C1q and tumor necrosis factorsuperfamily: modularity and versatilityUday Kishore1,2, Christine Gaboriaud3, Patrick Waters1, Annette K. Shrive4,

Trevor J. Greenhough4, Kenneth B.M. Reid5, Robert B. Sim5 and Gerard J. Arlaud3

1Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford, UK, OX3 9DS2Institute for Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, D-35392 Giessen, Germany3Laboratoire de Cristallographie et Cristallogenese des Proteines and Laboratoire d’Enzymologie Moleculaire,

Institut de Biologie Structurale Jean Pierre Ebel, CEA-CNRS-Universite Joseph Fourier, 41, rue Jules Horowitz,

38027 Grenoble Cedex 1, France4School of Life Sciences, Keele University, Staffordshire, UK, ST5 5BG5Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford,

UK, OX1 3QU

C1q is the target recognition protein of the classical

complement pathway and a major connecting link

between innate and acquired immunity. As a charge

pattern recognition molecule of innate immunity, C1q

can engage a broad range of ligands via its globular

(gC1q) domain and modulate immune cells, probably via

its collagen region. The gC1q signature domain, also

found in many non-complement proteins, has a compact

jelly-roll b-sandwich fold similar to that of the multi-

functional tumor necrosis factor (TNF) ligand family. The

members of this newly designated ‘C1q and TNF super-

family’ are involved in processes as diverse as host

defense, inflammation, apoptosis, autoimmunity, cell

differentiation, organogenesis, hibernation and insulin-

resistant obesity. This review is an attempt to draw

structural and functional parallels between the mem-

bers of the C1q and TNF superfamily.

C1q is the key subcomponent of the classical pathway ofcomplement activation and a major connecting linkbetween classical pathway-driven innate immunity andIgG- or IgM-mediated acquired immunity [1]. Binding ofC1q to IgG- or IgM-containing immune complexes via theligand-recognition gC1q domain induces a conformationalchange in the collagen region (Figure 1), leading to theautoactivation of C1r, which, in turn, activates C1s. C1rand C1s, the two serine protease proenzymes, togetherwith C1q constitute C1, the first component of the classicalcomplement pathway [2]. The activation of the C1 complexsubsequently leads to the activation of the C2–C9components of the classical pathway and formation ofthe terminal membrane attack complex.

In addition to being the key component of the classicalcomplement pathway, which is aimed at antimicrobialdefense, C1q is involved in several other immunologicalprocesses (Table 1), including maintenance of immunetolerance via clearance of apoptotic cells, phagocytosis ofbacteria, neutralization of retroviruses, cell adhesion, and

Corresponding authors: Uday Kishore (ukishore@hammer.imm.ox.ac.uk;u.kishore@rediffmail.com).

Available online 20 August 2004

www.sciencedirect.com 1471-4906/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved

modulation of dendritic cells (DCs), B cells and fibro-blasts [1,3]. Its ability to carry out such diversefunctions is aided by its capacity to engage a broadrange of ligands, such as envelope proteins of certainretroviruses, b-amyloid fibrils, lipopolysaccharides(LPS), porins from Gram-negative bacteria, phospho-lipids (PL), apoptotic cells and some acute phasereactants, including pentraxins (Table 1) [1,3]. Themajority of C1q ligands are recognized via the hetero-trimeric gC1q, which is an extremely efficient andversatile charge pattern recognition domain.

The gC1q signature domain is also found in a varietyof non-complement proteins, including collagen VIII andX, precerebellin, hibernation proteins, multimerin,adiponectin, saccular collagen and elastin microfibrilinterface-located protein (EMILIN) [3,4] (Table 2). Amajor advance in the understanding of the gC1qstructure–function relationship came with the elucida-tion of the crystal structure of the homotrimeric gC1qdomain of mouse ACRP30 [5]. It revealed a structuraland evolutionary link between tumor necrosis factor(TNF) and gC1q-containing proteins, and hence recog-nition of a C1q and TNF superfamily.

Studies involving transgenic and genetically deficientmice, together with gene mutations found in patients,have suggested aspects of immunity and energy homeo-stasis where members of C1q and TNF superfamily crossover. Here, we discuss the overall structural and func-tional aspects of the C1q and TNF superfamily proteins,and speculate on how modularity within the gC1q domainhas been exploited to achieve functional versatility by thesuperfamily members.

Modular organization of the gC1q domain

The gC1q domain of C1q is composed of the C-terminalregions of its A (ghA), B (ghB) and C (ghC) chains [4](Figure 1). Given the heterotrimeric organization of thegC1q, it has been debated whether modules ghA, ghB andghC have distinct ligand-binding properties, or that theability of C1q to bind its ligands is dependent upon a

Review TRENDS in Immunology Vol.25 No.10 October 2004

. doi:10.1016/j.it.2004.08.006

TRENDS in Immunology

CC

CC

Unit

A–chain

B–chain

C–chain

A–B dimer

C–C dimer

Structural unitA–B C–C B–A (d)

5 nm

7 nm11.5 nm

226 aa

217 aa

(a)

(b)

(c)

N-term3-9 aa

CLR~81 aa

gC1q~135 aa

(e)

223 aa

11.2 nm

A

B

C

A

B

C

C

Figure 1. Structural organization of the C1q molecule. C1q (460 kDa) is composed of 18 polypeptide chains (6A, 6B and 6C). (a) The A, B and C chains each have a short

