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The Journal of Immunology, 2001, 167: 6374-6381.
Copyright © 2001 by The American Association of Immunologists

{beta}-Amyloid Fibrils Activate the C1 Complex of Complement Under Physiological Conditions: Evidence for a Binding Site for A{beta} on the C1q Globular Regions1

Pascale Tacnet-Delorme, Sylvie Chevallier and Gérard J. Arlaud2

Laboratoire d’Enzymologie Moléculaire, Institut de Biologie Structurale, Grenoble, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies based on the use of serum as a source of C have shown that fibrils of {beta}-amyloid peptides that accumulate in the brain of patients with Alzheimer’s disease have the ability to bind C1q and activate the classical C pathway. The objective of the present work was to test the ability of fibrils of peptide A{beta}1–42 to trigger direct activation of the C1 complex and to carry out further investigations on the site(s) of C1q involved in the interaction with A{beta}1–42. Using C1 reconstituted from purified C1q, C1r, and C1s, it was shown that A{beta}1–42 fibrils trigger direct C1 activation both in the absence of C1 inhibitor and at C1 inhibitor:C1 ratios up to 8:0, i.e., under conditions consistent with the physiological context in serum. The truncated peptide A{beta}12–42 and the double mutant (D7N, E11Q) of A{beta}1–42 did not yield C1 activation, providing further evidence that the C1 binding site of {beta}-amyloid fibrils is located in the acidic N-terminal 1–11 region of the A{beta}1–42 peptide. Binding studies performed using a solid phase assay provided strong evidence that C1q interacts with A{beta}1–42 fibrils through its C-terminal globular regions. In contrast to previous studies based on a different experimental design, no significant involvement of the C1q collagen-like domain was detected. These findings were confirmed by additional experiments based on C1 activation and C4 consumption assays. These observations provide direct evidence of the ability of {beta}-amyloid fibrils to trigger activation of the classical C pathway and further support the hypothesis that C activation may be a component of the pathogenesis of Alzheimer’s disease.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Alzheimer’s disease (AD)3 is a common dementia resulting from neuronal loss. It is characterized by excessive deposition in the CNS of the {beta}-amyloid peptide, a 40- to 42-residue peptide derived from a larger precursor protein (1, 2). The {beta}-amyloid peptide accumulates around cerebral blood vessels and is the major protein component of the senile (or neuritic) plaques, a hallmark of pathology in the AD brain. There are several lines of evidence that accumulation of this peptide may cause neurotoxicity through different direct or indirect mechanisms (3, 4).

In addition to the direct role of the {beta}-amyloid peptide, a number of studies in the past few years have focused on the possible role of inflammation and immune-mediated damage in the progression of AD and have investigated the implication of factors such as cytokines and C proteins (5, 6, 7). It has been shown by immunohistochemical techniques that C proteins of the classical pathway are present in the AD brain, where they often appear associated with senile plaques (5, 8, 9). Whereas originally no evidence was found for the occurrence of components of the alternative pathway, more recent studies suggest that some alternative pathway activation or amplification does occur (10, 11). Levi-Strauss and Mallat (12) were the first to show that astrocytes are able to synthesize C proteins, a finding that was later extended to microglia and, to some extent, neurons and oligodendrocytes, providing a body of evidence that brain cells express a full arsenal of C factors and inhibitors (see review by Gasque et al. (13)). These observations along with the finding that {beta}-amyloid was able to enhance production of the C protein C3 by microglia (14) demonstrated that C proteins could be produced in the CNS and thus contribute to responses to {beta}-amyloid.

Experimental support to the hypothesis that C activation may play a role in the pathogenesis of AD came from the work by Rogers et al. (15), who provided evidence that the {beta}-amyloid peptide activates the classical C pathway in vitro. Further studies based on C activation assays, C1q binding assays, and/or electron microscopy studies provided further evidence of the ability of {beta}-amyloid fibrils to activate the classical C pathway and to bind C1 through its C1q subcomponent (16, 17, 18, 19, 20).

Triggering of the classical C pathway results from binding of the C1 complex, via its C1q subunit, to immune and nonimmune activators and leads to activation of its associated proteases C1r and C1s, a two-step process involving C1r autoactivation, then C1r-mediated activation of C1s (reviewed in Refs. 21 and 22). C1q, the recognition unit of C1, is a hexameric protein comprising six subunits, each consisting of three homologous and yet distinct chains, A, B, and C, of ~220 residues (23). Each chain has an N-terminal collagen-like sequence giving rise to collagen-like triple helices, prolonged by C-terminal globular regions (GR) that belong to a superfamily of protein modules also found, e.g., in TNF or in adipocyte C-related protein-30 (reviewed in Ref. 23). It has long been known that C1q binds Ab-Ag immune aggregates via its C-terminal GRs, but there are conflicting reports as to whether binding to various nonimmune activators occurs through the GRs, the collagen-like fragments (CLF) of C1q, or both (21). In this respect Gewurz and collaborators (24, 25, 26) have provided evidence that C1q binding to serum amyloid P component, DNA, and C-reactive protein involves a site located at residues 14–26 of the collagen-like region of the C1q-A chain. Recent studies have provided support for the involvement of the above site within the C1q-A chain in the recognition of {beta}-amyloid fibrils by C1q (16).

