Báo cáo khoa học: Interaction of the C1 complex of Complement with sulfated polysaccharide and DNA probed by single molecule fluorescence microscopy pdf

Interaction of the C1 complex of Complement with sulfatedpolysaccharide and DNA probed by single molecule fluorescence microscopy Be´range`re Tissot1, Re´gis Daniel1and Christophe Place2

Interaction of the C1 complex of Complement with sulfated

polysaccharide and DNA probed by single molecule fluorescence microscopy

Be´range`re Tissot1, Re´gis Daniel1and Christophe Place2

1

Laboratoire Analyse et Environnement, Universite´ d’Evry, France;2Laboratoire de Physique, Ecole Normale Supe´rieure de Lyon, France

The complex C1 triggers the activation of the Complement

classical pathway through the recognition and binding of

antigen–antibody complex by its subunit C1q The globular

region of C1q is responsible for C1 binding to the immune

complex C1q can also bind nonimmune molecules such as

DNAand sulfated polysaccharides, leading either to the

activation or inhibition of Complement The binding site of

these nonimmune ligands is debated in the literature, and it

has been proposed to be located either in the globular region

or in the collagen-like region of C1q, or in both Using single

molecule fluorescence microscopy and DNAmolecular

combing as reporters of interactions, we have probed the

C1q binding properties of T4 DNAand of fucoidan, an algal

sulfated fucose-based polysaccharide endowed with potent

anticomplementary activity We have been able to visualize

the binding of C1q as well as of C1 and of the isolated

collagen-like region to individual DNAstrands, indicating that the collagen-like region is the main binding site of DNA From binding assays with C1r, one of the protease compo-nents of C1, we concluded that the DNAbinding site on the collagen-like region is located within the stalk part Com-petition experiments between fucoidan and DNAfor the binding of C1q showed that fucoidan binds also to the col-lagen-like region part of C1q Unlike DNA, the binding of fucoidan to collagen-like region involves interactions with the hinge region that accommodate the catalytic tetramer C1r2–C1s2of C1 This binding property of fucoidan to C1q provides a mechanistic basis for the anticomplementary activity of the sulfated polysaccharide

Keywords: fucoidan; C1q; complement system; single mole-cule fluorescence microscopy

Studies on the interactions between carbohydrates and

proteins represent a major and challenging topic in

glyco-biology, as it is now recognized that many crucial life

processes are dependent on their specific molecular

recog-nition Carbohydrate–protein interactions mediate

funda-mental biological mechanisms, encompassing growth

control, apoptosis, cell differentiation and proliferation, as

well as physiopathologic disorders like tumoral metastasis,

autoimmune diseases and inflammation [1,2] However the

mechanisms of these interactions involving carbohydrates

are still poorly understood, particularly with regard to the

molecular basis of the strength and specificity of binding to

targeted proteins [3] Difficulties mainly arise from the high

structural diversity and from the complex dynamic

proper-ties of polysaccharides [4] Anew approach based on

single-molecule detection is currently arousing great interest in

biology as it allows the direct observation and manipulation

of biomolecules [5,6] This approach has already been successfully applied to the study of the interaction of DNA and proteins [7,8] Comparatively few data have been reported on the study of carbohydrates and their interaction

by this approach [9,10], probably because of the difficulties

in manipulation of such structurally heterogeneous and polydisperse biopolymers at the single-molecule level Most

of the studies provide topographic images by atomic force microscopy of polysaccharide molecules adsorbed on a surface [6,11] Nevertheless, we think that new insights into the carbohydrate–protein interactions should be obtained

by studying them at a single-molecule level in terms of the protein partner

We have applied this strategy to the study of the bioactive polysaccharide fucoidan, one of the most potent inhibitors

of the human Complement system Fucoidan is a sulfated polysaccharide extracted from brown algae and present-ing a structural organization based on an

[fi4)-a-L-Fucp-(1fi3)-a-L-Fucp-(1fi4)-a-L-Fucp-(1fi3)-a-L-Fucp (1fi] backbone [12,13] It is assumed that its biological properties are related to its capacity to achieve specific interactions with targeted proteins We have recently shown that fucoidan inhibits the first steps of activation of the Complement cascade [14] In addition, affinity electrophor-esis experiments indicated interaction between fucoidan and the Complement protein C1q, and this interaction could result in the observed inhibition [14] The protein C1q, a subunit of the C1 complex, is involved in the recognition

Correspondence to R Daniel, Laboratoire Analyse et Environnement,

UMR 8587 CNRS, Universite´ d’Evry-Val-d’Essonne, Bd Franc¸ois

Mitterrand, 91025 Evry cedex, France.

