molecular dissection of interactions between components of the

Molecular Dissection Of Interactions Between Components Of The Alternative Pathway

Of Complement And Decay Accelerating Factor (CD55)

Claire L Harris1, Rachel JM Abbott2, Richard A Smith3, B Paul Morgan1, Susan M

Lea2.

1Complement Biology Group, Department of Medical Biochemistry and Immunology, School

of Medicine, Cardiff University, Heath Park, Cardiff, UK; 2Department of Biochemistry,

Laboratory of Molecular Biophysics, Oxford University, South Parks Road, Oxford, UK;

3Adprotech Ltd., Chesterford Research Park, Little Chesterford, Essex, UK.

Corresponding Author

Claire L Harris, Complement Biology Group, Department of Medical Biochemistry and

Immunology, School of Medicine, Cardiff University, Henry Wellcome Building, Heath

Park, Cardiff, CF14 4XN, UK.

Tel: (+44) 29 20745254

Fax: (+44) 29 20744001

E-mail: [email protected]

Running Title

Interactions in the alternative pathway of complement

JBC Papers in Press. Published on November 9, 2004 as Manuscript M410179200

Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.

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SUMMARY

The complement regulatory protein, decay accelerating factor (DAF, CD55), inhibits the

alternative complement pathway by accelerating decay of the convertase enzymes formed by

C3b and factor B. Using surface plasmon resonance we show that in the absence of Mg2+

DAF binds C3b, factor B and the Bb subunit with low affinity (KD: 14±0.1 µM, 44±10 µM

and 20±7 µM respectively). In the presence of Mg2+ DAF bound Bb or the von Willebrand

factor type A subunit of Bb with higher affinities (KD: 1.3±0.5 µM and 2.2±0.1 µM

respectively). Interaction with the proenzyme, C3bB, was investigated by flowing factor B

across a C3b-coated surface in the absence of factor D. The dissociation rate was dependent

on the time of incubation, suggesting that a time-dependant conformational transition

stabilised the C3b-factor B interaction. Activation by factor D (forming C3bBb) increased

the complex half-life, however, the enzyme became susceptible to rapid decay by DAF unlike

the proenzyme which was unaffected. A convertase assembled with cobra venom factor,

CVFBb, was decayed by DAF, albeit far less efficiently than C3bBb. DAF did not bind CVF

implying that Bb decay is accelerated, at least in part, through DAF binding this subunit. It is

likely that DAF binds the complex with higher affinity/avidity promoting a conformational

change in either or both subunits accelerating decay. Such analysis of component and

regulator interactions will inform our understanding of inhibitory mechanisms and the ways

in which regulatory proteins co-operate to control the C cascade.


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INTRODUCTION

Complement (C)1 plays a central role in innate immune defence with the abilty to rapidly

opsonise or destroy microorganisms or infected cells. The alternative pathway (AP) provides

an immediate, antibody-independent means to activate the C cascade whereas the classical

pathway (CP) relies in most cases on antibody to initiate activation of the first component,

C1. A third pathway, the lectin pathway, is initiated by binding of mannan binding lectin

(MBL) to sugar residues on bacterial cell walls. Other than the initiating factor (MBL and

associated proteases) this pathway is identical to the CP. The AP provides an immediate line

of defence to potential infection, it ‘ticks-over’ continuously in plasma through low level

activation of the central component, C3, probably via hydrolysis of the internal thioester bond

(1). Tight regulation of this tickover ensures that C is not activated to excess in plasma but

provides enough active C3b to bind foreign surfaces and amplify the cascade swiftly when

appropriate (2). The AP also amplifies the CP by formation of further active convertases on

C3b deposited during early amplification steps by the CP convertase. The initiating stimulus

of the AP is nucleophilic attack on the internal thioester bond in nascent C3b by an amine or

hydroxy group present on a foreign or ‘activating’ surface (3). This results in covalent

binding of C3b and forms the nidus for C amplification. The C3 convertase is formed by

binding of factor B (fB) to C3b in an interaction that requires a Mg2+ ion. The fB changes

conformation such that the C3bB complex is capable of activating the zymogen factor D

1 Abbreviations. AP: alternative pathway; CP: classical pathway; DAF: decay accelerating factor; MCP: membrane cofactor protein; vWFA: von Willebrand factor type A; Bb: larger cleavage subunit of factor B; Ba: smaller cleavage subunit of factor B; fB: factor B; fD: factor D; SCR: short consensus repeat; CVF: cobra venom factor; sDAF: soluble decay accelerating factor comprising four short consensus repeats; SPR: surface plasmon resonance; RU: resonance unit; ligand: moiety covalently coupled to the sensor chip surface; analyte: moiety flowed across the sensor chip surface; Rmax: maximum binding capacity of ligand; KA: association equilibrium constant; KD: dissociation equilibrium constant; ka: association rate constant; kd: dissociation rate constant; HBS: HEPES buffered saline.


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(fD), a serine protease present in plasma at about 2µg/ml (4). FD cleaves fB into Ba (amino-

terminal fragment) and Bb, the latter comprising an amino-terminal von Willebrand factor

type A domain (vWFA) and a carboxy terminal serine protease (SP) domain. Following

release of Ba, Bb binds with higher affinity to C3b. It is likely that conformational changes

in the vWFA domain transmit an allosteric signal to the SP domain resulting in its activation

and conferring it with the ability to cleave multiple molecules of C3 to nascent C3b thereby

amplifying the cascade (5,6).