N-terminal region (containing a half-cystine residue involved in interchain disulfide bond formation), followed by a collagen region (CLR) of w81 residues and a C-terminal

globular region (gC1q domain) of w135 residues. (b) The interchain disulfide bonding yields 6A–B dimer subunits and 3C-C dimer subunits. The triple-helical collagen region

in the A and B chains of an A–B subunit, together with one of the C-chains present in a C–C subunit, form a structural unit (ABC–CBA), which is held together by both covalent

and non-covalent bonds (c). Three of these structural units associate, via strong non-covalent bonds in the fibril-like central portion, to yield the hexameric C1q molecule that

has a tulip-like structure, as also found in mannose-binding protein, surfactant protein A and ficolins [77] (d). The crystal structure of the gC1q domain of human C1q (Protein

Data Bank code 1PK6, depicted as a ribbon diagram of ghA in blue, ghB in green, ghC in red, with the calcium ion shown as a yellow ball), has revealed a compact, spherical,

heterotrimeric assembly (50 A diameter), held together predominantly by non-polar interactions, with non-crystallographic pseudo-threefold symmetry (e). The three gC1q

modules show clear differences in their electrostatic surface potentials, which in part explains modularity in terms of ligand recognition [11].

Review TRENDS in Immunology Vol.25 No.10 October 2004552

combined, globular structure [6,7]. Studies using recom-binant forms of ghA, ghB and ghC have suggested thateach of the three modules of gC1q can bind its preferredligand independently [7]. ghA can bind heat-aggregatedIgG and IgM, in addition to HIV-1 gp41-derived looppeptide; ghB prefers aggregated IgG to IgM, in addition tob-amyloid peptide (bA1–42); whereas ghC shows prefer-ence for IgM as well as HTLV-I gp21 peptide. Both ghAand ghB can inhibit C1q-dependent hemolysis of IgG- andIgM-sensitized sheep erythrocytes, ghC being a betterinhibitor than ghB in the case of IgM-coated erythrocytes[7–10]. The crystal structure of the native gC1q hassupported the general view of the modular nature of theheterotrimer [11] (Figure 1e). Whereas ghA and ghC bothshow a combination of basic and acidic residues scatteredon their external face, ghB shows a predominance ofpositive charges, especially a continuous patch of Arg101,Arg114 and Arg129, involved in the C1q–IgG interaction[12]. Thus, the modular organization of the heterotrimericassembly, together with the different surface chargepattern and spatial orientation of individual modules,

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confers flexibility and versatility to the ligand recognitionof gC1q [6,11].

Structural modeling has suggested a predominant rolefor the ghB module in the C1q–IgG interaction [7,8,10,11].As the most accessible of the three modules within thegC1q domain, ghB seems best located for binding IgG. Themost attractive model attempting to describe the ghB–IgGinteraction positions the two molecules in such a waythat Asp270 and Lys322 of IgG form salt bridges with Arg129

and Glu162 of ghB, respectively, with Arg114 providing anadditional ionic interaction. In this orientation, Arg129

appears to ‘act as a wedge’ between the CH2 and lightchain constant domains. Therefore, the Fab/Fc orientationmight be a crucial factor in dictating access of the ghBmodule to the CH2 domain. A recent mutational study hasalso suggested a dominant role for Arg114 of ghB and asubsidiary role for Arg129, Arg163 and His117 of ghB in theC1q–IgG interaction [12].

The flexibility and diversity of C1q binding is furtherhighlighted by the interaction of gC1q with C-reactiveprotein (CRP), a major acute phase reactant that binds,

Table 1. Interactions between gC1qa and its ligands

Ligands Interacting sites/motifs Implications/comments Refs

IgG Glu318, Lys320, and Lys322 in mouse IgG2b, which is

highly conserved in different IgG isotypes. In human

IgG1 Asp270, Lys322, Pro329, Pro331 and residues Lys326

and Glu333 are implicated.

C1q also interacts with the Fab (light chain constant

domain), supporting observations that Fab/Fc

flexibility has a crucial role in C1q binding.

[11,45,46]

IgM Hexameric and pentameric IgM in the ‘staple’ form

(bound to antigen) bind gC1q via the Cm3 domain

involving His, Asp/Glu and Pro residues at 430–434.

Monomeric IgM, fixed to antigen, does not bind C1q.

Hexameric IgM generally activates the classical path-

way better than pentameric IgM, possibly reflecting

better symmetry for binding one entire C1q molecule.

[47]

CRP The gC1q binds to the central ‘pore’ in the planar

pentamer of CRP when CRP has bound to a target

such as chromatin or bacterial C-polysaccharide: one

C1q head binds per pentameric CRP through one of

the five available binding sites on CRP. A central role

for a series of residues including CRP Tyr175, Glu88 and

Asp112, and Lys114 from a neighboring protomer has

been described.

Modeling studies suggest a complementarity of

shape between the top of gC1q and the central pore of

the pentameric CRP ring, which involve ghA, ghB and

CRP Tyr175 and Asp112. CRP binds chromatin and

might have a major role in clearing chromosomal

material from necrotic cells, via C1q binding.

[3,11–15]

SAP and PTX3 Because SAP is a homolog of CRP, its binding might

be similar. However, CRP Asp112 is not conserved in

SAP or PTX3. PTX3 binds via gC1q.

Immobilized SAP binds C1q but there is controversy

as to whether this results in complement activation.

PTX3 mediates complement activation on apoptotic

cells.

[48,49]

Decorin Decorin might bind to the ‘neck’ region of C1q

(between gC1q and collagen domain), or to both

domains via decorin core protein.

Modulation of the classical pathway activation in the

tissue.

[50]

Outer membrane

proteins from

Gram-negative

bacteria, lipopoly-

saccharide (LPS),

lipid A

C1q binds directly to the surfaces of many Gram

negative bacteria without the need for antibody. The

gC1q binding is mediated by lipid A, LPS and porins.

