Complement C4bC2 complex formation: an investigation by surface plasmon resonance

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Abstract

Complex formation between the human complement proteins C4b and C2 was investigated by surface plasmon resonance. C4b was immobilised and C2 was used in the fluid phase to measure interaction at different ionic strengths (30–830 mM NaCl) and in the absence and presence of MgCl2. Maximum binding was observed at 30 mM NaCl, and was negligible above 300 mM NaCl. Binding was not greatly influenced by variation in Mg2+ in the range of 2.5–15 mM. C4bC2 affinity (Kd) was determined by steady-state analysis to be 7.2×10−8 M in physiological conditions (10 mM Hepes, 2.5 mM MgCl2, 0.75 mM CaCl2 and 140 mM NaCl, pH 7.4). For C4(H2O)C2 complex formation, a Kd of 4.0×10−8 M was calculated. As far as detected by the applied method, complex formation does not involve conformational changes of one of the binding partners. Consistent with previous reports, C4bC2 binding takes place as a multiple-site binding event in the presence of Mg2+. C4bC2 complex formation in 10 mM Hepes, 2.5 mM EDTA and 140 mM NaCl (pH 7.4) was also observed and the interaction showed characteristics of a single-site binding event. Kd was 1.5×10−8 M. Complement factor B (FB) was also tested for its binding to immobilised C4b. Weak interaction was observed at FB concentrations in the physiological range (500–1000 nM). Kd was 1.2×10−6 M, indicating possible cross-reactivity between classical and alternative pathways of the activation of the complement system.

Introduction

Human complement component C2 is a 100 kDa single-chain polypeptide consisting of three types of globular domains [1]. The N-terminal domain of C2 consists of three complement control protein modules (CCP1,CCP2 and CCP3). The middle region is a type A domain similar to those found in von Willebrand factor (C2 vWF-A) [2]. The C-terminal end of C2 is a serine protease (SP) domain [3]. In a Mg2+ dependent reaction C2 binds to surface-bound C4b to form a C4bC2 complex. Cleavage of bound C2 by activated C1s results in the formation of the C3 convertase of the classical pathway (CP) of complement, C4b2a. Cleavage of C2 by activated C1s occurs in the N-terminal region of the vWF-domain, resulting in the generation of a 70 kDa C2a fragment (most of the C2 vWF-A domain+SP domain) and a 30 kDa C2b fragment (consisting of CCP1-2-3).

The homologue of C2 in the alternative pathway (AP) is Complement FB [4]. FB is a 90 kDa single-chain polypeptide that carries the catalytic site of the C3 convertase. Like C2, FB binds in a Mg2+-dependent reaction to soluble or surface-bound C3b to form a C3bB complex which is activated by Factor D cleavage to form the AP C3 convertase C3bBb. The half-life of the activated AP C3 convertase is about 2 min [5].

Factor B, similarly to C2, consists of 3 CCP modules, a von Willebrand factor domain (FB vWF-A domain) in the middle of the polypeptide chain, containing a Mg2+-dependent C3b binding site and, a serine protease domain (SP) at the C-terminus. Factor D cleavage of FB also occurs in the N-terminal region of the vWF-domain, resulting in the generation of a 60 kDa Bb fragment (most of the FB vWF-A domain+SP domain) and a 30 kDa Ba fragment (consisting of CCP1-2-3). The difference in molecular mass between C2 (100 kDa) and FB (90 kDa) is due to glycosylation. It has been shown that Ba undergoes rapid dissociation from the C3bBb complex [5] whereas it is not clear if C1s cleavage of C4bC2 gives C4b2a2b or if the C2b fragment dissociates to give C4b2a.

Formation of AP and CP C3 convertase is highly specific. As far as is currently known, C2 does not form any strong interaction with C3b, nor FB with C4b. Little is known about the topology and structures of the sites on C2 that participate in the protein/protein interaction leading to the assembly of the CP C3 convertase. Studies have shown that an anti-C2 monoclonal antibody, which recognises the second CCP domain in C2b, inhibits the initial binding of C2 to C4b, indicating the presence of C4b binding site(s) on C2b [6], [7]. Contribution of the 3 CCP modules to C4b–C2 complex formation has been investigated by the construction of FB–C2 chimaeras by substituting intact or partial FB CCP modules for the corresponding ones of C2 [7]. The haemolytic activity of the chimaeras containing CCP1, CCP2 and a part of CCP3, respectively, was substantially decreased compared with that of wild-type C2. Chimaeras in which all of CCP module 3 or all three CCP modules of FB were substituted had no haemolytic activity, probably because these chimaeras are not cleavable by activated C1s. Data on interaction sites have been obtained previously by using iodine and thiol blocking reagents [8]. Treating C2 with I2 decreases the rate of decay of C4b2a complexes through more avid binding of C2a to C4b. The effect of I2 has been attributed to oxidation of the free thiol group of Cys241, which is located in the N-terminal region of the von Willebrand Factor-A (vWF-A) domain of C2 (i.e., within C2a). Von Willebrand Factor domains contain a highly conserved, unusual Mg2+ coordination side [9]. The motif DxSxS,T and D termed ‘metal ion-dependent adhesion site’ (MIDAS) is involved in Mg2+-dependent interaction C3b–FB and C4b–C2 interaction. The C2 polypeptide chain around residue Cys241, as shown by site-directed mutagenesis, appears to constitute at least part of the C4b binding site on C2a [10] and is part of the C2 vWF-A MIDAS motif.