The background tick-over of the AP and the potential for rapid amplification of all pathways

on self cells means that strict control in both biological fluids and on the membranes is

essential. Numerous C regulatory proteins (CReg) have evolved to meet the need to protect

‘self’ from the potentially destructive effects of C (7). These proteins police the body,

collaborating to control all pathways. The different CReg belong to various gene families,

those encoded in the RCA (‘Regulators of Complement Activation’) gene cluster on

chromosome 1 comprise factor H (fH), C4b binding protein (C4bp), decay accelerating factor

(DAF; CD55), membrane cofactor protein (MCP; CD46) and complement receptor 1 (CR1;

CD35) (8). All contain a structural module termed the short consensus repeat (SCR), a

domain comprising approximately 60 amino acids which confers C3b/C4b binding affinity to

the proteins and contains the regulatory function of the molecule (9). The amino-terminal

fragment of fB, Ba, is also comprised from three SCRs. The number of domains in CReg

varies from 4 (in MCP, DAF) to 37 (CR1, ‘B’ isoform). DAF is a GPI-anchored CReg

which acts to accelerate decay of the naturally labile enzymes of the AP and CP, C3bBb and

C4b2a (10,11). Other than the SCRs it comprises an elongated, heavily glycosylated ‘stalk’


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proximal to the membrane which projects the active site the correct distance from the

membrane allowing it to function efficiently (12,13).

The C components, from C1 through to C9, comprise a fascinating group of proteins whose

activities are characterised by differing conformations and presentation of continuously

changing faces or binding sites to other C components, receptors or regulators. C3 and fB are

no exception. C3 transformation starts following cleavage and release of C3a, it continues

following destruction of the internal thioester bond and moves on by presentation of binding

sites for components such as fB, Bb and eventually C5 (14,15). Along the way binding sites

for a multitude of regulators are formed, their sites may be distinct or overlapping and are

often transient as the cascade progresses or C3b becomes inactivated (16). Cleavage and

inactivation of C3b by fI results in the production of iC3b, C3c and C3dg, these inactivation

products no longer bind components such as fB and all differ in their ability to bind various C

receptors (17). FB also undergoes a multitude of conformational changes. Within the intact

protein all three domains interact with each other, possibly Ba holds the vWFA and SP

domains in a way which prevents autoactivation (5,6). FB changes conformation following

binding to C3b, once more following release of Ba and presumably again following release to

the fluid phase (18,19). The exact mechanisms of AP convertase assembly and regulation are

still being determined 50 years following discovery of this pathway (20).

Interaction of DAF with the AP convertase has largely been examined by mutagenesis

studies. These have sought to define entire domains in DAF or individual amino acids that

are crucial to decay of the convertase and protection of indicator cells (usually erythrocytes)

from lysis by C (21-23). Further information has been gained following resolution of the

structure of DAF, both in solution and in crystals (13,23,24). Various sites which extend


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through SCRs 2, 3 and 4 have been proposed to be important for convertase regulation. It is

not yet clear whether DAF undergoes a conformational transition following binding to its

ligands, most likely it acts purely to induce changes in other proteins to which it binds. It is

interesting to note that DAF not only binds the convertase enzymes but also a multitude of

pathogens, both bacteria and viruses, and also the leukocyte activation antigen CD97 (25). A

potential site of interaction for DAF on fB has also been identified by mutagenesis studies

and is located distal to the C3b interaction site in vWFA domain (26). Various studies have

addressed the binding site on C3b for the RCA proteins fH, MCP and CR1 and also for fB,

but no definitive sites of interaction have been determined and that for DAF has not been

examined (27,28). In this report we examine the interactions between DAF and the AP

convertase components by surface plasmon resonance (SPR). We define the affinity of these

associations and examine in real time the assembly and decay of the active AP convertase

and its proenzyme and their interaction with DAF. This study provides new insight into these

associations and further guides our understanding of the complex mechanisms and protein

interactions intrinsic to the C cascade.

EXPERIMENTAL PROCEDURES

Preparation of complement components and regulators

C3, cobra venom factor (CVF) and factor B were prepared by classical column

chromatography using established methods (29). Factor D was purchased from Quidel

Corporation (San Diego, MA). C3b was prepared from C3 using either of two methods. In

the first, CVF was coupled to Sepharose CL-4B using the manufacturer’s protocol

(Amersham Biosciences, Chalfont St.Giles, UK). A solid-phase convertase was formed by

incubating CVF-Sepharose in normal human serum to form CVFBb. The Sepharose was


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washed and incubated at 37°C with C3. C3b generated by cleavage was separated from C3a

and other minor contaminants by anion exchange on a Source Q column (Amersham

Biosciences). In the second method, C3, factor B and factor D were incubated in

complement fixation diluent (CFD; Oxoid, UK) until total C3 cleavage had occurred. C3b,

Bb and Ba were purified by anion exchange chromatography. The C3b-containing fractions

were pooled, concentrated and monomeric C3b was separated from dimeric C3b by size

exclusion on a Superose 6 column (Amersham Biosciences).

Recombinant human DAF comprising the four SCRs (soluble DAF; sDAF) was isolated and

refolded from E.coli as described previously (30). The structure and function of sDAF has

been previously studied and the purified, refolded, protein is known to consist of four

correctly folded SCR domains and demonstrates the full range of complement regulating and

pathogen binding activities associated with DAF purified from erythrocytes (13,30,31).