Considering that previous studies showing activation of the classical C pathway by {beta}-amyloid fibrils were all based on the use of serum as a source of C, the present study was initiated to test the ability of these fibrils to trigger direct activation of the C1 complex in vitro. In addition, further investigation of the region(s) of C1q involved in {beta}-amyloid fibril binding was conducted. Our data demonstrate the ability of {beta}-amyloid fibrils to activate C1 under conditions close to those encountered in serum and provide strong evidence, not previously detected, that recognition of {beta}-amyloid fibrils by C1 can be mediated by the GRs of C1q.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Proteins and reagents

C1 inhibitor was purified from human plasma essentially as described previously (27), except that Con A-agarose (Sigma, St. Louis, MO) was used instead of Con A-Sepharose. Collagenase from Achromobacter iophagus was obtained from Roche (Indianapolis, IN). Porcine pepsin was purchased from Sigma. Con A-Sepharose was obtained from Pharmacia Biotech (Piscataway, NJ). The strain of C4-deficient guinea pigs used as a source of C4-deficient serum for C4 consumption assays was a gift from Prof. P. Lachmann (Cambridge, MA). Sheep erythrocytes and anti-sheep erythrocyte Abs were obtained from BioMerieux (Marcy l’Etoile, France).

C1 subcomponents and C1q-derived fragments

The C1q subunit of C1 was purified from human plasma as described previously (28, 29). Isolation of the proenzyme form of the C1s-C1r-C1r-C1s tetramer was performed as described previously (30), except that the C1r and C1s mixture released by EDTA was dialyzed against 50 mM triethanolamine hydrochloride, 145 mM NaCl, and 2.5 mM CaCl2 (pH 7.4), and further purification of the Ca2+-dependent C1s-C1r-C1r-C1s tetramer was achieved by high pressure gel permeation chromatography on a TSK-G3000 SW column (LKB, Rockville, MD) in the same buffer (31). The concentrations of purified C1q and C1s-C1r-C1r-C1s were determined spectrophotometrically using values of A (1%, 1 cm) at 280 nm of 6.8 and 13.5, and Mr values of 459,300 and 330,000, respectively (28, 32). The CLF of C1q were obtained by pepsin digestion using two methods, as indicated in the text. Method 1 was based on the procedure described by Reid (33), as modified by Siegel and Schumaker (34). Method 2 was essentially as described previously (35), except that contaminant uncleaved C1q molecules were removed by a further affinity chromatography step on Con A-Sepharose. The concentration of purified CLF was determined using values of A (1%, 1 cm) at 275 nm = 2.1, and Mr = 189,900 (36). The fragments corresponding to the GRs of C1q were generated by treatment of C1q with collagenase (C1q:collagenase ratio, 15:1, w/w) for 16 h at 37°C in 250 mM NaCl, 5 mM CaCl2, and 50 mM Tris-HCl (pH 7.4), and purification was achieved by high pressure gel filtration chromatography on a TSK-G2000 SW column (LKB). The purified GR were quantified by using values of A (1%, 1 cm) at 280 nm = 7.0, and Mr = 48,000 (37). The homogeneity of the purified proteins and fragments was assessed by SDS-PAGE under reducing and nonreducing conditions.

{beta}-Amyloid and C1q-derived peptides

Human {beta}-amyloid peptides {beta}1–42 (sequence: D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A), {beta}12–42, and the double mutant (D7N, E11Q) of {beta}1–42, as well as the rat {beta}1–42 peptide (sequence: D-A-E-F-G-H-D-S-G-F-E-V-R-H-Q-K-L-V-F-F-A-E-D-V-G-S-N-K-G-A-I-I-G-L-M-V-G-G-V-V-I-A) were synthesized using solid-phase F-moc methodology and purified as described previously (38). The N-terminal segment {beta}1–16 of the human {beta}-amyloid peptide was synthesized on an Applied Biosystems 430 A automated synthesizer (Foster City, CA) using the t-Boc methodology as described previously (36). The amino group of the N-terminal Asp residue was acetylated before deprotection and cleavage of the peptide chain, and purification was achieved by reversed-phase HPLC as described previously (36). The C1q-A chain peptide 14–26 (A-G-R-P-G-R-R-G-R-P-G-L-K) and the C1q-B chain peptide 14–25 (P-G-I-P-G-T-P-G-P-D-G-Q) were synthesized as described previously (16). The purity of all synthetic peptides was assessed by analytical reversed-phase HPLC and electrospray mass spectrometry analyses and was >95% on the basis of mass spectrometry measurements.

