Fig 1.
Schematic diagrams of C4BP molecule, C4BP recombinant mutants and L. interrogans proteins LigA and LigB.
(A) Structure of human C4BP isoform α7β1 [4]. Each α-chain is composed of 8 complement control protein (CCP) domains while the β-chain is composed of 3 CCPs. CCP1 from the α and β-chains are localized at the N-terminus region and α-chain CCP8 and β-chain CCP3 are situated near the central core (C-terminus). (B) C4BP recombinant wild type and mutants (α6β0) used in this work. Each mutant is composed of 6 α-chains. Each wild type α-chains contains 8 CCPs while mutant α-chains are formed by only 7 CCP domains (Δ denotes which CCP is missing in each mutant). (C) Illustration of recombinant leptospiral immunoglobulin-like proteins (Lig)A (LigA) and B (LigB). LigA is composed of 13 bacterial immunoglobulin-like (Big) domain repeats while LigB is composed of 12 Big domains. The fragment corresponding to the first six and a half domains of LigA and LigB (residues 26–630; identical in both proteins) is named LigBN. The fragments that corresponding to the second half of Big domain 7 to the Big domain 13 of LigA (residues 631–1225), is named LigAC and fragments corresponding to the half of Big domain 7 to Big domain 12 of LigB (residues 631–1156), is named LigBC. (D) Schematic representation of the recombinant LigA and LigB fragments containing tandem pairs of Big domains.
Fig 2.
Binding of recombinant proteins LcpA, LigAC, LigBC and whole L. interrogans to recombinant mutant C4BP molecules.
Microtiter plates were coated with LigAC (A), LigBC (B), LcpA (C) or LIC10301 (as negative control). After adding of each C4BP recombinant mutant or wild type (rec C4BP WT) protein (described in Fig 1B), binding was measured using rabbit polyclonal anti-human C4BP and peroxidase-conjugated anti-rabbit IgG. Each point represents the mean absorbance value at 492 nm +/- the SD of 3 independent experiments each performed in triplicate. The interaction of recombinant proteins of L. interrogans with rec C4BP WT was set as 100% binding. (D) Binding of C4BP mutant proteins to whole L. interrogans. Leptospires (1x108) were incubated with rec C4BP WT, C4BP mutants or PBS (negative control). To detect the C4BP binding to leptospires, polyclonal mouse anti-C4BP and FITC-conjugated anti-rabbit IgG were used. Each point represents the geometric mean fluorescence intensity (GMFI) +/- SE of 3 independent experiments each performed in triplicate. Data were analyzed using ANOVA test; *p<0.05; ***p<0.0001.
Fig 3.
LigA and LigB Big domains are important for binding to C4BP.
(A) The interaction of C4BP with the conserved N-terminus region of LigA and LigB proteins (LigBN), the C-terminus region of LigA (LigAC) and the C-terminus region of LigB (LigBC) was measured (B-C) The binding of two-domain serial constructs of LigAC (B) or LigBC (C) (described in Fig 1D) to C4BP was studied using increasing concentrations of C4BP. BSA was used as negative control. Each value represents the mean ± SD of three independent experiments, each performed in triplicate and binding of C4BP to the recombinant Lig proteins was compared with the binding of these molecules to BSA by the ANOVA (*p<0.05; **p<0.01 ***p<0.001) (D) Estimation of the dissociation constant (Kd) for the interactions of LigAC and LigBC with C4BP. Kd was calculated by fitting the data to the equation Y = Bmax*X/(Kd + X) using GraphPad Prism 5.0 (GraphPad Software, Inc.). Each data point represents the mean +/- SD of three trials in triplicate. C4BP binding to leptospiral proteins was detected using rabbit anti-C4BP as primary antibody and anti-rabbit IgG conjugated with peroxidase as secondary antibody.
Fig 4.
Interaction between C4BP and leptospiral recombinant proteins LigAC and LigBC is heparin dependent.
(A) C4BP was incubated with different concentrations of heparin before adding to microtiter plates previously coated with LigAC or LigBC. Recombinant LIC 10301 was included as negative control. The binding of C4BP to the leptospiral proteins was detected using polyclonal rabbit anti-C4BP as primary antibody and anti-rabbit IgG conjugated with peroxidase as secondary antibody. Each point represents the mean absorbance value at 492 nm of 3 independent experiments +/- the SD, each performed in triplicate. The binding of recombinant proteins of L. interrogans to C4BP in the absence of heparin was considered 100% (** p<0.001; *** p<0.0001). (B) Microtiter plates were coated with heparin and then, different amounts of LigAC or LigBC (0.1 μg–1 μg) were added to the plates. Anti-LigA or Anti-LigB antibodies were used to detect the protein bound to immobilize heparin. BSA was used as negative control. Each point represents the mean absorbance value at 492 nm +/- the SD of 3 independent experiments each, performed in triplicate (*** p<0.0001).
Fig 5.
LigAC and LigBC interact with heparin mainly via Big 7 and Big 8.
(A) Using different LigA and LigB tandem domain constructs, we observed that Big7-8 are the major heparin binding sites on the C-terminus variable regions of LigB and LigA. (B) Binding of C4BP to different LigA and LigB tandem domain constructs is affected by heparin. Immobilized Lig protein constructs were incubated with C4BP mixed with different concentrations of heparin. C4BP bound to each Lig protein fragments in the absence of heparin was set as 100%. Each point represents the mean absorbance value at 492 nm or 630 nm +/- the SD of 3 independent experiments each performed in triplicate. Binding of heparin to LigA7-8, LigA8-13 and LigB 7–8, LigB7-12 were compared to LigA10-11 and LigB 9–10, respectively. ***p<0,001 (C) Estimation of the dissociation constant (Kd) for the interactions of LigAC and LigBC with heparin. Kd was calculated by fitting the data to the equation Y = Bmax*X/(Kd + X) using GraphPad Prism 5.0 (GraphPad Software, Inc.). Each data point represents the mean +/- SD of three trials in triplicate.
Fig 6.
Interaction of C4BP and recombinant proteins of L. interrogans are dependent on ionic strength.
LigA, LigB or LcpA were immobilized on microtiter wells. C4BP WT (commercial) diluted in 10 mM Na2HPO4 and 1.8mM KH2PO4 was prepared in different concentrations of NaCl and then added to the wells. To detect the binding of C4BP to recombinant proteins of L. interrogans, polyclonal rabbit anti-C4BP antibodies were used, followed by peroxidase-conjugated anti-rabbit IgG. Each point represents the mean absorbance value at 492 nm +/- the SD of 3 independent experiments, each performed in triplicate. The binding of recombinant proteins of L. interrogans and C4BP WT in the absence of NaCl was considered 100%. Data were analyzed using ANOVA test; *p<0.05; ***p<0.0001.
Fig 7.
Summary of protein-protein and heparin-protein binding sites identified in this study.
Schematic diagram of C4BP, Lig proteins and heparin binding sites. (A) LigAC, LigBC, LcpA and heparin binding sites on the C4BP molecule. C4BP CCP7 and CCP8 are binding sites for LigAC, LigBC, LcpA and whole L. interrogans. C4BP CCP4 is a binding site for only LigAC and whole L. interrogans. Heparin binds to the interface between CCP1 and CCP2 of C4BP [24]. (B) C4BP and heparin binding sites located on LigAC and LigBC proteins (numbers represent each Big domain). Note that C4BP and heparin compete for the same region on the C-terminus variable fragments of LigA and LigB molecules, respectively LigA7-8 and LigB7-8. C4BP also binds to LigA9-10, LigA10-11, LigB9-10 and LigB11-12.