C-reactive protein collaborates with plasma lectins to boost immune response against bacteria
2007; Springer Nature; Volume: 26; Issue: 14 Linguagem: Inglês
10.1038/sj.emboj.7601762
ISSN1460-2075
AutoresPatricia Miang Lon Ng, Agnès Le Saux, Chia M Lee, Nguan Soon Tan, Jinhua Lu, Steffen Thiel, Bow Ho, Jeak Ling Ding,
Tópico(s)Antibiotic Resistance in Bacteria
ResumoArticle21 June 2007free access C-reactive protein collaborates with plasma lectins to boost immune response against bacteria Patricia ML Ng Patricia ML Ng Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore Search for more papers by this author Agnès Le Saux Agnès Le Saux Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore Search for more papers by this author Chia M Lee Chia M Lee Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore Search for more papers by this author Nguan S Tan Nguan S Tan School of Biological Sciences, Nanyang Technological University, Singapore Search for more papers by this author Jinhua Lu Jinhua Lu Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Search for more papers by this author Steffen Thiel Steffen Thiel Department of Medical Microbiology and Immunology, Wilhelm Meyers Allé, Bartholin Building, University of Aarhus, Aarhus, Denmark Search for more papers by this author Bow Ho Bow Ho Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Search for more papers by this author Jeak L Ding Corresponding Author Jeak L Ding Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore Search for more papers by this author Patricia ML Ng Patricia ML Ng Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore Search for more papers by this author Agnès Le Saux Agnès Le Saux Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore Search for more papers by this author Chia M Lee Chia M Lee Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore Search for more papers by this author Nguan S Tan Nguan S Tan School of Biological Sciences, Nanyang Technological University, Singapore Search for more papers by this author Jinhua Lu Jinhua Lu Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Search for more papers by this author Steffen Thiel Steffen Thiel Department of Medical Microbiology and Immunology, Wilhelm Meyers Allé, Bartholin Building, University of Aarhus, Aarhus, Denmark Search for more papers by this author Bow Ho Bow Ho Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore Search for more papers by this author Jeak L Ding Corresponding Author Jeak L Ding Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore Search for more papers by this author Author Information Patricia ML Ng1, Agnès Le Saux1, Chia M Lee1, Nguan S Tan2, Jinhua Lu3, Steffen Thiel4, Bow Ho3,‡ and Jeak L Ding 1 1Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 2School of Biological Sciences, Nanyang Technological University, Singapore 3Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 4Department of Medical Microbiology and Immunology, Wilhelm Meyers Allé, Bartholin Building, University of Aarhus, Aarhus, Denmark ‡These authors contributed equally to this work *Corresponding author. Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore. Tel.: 65 6516 2776; Fax: 65 6779 2486; E-mail: [email protected] The EMBO Journal (2007)26:3431-3440https://doi.org/10.1038/sj.emboj.7601762 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Although human C-reactive protein (CRP) becomes upregulated during septicemia, its role remains unclear, since purified CRP showed no binding to many common pathogens. Contrary to previous findings, we show that purified human CRP (hCRP) binds to Salmonella enterica, and that binding is enhanced in the presence of plasma factors. In the horseshoe crab, Carcinoscorpius rotundicauda, CRP is a major hemolymph protein. Incubation of hemolymph with a range of bacteria resulted in CRP binding to all the bacteria tested. Lipopolysaccharide-affinity chromatography of the hemolymph co-purified CRP, galactose-binding protein (GBP) and carcinolectin-5 (CL5). Yeast two-hybrid and pull-down assays suggested that these pattern