C1 activation assay

The various {beta}-amyloid peptides were solubilized in 50 mM Tris-HCl and 150 mM NaCl (pH 7.4) and kept at 4°C for at least 20 h before use to allow fibril formation. The C1 complex (0.25 µM), reconstituted from equimolar amounts of C1q and C1s-C1r-C1r-C1s, was incubated in 50 mM triethanolamine-HCl, 145 mM NaCl, and 1 mM CaCl2 (pH 7.4) in the presence of varying concentrations of the fibrillar {beta}-amyloid peptides for various periods at 21 or 37°C in the presence or the absence of 1 µM C1 inhibitor, as indicated in the text. The reaction mixtures were submitted to SDS-PAGE analysis (39) under reducing conditions using 10% acrylamide gels. The bands corresponding to C1s were revealed by Western blot analysis using a rabbit polyclonal Ab after electrotransfer to a nitrocellulose membrane as described previously (40). Membranes were scanned using a Shimadzu model CS 9000 gel scanner (Tokyo, Japan), and C1 activation was determined from the amounts of the A and B chains of activated C1s relative to that of the proenzyme. In a control experiment it was verified that Western blot analysis and direct protein staining with Coomassie blue yielded superimposable C1 activation kinetics, indicating that protein electrotransfer did not alter the measurements under the conditions used.

C1q labeling

C1q was labeled with 125I using Iodobeads (Pierce, Rockford, IL) as recommended by the manufacturer. Unbound 125I was removed by centrifugation on a Sephadex G-50 (fine) column (Pharmacia LKB) equilibrated in 145 mM NaCl and 50 mM triethanolamine-HCl (pH 7.4) as described by Penefsky (41). The specific radioactivity was ~200,000 cpm/µg protein.

Solid-phase C1q binding assay

The human {beta}1–42 peptide (1–5 µg) in 50 mM sodium carbonate (pH 9.6) was coated onto microtiter Immulon II plates (Dynatech, Chantilly, VA) and left overnight at 4°C. The plates were washed four times with 145 mM NaCl and 50 mM triethanolamine-HCl (pH 7.4), and nonspecific binding was blocked by incubation for 1 h at room temperature with 100 µl of 3% (w/v) BSA in the same buffer. Binding was performed by incubation for 1 h at room temperature with 50 µl of 125I-labeled C1q (0.2–5 µg) in the above buffer containing 1% BSA. Unbound material was removed by four washes with the same buffer containing 0.05% Tween 20, and bound C1q was solubilized by addition of 100 µl of 1 M NaOH and was measured by counting 125I radioactivity. Nonspecific binding of C1q was estimated by coating the plates with the carbonate buffer only, and the value was subtracted from that obtained with {beta}1–42-coated wells.

To determine the effect of the C1q-A chain 14–26 and B chain 14–25 peptides and that of the CLF and GR fragments of C1q, C1q was preincubated with these peptides or fragments at various concentrations, and then the mixtures were transferred to {beta}1–42-coated microtiter wells. Each binding experiment was performed in triplicate.

C4 consumption assay

Human serum was diluted 1/7 in dextrose gelatin veronal buffer containing 2.1 mM Ca2+ and 0.5 mM Mg2+ (DGVB) and incubated for 30 min at 30°C with an equal volume of peptide {beta}1–42 (50 µM) in the presence or the absence of varying concentrations of the C1q-A chain 14–26 and B chain 14–25 peptides or the C1q GR as indicated. Serial dilutions (1/10–1/500) of each sample were prepared in DGVB and assayed by incubating 0.2 ml of diluted sample together with 0.2 ml of C4-deficient guinea pig serum (diluted 1/50), 0.2 ml of Ab-sensitized sheep erythrocytes (5 x 107 cells/ml), and 0.6 ml of DGVB for 1 h at 37°C. After centrifugation, the extent of hemolysis was determined by measurement of the A412 of the supernatant. A C4 titer of 100 corresponds to the dilution of untreated normal human serum that yields 50% hemolysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The ability of {beta}-amyloid fibrils to activate the C1 complex was initially tested at low temperature (21°C) to keep spontaneous C1 activation (42) to a minimal level. Under these conditions, incubation of proenzyme C1 for 30 min in the presence of increasing concentrations of human or rat {beta}1–42 led to dose-dependent C1 activation, with a maximal value of about 45–50% at 150 µM of either peptide (Fig. 1Go). In contrast, the amino-terminal truncated segment of the human peptide {beta}12–42 yielded no significant C1 activation in the same range of concentrations.



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  		FIGURE 1. Dose-dependent activation of the C1 complex by {beta}-amyloid fibrils at low temperature. C1 (0.25 µM) was incubated in 50 mM triethanolamine-HCl, 145 mM NaCl, and 1 mM CaCl2 (pH 7.4) for 30 min at 21°C in the presence of increasing concentrations of fibrils formed from human {beta}1–42 (•), rat {beta}1–42 ({circ}), or human {beta}12–42 ({blacksquare}). C1 activation was monitored by SDS-PAGE and Western blot analyses as described in Materials and Methods.