OmpK36 (from Klebsiella pneumoniae) competes

directly with IgG for binding to C1q. Binding to lipid A

and LPS is mainly via the phosphate groups of lipid A.

Many pathogenic Gram-negative bacteria evade C1q

fixation by steric hindrance of C1q binding.

[51,52]

Viral proteins, e.g.

gp41 of HIV-1,

gp21 of HTLV-I,

p15e of MuLV

C1q binds directly to many viruses, enveloped and

non-enveloped. The ghA binds residues 601–613

(loop region) within the envelope protein gp41 of

HIV-1 (especially Lys608, Leu609 and Ile610). The Ala

substitution of all hydrophobic residues (including the

four-carbon aliphatic moiety of the Lys608 side chain)

within the loop abolishes the C1q–gp41 interaction.

C1q also binds the HTLV-I gp21 peptide 400–429,

which is required for syncytium formation.

C1q binding to viruses might result in virus

neutralization. C1q–gp41 interaction leads to

enhanced infection of complement-receptor-bearing

cells, instead of viral lysis. Interaction between HTLV-I

peptide and gC1q might affect the fusion process

required for syncytium formation.

[3,7]

b-amyloid and

familial dementia

peptides

C1q globular heads bind to the acidic N-terminal 1–11

region of Alzheimer b-amyloid peptide and to the

N-terminal regions of familial dementia peptides.

Classical pathway activation leads to inflammation in

neuritic plaques.

[53–55]

Apoptotic cells C1q binds apoptotic cells directly and via pentraxins.

The sites of interaction might include anionic PLs and

surface proteins. Surface blebs of apoptotic keratino-

cytes and peripheral blood mononuclear cells, which

contain autoantigens are targeted in SLE.

C1q deficiency can cause SLE as a result of an

impaired clearance of apoptotic cells. In C1q knockout

mice, which have glomerulonephritis with immune

deposits, a large number of apoptotic bodies are also

present in diseased glomeruli. C1q might protect

against autoimmunity by serving as an opsonin in the

efficient recognition and physiological clearance of

apoptotic cells, hence be required to maintain

immune tolerance.

[7,24]

Phospholipids

(PLs)

C1q binds cardiolipin and other anionic PLs. Possible role in clearance of apoptotic and necrotic

cells.

[52]

aAbbreviation: CRP, C-reactive protein; gC1q, globular domain of C1q; PL, phospholipid; PTX3, pentraxin 3; SAP, serum amyloid protein; SLE, systemic lupus erythematosus.

Review TRENDS in Immunology Vol.25 No.10 October 2004 553

via the face of its pentameric ring, to the phosphocholinecomponent of membrane PL [13,14]. The overall dimen-sions of C1q and CRP molecules appear to suggest thatonly one gC1q can bind to each CRP pentamer through oneof the five available binding sites on CRP. This interactioninvolves a series of residues, including CRP Tyr175, Glu88

and Asp112, and Lys114 from a neighboring subunit [15].Molecular modeling has revealed a shape complementar-ity between the top of the gC1q and the central pore of thepentameric CRP ring, suggesting a central role for ghA,ghB and CRP Asp112 and Tyr175 in this interaction [11].However, given severe steric restraint between the twostructures, it is apparent that structural changes in CRP,rather than participation of bound ligand, is required for

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this interaction [3]. It is evident that the compactheterotrimeric structure of the gC1q domain clearlyfacilitates ligand recognition by two or even threesubunits, thus providing a structural basis for theextremely versatile recognition properties of C1q [11].

A novel C1q family

A variety of collagenous and non-collagenous non-complement proteins also contain a gC1q signaturedomain (w140 residues long) and thus constitute a novelC1q family [1] (Table 2; Figures 2–4). Except in pre-cerebellin and multimerin, the gC1q domain is alwayslocated at the C-terminus of a collagen-like sequence.Comparative sequence analysis between gC1q domains of

Table 2. Proteins with a gC1qa domain, their cell sources and functions

Protein Tissues of origin and presentation Function Refs.

ACRP30

(adiponectin)

Serum protein produced by adipocytes: 5–10 mg/ml

in normal plasma.

An antidiabetic and antiatherogenic adipokine;

insulin sensitivity, energy homeostasis, and lipid

and carbohydrate catabolism; reduced blood levels

in obesity, insulin resistance and type 2 diabetes;

resolution of inflammation.

[30,33–35,56]

C1q Serum protein produced by the liver: 70 mg/ml. See Table 1 and Figure 1. See references

in Table 1

EMILIN-1 Connective tissue, blood vessels, skin, heart, lung,

kidney, cornea, small intestine, aorta, uterus and

appendix; fetal heart and lungs.

Smooth muscle adhesion to elastic fibers, elasto-

genesis and regulation of vessel assembly.

[57]

EMILIN-2 Fetal heart and adult lung; intermediate levels in

peripheral leukocytes, placenta and spinal cord;

major component of the cochlear basilar membrane

in the mouse.

Development of heart chambers and cochlear basilar

membrane, and the compositional and functional

heterogeneity of the extracellular matrix.

[58]

EMILIN-3

(multimerin 2)

Blood vessel endothelial cell surface including

capillaries, veins, arterioles and muscular arteries.

Proposed role in vasculogenesis, angiogenesis or

hemostasis and cell-matrix adhesion.

[59]

EMILIN-4

(multimerin 1)

Platelet a-granules, endothelial cell Weibel–Palade

bodies, megakaryocytes, platelets, endothelium and

subendothelium of blood vessels; placenta, lung and

liver.