recognition receptors (PRRs) form pathogen recognition complexes. We show the conservation of PRR crosstalk in humans, whereby hCRP interacts with ficolin (CL5 homologue). This interaction stabilizes CRP binding to bacteria and activates the lectin-mediated complement pathway. We propose that CRP does not act alone but collaborates with other plasma PRRs to form stable pathogen recognition complexes when targeting a wide range of bacteria for destruction. Introduction C-reactive protein, CRP (Tillett and Francis, 1930; Macleod and Avery, 1941; Kaplan and Volanakis, 1974), is a plasma protein that is highly upregulated during acute phase response. CRP is an indicator of acute infection and inflammation (Pepys and Hirschfield, 2003; Marnell et al, 2005). Systemic infection by a broad range of Gram-positive and -negative bacteria raises CRP level by up to 1000-fold (Hengst, 2003; Black et al, 2004; Sierra et al, 2004), suggesting its involvement in the immune response against most pathogens. Direct binding of purified CRP to Streptococcus pneumoniae (Volanakis and Kaplan, 1971), Neisseriae lactamica (Serino and Virji, 2000) and Haemophilus influenzae (Weiser et al, 1998) via repetitive phosphorylcholine moieties on the lipoteichoic acid or the lipopolysaccharide (LPS) of these pathogens has been shown. Binding of CRP recruits phagocytic cells, and activates the classical complement pathway (Kaplan and Volanakis, 1974; Siegel et al, 1974), causing bacterial clearance. However, purified CRP failed to bind Escherichia coli, Salmonella enterica (This includes S. enterica Group D serotype and S. enterica subsp enterica serovar Typhimurium, previously named Salmonella typhimurium (Truper, 2005)), Klebsiella pneumoniae, Pseudomonas aeruginosa, Campylobacter fetus jejuni, Neisseria meningitidis and Neisseria gonorrhoeae (Mold et al, 1982; Szalai et al, 2000), when tested in vitro, although increased CRP was observed in systemic infection by many of these bacteria (Sierra et al, 2004). Thus, the in vivo role of CRP during systemic infection is not completely understood. Mice administered with human CRP (hCRP) and/or hCRP-transgenic mice that were infected with S. pneumoniae (Mold et al, 1981; Szalai et al, 1995) or S. enterica previously named S. typhimurium (Szalai et al, 2000) showed decreased bacteremia and increased survival, reflecting the efficacy of hCRP in vivo. However, conclusions from these findings are problematic because: (1) purified hCRP showed no binding to S. enterica (Szalai et al, 2000), (2) hCRP did not activate the classical complement pathway in mice during infection by S. pneumoniae (Suresh et al, 2006) and (3) the major acute phase protein in mice is the serum amyloid P component and not CRP (Ku and Mortensen, 1993). The discrepancy between in vitro and in vivo findings and problems with research using mice have prompted us to explore the in vivo role of CRP against infection, using the horseshoe crab, which harbors no adaptive immune system and naturally relies on CRP as a potentially important frontline defense protein (Ng et al, 2004; Iwanaga and Lee, 2005). Many pattern recognition receptors (PRRs) are evolutionarily conserved (Shrive et al, 1999; Kairies et al, 2001) from horseshoe crab to human, suggesting that these proteins play essential roles in innate immunity. Horseshoe crab CRP exists in families of multiple isoforms (Nguyen et al, 1986; Iwaki et al, 1999; Iwanaga, 2002). In the Japanese species, Tachypleus tridentatus, the CRP families are designated CRP-1, CRP-2 and CRP-3 (Iwaki et al, 1999; Iwanaga, 2002). In the American species, Limulus polyphemus, CRP-2 is known as limulin (Roche and Monsigny, 1974; Kaplan et al, 1977; Robey and Liu, 1981; Armstrong et al, 1996). CRP-1 and CRP-2 bind phosphorylcholine in a calcium-dependent manner (Robey and Liu, 1981; Iwaki et al, 1999), and thus fulfill the functional definition of CRP (Kaplan and Volanakis, 1974). Studies using purified horseshoe crab CRP-1, CRP-2 and CRP-3 against E. coli K12, Enterococcus hirae, Micrococcus luteus and Staphylococcus