Fax: + 33 1 69477655, Tel.: + 33 1 69477641,

E-mail: regis.daniel@chimie.univ-evry.fr

Abbreviations: CLR, collagen-like region; EDC,

1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride; GR,

globular region; PMMA, polymethylmethacrylate; sulfo-NHS,

sulfo-N-hydroxysuccinimide.

(Received 23 July 2003, revised 22 September 2003,

accepted 6 October 2003)

and the binding of antigen–antibody complexes that triggers

the activation of the Complement [15] C1q (460 kDa) made

of three polypeptide chains (A, B and C) exhibits unique

structural features (Fig 1) It consists of a C-terminus

presenting six globular head groups connected through a

hinge region to a long (approximately 11 nm) triple helical

collagen-like stalk that ends at the N-terminus [16] Because

of this structural organization, C1q is often pictured as a

bunch of six flowers The interaction of C1q with immune

complexes takes place at the globular region [17,18], but

C1q is known to also bind through its collagen-like region

(CLR) several nonimmune molecules [19], with

conse-quences which remain unclear Actually the binding of the

C-reactive protein [20], of the serum amyloid protein [21]

and of DNA[22] to C1q leads to an activation of

Complement On the other hand, the binding of sulfated

glycosaminoglycans [23–25], of proteoglycan dermatan

sulfate decorin [26], and of chondroitin 4-sulfate (i.e the C1q inhibitor) results in the inhibition of the classical pathway [27]

Our goal in this study is to ascertain the binding of fucoidan to C1q and to determine the site of interaction on the protein For this purpose we took advantage of the binding properties of C1q toward DNAand of an emerging technique allowing the molecular combing of DNAstrands and its observation by single-molecule spectroscopy Dou-ble- and single-stranded DNAhas been demonstrated to bind preferentially to the collagen-like region of C1q under physiological saline conditions [22,28,29] Acationic peptide sequence on the Achain of C1q has been identified as the major binding site of DNA[30] We have analyzed herein the binding of the human of the C1 complex and of its subunit C1q to T4 DNAby molecular combing which results in a large array of DNAstrands individually observed by fluorescence microscopy [8] This approach allowed us to implement an analytical tool useful to investigate the binding of fucoidan to C1q through compe-tition experiments between DNAand the polysaccharide

We have addressed the question of whether a C1q inhibitor (fucoidan) and a C1q activator (DNA) are able to bind to the same region of the protein by using not only native C1q but also the C1q isolated domains CLR and the globular region (GR)

Materials and methods

Buffers The following buffers were used: 250 mMBis/Tris, pH 6.47; 0.1MMes buffer, pH 6, containing 0.5MNaCl; and 1M sodium hydrogen carbonate (NaHCO3) buffer, pH 8.4 All buffers were prepared with ultrapure water (milliQ, Milli-pore)

Reagents and proteins The Complement proteins C1r, C1q and C1 as well as the depleted sera and the specific antibodies were obtained from VWR (Fontenay-sous-Bois, France) C1q designed as purified C1q in this study, and the derived collagen-like region CLR and globular heads region GR were a generous gift from G.J Arlaud (IBS, Grenoble, France) The CLR and GR were prepared as previously described [28] Double-stranded DNAfrom salmon testes (type III), T4 DNAand 2-mercaptoethanol were purchased from Sigma (Saint-Quentin Fallavier, France) 1-Ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride (EDC) and sulfo-NHS (sulfo-N-hydroxysuccinimide) were purchased from Pierce (Rockford, IL, USA) The fluorescent intercalating agent YOYO-1 (dimer yellow oxozalone) and the Alexa 488-fluorescent beads (27 nm) were obtained from Molecular Probes (Eugene, OR, USA) Fucoidan from the brown algae Ascophyllum nodosum was prepared as previously described [31] The fucoidan fraction used for the study herein was of low molecular weight (8000 gÆmol)1 as determined by high performance size exclusion chromato-graphy using heparin standards [32]) and of high sulfate content (30% w/w), and was endowed with a high anti-complementary activity as we have previously reported [14]