Human DAF-Ig fusion protein comprising four SCR domains fused to human IgG1 Fc (DAF-

Ig) was prepared as previously described (32). Recombinant vWF-A domain of human factor

B was expressed in E. coli as a fusion protein with glutathione S-transferase using published

methods (33) with the addition of a final gel filtration on a S75 column (Pharmacia) to yield

protein at a purify of >98%, as assessed by SDS PAGE electrophoeresis (data not shown).

Biosensor Analysis

All analyses were carried out on a Biacore 3000 machine (Biacore International SA,

Stevenage, UK) other than that in Figure 4c which was carried out on a Biacore 2000.

Proteins were coupled to the sensor chip surface using an amine coupling chemistry as

instructed by the manufacturers (NHS/EDC coupling kit). For all kinetic analyses a CM5

chip (carboxymethylated dextran surface) was used and data was collected at 25°C, the flow


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rate was maintained at 30µl/minute and data from a reference cell was subtracted to control

for bulk refractive index changes. The Rmax was kept low and the flow rate high to eliminate

mass transfer. However, this was controlled for where appropriate by varying flow rate and

ensuring observed association rates did not vary. Samples were injected using the KINJECT

command to ensure accurate association kinetics. Interactions were analysed in HEPES

buffered saline (HBS: 10mM HEPES pH7.4, 150mM NaCl), 0.005% surfactant P20 and

either 1mM MgCl2 or 3mM EDTA as stated in the text. Data were evaluated using

Biaevaluation software (Biacore International). Concentration of analytes was assessed using

absorbance at A280, molarities were calculated using the following extinction coefficients

(molecular masses and coefficients obtained using Protean software, DNAStar): sDAF (1.3),

C3b (1.03), factor B(1.43), Bb (1.29), Ba (1.74).

RESULTS

Interaction of DAF with C3b

In order to study the interaction of DAF with the individual AP convertase components, C3

was incubated with factors B and D until formation of C3b, Bb and Ba was complete.

Individual components were separated by anion exchange chromatography (Figure 1a). This

also removed C3i from the C3b preparation. We routinely found that approximately 50% of

the C3b formed by AP activation was in a dimeric form. In order to eliminate avidity effects

from SPR analysis, C3b was further separated into monomeric and dimeric forms by size

exclusion chromatography (Figure 1b). Initial SPR studies indicated that the interaction

between DAF and C3b was of low affinity (subµM) with very fast on and off rates typical of

DAF-ligand interactions (31,34,35). The affinity of the interaction was studied under steady

state conditions by immobilising either DAF or C3b to the sensor chip surface and flowing


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monomeric C3b or DAF respectively over the surface. The most accurate determination as

judged by smallest SE values for KA and Rmax was obtained by immobilising C3b and

studying the interaction with soluble DAF as this analyte was available at concentrations of

the same order of magnitude as the KDs determined (>10µM). C3b was coupled to the chip

surface either by amine coupling or through the thioester group. The latter method has the

advantage of capturing C3b on the surface in a physiologically relevant orientation. A typical

analysis is illustrated in Figure 2, reference cells were used to correct for any bulk shift

effects. Mean KDs are given in Table 1.

Interaction of DAF with fB and its cleavage products

Mutational analysis of fB has revealed two sites in the vWFA domain which are involved

with decay, whether these sites interact directly with DAF or alter the stability of the

convertase is not yet clear (26). It is likely however that there is a direct interaction of DAF

with the Bb portion of the convertase. We immobilised DAF on the chip surface and flowed

intact fB, Bb or Ba over the surface at high concentrations. A direct interaction was seen

with intact fB and the Bb subunit but not with Ba (Figure 3a). The affinity of the interaction

between DAF and intact fB was determined by immobilising either fB or DAF to the surface

via amine groups. As with the DAF/C3b interaction, the affinity was low and was analysed

at steady state. The binding profile and affinity data obtained were similar irrespective of the

protein partner immobilised (mean KD 44±10 µM in EDTA; Figure 3) and the presence or

absence of Mg2+ (not shown, mean KD 74±30 µM in 1mM Mg2+). The interaction of DAF

with intact fB was of lower affinity than with C3b (Table 1).

Whilst the interaction of fB was relatively unaffected by the presence of Mg2+, we noticed

that this cation had a marked effect on the interaction of DAF with the Bb subunit (Figure


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4a). In the absence of Mg2+ the affinity of the Bb subunit for DAF and the binding profile

was similar to that of fB (data not shown, average KD 20±7 µM). However in the presence of

Mg2+ the affinity of the interaction between Bb and DAF was higher. The kinetic profile was

different to that of fB, on and off rates were slower and the affinity was calculated as 1.3 µM

(using a 1: 1 Langmuir interaction model; Figure 4b, Table 1). Further analysis using the

isolated vWFA domain demonstrated a similar binding profile (Figure 4c) and affinity of

2.2±0.1 µM for this subunit (Table 1).

Interaction of fB with C3b

We sought to assemble the convertase on the sensor chip surface itself, the aim being to

assess the affinity of DAF for the intact enzyme in addition to individual components. Low

levels of C3b were immobilised to the chip surface and fB was flowed across in the presence

of Mg2+ (1mM). It was immediately apparent that the interaction did not fit a simple 1:1

association model (Figure 5a). Furthermore, interaction of identical concentrations of fB

with C3b for different lengths of time (1, 3 or 20 mins) demonstrated that the dissociation

became slower the longer the components interacted (Figure 5b), this can be indicative of a

conformational change which results in a tighter binding between components. Indeed,

fitting the data from Figure 5a to a conformational change model (BIAcore software) gave a

good fit, although some heterogeneity was evident at high concentrations of fB (Figure 5c).