As a factor V/Va binding protein, it might play a role

in platelet factor V storage and stability; adhesion

functions via an RGDS motif once released

extracellularly.

[60]

HP-20, -25, -27 Plasma protein produced by the liver. HP-20, -25 and –27 form a 140 kD complex with a

HP-55 serpin; this complex disappears from the

blood during hibernation.

[37,61]

Precerebellin Expressed in the cerebellum associated with post-

synaptic structures of the Purkinje cell membrane

and the dorsal cochlear nucleus; brain.

Potential role in the development and stability of the

Purkinje cell synapse.

[62,63]

Saccular

collagen

Produced by specialized secretory-supporting cells

at the outer perimeter of the saccular macula; also

found in saccular otolithic membrane.

Structural constituent of the otolithic membrane, a

sensory accessory membrane in the inner ear

involved in vestibular function.

[38,64]

Collagen VIII Produced by endothelial cells; major constituent of

Descemet’s membrane (specialized basement

membrane of corneal endothelial cells); optic nerve,

aorta, umbilical cord and tissues undergoing

remodeling; calvarium, eye and skin.

Vascular tissue development and remodeling,

cell migration, plaque stability and thrombus

organization.

[65–67]

Collagen X Produced by hypertrophic chondrocytes in the

epiphyseal growth plate of long bones, ribs and

vertebrae during endochondral bone formation,

bone fracture callus and osteoarthritic cartilage.

Secreted into the extracellular matrix of presumptive

ossification zones of cartilage; formation of a

hexagonal lattice and interaction with

proteoglycans; eventually replaced by bone

extracellular matrix.

[16,68,69]

CORS26 Prechondrocytes in developing cartilage; colon,

small intestine, placenta, fibroblasts, white adipose

tissue, blood and kidney; osteosarcoma, chondro-

blastoma and giant cell tumors.

Embryonic skeletal development; might act as a

growth factor, or be involved in signaling via three

possible phosphorylation sites; implicated in bone

tumor; a potential candidate gene in the

development of arthritis.

[70,71]

CTRP5 Retinal epithelium, lung, placenta, cerebrum and

liver.

Formation of an extracellular hexagonal lattice

between RPE and Bruch’s membrane in the retinal

epithelium.

[39]

aAbbreviations: ACRP30, adipocyte complement-related protein – 30 kD; CORS26, collagenous repeat containing sequence of 26 kDa protein; CTRP5, C1q and tumor necrosis

factor related protein 5; EMLIN-1, Elastin microfibril interface-located protein 1; gC1q, globular domain of C1q; HP, hibernation protein of Siberian chipmunk; RGDS, Arg-Gly-

Asp-Ser; RPE, retinal pigment epithelium.

Review TRENDS in Immunology Vol.25 No.10 October 2004554

C1q family members shows that whereas limited varia-bility occurs at several positions, only five residues arestrictly conserved throughout the C1q family (Figure 3).

Four structures of gC1q domains, namely those ofmouse ACRP30 [5], human collagen X [16], mouse colla-gen VIII (a1) [17] and human C1q [11], have been solvedby X-ray crystallography, at resolutions of 1.9–2.1 A. EachgC1q domain exhibits a ten-stranded b-sandwich foldwith a jelly-roll topology, consisting of two five-strandedb- sheets (A 0, A, H, C, F) and (B 0, B, G, D, E), each made ofantiparallel strands (Figure 4a–c). Each of the fiveconserved residues within C1q family proteins belongs tothe hydrophobic core of the gC1q domain. The b-strandsare strongly conserved in the different gC1q domains,both with respect to their relative orientation and size(Figure 3, 4a–c). By contrast, the loops connecting the

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b-strands exhibit significant variability, as is the case inthe extended segments A-A 0 and G-H, which exhibitmarkedly different sequences and structures in the ghA,ghB and ghC modules (Figures 3,4a).

All structures reveal similar tight assemblies withpseudo-threefold symmetry, resulting in globular, almostspherical domains (45–55 A in diameter). The N- andC-termini of the three subunits emerge on the same side ofthe trimer and are adjacent to one another (Figure 4d). Inthe case of C1q (Figure 4e), the subunits are arrangedclockwise in the order ghA (blue), ghB (green) and ghC(red) when viewed from the top. Structural homologyis very high between collagen VIII and X, with a rootmean square deviation (rmsd) of only 0.61 A, but is muchlower between distantly related collagen VIII andACRP30 (rmsdZ2.3 A). Trimerization involves a very

TRENDS in Immunology

600 700 800 900 1000400 5003000 100 200 1100 1200 1300

Residue number

NH2

NH2

Hibernation protein

C1q

ACRP-30/Adiponectin

Type X collagen

EMILIN - 1

Motifs

gC1q domain

Collagen like region

EGF

Leucine zipper

Coiled-coil

Partial EGF

RGDS domain

Possible glycosylation sites

Cysteine residues

Saccular collagen

Multimerin

Precerebellin

Type VIII collagen - 1

CORS-26NH2

NH2

NH2

NH2

NH2

NH2

NH2

NH2

Figure 2. Cartoon depicting the structural motifs of the C1q family members. Proteins belonging to the C1q family have a C-terminal gC1q signature domain [1,4,6]. Most

members of the C1q family have a triple-helical Gly-X-Y collagen repeat, with the exception of precerebellin, and multimerin. The gC1q are either homotrimeric (type VIII and

X collagen, multimerin, ACRP30 and saccular collagen) or heterotrimeric (C1q and hibernation proteins, and probably precerebellin) structures. Although collagen VIII is

considered to be composed of two distinct gene products (a1 and a2), both chains can also preferentially form pepsin-resistant homotrimers and exist as two distinct proteins

[67]. Elastin microfibril interface-located protein (EMILIN) has a region containing two leucine zippers and at least four heptad repeats, with a high potential for forming

amphipathic coiled-coil a-helices, and at the N-terminus, a partial epidermal growth factor (EGF)-like motif, as found in multimerin [57,58]. Multimerin also has an RGDS

(Arg-Gly-Asp-Ser) motif, EGF domain and a domain containing coiled-coil structures. Multimerin and EMILIN are unique in having a-helical coiled-coil structures, which

might be involved in trimerization or higher-order multimerization.