Fig 1 Schematic representation of the structural organization of human

C1 (A) Model of the C1 complex showing the catalytic tetramer C1r 2 –

C1s 2 interacting with C1q (adapted from [37]) (B) Representation of

the association between the A, B and C chains constituting the six ABC

heterotrimeric triple helices of C1q The cationic sequence 14–26 of the

Achain, as well as the sequence responsible for the kink of the

colla-gen-like region are shown.

Surface treatment

Glass surfaces were rendered hydrophobic by coating with

polymethylmethacrylate (PMMA, Mr 8000 gÆmol)1) A

droplet of PMMAin chlorobenzene (13% w/w) is put

down onto a clean glass cover-slide and spread with a spin

coater of in-house design, at 2500 r.p.m for 3 min Surfaces

are then baked at 165C for 40 min and stored at room

temperature in a dust-free environment

DNA preparation

T4 DNA(160 kb) was labeled as follows: 7.2 lL of 1.35 nM

T4 DNAwere incubated with 10 lMYOYO-1 (Molecular

Probes) in ultrapure water This respresents a ratio of 1

molecule of dye to 30 bases of DNA, and 150 lL Bis/Tris

pH 6.47, completed with 1.5 mL of ultrapure water during

at least 1 h at room temperature

Fluorescent beads preparation

Alexa 488-conjugated beads (Molecular Probes) were

activated following the manufacturers’ instructions Briefly,

50 lL of beads (3.25 lM in ultrapure water) were mixed

with 50 lL of sulfo-NHS (0.5M in ultrapure water) and

10 lL of EDC (0.2Min ultrapure water) in 100 lL of 0.1M

Mes buffer and completed with 400 lL of ultrapure water

After 60 min incubation at room temperature under gentle

agitation, reaction was stopped by addition of 3 lL of

2-mercaptoethanol Elimination of the excess of reactants

was performed on P6 Biospin Columns (Bio-Rad) The

concentration of used beads solutions in 0.1MMes buffer

ranged from 20 to 30 nM, as evaluated by

spectrophoto-metric determination at 505 nm

Protein labeling

Proteins C1q, C1r and the CLR fragments of C1q were

labeled with fluorescent beads The concentration of beads

was adjusted according to the type and the concentration of

protein in order to maximize the rate of the labeling reaction

without generating cross-linking of the beads Twenty

microliters of commercial C1q (2.46 lM) were added to

0.5 lL of beads solution (25 nM) and to 2.5 lL NaHCO3

buffer completed with 25 lL ultrapure water After 45 min

incubation at 20C under gentle agitation, reaction was

stopped by addition of 0.6 lL NH2OH (3M in ultrapure

water) Purified C1q (2.8 lM; 17.5 lL) were added to 0.5 lL

of beads solution (25 nM) and to 25 lL NaHCO3 buffer

completed with 100 lL ultrapure water After 45 min at

20C under gentle agitation, reaction was stopped by

addition of 2.4 lL NH2OH (3M in ultrapure water) Six

microliters of CLR (7.8 lM) were added to 0.5 lL of beads

solution (25 nM) and to 12.5 lL of NaHCO3 buffer

completed with 100 lL ultrapure water After 45 min at

20C under gentle agitation, reaction was stopped by

addition of 2.4 lL of NH2OH (3M in ultrapure water)

Twenty microlitres of C1 (0.27 lM) were added to 0.5 lL

of beads solution (20 nM) and to 2.5 lL NaHCO3 buffer

completed with 25 lL ultrapure water After 45 min at

20C under gentle agitation, reaction was stopped with

1 lL of NHOH (3Min ultrapure water) Two microliters

of C1r (10.5 lM) were added to 1 lL of bead solution (25 nM) and to 4 lL NaHCO3buffer completed with 40 lL ultrapure water After 45 min at 20C under gentle agitation, reaction was stopped by addition of 2 lL

