27, P = 0.016; P2 2μg vs P2 6 μg, t = 2.217, df = 27, P = 0.035). Six micrograms of P2 possessed a similar inhibitory effect to 2 μg of full-length TsPmy (P2 6 μg vs TsPmy 2 μg, t = 1.069, df = 27, P = 0.295). The same amount of BSA (6 μg) had no any inhibitory effect on C4 deposition (BSA 6 μg vs P2 0 μg, t = -0.496, df = 27, P = 0.624) (Fig. 5a). At a similar level to the full-length TsPmy (P2 1 μg vs TsPmy 0.5 μg, t = 0, df = 22, P = 1.000), the inhibitory effect of P2 on C1q-induced classical complement-mediated hemolysis was observed (P2 0 μg vs P2 0.5 μg, t = 3.801, df = 22, P = 0.001; P2 0.5 μg vs P2 1 μg, t = 2.110, df = 22, P = 0.046) (Fig. 5b). There was no significant hemolysis in the presence of C1q D serum because the classical pathway could not be activated without C1q. BSA had no inhibitory effect on complement-mediate hemolysis (BAS 1 μg vs P2 0 μg, t = -1.247, df = 22, P = 0.226) (Fig. 5b).
Inhibition of C1q-induced chemotaxis of THP-1-derived macrophages by peptide P2
As shown in Fig. 6, both LPS and C1q significantly attracted THP-1-derived M2 macrophages migration through the membrane (F(7,103) = 151.081, P < 0.001). However, pre-incubation with peptide P2 significantly inhibited C1q-induced M2 macrophage migration through the membrane in a dose-dependent manner (P2 0 μg vs P2 2 μg, t = 9.813, df = 103, P < 0.001; P2 2 μg vs P2 6 μg, t = 9.813, df = 103, P = 0.001) (Fig. 6) as did full-length TsPmy. The same amount of BSA did not show any inhibitory effect on M2 migration (BSA 2 μg vs P2 0 μg, t = 0.629, df = 103, P = 0.531).
Due to the importance of the complement system in the clearance of invaded pathogens, pathogens evolve different strategies to interfere with the activation and functions of complement including the expression of some complement regulatory proteins . C1q is the key target of these pathogen expressed complement regulatory proteins. In addition to TsPmy, other complement regulatory proteins include human astrovirus coat protein ; paramyosin from Taenia solium ; calreticulins from Trypanosoma carassii , Brugia malayi  and T. spiralis ; scabies mite inactive protease ; and GAPDH from Haemonchus contortus . All of them can bind to C1q and inhibit its ability to escape C1q-initiated complement attack and to survive in the host. C1q is not only the initiation factor for the classical complement pathway, but is also involved in a number of other immunological processes through binding to immunocyte surface receptors [31,32,33]. A study on infective larvae of Strongyloides stercoralis identified that activation of the classical complement pathway promoted the adhesion of monocytes to the larval surface and reduced the motility of the larvae; notably complement C1q was a vital component . However, it is still unclear how parasitic helminths evade complement attack through acting on C1q.
Paramyosin is also expressed on the surface of some helminths with key role in immunomodulation [35, 36]. The structure of paramyosin contains an alpha-helical coil with repetitive sequences of hydrophobic and charged amino acids that contributes to its strong binding affinity to many other proteins such as human collagen [9, 37, 38], calgranulin , IgG [37, 38], IgA , C1q , C8  and C9 [35, 38], which may be related to immunomodulatory functions.
In our previous study, we identified that TsPmy of T. spiralis plays important roles in immunomodulation and complement evasion mainly through binding to C8/9 [5, 18] and C1q  as a survival strategy in the host. Binding of TsPmy to human C1q inhibited C1q-initiated classical complement activation and C1q-induced macrophages migration . The present study describes the identification of the C1q binding site in TsPmy241-280aa by sequential fragment expression. Other synthesized peptides containing more amino acids (P1) or fewer amino acids (P3-P9) than TsPmy241-280aa did not show any binding activity, indicating the C1q binding domain may need both the linearized sequence and conformational structure required for binding to C1q. It is possible that P2 peptide contains fragments of 241Val and 280Ile that forms the proper conformational structure necessary for its binding activity to C1q. The subsequent classical activation pathway inhibitory assay with P2 confirmed that this peptide was able to inhibit the formation and deposition of C4, the intermediate product of C1q-induced classical activation. It also inhibited C1q-induced chemotaxis of macrophages, further confirming that P2 is not only able to bind to C1q, but also inhibit C1q induced complement activation and macrophage migration.