aureus 209P showed neither agglutination nor growth inhibition activity (Iwaki et al, 1999). These unexpected observations raised doubts on the role of horseshoe crab CRP as a potentially important frontline immune response protein. However, our study on the horseshoe crab showed its remarkable ability to clear P. aeruginosa in vivo, and suggested that CRP plays an effective role in frontline defense against infection (Ng et al, 2004). We found that in the presence of plasma or hemolymph, CRP binds to a wider range of bacteria than when CRP was tested in isolation (Mold et al, 1982; Iwaki et al, 1999; Szalai et al, 2000). Co-purification results and yeast two-hybrid analysis suggest that CRP collaborates with other PRRs to form stable complexes as they bind to the pathogens. Significantly, such PRR–PRR crosstalk is conserved from horseshoe crab to human, where the collaboration between hCRP and ficolin (another PRR) was found to consequently activate downstream components in the lectin-mediated complement pathway. Results Plasma factors enhance the binding of CRP onto bacteria As hCRP does not bind S. enterica in vitro, but protects against blood infection by this pathogen (Mold et al, 1982; Szalai et al, 2000), we reasoned that in vivo, other plasma factors probably enable hCRP to interact with the pathogen effectively. To test this, we incubated S. enterica with hCRP in the presence of either 10% human plasma or human serum albumin (HSA, as control). In the presence of HSA, hCRP binds to bacteria (Figure 1A, lane 1). This binding is enhanced in the presence of other plasma proteins (lane 2). The enhancement is not due to endogenous CRP in the plasma, since control using plasma alone showed negligible binding of CRP (lane 3). Although calcium is required for hCRP binding, controls with and without ethylenediaminetetraacetic acid (EDTA) showed that endogenous calcium in the plasma is not the cause of the enhancement (Figure 1B). These results suggest that there are plasma factors other than calcium ions that enhance the binding of hCRP to bacteria. Figure 1.Plasma factors enhance the binding of CRP onto bacteria. (A) Immunodetection of hCRP bound to S. enterica. S. enterica were incubated with (1) purified hCRP in human serum albumin (HSA, control), (2) purified hCRP in 10% plasma or (3) directly with 10% plasma. The experiment was performed in the presence of 1 mM CaCl2. Proteins bound to bacteria were eluted (lanes 1–3) and analyzed by Western blot using anti-hCRP antibody. Purified hCRP (50 ng) and plasma (100 μg) were loaded as controls. (B) Immunodetection of hCRP that was bound to S. enterica (in VBS), with and without 2 mM EDTA, showed no difference in that both lacked hCRP binding, confirming that the purified hCRP and the plasma samples did not have any calcium that would have interfered with the study of hCRP binding in these experiments. (C) Immunodetection of hCRP bound to S. enterica during pretreatment, co-treatment or post-treatment with plasma and/or HSA in the indicated order of incubation. (D) SDS–PAGE of hemolymph proteins (e.g., 35, 52, 74 and 75 kDa) binding to bacteria and immunodetection of CrCRP (lower panels). Hemolymph was incubated with E. coli, P. aeruginosa, S. aureus and S. enterica. The bound proteins were eluted after various time periods of incubation with the bacteria. Lane H shows the relative abundance of hemocyanin and CRP in untreated hemolymph. Lane C shows proteins eluted from bacteria after incubation with buffer alone. (E) Immunodetection of CrCRP and GBP bound to S. enterica. Purified CrCRP, 'hemolymph CrCRP' with an equal amount of CrCRP, or BSA alone (negative control) was incubated with S. enterica. In each treatment, either 1 mM CaCl2, 2 mM EDTA or neither was included in the buffer. Proteins bound to the bacteria were eluted and analyzed by Western blot using anti-CrCRP and anti-GBP antibodies. Hemolymph, purified CrCRP and GBP were loaded as controls. Download figure Download PowerPoint To test if plasma factors enhance the binding of hCRP, we pretreated, co-treated