NH2OH (3Min ultrapure water)

Fluid phase incubations of DNA with complement proteins

Fluorescent T4 DNAwas incubated with the labeled proteins, either in the presence of fucoidan or not, for 45–60 min at room temperature under gentle agitation in the following conditions: 150 lL of 6.5 pMT4 DNAwere mixed with 1 lL of labeled C1q prepared as above, and according to case with 5 lL of 45 lM fucoidan; when the purified C1q was used, 400 lL of 6.5 pM T4 DNAwere mixed with 10 lL of labeled purified C1q One hundred and fifty microliters of 6.5 pMT4 DNAwere mixed with 5 lL of labeled CLR or 2.5 lL of labeled C1, and according to case with 1–2 lL of 45 lMfucoidan Then, the treated DNAwas combed as described below For the study of the interaction between C1r and C1q bound to combed T4 DNA, 150 lL

of 6.5 pMT4 DNAwere preincubated with 1 lL of 2 lM unlabeled C1q for 45 min, then 6 lL of labeled C1r were added, and according to case with 9 lL of 45 lMfucoidan Molecular combing

The combing process consists in the stretching of the DNA

by the passage of an air/water meniscus [33,34] Adroplet of 6.5 pMT4 DNAin Bis/Tris buffer, pH 6.47, is deposed on

a hydrophobic surface, incubated for 2 min and then removed This procedure is sufficient to stretch the DNA strands when DNAis brought in small droplet The interaction between DNAand the hydrophobic surface is very strong, so that DNAcan be considered as grafted onto the surface

Fluorescent microscopy Samples were observed using an inverted microscope (Leica

DM IRBE) by epifluorescence An X100 infinity-corrected 1.4 NAoil objectives (Leica) was used and a cooled CCD camera (C4880 Hamamatsu, and Ixon-Andor) was moun-ted on the microscope For fluorescence observations, a mercury lamp was used in combination with a filters set for fluorescein (Leica I3) The images were acquired using the HIPICsoftware (Hamamatsu) and IXONsoftware (Andor), with an exposition time ranging from 100 ms to 1 s

Results

Interaction between DNA and C1q DNAis described as an activator of human complement system [30] The DNA-dependent activation of Comple-ment may result from the formation of a complex between DNAand the first Complement protein C1q as proposed in the literature [20,22,28] The possibility to visualize individ-ual DNAmolecules prompted us first to investigate the binding properties between C1q and DNAthrough the observation of their complex

T4 DNA(160 kbps) labeled with the fluorescent

inter-calating agent YOYO-1 was combed on a PMMAsurface

Fluorescent strands were observed corresponding to

indi-vidual linear DNAchains stretched on the surface as

previously described (Fig 2A) [8] Fluorescent T4 DNA

was incubated in solution with labeled C1q For that

purpose, C1q was covalently attached through its amino

groups to fluorescent beads (27 nm diameter) C1q is

estimated to have an overall size of 35 nm based on

literature data [15] Given their similar sizes, we can

reasonably expect that one or two molecules of C1q are

bound per fluorescent bead After incubation with such

labeled C1q (C1q*), fluorescent T4 DNAwas combed as

described above The fluorescent individual DNAmolecules

then observed were clearly decorated with a succession of

fluorescent spots (Fig 2B) The fluorescence of these spots

is easily distinguishable from the YOYO-1-induced

fluores-cence of the combed DNA, by its color and by its strong

intensity corresponding to the high density of the Alexa

fluor contained in the beads These fluorescent spots were

then due to the presence of the beads along the DNA

strands When the beads were noncoupled to C1q, no such

binding was observed on DNAstrands (data not shown)