Even though the full-length TsPmy has been determined as a good vaccine candidate for trichinellosis [16, 19], the difficulty in expressing TsPmy as a soluble recombinant protein, in scaling-up product development, and the functional multiplicity and complexity of the whole protein, prevent TsPmy from being developed as a recombinant protein vaccine . Much effort has been made to identify the protective epitope(s) of TsPmy to streamline the design process of the TsPmy epitope or multi-epitopes vaccine against trichinellosis. Two B-cell epitopes were identified on TsPmy by phage display screening with protective monoclonal antibodies against TsPmy [20, 22] and several T-cell epitopes were predicted . Vaccinating with individual B-cell or T-cell epitopes induced a limited amount of protection in mice against T. spiralis infection [22, 41]. Strikingly, the multi-epitope subunit vaccine with a combination of B and T cell epitopes of TsPmy  or with an epitope from another protective antigen, Ts87 , induced much higher protection than an individual epitope or the recombinant full-length protein which correlated with higher Th1 and Th2 immune responses [22, 24]. The identification of C1q binding domain in the TsPmy N-terminus in this study, together with the C9 binding domain that has been identified within C-terminal 14 amino acid residues (866Val-879Met) , provides the feasible design of a multivalent peptide vaccine for preventing infection of T. spiralis in immunized host through efficiently interrupting worm’s ability to evade complement attack. A multi-epitope vaccine is a promising approach for vaccine design and application to prevent infections and cancer therapy [24, 42, 43], since it has been shown to increase safety and protective efficacy, with decreased side effects, immunological competition and interference of the whole vaccine molecule. TsPmy-induced protection in immunized animal should be a consequence of protective immunity induced by multiple protective epitopes. Due to the importance of TsPmy involved in the immunomodulation of host complement activation as an immune evasion strategy, inhibition of the complement binding activity of TsPmy could possibly reduce the viability of parasites in the host by vaccinating with the C1q binding site identified in this study or/and the C9 binding domain . The finding of the C1q binding site in TsPmy provides another target for the design of a multivalent peptide vaccine for preventing infection of T. spiralis in an immunized host. The multivalent vaccine containing C1q-binding and C9-binding epitopes (domains) against T. spiralis infection is under investigation.
Our study has identified the C1q binding domain within 241Val - 280Ile region of TsPmy. The synthesized peptide P2 based on this region was able to interfere with C1q-initiated complement classical activity and C1q-induced macrophage chemotaxis at a similar level to recombinant full-length TsPmy protein. This study provides molecular insight into the interaction between TsPmy and human C1q.
Biotinylated peptide P2
Bovine serum albumin
- C1q D:
Enzyme-linked immunosorbent assay
Normal human serum
Standard error of the mean
Trichinella spiralis paramyosin
Pozio E. The broad spectrum of Trichinella hosts: from cold- to warm-blooded animals. Vet Parasitol. 2005;132:3–11.
Murrell KD, Pozio E. Worldwide occurrence and impact of human trichinellosis 1986–2009. Emerg Infect Dis. 2011;17:2194–202.
Zhao L, Shao S, Chen Y, Sun X, Sun R, Huang J, et al. Trichinella spiralis calreticulin binds human complement C1q as an immune evasion strategy. Front Immunol. 2017;8:636.
Sun R, Zhao X, Wang Z, Yang J, Zhao L, Zhan B, et al. Trichinella spiralis paramyosin binds human complement C1q and inhibits classical complement activation. PLoS Negl Trop Dis. 2015;9:e0004310.
Zhang Z, Yang J, Wei J, Yang Y, Chen X, Zhao X, et al. Trichinella spiralis paramyosin binds to C8 and C9 and protects the tissue-dwelling nematode from being attacked by host complement. PLoS Negl Trop Dis. 2011;5:e1225.
Sorci G, Cornet S, Faivre B. Immune evasion, immunopathology and the regulation of the immune system. Pathogens. 2013;2:71–91.
Bruschi F, Chiumiento L. Immunomodulation in trichinellosis: does Trichinella really escape the host immune system? Endocr Metab Immune Disord Drug Targets. 2012;12:4–15.
Laclette JP, Shoemaker CB, Richter D, Arcos L, Pante N, Cohen C, et al. Paramyosin inhibits complement C1. J Immunol. 1992;148:124–8.