or post-treated the S. enterica with plasma before, during or after addition of hCRP, respectively. Addition of plasma in any order to hCRP enhanced its binding to bacteria (Figure 1C). However, pretreatment and co-treatment of S. enterica with plasma gave the most effective hCRP binding. These results suggest that plasma enhances the ability of hCRP to bind bacteria. This is possibly due to either (a) interaction of hCRP with other pathogen-bound plasma PRRs or (b) exposure of hCRP-binding sites on the bacteria by the plasma treatment. To study the role of CRP in an innate immune model, we incubated horseshoe crab hemolymph with E. coli K12, P. aeruginosa, S. aureus and S. enterica R595, and examined the proteins that associated with the bacteria. Figure 1D shows that from 1 min to 18 h, the C. rotundicauda CRP (CrCRP) binds rapidly and incrementally to all the bacteria tested, confirming that CrCRP is a PRR that recognizes different bacteria. Differences in the accumulation of CrCRP are probably due to different chemostructures of the bacterial outer membrane. To ascertain that the increase in CrCRP binding is not due to bacterial growth over time, the bacteria were pretreated with 5% acetic acid. The fixation process did not affect CrCRP binding to bacteria (Supplementary Figure 1). Addition of the serine protease inhibitor, phenylmethylsulfonylfluoride (PMSF) also did not inhibit the deposition of CrCRP (Supplementary Figure 2), suggesting that the binding and accumulation of CrCRP is independent of a serine protease. Although it is unclear how the other components of the hemolymph might enhance binding of CrCRP to bacteria, various possible processes might be involved, including the formation of a protein complex that binds to the bacterial surface and/or the involvement of ancillary proteins that can modify the bacterial surface (e.g. via limited proteolysis) to promote binding of CrCRP. To study the influence of hemolymph factors, purified CrCRP or endogenous CrCRP in hemolymph (henceforth named 'hemolymph CrCRP', present at the same level as the purified CrCRP) was added to S. enterica. In the presence of calcium, purified CrCRP and 'hemolymph CrCRP' bind the bacteria (Figure 1E, lanes 1 and 2). With EDTA, purified CrCRP does not bind (lane 7), but 'hemolymph CrCRP' still binds the bacteria (lane 8). This suggests that other hemolymph proteins can collaborate with CrCRP to bind bacteria under conditions where cations are chelated by EDTA. The lack of binding of purified CrCRP and 'hemolymph CrCRP' to bacteria in the absence of both calcium and EDTA (lanes 4 and 5) further indicates that the chelation effect of EDTA on other hemolymph proteins is necessary for these protein–protein interactions to occur. Figure 1E shows the difference in CrCRP binding to bacteria without the addition of calcium or EDTA (lane 5) and with the addition of EDTA (lane 8). Thus, like human plasma proteins, the horseshoe crab hemolymph proteins enhance CrCRP to bind to bacteria, although in the horseshoe crab, a 'chelation of cations from the hemolymph proteins' (which may occur in the microenvironment of pathogen invasion) seems to be necessary for hemolymph proteins to enable CrCRP bind bacteria. Supplementary Figure 3 illustrates and further explains the result of Figure 1E. Conceivably, infection that can result in the chelation of cations from hemolymph proteins due to cation uptake by the bacteria (Blackwell et al, 2000; Maguire, 2006; Ong et al, 2006; Papp-Wallace and Maguire, 2006) would have some effects on protein interactions and consequently, regulate the immune response. However, this mechanism remains to be confirmed. Identification of hemolymph proteins that collaborate with CRP in LPS binding To identify proteins that collaborate with CrCRP in the recognition of bacteria, we used LPS-affinity chromatography. ReLPS, a truncated form of LPS, containing only the conserved core elements of two 2-keto-3-deoxyoctonate (KDO) and the lipid A moiety (Figure 2A) was conjugated to the Sepharose. As ReLPS lacks the O-polysaccharide