These results indicate that C1q mediates the attachment of

the spheres to DNA, hence evidencing interaction between

C1q and DNA

In order to identify the preferential DNAbinding region

on C1q, we have carried out experiments with the separated

domains of C1q, i.e the collagen-like region CLR and the

globular region GR CLR and GR were prepared by

enzymatic digestion by pepsin and collagenase, respectively,

as previously described [28] The C-terminal domain of the

CLR, named the hinge region and joining the CLR to the

GR, is resistant to both proteases so that the

conforma-tional organization of CLR is conserved, whereas the GR

was obtained as individual globular heads [35] In a first set

of experiments, CLR and GR were studied for their

capacity to compete with purified C1q* for the binding to

DNA When incubation of T4 DNA and C1q* was

performed with various amounts of CLR, the analysis of

the resulting combed DNAshowed that the binding of

C1q* to DNAstrands started to decrease for a C1q/CLR

ratio of 1 : 10 and was totally suppressed from a 1 : 50 molar ratio On the other hand, a higher amount of GR was required to observe a similar inhibition as a C1q*/GR molar ratio of 1 : 3000 was at least necessary (data not shown) Such a large difference in the amount required to efficiently compete for the binding of C1q* indicates that the collagen-like region of C1q contains the preferential site for the interaction and the binding to DNA

In order to confirm this result, we checked for the binding

of the CLR to DNAusing CLR preparation labeled with the fluorescein-conjugated beads (CLR*) CLR* was incu-bated with T4 DNAin fluid phase, after which the DNA was combed on a PMMAsurface The analysis of the images showed that CLR* was colocalized like C1q* with the combed DNA(Fig 2C), proving the binding of CLR

to DNA

The C1 complex, which triggers the classical pathway of Complement, comprises the subunit C1q and the two serine proteases C1r and C1s associated into a C1r2–C1s2 tetramer Several lines of evidence in literature indicate that the binding site of the tetramer on C1q is located within the collagen-like region of C1q [36] As our results show that this region also contains the binding site of DNA, we wondered whether the association of the tetramer C1r2– C1s2 to C1q could interfere with the binding of C1q to DNA We studied the binding to DNA of the C1 complex labeled with the fluorescent beads (C1*) We observed that C1* and the individual DNAstrands were colocalized (Fig 2D), indicating the binding of C1 to DNA Hence the presence of the tetramer bound to the collagen-like region of C1q does not impede the binding of C1q to DNA Conversely, in a subsequent experiment, we checked the ability of C1r to associate on DNA-bound C1q For that purpose, T4 DNAwas preincubated with nonlabeled C1q,

in order to form a DNA–C1q complex The mixture was then incubated with C1r labeled with fluorescent beads (C1r*), and finally DNAwas combed We observed that C1r* spots were aligned along the DNAstrands (Fig 2E) Thus C1r binds to the DNA–C1q assembly, whereas C1r does not bind to DNAstrands in the absence of C1q (data not shown) Therefore the binding of C1r is a direct consequence of the presence of C1q on DNAstrands

Fig 2 Molecular combing of the T4 DNAon a

PMMAsurface after incubation with

fluores-cent Complement proteins and fucoidan (A)

Individual strands of fluorescent 160 kbps T4

DNAcombed on the PMMAsurface (B–D)

Molecular combing of the T4 DNAafter

incubation with fluorescent (Alexa

488-fluor-escent beads) C1q, CLR and C1 proteins,

respectively (E) Molecular combing of the T4

DNAafter incubation, first with nonlabeled

C1q and then with fluorescent C1r (F)

Molecular combing of the T4 DNAafter

incubation with fluorescent C1q in presence

of fucoidan.

Altogether these results indicate that the collagen-like region

of C1q contains distinct sites for the binding of DNAand

for the binding of the catalytic tetramer C1r2–C1s2

Interactions between fucoidan and C1q

We have previously reported that the sulfated

polysaccha-ride fucoidan interacts with C1q In order to ascertain this

binding property of fucoidan, we performed competition

experiments between fucoidan and DNAfor the binding to

C1q Fluorescent T4 DNAwas incubated with C1q* and

fucoidan in the C1q/fucoidan molar ratio 1 : 100 (i.e the

amount which leads to 30% inhibition of the hemolytic

activity of C1q as we have previously reported [14]) After

combing, the individual fluorescent DNAstrands clearly

appeared without any decoration by C1q* (Fig 2F) This

result shows that fucoidan is able to compete with DNAfor

the binding to C1q, suggesting that interactions between

fucoidan and C1q probably occur through the collagen-like

region This hypothesis was confirmed by performing the

same competition experiment using labeled-CLR (CLR*)