Landa A, Laclette JP, Nicholsonweller A, Shoemaker CB. Cdna cloning and recombinant expression of collagen-binding and complement inhibitor activity of Taenia solium paramyosin (Agb). Mol Biochem Parasit. 1993;60:343–7.
Bonaparte RS, Hair PS, Banthia D, Marshall DA, Cunnion KA, Krishna NK. Human astrovirus coat protein inhibits serum complement activation via C1, the first component of the classical pathway. J Virol. 2008;82:817–27.
Oladiran A, Belosevic M. Trypanosoma carassii calreticulin binds host complement component C1q and inhibits classical complement pathway-mediated lysis. Dev Comp Immunol. 2010;34:396–405.
Reynolds SL, Pike RN, Mika A, Blom AM, Hofmann A, Wijeyewickrema LC, et al. Scabies mite inactive serine proteases are potent inhibitors of the human complement lectin pathway. PLoS Negl Trop Dis. 2014;8:e2872.
Yadav S, Gupta S, Selvaraj C, Doharey PK, Verma A, Singh SK, et al. In silico and in vitro studies on the protein-protein interactions between Brugia malayi immunomodulatory protein calreticulin and human C1q. PLoS One. 2014;9:e106413.
Vedamurthy GV, Sahoo S, Devi IK, Murugavel S, Joshi P. The N-terminal segment of glyceraldehyde-3-phosphate dehydrogenase of Haemonchus contortus interacts with complements C1q and C3. Parasite Immunol. 2015;37:568–78.
Scietti L, Sampieri K, Pinzuti I, Bartolini E, Benucci B, Liguori A, et al. Exploring host-pathogen interactions through genome wide protein microarray analysis. Sci Rep. 2016;6:27996.
Yang J, Yang Y, Gu Y, Li Q, Wei J, Wang S, et al. Identification and characterization of a full-length cDNA encoding paramyosin of Trichinella spiralis. Biochem Biophys Res Commun. 2008;365:528–33.
Deng J, Gold D, LoVerde PT, Fishelson Z. Mapping of the complement C9 binding domain in paramyosin of the blood fluke Schistosoma mansoni. Int J Parasitol. 2007;37:67–75.
Zhao X, Hao Y, Yang J, Gu Y, Zhu X. Mapping of the complement C9 binding domain on Trichinella spiralis paramyosin. Parasit Vectors. 2014;7:80.
Yang J, Gu Y, Yang Y, Wei J, Wang S, Cui S, et al. Trichinella spiralis: immune response and protective immunity elicited by recombinant paramyosin formulated with different adjuvants. Exp Parasitol. 2010;124:403–8.
Wei J, Gu Y, Yang J, Yang Y, Wang S, Cui S, et al. Identification and characterization of protective epitope of Trichinella spiralis paramyosin. Vaccine. 2011;29:3162–8.
Chen X, Yang Y, Yang J, Zhang Z, Zhu X. RNAi-mediated silencing of paramyosin expression in Trichinella spiralis results in impaired viability of the parasite. PLoS One. 2012;7:e49913.
Gu Y, Wei J, Yang J, Huang J, Yang X, Zhu X. Protective immunity against Trichinella spiralis infection induced by a multi-epitope vaccine in a murine model. PLoS One. 2013;8:e77238.
Wang L, Wang X, Bi K, Sun X, Yang J, Gu Y, et al. Oral vaccination with attenuated Salmonella typhimurium-delivered TsPmy DNA vaccine elicits protective immunity against Trichinella spiralis in BALB/c mice. PLoS Negl Trop Dis. 2016;10:e0004952.
Gu Y, Sun X, Li B, Huang J, Zhan B, Zhu X. Vaccination with a paramyosin-based multi-epitope vaccine elicits significant protective immunity against Trichinella spiralis infection in mice. Front Microbiol. 2017;8:1475.
Wang L, Sun X, Huang J, Zhan B, Zhu X. Heterologous prime-boost vaccination enhances TsPmy’s protective immunity against Trichinella spiralis infection in a murine model. Front Microbiol. 2017;8:1394.
Song YY, Zhang Y, Ren HN, Sun GG, Qi X, Yang F, et al. Characterization of a serine protease inhibitor from Trichinella spiralis and its participation in larval invasion of host’s intestinal epithelial cells. Parasit Vectors. 2018;11:499.