Review TRENDS in Immunology Vol.25 No.10 October 2004 555

tight association of the subunits, although with significantvariations, as judged from total buried surface, which aremuch lower in ACRP30 (5324 A2) and C1q (5490 A2) thanin collagen VIII (6150 A2) and X (7360 A2), accounting forthe exceptional stability of the collagen VIII and Xmolecules [16]. Trimerization also involves a centralinterface and lateral contacts which are hydrophobicnear the base, becoming progressively more polar towardsthe top. These interactions involve residues contributed bythe b-sheet formed by strands A 0, A, H, C and F, with amajor contribution from strands C and F (Figure 4e). Thisresults in the creation of a central solvent-filled channel,which is discontinuous in C1q and whose access is blockedat both ends. Irrespective of whether the structures arehomotrimeric (ACRP30 and collagen VIII and X) orheterotrimeric (C1q), all trimers assemble in a similarway. However, tentative assembly of C1q homotrimersin silico has been shown to result in severe steric

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hindrances, providing a structural basis for theirassembly only as heterotrimers [11].

Near the top of the solvent channel, the collagen Xstructure has a buried cluster of three Ca2C ionssurrounding the one located on the threefold symmetryaxis of the trimer (Figure 4f). It is coordinated by acidicresidues and other residues (Figure 3), which togetherform an intricate network of ionic bonds which probablycontribute to the high stability of the collagen X gC1qtrimer [16]. This cluster is absent from the collagen VIIIstructure, due to the presence of Lys692 in place of Thr629

in its solvent channel [17]. This enables the amino groupsto substitute for the peripheral Ca2C ions while main-taining a similar network of interactions near the apex ofthe trimer. Whereas loops A-A 0, G-H and E-F are partlydisordered in the Ca2C-free form of ACRP30 [5], they arestructured in the Ca2C-bound form (Figure 4b), providingsupport for a stabilizing effect of Ca2C. A single Ca2C ion,

Figure 3. Sequence alignment of the gC1q domains of human collagen X (Col_X), mouse collagen VIII (Col_VIII), mouse ACRP30, human C1q, human C1q-related factor

(C1q_RF), chipmunk hibernation proteins (HP_27, HP_25, HP_20), CORS26, human endoglyx-1/elastin microfibril interface-located protein (EMILIN)-3 (Endo_1), human

precerebellin (Precer) and saccular collagen from bluegill sunfish (Saccol). Location of the b-strands is indicated on top of the sequences. Residues showing minimal

variability are colored yellow and those strictly conserved in all known gC1q sequences are indicated underneath the alignment as a consensus sequence. Residues involved

in intermodular interfaces are marked by #. Ca2C ligands are colored red, and those involved through their side chain are shown in bold. The free cysteine residue present in

most gC1q domains is colored green, and Lys692 of collagen VIII is colored purple.

Review TRENDS in Immunology Vol.25 No.10 October 2004556

also located at the upper end of the central channel, ispresent in the C1q structure [11]. In contrast to the Ca2C

cluster of collagen X, the Ca2C ion of C1q is well exposed tothe solvent (Figure 4d), suggesting that, in addition to astabilizingrole, itmightalsoparticipate in ligandrecognition[11]. The surfaces of the collagen VIII and X structures havethree strips, each containing eight aromatic residuespartiallyexposed to thesolvent, extendingacross theshallowgrooves between the subunits [16,17]. In both cases, adetergent molecule is bound near the top of the grooves(Figure 4f), highlighting the hydrophobic nature of thestrips, which probably initiate supramolecular assembly.

Thus, the members of the C1q family exhibit subtlevariations in the three-dimensional structure of thegC1q domain according to their functional requirements.As discussed below, the structural fold of the gC1qdomain is homologous to the one described initially forTNF [18,19], suggesting an evolutionary link between theC1q and TNF families.

The C1q and TNF superfamily

The ACRP30 gC1q structure revealed a symmetricaltrimer of b-sandwich subunits, each having a ten-strand

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jelly-roll-folding topology, which is also present in theconserved C-terminal TNF homology domain (THD)within the TNF ligand family proteins [18,19]. Membersof the TNF family are involved in a range of biologicalprocesses such as inflammation, adaptive immunity,apoptosis, energy homeostasis and tissue regeneration(Table 3). Each of the ten b-strands of ACRP30 can besuperimposed with the ten strands of TNF-a, TNF-b andCD40L [5]. The relative positions and lengths of theseb-strands are almost identical between ACRP30 and theTNF ligands [5]. The C1q and TNF family proteins alsohave similar gene structures: their gC1q or THD domainsare each encoded within one exon, whereas introns inboth families are restricted to respective N-terminalcollagen or stalk regions, suggesting divergence from acommon precursor molecule of the innate immune system,and thus establishing a C1q and TNF molecular super-family [5]. This jelly-roll structure is remarkably similarto the capsid proteins of plant viruses and mammalianpicornaviruses including foot-and-mouth and poliovirus[18,19]. The presence of oligomerizing jelly-roll motifsin viruses is interesting because capsid proteins andmembers of the C1q and TNF superfamily are unrelated