component and is expected to offer no cognate ligands for most of the other lectin PRRs, it is an ideal molecule to isolate PRRs, which may interact with each other via protein–protein rather than ligand–protein interactions. Previously, we have isolated a repertoire of calcium-independent proteins bound to ReLPS (Ng et al, 2004), and identified CrCRP among these proteins. Since 'hemolymph CrCRP' and other proteins bind bacteria when EDTA is present (Figure 1E), we washed the ReLPS-bound proteins with EDTA before elution to retrieve CrCRP-interacting proteins. Proteins associated to the ReLPS-Sepharose and not to the control Sepharose (Figure 2B) were identified by mass spectrometry (MS). Results show isoforms of CrCRP-1 and CrCRP-2 (Figure 2C), carcinolectin-5 (CL5) (Figure 2D and Supplementary Figure 4) and the 26 and 52 kDa forms of galactose-binding protein (GBP) also known as Tachypleus lectin (Figure 2E and F). Next, we determined whether GBP also binds bacteria when incubated with hemolymph. Figure 1E shows that GBP binds more strongly without addition of calcium (lane 5), and best in the presence of EDTA (lane 8). These data support the notion that GBP and possibly CL5 could potentially interact with CrCRP to enhance its binding to bacteria. Figure 2.GBP and CL5 co-purified with CRP as an LPS-binding complex. (A) Schematic representation of LPS showing the O-polysaccharide and core polysaccharide and lipid A regions. Hexagons represent monosaccharides. KDO is shaded. The lipid A has acyl chains (wavy lines) attached to a disaccharide. Filled circles represent phosphate. The common substituents that are associated with the core region and lipid A moiety include phosphorylethanolamine (filled triangle) and 4-amino-4-dehydroarabinose (open triangles). The O-polysaccharide has a variable number (n) of the repeating units (in parentheses). ReLPS has only the lipid A region and two KDO residues. (B) SDS–PAGE of the plasma proteins purified with ReLPS-conjugated Sepharose or control Sepharose. Fractions from the EDTA wash and urea elution are shown. The proteins eluted from the ReLPS column (arrowed) are CRP, GBP and isoforms of CL5. Lanes 'm' represent protein markers. (C) Peptide mass fingerprint (PMF) of trypsin-digested proteins from the p26–28 shows peaks corresponding to CRP (white box) and GBP (black box). The 1205 and 1432 peaks were common to CRP-1 and CRP-2. The 1350 and 1381 peaks were from CRP-1 and the 985 was from CRP-2. The m/z values of peaks that are unidentified are set in smaller font. A representative CrCRP sequence (CrCRP-1 hp1, GenBank accession no. AAV65022) is shown. Shaded regions represent parts of the sequences detected by peptide sequencing via ESI-Q-TOF. (D) PMF of the CL5 isoforms p35 and p40 and the peptide sequences obtained by MS sequencing of the peptide fragments show that they belong to CL5s. The CL5 peaks are set in bigger and bolder fonts. The unidentified peaks are set in smaller font. (E) PMF of p52 is similar to the fingerprint of the p26, which was confirmed to be GBP by peptide sequencing and cDNA cloning. (F) Alignment of the cloned C. rotundicauda GBP sequence against T. tridentatus GBP (TtGBP) sequence. Shaded sequences represent the fragments detectable by MS (via m/z peaks and/or peptide sequencing). In silico trypsin digestion of the CrGBP protein sequence yielded m/z values corresponding to the peaks ((C) and (E), black box) in p26–p28 and p52. Download figure Download PowerPoint CRP interacts with GBP, which interacts with CL5 To test the interaction between CRP, GBP and CL5, we used the yeast two-hybrid system. CRP interacts with GBP but not CL5 (Figure 3A). However, GBP interacts with CL5. Hence, a bacteria-binding complex consisting of CRP, GBP and CL5 is mediated by direct interaction between CRP and GBP, and interaction of the latter with CL5. The relative growth rate of the transformed yeast suggests that interaction between CRP and GBP is stronger than that between GBP and CL5 (Supplementary Figure 5). Figure 3.CRP interacts with GBP, which interacts with