 
{beta}-Amyloid fibrils activate C1 under conditions close to the physiological situation

Further C1 activation experiments were conducted at 37°C in the presence of 0.25 µM C1 and 1 µM C1 inhibitor, i.e., under conditions closer to the physiological situation, where normal concentrations of C1 and C1 inhibitor are ~0.2 and 1.4 µM, respectively (42). As expected, C1 alone activated spontaneously in the absence of C1 inhibitor, reaching nearly complete activation after 90 min, whereas the activation process was totally prevented in the presence of C1 inhibitor (Fig. 2Go). In contrast, incubation of C1 in the presence of both C1 inhibitor and human {beta}1–42 fibrils led to significant C1 activation, with about 55% activation in the presence of 150 µM {beta}1–42, indicating that {beta}-amyloid fibrils were able to release the control exerted by C1 inhibitor.



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  		FIGURE 2. Kinetic analysis of {beta}-amyloid fibril-mediated C1 activation in the presence of C1 inhibitor. C1 (0.25 µM) was incubated in 50 mM triethanolamine-HCl, 145 mM NaCl, and 1 mM CaCl2 (pH 7.4) for varying periods at 37°C alone ({square}), in the presence of 1 µM C1 inhibitor ({blacksquare}), or in the presence of both 1 µM C1 inhibitor and 150 µM human {beta}1–42 (•). C1 activation was monitored by SDS-PAGE and Western blot analyses as described in Materials and Methods.

 
To further check the relevance of the activating effect of the {beta}-amyloid fibrils, C1 activation experiments were conducted in the presence of increasing concentrations of C1 inhibitor. As shown in Fig. 3Go, spontaneous C1 activation measured in the absence of {beta}-amyloid fibrils was abolished at a C1 inhibitor/C1 molar ratio of 2. In contrast, in the presence of 150 µM {beta}1–42, C1 activation gradually decreased with increasing C1 inhibitor concentrations, but reached a plateau at 45–50% activation for a C1 inhibitor/C1 ratio of ~6. This activation value was comparable to that determined in the previous experiment (see Fig. 2Go) and was not decreased further at higher C1 inhibitor:C1 ratios, indicating that at relative concentrations of C1 and C1 inhibitor close to the physiological values the {beta}1–42 amyloid fibrils were able to circumvent the inhibitory effect of C1 inhibitor. Comparable results were obtained when the concentration of {beta}1–42 was decreased from 150 to 50 µM, although somewhat greater inhibition was achieved at this lower concentration of activator when the C1 inhibitor:C1 ratio exceeded that normally present in serum.



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  		FIGURE 3. Effect of C1 inhibitor on {beta}-amyloid fibril-mediated C1 activation. C1 (0.25 µM) was incubated in 50 mM triethanolamine-HCl, 145 mM NaCl, and 1 mM CaCl2 (pH 7.4) for 90 min at 37°C in the presence of increasing concentrations of C1 inhibitor in the absence of {beta}-amyloid fibrils ({square}) or in the presence of 150 µM human {beta}1–42 (•). C1 activation was monitored by SDS-PAGE and Western blot analyses as described in Materials and Methods.

 
C1-activating ability of {beta}-amyloid fibrils depends on acidic residues in the N-terminal region of peptide {beta}1–42

To define the region of the human {beta}1–42 peptide involved in C1 activation, amyloid fibrils were prepared from the double mutant (D7N, E11Q) of {beta}1–42 and from the amino-terminal truncated peptide {beta}12–42, and their ability to activate C1 in the presence of C1 inhibitor was compared with that of the wild-type peptide. As illustrated in Fig. 4Go, the human and rat {beta}1–42 peptides yielded comparable C1 activation kinetics, with about 50% activation after 90 min when both peptides were used at a concentration of 150 µM. In contrast, the double mutant (D7N, E11Q) only induced weak C1 activation at the same concentration, whereas the truncated peptide {beta}12–42 yielded no activation. In full agreement with previous data based on activation of the classical pathway of C (19), this provided direct evidence that the N-terminal segment 1–11 of peptide {beta}1–42, and particularly the acidic residues Asp7 and Glu11, play a crucial role in C1 activation and therefore in C1 binding by {beta}-amyloid fibrils.



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  		FIGURE 4. {beta}-Amyloid fibrils of the truncated peptide {beta}12–42 and the double mutant (D7N, EQ11) of human {beta}1–42 do not activate C1. C1 (0.25 µM) was incubated in the presence of 1 µM C1 inhibitor in 50 mM triethanolamine-HCl, 145 mM NaCl, and 1 mM CaCl2 (pH 7.4) for varying periods at 37°C in the absence of {beta}-amyloid fibrils ({square}) or in the presence of human {beta}1–42 (•), rat {beta}1–42 ({circ}), human {beta}12–42 ({blacksquare}), or the (D7N, EQ11) mutant of human {beta}1–42 ({blacktriangleup}), each at 150 µM. C1 activation was monitored by SDS-PAGE and Western blot analyses as described in Materials and Methods.