N

C

B′B

A′A

H

G

C

DE

F

SH

C

NC

N N

C

B

B′

A′G

ED

H

F

C

A

(a) (b) (c)

(d) (e) (f)

TRENDS in Immunology

Figure 4. Structural features of C-terminal globular region (gC1q) signature domains in the C1q family. (a) Superimposed structures of modules A, B and C of human C1q

Protein Data Bank (PDB) code 1PK6. The modules are colored blue (ghA), green (ghB) and red (ghC). The free cysteine and the disulfide bond connecting b-strands D and E are

shown in the case of module C. (b) Superimposed structures of the Ca2C-free (green) and Ca2C-bound (blue) forms of the mouse ACRP30 gC1q domain (PDB codes 1C28 and

1C3H). (c) Superimposed structures of the gC1q domains of mouse collagen VIII (pink) and human collagen X (blue) (PDB codes 1O91 and 1GR3). The side chain of Lys692 of

collagen VIII is shown. (d) Side view and (e) top view of the gC1q domain of human C1q. Color coding is the same as in (a). N and C indicate the N- and C-terminal ends of each

module. (f) Top view of the collagen X NC1 domain. The detergent molecules bound to the structure are shown in ball and stick format. Ca2C ions are represented as yellow

balls.

Review TRENDS in Immunology Vol.25 No.10 October 2004 557

and without any functional conservation. Given similarintracellular signaling domains of some TNF receptorsand toll-like receptors (TLR), which function as germline-encoded receptors for surface proteins of pathogens, C1qand TNF proteins are likely to have descended viahorizontal capture of a gene encoded by an ancient viralpathogen [5,20].

Functional overlaps between the members of the C1q

and TNF families

The TNF ligands are synthesized as type II membraneproteins which serve as cell contact-mediated regulators.They can be proteolytically cleaved as soluble homo-trimers, with the exception of membrane-bound lympho-toxin (LT; which contains one LTa subunit and twosubunits of LTb), B-cell-activating factor (BAFF) and aproliferation-inducing ligand (APRIL) that can also formheterotrimers. The soluble forms can act as either agonistsor antagonists of membrane-bound forms. TNF contrib-utes towards inducing inflammation transiently andreversibly to foster immune cell networking (Table 3).

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Some members induce apoptosis, others promote cellsurvival and some induce cell proliferation via activationof pathways involving nuclear factor-kB (NF-kB), C-JunN-terminal kinase (JNK), p42 or p44 mitogen-activatedprotein kinase (MAPK) and p38 MAPK. The multiplefunctions assigned to TNF ligands have their genesis inthe expression of TNF receptor (TNFR) by all cell types[20–22].

There are aspects of immune mechanisms and energyhomeostasis in which members of the C1q and TNFsuperfamily cross over and effect similar or almostopposite pathophysiological outcomes [5,6]. TNF ligandshave been implicated in the development of autoimmun-ity. Mice transgenically overexpressing BAFF secrete highlevels of autoantibodies, such as rheumatoid factors andantinuclear antibodies, similar to the situation in systemiclupus erythematosus (SLE), and develop severe glomer-ulonephritis leading to kidney failure [23]. C1q deficiencyis a likely cause of SLE, resulting from an impairedclearance of apoptotic cells. In C1q knockout mice, whichhave glomerulonephritis with immune deposits, a large

Table 3. TNF ligands, their cell sources and functionsa

TNF ligands Cell source Functions

LTa/bb NK, T and

B cells; DCs, macrophages

Central to organogenesis of secondary lymphoid tissue; LTa ko mice are deficient in

peripheral lymph nodes and Peyer’s patches; LTb ko mice are deficient in peripheral

lymph nodes, Peyer’s patches, splenic germinal centers and FDCs.

TNFa Macrophages, NK, T and B

cells, adipocytes

Multifunctional cytokine involved in inflammation, apoptosis and survival, cytotoxicity,

production of IL-1 and IL-6, induction of insulin resistance; ko mice are susceptible to

Listeria and resistant to LPS; deficient in splenic primary B cell follicle and organized FDC

and germinal centers.

FasL (CD95L) Activated splenocytes,

thymocytes, eyes, testis

Induction of cell-mediated cytotoxicity; death-inducing ligand (apoptotic suicide);

involvement in angiogenesis and tumor progression; role in immune homeostasis;

prevention of autoantibody production; ko mice have lymphadenopathy and systemic

autoimmunity; immune complex glomerulonephritis.

TRAIL NK and T cells, DCs Induction of apoptosis selectively in tumor cells via death receptors; activation of NK

cells, cytotoxic T cells and DCs; a potential tumor-specific cancer therapeutic; ko mice

susceptible to tumor metastasis.

TWEAK Monocytes Stimulation of cell growth and angiogenesis; induction of inflammatory cytokines;

stimulation of apoptosis.

CD27L NK, T and B cells Expansion and survival of T cells; T cell priming.

CD30L T cells and monocytes Important for processes mediated by Th2 cells.

CD40L T and B cells Proliferation, differentiation, germinal center development, isotype switching, memory;

ko mice do not develop germinal centers to thymus-dependent antigens, have impaired

virus-specific CD4C T cell response, fail to develop memory cell response and have

impaired T-cell-dependent macrophage activation.

4-1BBL B cells, DCs and

macrophages

Expansion and survival of T cells; T-cell priming; ko mice have decreased CD8C T-cell

expansion.

OX40L T and B cells, DC,

macrophages

Expansion and survival of T cells; T-cell priming; ko mice have DCs defective in

costimulating Th cytokine production (impaired APC function).