Kinetic analysis following a 4 minute association phase gave the following information: ka1:

1.36x104M-1s-1; kd1: 0.115 s-1; ka2: 5.53x10-3M-1s-1; kd2: 1.78x10-3s-1 (χ2: 4.2). In this model

the off rate following the conformational change (kd2) is much lower.


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Formation of the AP convertase and interaction with DAF

Either the active or inactive convertase was assembled on the chip surface by flowing fB over

C3b in the presence or absence of fD. Either fB (46µg/ml) or a mixture of fB (46µg/ml) and

fD (8µg/ml) was flowed across the surface as indicated in Figure 6a. Several differences in

the two complexes were noted. Firstly, cleavage of fB by fD altered the rate of decay of the

active enzyme, prolonging its half-life. Secondly, the Mg2+ ion in the active enzyme was

'locked' in place, flowing 10mM EDTA over C3bB(Mg2+) dissociated fB from C3b whereas it

had no effect on decay of C3bBb(Mg2+).

C3b was also bound to the surface using the AP, this resulted in coupling of nascent C3b via

its thioester to hydroxy groups present on the dextran-coated surface. We used a

modification of a previously described technique in which repetitive cycles of fB/fD with C3

resulted in AP amplification on the chip surface (36). To increase efficiency of activation we

deposited the first nidus of C3b on the chip surface using a fluid phase convertase, CVFBb

(Figure 6b). Each subsequent ‘cycle’ illustrated in Figure 6b comprised incubation with fB

and fD to form active convertase followed by incubation with native C3. Following C3b

deposition, buffer was flowed across the surface for 16 hours to allow dissociation of non-

covalently bound C3b and of any active convertase. In order to study the interaction of DAF

with the convertase, factors B and D were flowed over the surface. The amount of

active/inactive convertase on the chip surface was varied by titrating the amount of fD in the

incubation (Figure 6c). In the presence of fD, the surface reached an equilibrium (Figure 6c

(E)), unlike the proenzyme which displayed complex kinetics during formation (Figures 5, 6c

(A)). When soluble DAF was flowed across the active convertase, decay of the Bb subunit

was virtually instantaneous (Figure 6c (B-E)). Decay of the enzyme was so efficient that it

was impossible to measure binding and an affinity of DAF for the intact convertase. This


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contrasted with flow of DAF across the inactive enzyme in which rapid and total decay was

not apparent (Figure 6b (A)). Uncleaved fB was dissociated from the surface at the end of

the incubation using EDTA as shown in Figure 6. Alignment of individual sensorgrams at

either the fB/fD inject or DAF inject demonstrates the variation in decay and identical

binding of DAF to C3b on the chip surface (Figure 6d).

Formation of CVFBb and interaction with DAF

We have demonstrated that DAF interacts with both subunits of the active enzyme and that

decay is rapid and efficient. The convertase enzymes formed from CVF (C3b-like molecule

found in cobra venom) and fB are very stable and are assumed to be resistant to decay by

human RCA proteins. To analyse the interaction with DAF, CVF was immobilised on the

sensor chip surface and DAF was flowed across, no interaction between CVF and DAF was

evident even at high concentrations (47µM) (Figure 7a). To assess whether DAF could decay

the CVF-containing convertase, fB and fD were flowed sequentially across the CVF surface

and a C3b-coated surface (Figure 7b). In the absence of fD the CVFB complex dissociated

rapidly and the surface could be regenerated with EDTA. Inclusion of fD resulted in an

EDTA-stable surface which efficiently cleaved C3 to C3b (data not shown). Formation of

the active enzyme was less efficient than with human C3b but that which did form was very

stable (Figure 7b). When DAF was flowed across the surface at concentrations (15µM) much

higher than those which brought about immediate decay of C3bBb, it was apparent that it

could indeed accelerate decay of CVFBb. However, this was far less efficient than with the

native, human enzyme. The large concentration of DAF used in this experiment is apparent

in Figure 7b where binding to the C3b-coated surface can be seen during the DAF inject

(indicated in the figure).


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DISCUSSION

We have used SPR technology to analyse the interaction of DAF with individual components

of the AP convertase and also with the intact (pro)enzyme and to visualise assembly and

decay of the convertase in real time. We demonstrate that DAF interacts with the individual

components C3b and fB with low affinity. The equilibrium dissociation constant (KD) for the

interaction of DAF with fB is 44±10µM demonstrating that this is a significantly weaker

interaction than that of DAF with C3b where the interaction with amine-coupled C3b is

characterised by a KD of ~14±1µM and with thioester-coupled C3b with a KD of ~7±1µM

(Table 1 and Figures 2 and 3). The small differences between the KDs for DAF interactions

with differently coupled C3b probably reflect the more optimal presentation of binding sites

in the natively coupled C3b as compared to the amine coupled protein where steric hindrance

is more likely to occur.

DAF did not interact with the Ba subunit of fB but did interact with the Bb subunit. The

affinities of DAF for fB and Bb in EDTA were comparable (44±10 µM and 20±7 µM

respectively). However, the affinity of DAF for the Bb subunit was increased >10-fold to

1.3µM (KD) in the presence of Mg2+; a similar affinity was obtained for the isolated vWFA

domain (2.2µM ) suggesting that this domain mediates the contact with DAF (Figure 4).