Yang Y, Zhang Z, Yang J, Chen X, Cui S, Zhu X. Oral vaccination with Ts87 DNA vaccine delivered by attenuated Salmonella typhimurium elicits a protective immune response against Trichinella spiralis larval challenge. Vaccine. 2010;28:2735–42.
Sun GG, Ren HN, Liu RD, Song YY, Qi X, Hu CX, et al. Molecular characterization of a putative serine protease from Trichinella spiralis and its elicited immune protection. Vet Res. 2018;49:59.
Qaddoori Y, Abrams ST, Mould P, Alhamdi Y, Christmas SE, Wang G, et al. Extracellular histones inhibit complement activation through interacting with complement component 4. J Immunol. 2018;200:4125–33.
Huang X, Li Y, Fu M, Xin HB. Polarizing macrophages in vitro. Methods Mol Biol. 1784;2018:119–26.
Liu G, Pang Y, Liu X, Li QW. Structure, distribution, classification, and function of C1q protein family: a review. Yi Chuan. 2013;35:1072–80 (In Chinese).
Son M, Diamond B, Santiago-Schwarz F. Fundamental role of C1q in autoimmunity and inflammation. Immunol Res. 2015;63:101–6.
Thielens NM, Tedesco F, Bohlson SS, Gaboriaud C, Tenner AJ. C1q: A fresh look upon an old molecule. Mol Immunol. 2017;89:73–83.
de Messias IJ, Genta RM, Mohren WD. Adherence of monocytes and polymorphonuclear cells to infective larvae of Strongyloides stercoralis after complement activation. J Parasitol. 1994;80:267–74.
Deng J, Gold D, LoVerde PT, Fishelson Z. Inhibition of the complement membrane attack complex by Schistosoma mansoni paramyosin. Infect Immun. 2003;71:6402–10.
Matsumoto Y, Perry G, Levine RJ, Blanton R, Mahmoud AA, Aikawa M. Paramyosin and actin in schistosomal teguments. Nature. 1988;333:76–8.
Ferreira CA, Barbosa MC, Silveira TC, Valenzuela JG, Vaz ISJ, Masuda A. cDNA cloning, expression and characterization of a Boophilus microplus paramyosin. Parasitology. 2002;125:265–74.
Strube C, Buschbaum S, von Samson-Himmelstjerna G, Schnieder T. Stage-dependent transcriptional changes and characterization of paramyosin of the bovine lungworm Dictyocaulus viviparus. Parasitol Int. 2009;58:334–40.
Akpek EK, Liu SH, Thompson R, Gottsch JD. Identification of paramyosin as a binding protein for calgranulin C in experimental helminthic keratitis. Invest Ophthalmol Vis Sci. 2002;43:2677–84.
Hernandez MG, Hafalla JC, Acosta LP, Aligui FF, Aligui GD, Ramirez BL, et al. Paramyosin is a major target of the human IgA response against Schistosoma japonicum. Parasite Immunol. 1999;21:641–7.
Gu Y, Huang J, Wang X, Wang L, Yang J, Zhan B, et al. Identification and characterization of CD4+ T cell epitopes present in Trichinella spiralis paramyosin. Vet Parasitol. 2016;231:59–62.
Correale P, Botta C, Martino EC, Ulivieri C, Battaglia G, Carfagno T, et al. Phase Ib study of poly-epitope peptide vaccination to thymidylate synthase (TSPP) and GOLFIG chemo-immunotherapy for treatment of metastatic colorectal cancer patients. Oncoimmunology. 2016;5:e1101205.
Higashihara Y, Kato J, Nagahara A, Izumi K, Konishi M, Kodani T, et al. Phase I clinical trial of peptide vaccination with URLC10 and VEGFR1 epitope peptides in patients with advanced gastric cancer. Int J Oncol. 2014;44:662–8.
We thank Ran Sun, Xi Zhao, Jing Yang, Yuan Gu, Ximeng Sun, Yuli Cheng and Kai Guo for their technical assistance and helpful suggestions.
This study was supported by grants from the Beijing Natural Science Foundation of China (7162017) and National Natural Science Foundation of China (81672042).
Availability of data and materials
The data supporting the conclusions of this article are included within the article.
Ethics approval and consent to participate
Normal human serum (NHS) was selected from healthy human volunteers with written consent forms. The project was approved by the Institutional Review Board (IRB) of Capital Medical University (approval number: 2016SY01). Human C1q-deficient serum (C1q D) was purchased from Merck (Merck Darmstadt, Germany).
Consent for publication
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.