APRIL Macrophages, lymphoid

cells, tumor cells

Role in T-cell-independent type II antigen response and T-cell survival; can induce

proliferation and survival of non-lymphoid cells; role in lupus-like syndromes.

BAFF T cells, DCs, monocytes,

macrophages

Important survival and maturation factor for peripheral B cells; role in lupus-like

syndromes; overexpression linked to autoimmune disease and B-cell neoplasia; ko mice

have reduced IgG and IgM; blocked B-cell development and reduced mature circulating

B cells,

LIGHT T cells, granulocytes,

monocytes, DCs

Initiator of T-cell costimulation signals causing CTL-mediated tumor and allograft

rejection; a potential target for regulating cellular immunity; ko mice have reduced

proliferative response of Vb8C CD8C to enterotoxin B.

VEGI Endothelial cells Potent inhibitor of endothelial cell proliferation (early G1 arrest) and inducer of

apoptosis, angiogenesis and tumor growth.

GITRL DCs, macrophages, B cells A regulator of regulatory T cells.

RANKL Activated T cells and

osteoblasts

Survival factor for activated DCs; maintenance of immune tolerance; also involved in

osteoclast differentiation and activation, hence crucial for bone homeostasis; potential

therapeutic target in osteoporosis,

osteolytic metastatic cancer, arthritis and periodontitis; mammary gland lactation; ko

mice have retarded growth after weaning, osteoporosis, deficient osteoclasts, defective

tooth eruption and lack lymph nodes, but have normal spleen and Peyer’s patches.

EDA1,2 Skin Hair follicle and sweat gland development; ko mice have no primary hair follicles or

sweat glands and have malformed teeth; they also have hypoplastic hair, teeth and

eccrine sweat glands.aBased on Refs [20–22,72–76].bAbbreviations: APC, antigen-presenting cell; APRIL, a proliferation-inducing ligand; BAFF, B-cell-activating factor; CTL, cytotoxic lymphocyte; DC, dendritic cell; EDA,

ectodermal dysplasin; FDC, follicular dendritic cell; GITRL, glucocorticoid-induced TNFR family receptor ligand; IL, interleukin; ko, gene-deficient or gene knock-out; LIGHT,

lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by

T lymphocytes; LPS, lipopolysaccharide; LT, lymphotoxin; RANKL, receptor activator of nuclear factor-kB ligand; TNF, tumor necrosis factor; TNFR, tumor necrosis factor

receptor; TRAIL, TNF-related apoptosis-inducing ligand; TWEAK, TNF-like weak inducer of apoptosis; VEGI, vascular endothelial cell growth inhibitor.

Review TRENDS in Immunology Vol.25 No.10 October 2004558

number of apoptotic bodies are present in diseasedglomeruli [24]. CD40L deficiency impairs CD4C T-cellpriming, follicular dendritic cell (FDC) differentiation,germinal center formation and class switching. TheIgM-expressing cells cannot undergo isotype conversion toIgG expression, leading to hyper-IgM syndrome [25]. C1qknockout mice also show significantly reduced productionof T-cell-dependent antigen-specific IgG2a and IgG3isotypes owing to reduced interferon-g production byT cells [26]. C1q therefore might be crucial to antigendelivery to FDCs and subsequent generation of a normalsecondary antibody response. Furthermore, mutationsin FasL are known in patients with autoimmune lympho-proliferative syndrome, which causes a lack of

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apoptosis, massive lymphoid hyperplasia and autoanti-body production [27].

C1q and TNF-a are known to be produced in response toinfection as inducers of proinflammatory activators [28].Curiously, C1q has also been shown to suppress LPS- andCpG-induced interleukin-12 p40 and TNF-a production inbone-marrow-derived DCs [29]. As a regulator of LPS andthe TLR pathway, C1q inhibits the LPS-induced MyD88-dependent pathway, leading to reduced NF-kB activityand delayed phosphorylation of MAPK [29]. Adiponectincan also suppress mature macrophage function by sig-nificantly inhibiting their phagocytic activity and theirLPS-induced production of TNF-a, and thus might resolveinflammation [30]. C1q is also known to be

Review TRENDS in Immunology Vol.25 No.10 October 2004 559

antiproliferative (G1 mitotic arrest) and proapoptoticfor human fibroblasts [31]. However, this effect of C1q,which involves p38 MAPK, is mediated by the collagenregion and not the gC1q domain [31]. TNF-relatedapoptosis-inducing ligand (TRAIL) also inhibits cell-cycle progression by T cells and mediates thymocyteapoptosis, and hence is important in the induction ofautoimmunity [32].

Adiponectin has been shown to reverse insulin resist-ance associated with obesity by decreasing triglyceridecontent in the muscle and liver of obese mice [33,34]. Thiseffect is mediated by activation of AMP-activated proteinkinase (AMPK) and phosphorylation of acetyl-CoA car-boxylase (ACC). Decreased adiponectin has been impli-cated in the development of insulin resistance in mousemodels of obesity and type 2 diabetes [35]. A collagen-freeform of ACRP30 has been shown to be present in serum,suggesting that full-length adiponectin can undergoproteolytic processing [33]. Interestingly, the gC1qdomain of adiponectin can ameliorate hyperglycemia andhyperinsulinemia much more potently than full-lengthadiponectin [33]. TNF-a, a major secretory product ofadipocytes, induces insulin resistance via NF-kB byinterfering with an insulin-signaling mechanism: itinhibits tyrosine kinase activities of the insulin receptorand serine phosphorylation of insulin receptor substrate 1[36]. TNF-a also contributes to insulin resistance byreducing the secretion of adiponectin by adipocytes [36].By analogy, a set of hibernation proteins are downregulatedin the serum of hibernating chipmunk, suggesting a rolein energy homeostasis [37].