CL5. (A) Yeast two-hybrid analysis shows that CRP1 interacts with GBP, which in turn interacts with CL5. As the dominant isoform on the p35 spectrum from ReLPS-affinity chromatography, CL5c, was used for analysis (see Figure 2B). Growth on SC-Trp-Leu (Trp- and Leu-dropout) agar indicates the presence of both plasmids. Growth on QDO (quadruple dropout lacking Trp, Leu, His and Ade) agar indicates interaction. Empty denotes AH109 yeast strain co-transformed with either pGBKT7 or pGADT7-Rec vector without cDNA inserts. (B) One- and two-dimensional SDS–PAGE analyses show that CRP and CL5 co-purified with GBP from hemolymph. Western analysis using anti-CrCRP antibody showed that co-purification of CrCRP and GBP occurred only with 6 hpi hemolymph. The p74 and p75 (not identified) are probably hemocyanin, which, due to its sheer abundance in the hemolymph, commonly associates non-specifically onto Sepharose. (C) A first-dimensional native PAGE followed by second-dimensional denaturing SDS–PAGE of the complex co-purified with GBP from naïve hemolymph. (D) SDS–PAGE of proteins co-immunoprecipitated by anti-CRP antibody from naïve hemolymph, 6 hpi hemolymph or buffer alone (negative control). Control using an unrelated antibody (anti-IgG) for co-immunoprecipitation showed that the 74 kDa protein was nonspecific. The untreated hemolymph (naive) used for co-immunoprecipitation is shown. The 26 kDa proteins (P1 and P2) were excised for MS. (E) Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) spectra of P1 and P2 show CRP (white boxes) and GBP (black boxes) peaks. Download figure Download PowerPoint To test if GBP binds CRP and CL5 in native hemolymph conditions, we used CNBr-activated Tris-reacted Sepharose, which can bind GBP, to co-purify GBP-interacting proteins from the hemolymph. CL5 is co-purified with GBP when naïve or infected hemolymph is used, whereas CRP is only co-purified with GBP when infected hemolymph is used (Figure 3B). Under native conditions, GBP co-migrated with CL5, suggesting that GBP and CL5 is a complex (Figure 3C). Densitometric analysis of the CRP–GBP–CL5 complex resolved by two-dimensional electrophoresis gives an estimated stoichiometric ratio of 1 CRP:19 GBP:3 CL5 (Figure 3B). These results suggest that during infection, oligomers of GBP interact with CL5 and CRP to interlock the PRR network on the surface of the invading bacteria. GBP is reciprocally co-immunoprecipitated with CRP when hemolymph is incubated with anti-CRP antibody. This is not seen in a control where an unrelated antibody (anti-rabbit IgG antibody) is used (Figure 3D and E). This confirms the specificity of interaction between CRP-GBP, despite the ability of GBP to bind unconjugated-Sepharose (Chen et al, 2001). Interestingly, GBP interacts with CRP immobilized via anti-CRP even when naïve hemolymph is used. This suggests that immobilization of CRP can facilitate its interaction with GBP, although conversely, immobilized GBP requires additional infection-triggered factors before it could bind soluble CRP. These findings reveal intricate protein–protein interactions between three PRRs that have been previously shown to individually bind certain pathogen-associated molecular patterns (PAMPs) or pathogens on their own (Ng et al, 2004; Kuo et al, 2006; Zhu et al, 2006). hCRP collaborates with ficolin The human ficolins (L-, M- and H-isoforms) share 35% homology with horseshoe crab TL5s and CL5s (Supplementary Figure 6), and both harbor a fibrinogen-like (FBG) domain (Holmskov et al, 2003). Although CrCRP cannot directly bind CL5, hCRP has been shown to bind fibrinogen (Salonen et al, 1984). We therefore reasoned that hCRP could possibly interact with ficolin. As hCRP is mainly expressed by the liver and secreted into the plasma, we tested for interaction of hCRP with L-ficolin, a ficolin isoform found in the plasma. L-ficolin shows binding to immobilized hCRP and acetylated bovine serum albumin (BSA) (positive control). The minimal binding to L. polyphemus CRP (LpCRP) (Figure 4A) indicates that the co-evolution of these proteins