 
Evidence for a major {beta}-amyloid binding site in the globular regions of C1q

Additional experiments were aimed at identifying the region(s) of C1q responsible for C1 binding to {beta}-amyloid fibrils. For this purpose, a solid-phase assay was used, in which 125I-labeled C1q was allowed to bind to microtiter plates coated with fibrils of the human {beta}1–42 peptide. In preliminary experiments (data not shown), it was verified that coating the plates with increasing amounts (0–5 µg) of the {beta}1–42 peptide led to increased binding of radioactive C1q, with maximal binding at ~2.5 µg of {beta}1–42. In the same way, increasing the amount of C1q added to the wells led to a dose-dependent increase in bound radioactivity.

With a view to locate the {beta}-amyloid binding site(s) within C1q, GR and CLF were prepared by collagenase and pepsin digestion of the protein, respectively, and tested for their ability to compete with 125I-labeled C1q for binding to fibrils of the {beta}1–42 peptide. As expected, unlabeled C1q competed efficiently for binding, yielding nearly complete inhibition of the binding of radioactive C1q at a molar excess of 50/1 (Fig. 5GoA), indicating that 125I-labeled C1q shared the same binding ability as the unlabeled molecule. As shown in Fig. 5GoB, the GR of C1q also competed for binding, although less efficiently than intact C1q, with nearly complete inhibition of 125I-labeled C1q binding at GR:C1q ratios above 200. Comparative fitting of these data indicated that the ability of the GR to inhibit binding of 125I-labeled C1q was about 24- to 26-fold lower than that of intact C1q. The same type of competition experiment was performed with the C1q CLF obtained by two different methods (see Materials and Methods). As illustrated in Fig. 5GoC, increasing the CLF:C1q ratio to values up to 100:1 yielded no significant inhibition of radioactive C1q binding whichever CLF preparation was used, indicating that this fragment of C1q did not bind specifically to {beta}-amyloid fibrils under the conditions of the solid-phase binding assay used in this study.



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  		FIGURE 5. Binding of 125I-labeled C1q to {beta}1–42 amyloid fibrils: competition by intact C1q, the C1q GR and CLF, and peptides C1q-A 14–26 and C1q-B 14–25. 125I-labeled C1q (1 µg) was allowed to bind to microtiter wells coated with {beta}1–42 fibrils (2.5 µg/well) in the presence of increasing amounts of unlabeled C1q (A), the C1q GR (B), the C1q CLF (C), or the C1q-A 14–26 and C1q-B 14–25 (D) peptides. The filled bars and open bars used in C correspond to two different CLF preparations (see Materials and Methods). Error bars represent the SD of triplicate experiments.

 
The above data were unexpected, since previous studies had provided support for the occurrence of a {beta}1–42 binding site in the collagen-like region of C1q, and a peptide overlapping residues 14–26 of the C1q-A chain had been shown to inhibit A{beta}-dependent C activation in serum (16). This prompted us to test the ability of the C1q-A chain 14–26 peptide to inhibit binding of 125I-labeled C1q to {beta}1–42 amyloid fibrils. As illustrated in Fig. 5GoD, the synthetic peptide C1q-A 14–26 showed no ability to inhibit C1q binding at concentrations up to 250 µM. Indeed, increasing the concentration of the C1q-A peptide slightly increased C1q binding, an effect that was probably nonspecific, as it was also observed with the corresponding peptide 14–25 from the C1q-B chain (Fig. 5GoD). Thus, in contrast with what was previously observed in studies based on C4 consumption and C4d deposition assays (16, 18), the C1q-A chain 14–26 peptide did not compete with C1q for binding to {beta}-amyloid fibrils, providing no evidence for a significant involvement of the N-terminal region of C1q in the binding process under the conditions used in the present study.

We next tested the effect of the C1q-A chain 14–26 peptide on {beta}-amyloid fibril-mediated C1 activation. As shown in Fig. 6GoA, C1 incubated for 90 min at 37°C in the presence of both C1 inhibitor and fibrils of the {beta}1–42 peptide (100 µM) underwent ~50–55% activation, and this value remained unchanged when incubation was performed in the presence of increasing concentrations of the C1q-A chain peptide up to 250 µM. Similarly, the control peptide C1q-B 14–25 had no effect on {beta}-amyloid fibril-mediated C1 activation. None of the C1q-A and -B chain peptides significantly influenced spontaneous C1 activation measured in the absence of C1 inhibitor (Fig. 6GoA). Additional experiments were conducted at a lower concentration of {beta}1–42 (25 µM), and again the C1q-A and -B chain peptides had no significant effect on {beta}-amyloid fibril-mediated C1 activation under these conditions (data not shown).