Indeed, a previous report has defined two surface patches on opposing surfaces of the vWFA

domain that may be involved in the decay of C3bBb by DAF; one of these may mediate

binding to C3b whilst the other may be involved in binding DAF (26). It is interesting that

DAF has a higher affinity for Mg2+-bound Bb. It is known that the Mg2+ coordination site of

fB is in the active site cleft of the vWFA domain, a site that is involved in the binding to C3b,

and that the structure of this domain and of Bb is more stable in the presence of the cation. A


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conformational dependence of the vWFA domain on the presence of a metal ion has been

demonstrated by various spectroscopic techniques (6), in particular the α-helix A7 is

conformationally mobile between the metal-free and metal-bound forms of the vWFA

domain. Our data show that DAF may bind distinct conformational states of this molecule

with different affinities. Whilst we have demonstrated a direct interaction of DAF with the

isolated Bb fragment, the physiologically relevant complex is Bb in association with C3b. Bb

has little or no affinity for C3b following dissociation indicating that fluid phase Bb differs in

conformation from C3b-bound Bb ((18) and our unpublished SPR data). It is known that the

vWFA domain in Bb, in common with similar domains in other molecules such as CR3 or

von Willebrand factor, exists in high affinity and low affinity conformations (37). Following

cleavage of fB by fD, Bb binds to C3b with higher affinity. The vWFA domain likely

transmits an allosteric signal resulting in proteolytic activity in the SP domain of Bb (6). We

cannot determine whether the affinity of DAF that we demonstrate here is for a low or high

affinity conformation of vWFA domain.

The initial binding of fB to surface bound C3b is complex and clearly does not follow a

simple 1:1 Langmuir interaction (Figure 5). The data fit a model in which a conformational

change occurs resulting in a higher affinity interaction with a slower dissociation rate (kd2).

This model can only be applied when the change is slow and occurs over the period of the

association phase, extremely rapid changes are more likely to fit a simple 1:1 interaction. It

is known that fB changes conformation upon binding C3b allowing it to induce a

proteolytically active conformation of fD (4). However inclusion of fD in the incubation

resulted in a very different binding profile (Figure 6), the binding rapidly reached a plateau

and an equilibrium was obtained. Interestingly, the resulting complex was resistant to EDTA

(Figure 6a), this is in agreement with previous work which has shown that a transition in Bb


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and tight binding of the vWFA active site cleft to C3b protects the Mg ion from chelation by

EDTA (19,38). The binding profile of fB in the presence of fD was more typical of a simple

interaction implying that if conformational changes have occurred they were rapid and an

'end-point' was swiftly reached. Others have previously reported that fB can bind C3b (and

CVF) and form an active enzyme in the absence of fD (39-41). It is possible that the

conformational change evident in the absence of fD in Figure 5 represents a slow transition to

an active conformation in which the SP domain of fB can cleave C3b, fD-mediated cleavage

of fB to Bb may act to expedite this transition.

We have shown that DAF binds individual components of the AP convertase with low

affinity, the crucial question is how does DAF bind the intact convertase? DAF must

'recycle' in order to protect self cells from C attack. Clearly if DAF bound to C3b with the

same affinity as to the convertase it would rapidly become saturated with C3b at a site of C

activation and would not be available for decay of the active convertase. In 1986, Pangburn

examined the ability of DAF and other CReg to decay the zymosan-bound convertases in the

presence of fluid phase competitors such as C3b, Bb, C3bB and C3bBb (42). Apparent

association constants (appKA) of DAF with C3b and Bb were 0.045µM-1 and 0.067µM-1

respectively. The affinity of DAF for C3bB or C3bBb was >10 fold higher (0.71µM-1,

0.91µM-1) suggesting that following decay of the enzyme DAF would be 'released' enabling it

to bind the next convertase with high affinity. Despite the fact that DAF appeared to bind

C3bB almost as well as C3bBb in this study, it was not clear whether DAF decayed both the

active convertase and the proenzyme. Early studies using DAF incorporated into erythrocyte

membrane showed that DAF did not prevent fB binding surface bound C3b or C4b, but

rapidly decayed the activated fragments Bb (43). More recently it has been demonstrated

using an ELISA-based decay assay that DAF decays C3bBb(Ni2+) but not C3bB(Ni2+) (44),


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this selective decay may be related to the >10-fold higher affinity of DAF for Bb compared to

fB in the presence of Mg2+. Our data support the suggestion that DAF only decays the active

enzyme. In order to monitor the decay of the convertase using SPR, we formed the

convertase on the sensor chip surface in the presence or absence of fD (Figure 6). DAF-

mediated decay of the active convertase, C3bBb, was visualised in real time. It was

immediately apparent that dissociation of Bb from C3b was virtually instantaneous and it was

not possible to measure an affinity of DAF for the active convertase directly. In support of

the ELISA-based assay it was evident that DAF decayed the active enzyme and had little

effect on the inactive convertase. By titrating the amount of active convertase on the chip

surface it was possible to see this differential decay (Figure 6c). In the absence of any fD a

small amount of DAF-mediated decay was evident (Figure 6c(A)), we do not know what this

represents but it is possible that a portion of C3bB has undergone a suitable transition such

that it is in an ‘active’ conformation and the affinity for DAF is enhanced as discussed above

rather than this being the observation of decay of the inactive convertase. When DAF was

flowed across the C3b-coated surface in the absence of any fB or fD an increase in RU was

evident (Figure 6d, alignment at DAF inject). This represented binding of DAF to C3b and

decreased again rapidly after the DAF inject was completed, the off rate for this interaction is

extrememly rapid as illustrated in Figure 2. Following an identical inject of DAF over the

C3bB or C3bBb coated surface the same decrease in RU was evident at the end of the inject

and dissociation was rapid (Figure 6d). The dissociation of DAF from the surface was

evident above the background decay of fB from C3b which remained unaltered. These

preliminary data imply that in our assay system the affinity of DAF for C3b was unaffected

by the presence or absence of proteolytically inactive fB, we are currently investigating the

kinetics of this interaction further. Attempts to stabilise the C3bBb surface using chemical

crosslinking in order to measure the affinity of DAF for the active enzyme were not


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successful. Whilst the release of Bb from the surface was prevented, an interaction with DAF

could not be visualised. Either the crosslinking reagent destroyed the interaction sites with

the convertase or DAF did indeed decay the components but they remained loosely tethered

to each other via the crosslinker without any physical protein/protein interaction. There are

various reasons why DAF may decay the active but not the inactive convertase. Firstly the

SCR-containing subunit of fB, Ba, may directly compete for the same binding site on C3b.