Incubation of T4 DNAwith CLR* in the presence of

fucoidan (CLR/fucoidan molar ratio of 1 : 20 and 1 : 40)

leads to an inhibition of the binding of CLR* to DNAfrom

the molar ratio 1 : 20 Altogether these results confirm that

fucoidan interacts with C1q through the same binding

region than DNA, i.e the collagen-like region that includes

in our CLR preparation the stalk region and partially the

hinge region

At this stage, it is worthwhile to note that, when C1

instead of C1q was used in this competition experiment with

fucoidan, a lower inhibition of the protein binding to DNA

was obtained (for the same C1/fucoidan molar ratio

1 : 100), as colocalization of C1* was still observed with

some DNAstrand (Fig 3) This lower inhibition may be

due to the presence of the catalytic tetramer C1r2–C1s2in

the C1 complex that hinders the interaction of the C1q

subunit with the polysaccharide According to this

hypo-thesis fucoidan should then interact with the hinge region

containing the binding site of the tetramer C1r2–C1s2, in

addition to the stalk region of CLR In order to check this

hypothesis, we tested the effect of fucoidan on the binding of

C1r to DNA-bound C1q For this purpose, T4 DNA and

nonlabeled C1q were firstly preincubated, before the

addition of C1r* and fucoidan (molar ratio C1q/fucoidan

1 : 100) The resulting molecular combing appeared as in Fig 2F, exhibiting the absence of C1r* spots on the T4 strands We have seen above that C1r is able to associate to DNA-bound C1q Furthermore, we have previously repor-ted an affinity capillary electrophoresis study evidencing no interaction between either C1r or C1s and fucoidan [14] Hence this result proved that the binding of C1r to C1q is inhibited by fucoidan, consistent with the interaction of the polysaccharide with the hinge region of the collagen-like region of C1q

Discussion

C1q can bind several polyanionic molecules like sulfated polysaccharides and also DNA, but with the opposite effects of either inhibition or activation, respectively Using single-molecule observation of immobilized DNAstrands,

we have been able to visualize the binding of C1q to individual DNAstrands This single molecule approach appeared as a valuable tool with which to investigate the binding properties of fucoidan, a sulfated fucose-based polysaccharide known as one of the most potent inhibitor of IgG-dependent activation of Complement [14]

C1q binding to DNAwas deduced in literature from data based either on C1q–DNAprecipitation experiments [22] or

on solid-phase assays with immobilized C1q [28,29] In the present study, we obtained the direct evidence of the DNA– C1q binding through the observation of the colocalization

of C1q and DNAstrands It is worthwhile noting that this interaction was performed in the fluid phase In these conditions, the resulting C1q-DNAcomplex was resistant

to the stretching and combing of DNAon PMMAsurfaces, indicating the strength of the interaction Acontinuous succession of fluorescent C1q could be observed on some DNAstrands, corresponding to a high density of binding Although the optical resolution does not allow the deter-mination of this density, the obtained images are consistent with previous estimates of one C1q per 34 nm of double-stranded DNAfor the highest density [22] C1q is usually described as constituted of two main domains, the N-terminal collagen-like region CLR, and the C-terminal globular region GR Divergent data have been reported in the literature concerning the binding site of DNAon C1q DNAhas been proposed to bind to either the GR or the CLR, or to both of the two domains, according the method used Here, we have individually used each of these domains

in the single molecule approach to unambiguously identify the CLR as the main binding site of DNA Indeed, we have shown that CLR binds to DNAas well as C1q does, and that, compared with GR, CLR competes much more efficiently with C1q for binding to DNA The dissociation

of the globular region of C1q into individual structures could decrease the affinity of the GR, but to an extent that could not lead to the observed difference between CLR and