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  		FIGURE 6. Effects of the C1q-A chain 14–26 peptide and C1q GR on {beta}-amyloid fibril-mediated C1 activation. A, C1 (0.25 µM) was incubated for 90 min at 37°C in 50 mM triethanolamine-HCl, 145 mM NaCl, and 1 mM CaCl2 (pH 7.4) together with 1 µM C1 inhibitor and {beta}1–42 fibrils (100 µM) in the presence of increasing concentrations of peptides C1q-A 14–26 ({square}) and C1q-B 14–25 ({circ}). In both cases the fibrils were preincubated for 20 min at 0°C with the peptides before addition of the other reagents. In a control experiment, C1 alone was incubated under the same conditions in the presence of increasing concentrations of peptides C1q-A 14–26 ({blacksquare}) and C1q-B 14–25 (•). B, C1 (0.25 µM) was incubated for 90 min at 37°C in 50 mM triethanolamine-HCl, 145 mM NaCl, and 1 mM CaCl2 (pH 7.4) together with 1 µM C1 inhibitor and {beta}1–42 fibrils (150 µM) in the presence of increasing concentrations of C1q GR.

 
We also tested the effect of C1q GR on C1 activation by {beta}-amyloid fibrils. As illustrated in Fig. 6GoB, increasing the GR:C1 ratio resulted in a gradual decrease in {beta}-amyloid fibril-mediated C1 activation from 57% in the absence of GR to the minimal level of about 10% at a GR:C1 molar ratio of 600:1. Thus, consistent with their ability to compete for C1q binding to {beta}-amyloid fibrils (see Fig. 5GoB), GR also inhibited the resulting C1 activation. The fact that the latter effect required significantly higher GR:C1 ratios is possibly accounted for by basic differences between the binding and activation assays. Thus, the solid phase binding assay is expected to give an estimate of the fraction of the C1q population that remains bound to the amyloid fibrils at a given GR:C1q ratio, thereby reflecting the state of the equilibrium between bound and free C1q molecules under these conditions. The activation assay, in contrast, is performed in the fluid phase and measures conversion of proenzyme C1 into its active form over an incubation period of 90 min. During this period, the binding equilibrium is expected to allow a continuous turnover of C1 molecules at the surface of the fibrils, with a substitution of proenzyme molecules for active ones. Such a process should result in a C1 activation level higher than that expected from the fraction of C1 molecules actually bound at a given GR:C1q ratio. This hypothesis appears consistent with the observation that complete inhibition of C1 activation requires higher amounts of GR than does complete inhibition of C1q binding, i.e., concentrations that prevent any contact, even transient, between C1q and the amyloid fibrils.

A further series of experiments was based on the use of a C4 consumption assay. In keeping with previous data (16), incubation of normal human serum for 30 min at 30°C in the presence of increasing concentrations of peptide {beta}1–42 induced progressive depletion of C4 hemolytic activity, with ~80% C4 consumption at a peptide concentration of 50 µM (data not shown). We next tested the ability of peptide C1q-A 14–26 to interfere with {beta}-amyloid fibril-induced C4 consumption. As shown in Fig. 7GoA, incubation of serum with peptide {beta}1–42 alone (25 µM) decreased the C4 titer from 100 to 25. Increasing the C1q-A peptide concentration up to 250 µg/ml led to a progressive increase in the C4 titer. However, the C4 titer also increased in comparable proportions as a function of C1q-A 14–26 concentration when serum was initially incubated in the absence of peptide {beta}1–42. Similar effects were observed when the C1q-B peptide 14–25 was used instead of C1q-A peptide 14–26 (Fig. 7GoB). It was concluded therefore that the observed increase in C4 titer induced by the C1q-A and C1q-B peptides did not result from an inhibition of the initial {beta}-amyloid fibril-induced C4 consumption (and therefore did not result from an inhibition of C1 activation), but was due to a facilitation of the subsequent hemolytic assay used to measure residual C4. The same protocol was used to test the effect of the C1q GR on {beta}-amyloid fibrils-induced C4 consumption. As shown in Fig. 7GoC, increasing the GR concentration relative to the theoretical C1 concentration in serum led to a progressive restoration of the C4 titer. In contrast, when serum was initially incubated in the absence of peptide {beta}1–42, increasing the GR:C1 ratio slightly decreased the C4 titer, presumably through slight inhibition of the subsequent hemolytic assay. These data clearly demonstrated the ability of the C1q GR to inhibit {beta}-amyloid fibril-induced C4 consumption in a range of GR:C1 ratios comparable to that required to inhibit C1 activation (see Fig. 6GoB).