Formation of AP C3 convertase C3bBb has been more extensively investigated, as C3b and FB are stabler and more abundant than C4b and C2. A specific, metal ion-independent interaction between Ba and C3b was demonstrated during formation of fluid phase and surface bound AP C3 convertase. Purified Ba was able to inhibit C3bFB complex formation [5]. Pre-incubation with monoclonal antibodies, raised against distinct epitopes on Ba or Bb down-regulated or inhibited the haemolytic activity of FB. Monoclonal antibodies recognising CCP1 or CCP2 of Ba prevent binding of FB to C3b [7], [11], [12]. Individual CCP domains have been reported to contribute to the initial C3b–FB interaction. Substitution of parts of CCP2 and CCP3 by C2 sequences resulted in the loss of haemolytic activity and in inhibition of complex formation [12].

Similar approaches have been taken to investigate the role of the FB vWF-A domain in C3b–FB binding. Generation of chimaeras by exchanging parts of the FB vWF-A domain surrounding the MIDAS by corresponding C2 vWF-A sequences inhibited complex formation substantially [13]. FB vWF-A is similar in structure to the type A domain of the complement receptor and integrin, CR3. Substituting the FB type A domain amino acids with homologous ones derived from the type A domain of CR3, increased haemolytic activity and C3bBb stability [14]. The presence of a C3b binding site in this domain was directly demonstrated by binding experiments with a recombinant, functionally active FB vWF-A domain [15].

Two papers independently suggest that the serine protease domain of FB has affinity for C3b. Treatment of human FB with porcine elastase resulted in generation of a 33 kDa fragment, which expressed esterolytic activity and restored alternative pathway activity in Factor B-depleted serum. The 33 kDa fragment bound to C3b in the presence of Ni2+ but not in the presence EDTA. The fragment was shown to be derived from the C-terminal end of Bb, containing parts of the FB vWF-A and the SP domains [16], [17].

Although nearly all experimental work on AP C3 convertase formation is done with buffers containing Mg2+, there are reports that fluid-phase C3bBb complexes are formed in the absence of Mg2+. Using a sensitive haemolytic assay, it was estimated that the ability of FB to form a convertase with surface-bound C3b was 80-fold greater in the presence of 0.5 mM Mg2+ than in 2 mM EDTA [18].

As noted above, much more work has been done on C3b–FB interaction than on C4b–C2 complex formation. To overcome this imbalance, the aim of the present investigation was to study C4b/C2 interaction quantitatively. Recombinant expression of C2 or its fragments proved to be difficult and strategies similar to those used for the expression of recombinant FB fragments [15] were only partially successful. Instead a new method for the purification of C2 from human plasma was developed which gave reasonable quantities of active and pure protein to study complex formation by surface plasmon resonance.

Section snippets

Materials

Frozen, outdated human plasma for the purification of complement proteins C1s, C4, C2, C3 and FB was from HD Supplies, High Wycombe, UK. Ammonium acetate, barium chloride, dithiothreitol (DTT), diethylamine, ϵ-aminocaproic acid (EACA), ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethylether) N,N′-tetraacetic acid (EGTA), 2[N-morpholino]ethanesulfonic acid (MES), polyethyleneglycol (PEG), (N-[2-Hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]) (Hepes), iodoacetamide (IAM),

Binding in response to ionic strength

C2 as analyte in the fluid phase was passed over a mixture of immobilised C4(H2O) and C4b. To find suitable buffer conditions for binding, preliminary experiments with one concentration of C2 (180 nM) in buffers of different ionic strength were carried out. First a basic buffer of 10 mM Hepes, 5 mM MgCl2 and 5 mM CaCl2 was made 30–830 mM NaCl and binding was monitored. Because of the low flow rate of 5 μl/min, the high amount of immobilised ligand and only one concentration of C2, the binding

Discussion

It was demonstrated that C4C2 complex formation is ionic strength dependent as the total amount of complexes formed decreased under increasing NaCl or Mg2+ concentrations. As maximum binding was observed at 30 mM NaCl and as almost no interaction was observed above 300 mM NaCl, interactions between analyte and ligand are electrostatic rather than hydrophobic. NaCl concentrations lower than 30 mM were not tested.

C4bC2 complex formation in MC- or ME-buffer is a multiphasic, very fast reaction

Acknowledgements

We wish to thank B. Moffatt for assistance in purifying complement components FB, C2 and C3, and A.W. Dodds for purifying C4b. We are grateful to Professor P. Nuttal, NERC Institute of Virology and Environmental Microbiology, Oxford, for the use of a BIACORE 2000 system (BIACORE is a registered trademark of BIAcore AB). This work was supported by Grants J1767-med and J1572-med by the Austrian Science Fund (Fonds zur Förderung der wissenschaftlichen Forschung; FWF) and by the UK Medical Research

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