Secondly, the presence of Ba may stabilise the C3bB complex, a direct interaction of Ba with

C3b has been demonstrated ((45) and our unpublished SPR data), several points of contact

will increase the avidity of the interaction. Thirdly, as suggested above Bb may adopt a

conformation in the absence of Ba which binds DAF with a higher affinity. Finally, it is also

possible that the Ba domain partially blocks the binding site on Bb for DAF, it has been

shown previously that Ba contacts both the vWFA and SP domains in inactive fB (5).

It is interesting that DAF accelerates decay of the CVFBb convertase, albeit with much lower

efficiency than decay of C3bBb (Figure 7b). DAF had no affinity for the isolated C3b-like

component of the enzyme, CVF (Figure 7a). In the study described above Pangburn

demonstrated that the affinity of DAF for CVFBb was identical to that of DAF for Bb (42).

Although we show here that the accelerated decay of CVFBb mediated by DAF was very

inefficient, it was the only way in which we could regenerate the surface of the chip, neither

EDTA nor another powerful CReg, soluble recombinant CR1 (sCR1), could regenerate the

surface back to uncomplexed CVF. The inefficiency of this decay implies that either the

conformational change in Bb resulting in its release is slowed by the presence of CVF, or that

DAF works by also inducing a transition in C3b (not seen with CVF) that accelerates decay

of the enzyme.


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Current concepts regarding AP convertase assembly and decay are as follows. Ba mediates

initial binding of fB to C3b, the avidity of binding may be enhanced by sites on the Bb

subunit which interact with C3b. If sites in Bb interact with C3b the conformation differs

from that of the dissociated subunit, Bb, which has much decreased affinity for C3b.

Following binding of fB a conformational change occurs such that the complex activates the

zymogen fD, which in turn cleaves fB into Bb and Ba. Ba is released from the complex

(although this interaction is reversible). Release of Ba results in a higher affinity complex

between C3b and Bb, the Mg2+ ion in the active site cleft of the vWFA domain is 'locked' into

the enzyme through association with C3b, the complex becomes more stable with a longer

half-life and the SP domain in Bb is activated such that it can proteolytically cleave C3.

However, this complex is subject to decay by DAF. We show that DAF binds individual

components of the convertase in the following order of affinity: Bb>C3b>fB. Presumably it

associates weakly with C3b deposited covalently on a cell surface. However, in order to

selectively bind and decay active convertase, binding to C3bBb must be greater than that to

C3b. A simple explanation would be that a second point of contact with the Bb subunit

increases the avidity of the interaction, we have noted that apparent affinities of bivalent

interactions can be 10-fold that seen with the same monovalent interaction (unpublished

data). It is also possible that complexing of C3b and Bb favours a particular conformation in

one or both of the components that bind DAF with higher affinity. Increased avidity of DAF

binding to the multi-molecular complex would in effect stabilise the complex further and

prevent decay/dissociation. It is likely therefore that DAF promotes a further change in

conformation resulting in dissociation of Bb from C3b, possibly by inducing the 'low affinity'

conformation of the vWFA domain. Decay of the complex results in a low (subµM)

interaction with the individual components allowing DAF to recycle to other active

convertase. It is of note that the AP convertase is stabilised in vivo by binding of properdin


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to the C3bBb complex (46). We are currently examining the effect of properdin on DAF

binding to the AP convertase and to individual components. Whilst the exact mechanisms of

assembly and decay of the AP convertase are still unclear, we have come a long way in

deciphering the ways in which C is activated and regulated. Data presented here and similar

analyses of other C regulators will provide a more informed picture of their co-operation in

inhibiting the complement activation pathways and the mechanisms responsible for release

from the convertase and ‘recycling’ of the regulatory proteins.

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ACKNOWLEDGEMENTS

This work was supported by the Wellcome Trust (grant reference 068823/Z). We

acknowledge the support of the MRC (studentship to RJMA).


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FIGURE LEGENDS

Figure 1. Isolation of activated C components. C3 was incubated with fB and fD in CFD.

Activated components were separated by anion exchange (a), C3b monomers and dimers

were further fractionated by size exclusion (b).

Figure 2. Interaction between C3b and DAF. (a) C3b (monomeric) was immobilised on

the chip surface via amine coupling. Interaction with sDAF was analysed in HBS-EDTA at

the indicated concentrations and sensorgram traces were obtained (grey/black lines are

duplicates and virtually overlie each other). The affinity of the interaction was analysed by

steady state analysis (see inset). Values for KD and Rmax and standard errors associated with

the fit illustrated here are shown.

Figure 3. Interaction between DAF and factor B, Bb and Ba. (a) Differing amounts of

DAF (either sDAF or DAF-Ig) were immobilised on the chip surface via amine groups.