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  		FIGURE 7. {beta}-Amyloid fibril-induced C4 consumption: effect of the C1q-A chain 14–26 peptide, the C1q-B chain 14–25 peptide, and the C1q GR. Human serum was incubated with either {beta}1–42 fibrils (25 µM; {blacksquare}) or buffer ({square}) for 30 min at 30°C in the presence of increasing concentrations of peptide C1q-A 14–26 (A), peptide C1q-B 14–25 (B), or C1q GR (C) as indicated. The residual C4 titer of each sample was then measured by a hemolytic assay as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study provides the first direct experimental evidence of the ability of {beta}-amyloid fibrils to trigger activation of the C1 complex of C and shows that {beta}-amyloid fibril-induced C1 activation takes place both in the absence of C1 inhibitor and at C1 inhibitor:C1 ratios up to 8:0, i.e., under conditions close to the physiological situation in plasma. In keeping with previous studies based on the use of serum as a source of C (15, 16, 19), these data provide strong support to the biological relevance of the activation of the classical pathway of C by {beta}-amyloid fibrils.

It is noteworthy that the {beta}-amyloid fibril-mediated C1 activation did not proceed to completion under the experimental conditions used, even in the presence of high amounts of fibrils and/or upon prolonged incubation. Therefore, in terms of C1 activation, {beta}-amyloid fibrils appear significantly less efficient than immune complexes, but exhibit efficiency similar to that of other known nonimmune C1 activators (21, 42, 43). However, contrary to several nonimmune C1 activators, such as DNA and heparin (42), {beta}-amyloid fibrils clearly have the ability to overcome the negative control exerted by C1 inhibitor and hence may be considered as medium-strong C1 activators.

To test whether this inability to activate C1 to completion could be explained by aggregation of the fibrils during the activation process, {beta}-amyloid fibrils were prepared as described in the present study as well as by two other methods previously shown to yield a high percentage of isolated fibrils (20, 44). All three preparations were allowed to activate C1 in the presence of C1 inhibitor under conditions similar to those used in Fig. 2Go, and they yielded comparable activation kinetics, with, again, maximalactivation rates of ~40–45% (data not shown). Each fibril preparation was also examined by electron microscopy under the salt conditions used for the C1 activation assay (i.e., 145 mM NaCl), and in each case fibrils were found to associate into large aggregates within a few minutes. It is likely therefore that fibril aggregation occurred rapidly during the C1 activation assays, and it may be hypothesized that aggregate formation decreased the accessibility of potential C1 binding sites, and thereby limited the C1 binding efficiency of the {beta}-amyloid fibrils. Nevertheless, although our data suggest that isolated {beta}-amyloid fibrils may be more efficient in terms of C1 binding and activation than large aggregates, they also provide evidence that the latter, which are probably the major component of neuritic plaques (45), activate C1 to a significant extent. Taken together, the above observations appear consistent with a role of C activation in AD, a slow degenerative disease.

In keeping with previous studies (19, 20), our data based on the use of truncated and mutant forms of {beta}1–42 provide further evidence that the C1 binding site in {beta}-amyloid fibrils is located in the N-terminal 1–11 region of the {beta}1–42 peptide and specifically involves acidic residues Asp7 and/or Glu11. Based on previous analyses by electron microscopy and circular dichroism (46), the fact that peptide {beta}12–42 is devoid of C1-activating ability is not due to an inability to form {beta}-amyloid fibrils. In addition, previous studies have provided strong evidence for a crucial role of residue Asp7 of the {beta}-amyloid peptide in activation of the classical pathway of C (19). On the other hand, it is noteworthy that the N-terminal segment 1–16 of {beta}1–42 (which is not expected to form amyloid fibrils due to the lack of the C-terminal region (47, 48)) had no effect on C1 activation by fibrillar {beta}1–42 (data not shown). This is in agreement with the observation that the ability of peptide {beta}1–40 to activate C is correlated with fibril formation (16, 49). A likely hypothesis is therefore that formation of the fibrillar structure, through the C-terminal moiety of {beta}1–42, leaves the N-terminal region 1–11 of the peptide exposed to the solvent and allows this region to adopt the appropriate conformation and/or to provide the appropriate charge pattern for efficient recognition by C1q, as demonstrated previously by cross-linking experiments (50).

Our binding data provide strong evidence that under the conditions of the solid phase binding assay used in this study, C1q interaction with fibrils of the {beta}1–42 peptide takes place primarily through the C-terminal GR of the protein. This conclusion is based on the following observations. 1) Isolated GRs obtained by collagenase digestion compete efficiently with intact C1q for binding to {beta}1–42 fibrils, with an efficiency that is only about 25 times less than that of intact C1q. If one takes into account that C1q has six GRs and is therefore hexavalent, whereas the GRs are monomeric, it can be deduced that isolated GRs retain a surprisingly high affinity for {beta}1–42 fibrils. In addition, it should be emphasized that direct evidence of the ability of the isolated GRs to bind to {beta}1–42 fibrils, showing dose-dependent binding of the GRs to the fibrils, was obtained using microtiter plates coated with fibrils and measurement of GR binding by ELISA (data not shown). 2) There is no significant competition for binding to {beta}1–42 fibrils by the remainder part of C1q, the N-terminal CLF, even at CLF:C1q ratios up to 100:1, a value that is quite high, considering that the CLF moiety of C1q retains the hexameric structure of C1q and is therefore potentially also multivalent in terms of binding. 3) We found no significant effect of the C1q-A 14–26 peptide on the C1q/{beta}1–42 interaction at peptide concentrations up to 250 µg/ml.