GR in these competition experiments The low competition effect observed only when a very large excess of GR is used could be due to non-specific interactions or to interaction with the residual GR tail present in the GR preparation The collagen-like region can be divided into an N-terminal domain organized into a triple-helical stalk, which diverges into six arms constituting the C-terminal Fig 3 Molecular combing of the T4 DNAon a PMMAsurface after

incubation with fluorescent C1 complex and fucoidan.

domain of the CLR; this has been named the hinge region

[15] This hinge region contains the binding site for the

catalytic tetramer C1r2–C1s2, essential for the C1 activity

[35] Our results show that C1q as well as C1 bind to DNA,

and that C1r can bind to C1q bound to DNA Thus DNA

binding site on CLR does not overlap with the binding site

of the tetramer in the hinge region It is likely that DNA

binds to the stalk domain of CLR, consistent with previous

findings showing that a synthetic peptide of the N-terminal

portion of the Achain (residues 14–26) binds to DNAand

inhibits its binding to C1q [20,30]

During the course of our studies of the

anticomplemen-tary activity of fucoidan, we have previously shown by

affinity electrophoresis that this sulfated polysaccharide

binds also to C1q [14] The results obtained here clearly

showed its ability to inhibit the binding of C1q, as well as

that of CLR, to DNA These data indicate that, like DNA,

fucoidan binds to C1q through the collagen-like region The

C1q Achain that appeared to be essential for the binding of

nonimmune substance contained a cationic region within

residues 14–26 of stalk [20] This positively charged sequence

contained five basic proximal residues, arginine and lysine,

that are assumed to be involved in the binding of polyanions

like DNAand fucoidan (Fig 1B) Consistent with these

data, we have previously shown that fucoidan protects the

lysine residues of C1q from chemical modification by

specific reagent [14] However fucoidan exhibits a major

difference with DNAin that the polysaccharide is able to

block the association of C1r to C1q This result is in

agreement with our previous finding where the

polysaccha-ride was shown by ELISAto inhibit the reconstitution of C1

from C1r, C1s, and C1q [14] Thus, unlike DNA, fucoidan

also interacts with the arms domain of CLR, which

contains the binding site of the tetramer C1r2–C1s2 It has

been reported that basic residues lysine and arginine in the

hinge region are involved in the assembly of C1 through

specific interaction with acidic residues of C1r [35]

Furthermore, the structural model of C1q shows a cluster

of basic residues that are located in the hinge region at the

junction between the stalk and the arms We assumed that

fucoidan interacts with these positively charged residues in

the hinge region, leading to the observed blockage of the C1

assembly As a consequence, the inhibiting activity of

fucoidan on the classical pathway activation should result

from this binding property to the hinge region, hampering

the activation of the two proteases C1r and C1s This

mechanism is probably related to the inhibiting property of

endogenous C1q inhibitors of Complement, like the

chon-droitin 4-sulfate proteoglycan, which is secreted by the

human B lymphocytes This glycosaminoglycan has been

proposed as a potential physiologic C1q inhibitor, through

the inhibition of the C1q– (C1r2–C1s2) assembly [37]

Other C1q binding substances that are not C1q

inhibi-tors, like DNA, C-reactive protein [20], and amyloid

protein, do not bind to the hinge region but rather to the

stalk domain of the CLR [38] It has also been proposed that

the collagen-like stalk is involved in the binding of C1q to

different cell types and to liposomes [39] Strikingly, this

binding leads to the activation of Complement, as we

observed for DNA(data not shown) The mechanism of

this activation, independent of the recognition of the

immune complex by the globular heads, remains unclear

and is debated in the literature [40] Recently, it has been reported from the structural model of C1r that the activation of the C1 complex could result from mechanical constraints upon C1q binding, which affect the flexible hinge region [41] Further investigations are required to determine whether the binding to the stalk region also results in such mechanical stresses that could be transmitted

to the hinge region

Acknowledgements

We thank J Ratiskol and C Sinquin for the extraction and the preparation of the fucoidan fraction and for their experimental advices.

We are grateful to Professor G J Arlaud for his generous gift of purified C1q and of GR and CLR preparations This work was supported by CNRS and the county Pays de La Loire, as well as the program Physique et Chimie du Vivant, from CNRS and the Ministe`re

de la Recherche, France.

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