Factor B, Bb or Ba was flowed at the indicated concentrations in HBS-EDTA and

sensorgram traces were obtained. (b) sDAF was immobilised on the chip surface via amine

groups. Interaction with factor B was analysed in HBS-EDTA at the indicated concentrations

and sensorgram traces were obtained. The affinity of the interaction was revealed by steady

state analysis to yield the values shown. (c) FB was immobilised on the chip surface via

amine groups and interaction with sDAF was analysed in HBS-EDTA at the indicated

concentrations.

Figure 4. Interaction between DAF, Bb and vWFA domain. sDAF was immobilised on

the chip surface via amine groups. FB or Bb in either EDTA or 1mM Mg2+ was flowed


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across the surface (a). Further kinetic analysis of either (b) Bb or (c) vWFA domain was

performed at the indicated concentrations in HBS-Mg2+ and sensorgram traces were obtained.

The affinity of the interactions were analysised by 1:1 Langmuir binding. Black traces

represent the actual data and grey traces the modelled data. Values derived from and errors

associated with these fits are also shown.

Figure 5. Interaction between C3b and factor B. (a) C3b was immobilised on the chip

surface via amine coupling. Interaction with factor B was analysed at the indicated

concentrations in HBS-Mg2+ allowing association for 4 minutes. (b) Factor B was flowed

over C3b for different lengths of time (1, 3 and 20 minutes), data were normalised using

BIAcore evaluation software and sensorgrams overlaid at the start of the dissociation curve.

(c) The affinity of the interaction was analysed using the ‘conformational change’ model;

dashed lines are modelled data, χ2=4.2; grey lines are actual data.

Figure 6. Assembly and decay of the AP C3 convertase in real time. (a) C3b was amine-

coupled to the chip surface. fB was flowed across in HBS-Mg2+ as indicated, incubation in

10mM EDTA decayed the complex. In contrast C3bBb was not decayed by 10mM EDTA.

(b) C3b was deposited on to the acceptor surface using a method similar to that already

described (36). The AP was amplified by repeated cycling (in HBS-1mM Ni2+) at 30ºC of C3

followed by fB and fD. Convertases and non-covalently bound C3b was allowed to decay for

16 hours before using the surface. (c) Different amounts of active convertase were assembled

on the chip surface by varying the concentration of factor D in the incubation. As the

quantity of active convertase increased, the ability of sDAF to decay the components was

greater. FB, fD, sDAF and EDTA were injected at the indicated timepoints. Decay mediated

by (0.8µM) sDAF is indicated by a double-headed arrow. FB was injected at 450µg/ml and


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fD at 0, 2.5, 5, 10 and 20ng/ml (numbered A-E respectively). (d) Sensorgrams from (c) are

aligned at the start of the fB/fD inject to allow for comparison of convertase assembly or at

the end of the DAF inject to allow comparison of DAF-mediated decay.

Figure 7. Interaction of DAF with cobra venom factor (CVF). CVF was immobilised on

the chip surface via amine groups. (a) CVF did not bind sDAF although it formed a C3

convertase when factor B and factor D were flowed across the chip surface (b). sDAF was

injected as indicated during which time the rate of decay of the convertase increased. The

C3bBb sensorgram is indicated by the dashed line, that of CVFBb by the solid black line.

CVF convertase was assembled in HBS-Mg2+.


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Table 1. Parameters measured that define DAF-ligand interactions. Values given are

mean ± SD of multiple experiments (n) other than Bb (1mM Mg2+) where individual datasets

are illustrated. Concentration ranges of analytes are as indicated in previous figures. ND:

not determined.

Protein interacting with DAF

ka

(mol-1

s-1

)

kd

(ms-1

)

KD

(µM)

χ2

C3b monomer amine coupled (n=5) ND ND 14 ± 1 1.2 ± 0.7

C3b dimer amine coupled (n=3) ND ND 13 ± 1 2.5 ± 1.3

Thioester coupled C3b (n=4) ND ND 7 ± 1 6.1 ± 6.4

fB (no Mg2+) (n=4) ND ND 44 ± 10 1.2 ± 0.6

fB (1mM Mg2+) (n=4) ND ND 74 ± 30 2.3 ± 3.2

Bb (no Mg2+) (n=4) ND ND 20 ± 7 2.3 ± 0.8

Bb (1mM Mg2+) (n=2) 824, 1420

1.4, 1.2 1.7, 0.8

mean 1.3

3.7, 1.8

vWF-A (1mM Mg2+) (n=6) 830 ± 30 1.8 ± 0.03 2.2 ± 0.1 0.4 ± 0.3


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0

200

400

600

800

1000

1200

0 50 100 150 200 250 300 350

(a)

volume

A28

0 (m

AU

)anion exchange

BbBa

C3b

C3b aggregatesC3i

(b)

-20

30

80

130

180

230

280

0 5 10 15 20 25volume

A28

0 (m

AU

)

size exclusion; C3b

C3bmonomer

C3bdimer

Figure 1


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-100

-50

0

50

100

150

200

100 150 200 250 300 350Time (s)

Res

pons

e D

iffer

ence

(RU

)

1.28µM

40.8µM20.4µM10.2µM5.11µM2.55µM

Amine coupling Req

10

30

50

70

90

110

concentration DAF (µM)0 5 10 15 20 25 30 35 40 45

KA = 7.2x104± 2.4x103 M-1

Rmax =135 ± 2KD = 13.9µΜχ2 = 1.3

Figure 2


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(a)