Consistent with the above binding studies, at concentrations up to 250 µg/ml the C1q-A 14–26 peptide did not inhibit C1 activation by {beta}1–42 fibrils. In contrast, the isolated GRs of C1q were shown to exert a significant inhibitory effect on {beta}1–42 fibril-mediated C1 activation, even though this required a relative amount of GR about 3-fold that needed to effectively block stable C1q binding. As discussed in Results, this finding probably arises from differences between the binding and activation assays.

Our experiments based on the use of a C4 consumption assay are also consistent with the hypothesis that recognition of {beta}-amyloid fibrils by C1q is mediated by its C-terminal GRs. 1) As observed previously (16), peptide C1q-A 14–26 was found to inhibit {beta}-amyloid fibril-induced C4 consumption assays. However, in our hands, similar inhibitory effects were observed whether serum was incubated in the presence or the absence of peptide {beta}1–42. In addition, the control C1q-B 14–25 peptide had a comparable effect. We conclude from these observations that the apparent inhibition exerted by peptide C1q-A 14–26 peptide on {beta}-amyloid fibril-induced C4 consumption is mainly due to nonspecific effects on the hemolytic assay and does not arise from inhibition of the initial C1 activation step. 2) The data obtained with the C1q GRs show unambiguously that these inhibit {beta}-amyloid fibril-induced C4 consumption in a range of GR/C1 ratios comparable to that required to inhibit C1 activation.

With respect to the region of C1q responsible for binding to {beta}1–42 fibrils, it is clear that our data differ from previous results that provided support for the involvement of residues 14–26 from the N-terminal collagen-like region of the C1q-A chain (16, 18). Several methodological differences may account for the observed discrepancies. First, in contrast with the binding assay used in our study, in the assay used by Jiang et al. (16) C1q or its CLF part was coated first on microtiter plates, and then fibrillar {beta}1–42 was allowed to interact with the immobilized protein. In view of the particular structure of C1q and the known binding ability of its peripheral GRs, C1q coating may be expected to take place mainly through these regions, which, once C1q is coated, would therefore exhibit a decreased accessibility to secondary ligands. If this hypothesis is correct, this may favor binding of the N-terminal acidic segment of {beta}1–42 to low-affinity binding sites in C1q, such as the polycationic sequence A-G-R-P-G-R-R-G-R-P-G-L-K corresponding to segment 14–26 of the C1q-A chain. It should be kept in mind that this 13-residue segment contains as many as five basic residues that may bind to the acidic portion of {beta}1–42 through ionic interactions. In this respect it should be emphasized that Chen et al. (18) found that tetra-lysine inhibits C activation by {beta}1–42 at concentrations and to an extent comparable to those observed with C1q-A peptide 14–26 (16), which casts further doubt on the specificity of the inhibitory effect yielded by the latter peptide. Indeed, a body of evidence indicates that C1q is a charge pattern recognition protein with the ability to bind acidic residues in several ligands, including IgG (51, 52), C-reactive protein in complex with phosphocholine (53), and a variety of polyanionic molecules, including heparin and DNA (21, 54). Although there are conflicting reports on this matter (24, 25, 26), there is convincing evidence that recognition of these ligands by C1q is mediated in many instances by its C-terminal GRs.

In summary, in keeping with previous findings our study brings strong evidence that amyloid fibrils of the {beta}1–42 peptide trigger direct activation of the C1 complex of C under conditions close to the physiological situation. The data obtained here using C1q binding assays, C1 activation assays, and C4 consumption assays are all consistent with the hypothesis that C1q recognizes {beta}1–42 fibrils through its C-terminal GRs and provide no evidence for a significant involvement of the collagen-like region of the molecule. At variance with previous findings (16), we propose that C1q binds to {beta}-amyloid fibrils through multivalent interaction with recognition sites located in its peripheral GRs.


   Acknowledgments
 
We are grateful to Dr. Andrea Tenner for the gift of human and rat {beta}-amyloid peptides, C1q-derived peptides, and C1q collagen-like fragments.


   Footnotes
 
1 This work was supported in part by the Centre National de la Recherche Scientifique and the Commissariat à l’Energie Atomique. Back

2 Address correspondence and reprint requests to Dr. Gérard J. Arlaud, Institut de Biologie Structurale Jean-Pierre Ebel, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France. E-mail address: arlaud{at}ibs.fr Back

3 Abbreviations used in this paper: AD, Alzheimer’s disease; CLF, collagen-like fragments of C1q; DGVB, dextrose gelatin veronal buffer; GR, globular regions of C1q;. Back

Received for publication January 17, 2001. Accepted for publication September 19, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
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