has ensured species-specific interaction. Interestingly, M-ficolin FBG domain can bind to hCRP better than to acetylated BSA (Figure 4B). M-ficolin is found in the secretory granules of neutrophils, monocytes and type II alveolar epithelial cells of the lung (Liu et al, 2005). Since expression of hCRP by alveolar macrophages has been reported (Dong and Wright, 1996), hCRP may interact with M-ficolin in the lung surfactant during infection. Affinity analysis suggests that M-ficolin FBG domain binds to immobilized hCRP with a kD of 4 × 10−8 M (Figure 4C). It remains to be confirmed if such observations are consistent when full-length M-ficolin is used. In the reverse orientation, we observed preferential binding of soluble hCRP to immobilized L-ficolin, rather than to immobilized M-ficolin FBG domain (Figure 4D). It appears that binding of both the L- and M-ficolins to immobilized hCRP (Figure 4A and B) and the reverse binding of hCRP to immobilized L-ficolin (Figure 4D) could be more predominant than the binding of hCRP to immobilized M-ficolin FBG domain (Figure 4D). The differential binding of hCRP to the ficolin isoforms under different conditions reveals an intricacy of PRR–PRR interactions. Figure 4.Interaction of hCRP with L-ficolin or myc-tagged M-ficolin FBG domain. (A) Relative binding of L-ficolin or (B) M-ficolin FBG domain to immobilized hCRP. L-ficolin or M-ficolin FBG domain bound to hCRP, acetylated BSA (AcBSA) or L. polyphemus CRP (LpCRP) that was coated on wells, was detected by anti-L-ficolin or anti-myc antibody, respectively. The positive control was directly coated L-ficolin or M-ficolin FBG domain. (C) Binding curve of M-ficolin FBG domain to immobilized hCRP. Addition of an increasing amount of M-ficolin FBG domain results in a saturable increase in its binding. The kD of 4 × 10−8 M was determined from non-linear regression analysis of the binding curve using Sigma plot (version 8.0). (D) Relative binding of hCRP to immobilized ficolins. M-ficolin FBG domain (FBG) or L-ficolin was coated onto wells. hCRP binding was detected by goat anti-CRP antibody. Direct coating of hCRP was used as the positive control for 100% binding. For panels A, B and D, 0.5 μg of proteins was used for coating and adding of interaction partner. Readings were subtracted off negative controls (BSA-blocked wells with corresponding treatment) and expressed as a percentage of the corresponding positive control. The means±s.e.m. of three independent experiments are plotted. Download figure Download PowerPoint Crosstalk between hCRP and ficolin enhances the recognition of Salmonella and activates the lectin-mediated complement pathway To test if M-ficolin FBG domain augments the binding of hCRP to bacteria, we incubated S. enterica (1) with hCRP, followed by M-ficolin FBG domain, (2) in the reverse order or (3) concurrently. M-ficolin FBG domain added before or concomitantly with hCRP enhanced the amount of hCRP bound to bacteria, compared with the reaction without M-ficolin FBG domain (Figure 5A). This suggests that M-ficolin FBG domain aids the deposition of hCRP on the bacteria. Figure 5.hCRP–ficolin crosstalk enhances pathogen recognition and activates the lectin-mediated complement pathway. (A) S. enterica incubated with CRP followed by M-ficolin FBG domain (FBG), in the reverse order or concurrently were analyzed by Western blot using anti-hCRP antibody. Addition of M-ficolin FBG domain (with myc tag) either before or together (C+F) with CRP enhanced the amount of pathogen-bound CRP. (B) CRP and L-ficolin (FL) collaboration triggered MASP-2 and C4b deposition. hCRP or BSA was coated onto the 96-well plate and incubated with L-ficolin/MASP-2 complex, followed by C4. C4b deposition was detected by anti-C4c antibody. Results are the means of triplicates. * indicates a significant difference of P 50% of the total C4 activation. These findings confirm that the collaboration between CRP and other PRRs is evolutionarily conserved in human. Moreover, collaboration between C
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