Analogous to the structural role of collagen VIII, a mildautosomal disorder associated with growth plate abnor-malities, called ‘Schmid metaphyseal chondrodysplasia’,has been associated with missense mutations in the gC1qdomain of collagen X which disrupt the hydrophobic coreand perturb trimer assembly [16]. The receptor activatorof NF-kB ligand has been linked with an autosomaldominant bone disease, ‘familial expansile osteolysis’,characterized by increased bone remodeling. Anothergenetic disease, called ‘hypohydrotic ectodermal dys-plasia’, which is linked to ectodermal dysplasin (EDA),affects ectodermal tissue [22]. EDA is also required for thedevelopment of fish scales, suggesting a highly conservedfunction through evolution, reminiscent of saccularcollagen, which probably has a vestibular function in theinner ear of the bluegill sunfish [38]. A Ser163 mutation toArg163 in the gC1q domain of another C1q family member,CTRP5, has recently been associated with late-onsetretinal degeneration [39]. The mutation causes formationof higher-order aggregates of the CTRP5 molecule, whichleads to impairment of adhesion between retinal pigmentepithelium (RPE) and Bruch’s membrane [39]. It is thusevident that the members of the C1q and TNF familieshave certain overlapping functions. This heralds excitingtimes ahead in the field, which could have significantimplications for human health and disease.

Perspectives and concluding remarks

Members of the C1q and TNF superfamily are active asself-assembling non-covalent trimers whose individual

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chains fold as compact ‘jelly-roll’ b-sandwiches. Thetopology and hydrophobic character of this b-core areconserved throughout both families. Sequence homologywithin the two families is relatively modest overall(w20–30% within the TNF family) but is high within thestructurally conserved hydrophobic core and for residuesresponsible for trimer assembly through conservedhydrophobic surfaces. Sequence homology between thetwo families is low to the point of being undetectable. Theinternal structural homology is not mirrored on thesurface of the proteins, with markedly different sequencesand structures throughout both families in the loopswhich connect the conserved framework. In the TNFside of the superfamily, these differences give rise todiverse regions of contact between TNF and TNFR(3:3 stoichiometry) and contribute to the specific inter-action with TNFR [20,40,41]. Such modes of ligand–receptor interaction have not been reported for the C1qfamily. Within the C1q family, the diversity of sequenceand structure in the protruding loops, and hence ofC1q–ligand interactions, is exemplified by the variabilitywithin C1q itself [3,11].

Although the overall trimeric structure appears to becentral to the function of the TNF family, it is not yet clearthat this is necessarily the case throughout the C1qfamily. It is also not known if any of the C1q family trimersengage the TNFR. The TNFR crossutilization (19 TNFligands signal through 29 TNFRs) and crosstalk, whichsuggest an overlap in cell-signaling pathways, probablyoffer certain clues to future research aimed at the C1qfamily. The most ‘respected’ receptor for C1q, the cal-reticulin-CD91 complex, operates via the collagen regionof C1q [42]. This interaction enhances p38 MAPKactivation, NF-kB activity and production of proinflam-matory cytokines and chemokines in macrophages [42].Paradoxically, reduced NF-kB activity has been shown tobe mediated by both gC1q and collagen regions using DCs[29], which points at the existence of a gC1q-specificreceptor for C1q. The dichotomy of the gC1q–receptorinteraction is further highlighted by two low- and high-affinity receptors for gC1q and full-length moleculesof ACRP30, AdipoR1 and AdipoR2, which can mediategC1q-stimulated AMPK, p38 MAPK activation and ACCphosphorylation [43]. Interestingly, AdipoR1 and AdipoR2can form both homo- and heteromultimers, reminiscentof certain TNFRs [43].

Irrespective of the possibility of an evolutionary linkbetween C1q and TNF families [5,20,41], the formation ofa trimer with the ability to participate in protein–proteininteractions through diverse external loops is well servedby the adoption of a common core framework. The ten-stranded b-sandwich core of the THD and gC1q domainsprovides not only the scaffolding for variable protrudingloops and diverse functions but also the hydrophobiccharacter required for formation and stabilization of thetrimer. Given the involvement of the members of the C1qand TNF superfamily in such broad and diverse processes,it is tempting to envisage certain common themes thatmight unite their actions in different tissues, therebyjustifying the evolutionary success of this superfamily.The TNF ligand family is well characterized in terms of

Review TRENDS in Immunology Vol.25 No.10 October 2004560

receptor interaction, signal transduction pathways andeffector and regulatory mechanisms. The C1q familyseems to be a growing branch of the C1q and TNFsuperfamily tree, which requires rigorous dissection of itsstructure–function relationships and signaling pathways:clearly an area to watch in coming years. It is likely thatthe TNF family became more specialized and expandedduring the process of orchestrating adaptive immunity[44], whereas C1q remained a versatile, scavenging innateimmune molecule on the fringes of adaptive immunity.Whether an innate immune molecule like C1q has aTNF-related function, remains to be addressed.

AcknowledgementsWe are funded by the European Commission (U.K., P.W., K.B.R.), theGerman National Genome Network (U.K.), the Alexander von HumboldtFoundation (U.K.), the Medical Research Council (K.B.R., R.B.S.), theWellcome Trust (A.K.S., T.J.G.), the Commissariat a l’Energie Atomique,the CNRS and the Universite Joseph Fourier, Grenoble (C.G., G.J.A.). Wethank Rohit Ghai for his comments on the manuscript. We sincerelyregret being unable to cite many seminal contributions due to restrictionson the number of references.

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