-100

-50

0

50

100

150

200

250

300

350

2200 2400 2600 2800 3000 3200 3400 3600

Time (s)

Res

pons

e D

iffer

ence

(RU

)

Ba at 7.5µMfB at 3.2µM

Bb at 5.0µMLigand:

CD55-Ig low RUCD55-Ig high RUsDAF

(b) (c)

350 400

Time (s)

39.5µM

1.2µM2.5µM4.9µM9.9µM

19.8µM

100 150 200 250 300 350-40

-200

2040

6080

100120

140

Res

pons

e D

iffer

ence

(RU

)

-100

-50

0

50

100

150

200

250

100 150 200 250

Time (s)

Res

pons

e D

iffer

ence

(RU

)

300

13.7µM

0.9µM1.7µM3.4µM6.9µM

27.4µM

FB flowing over CD55 CD55 flowing over fB

KA = 1.8x104± 0.8x103 M-1

Rmax =413 ± 13KD = 53µMχ2 = 0.8

KA = 3.0x104± 2.4x103 M-1

Rmax =140 ± 6KD = 33µMχ2 = 1.8

Figure 3


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(a)R

espo

nse

Diff

eren

ce (R

U)

-40-20

020406080

100

Time

fB (11µM)EDTA

Bb1mM Mg2+

Bb (17µM)EDTA

fB1mM Mg2+

(b) Bb flowing over DAF in 1mM Mg2+

(c)

-100

-50

0

50

100

150

200

-500 -200 100 400 700 1000 1300

Res

pons

e D

iffer

ence

(RU

)

Time (s)

ka = 824 ± 5 M-1s-1

kd = 1.4x10-3 ± 4x10-6s-1

Rmax =155 ± 0.6KD = 1.7µMχ2 = 3.7

7.9µM

4.0µM2.0µM1.0µM

0.25µM0.5µM

vWFA flowing over DAF in 1mM Mg2+

1600 1900 2200 2500

7.2µM

4.5µM

2.8µM

1.8µM1.1µM

0.43µM0.27µM0.68µM

-200 100 400 700 1000 1300 1600 1900 2200 2500-40

-20

0

20

40

60

-500

Res

pons

e (R

U)

ka = 851 ± 1 M-1s-1

kd = 1.9x10-3 ± 1x10-6s-1

Rmax =106 ± 0.1KD = 2.2µMχ2 = 0.2

140

120

100

80

Time (s)

Figure 4


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(a)

-150

-100

-50

0

50

100

150

200

250

300

350

0 200 400 600 800 1000 1200 1400Time (s)

Res

pons

e di

ffere

nce

(RU

)55µM27µM14µM

7µM3.4µM1.7µM

fB flowing over C3b in 1mM Mg2+

(b)

0

20

40

60

80

100

120

-2000 -1400 -800 -200 400 1000

Res

pons

e D

iff.e

renc

e(R

U)

1min

3mins

20mins

increasing off rate

Time (s)

association dissociation

(c)

-50

0

50

100

0

Res

pons

e D

iffer

ence

(RU

200 400 600 800 1000 1200

Time (s)

14µM

7µM

3.4µM

1.7µM

Figure 5

conformational change fit

200

)

150

1400


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(a)

-1000

100200300400500600700

500 1000 1500 2000 2500 3000

Time

Res

pons

e D

iffer

ence fB

10mMEDTA

fB+fD10mMEDTA

Decayof fB

18000

20000

22000

24000

26000

28000

30000

32000

34000

4000 5000 6000 7000 8000 9000 10000 11000 12000 13000 14000

Time (s)

Res

pons

e (R

U)

CVFBb+ C3

fB+fD

C3

Cycle 1

Cycle 2Cycle 3

Cycle 4

(b)

(c)increasing [fD]

-2000

200400600800

1000120014001600

0 500 1000 1500 2000 2500

Res

pons

e D

iffer

ence

(RU

)

EDTADAF

(A) (B) (C)

fB/fD

D

E

D

E

fB/fD

fB

3000 3500

(D)

D

E

fB/fD

4000 4500

(E)

D

E

fB/fD

5000Time (s)

Figure 6


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(d)

Res

pons

e D

iffer

ence

(RU

)

Time (s)

RU-alignment at fB/fD inject

-600-500-400-300-200-100 0 100 200 300 400-200

200400600800

1000120014001600

0

fB/fD

DAF(0.8µM)

EDTADAFonly

RU-alignment at end DAF inject

-400-300-200-100 0 100 200 300 400 500-600-400-200

200400600800

100012001400

-500

0

Res

pons

e D

iffer

ence

(RU

)Time (s)

fB/fD

DAF(0.8µM)

Figure 6


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DAF flowing across CVF

(a)

-100

-80

-60

-40

-20

0

20

40

2060 2080 2100 2120 2140 2160 2180 2200 2220 2240

Res

pons

e D

iffer

ence

(RU

)

Time (s)

DAF (47µM)

Figure 7

(b)enzyme

formation enzyme

-100

0

100

200

300

400

500

500

Res

pons

e D

iffer

ence

(RU

)

DAF (15µM)

C3bBb

CVFBbDAF

bindingC3b

accelerated decay of CVFBb

decay

DAFdecay of C3bBb

1000 1500 2000 2500 3000 3500

Time (s)


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LeaClaire L. Harris, Rachel J. M. Abbott, Richard A. Smith, B. Paul Morgan and Susan M.

pathway of complement and decay accelerating factor (CD55)Molecular dissection of interactions between components of the alternative

published online November 9, 2004J. Biol. Chem.

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