C1q and its growing family

ARTICLE IN PRESS Immunobiology 212 (2007) 253–266 www.elsevier.de/imbio C1q and its growing family Rohit Ghaia, Patrick Watersb, Lubka T. Roumeninac, Mihaela Gadjevad, Mihaela S. Kojouharovac, Kenneth B.M. Reide, Robert B. Sime, Uday Kishoref, a Institute of Medical Microbiology, Justus-Liebig-University, Frankfurter Strasse 107, 35392 Giessen, Germany Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, UK c Department of Biochemistry, Sofia University, St. Kliment Ohridski, 8 Dragan Tsankov St., Sofia 1164, Bulgaria d Brigham and Women’s Hospital, Department of Pathology, Harvard Medical School, Boston, MA 02115, USA e Medical Research Council Immunochemistry Unit, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK f Laboratory of Human Immunology, CCCB/Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge UB8 3PH, West London, UK b Received 7 September 2006; accepted 1 November 2006 Abstract 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 self and non-self ligands via its heterotrimeric globular (gC1q) domain and thus trigger the classical pathway. The trimeric gC1q signature domain has been identified in a variety of non-complement proteins that can be grouped together as a C1q family. The X-ray crystal structures of the gC1q domain of a few members of the C1q family reveal a compact jelly-roll b-sandwich fold similar to that of the multifunctional tumor necrosis factor (TNF) ligand family, hence the C1q and TNF superfamily. This review is an update on the structural and functional aspects of the gC1q domain of human C1q. We also mention the diverse range of proteins that utilize a gC1q domain in order to reflect on its importance as a versatile scaffold to support a variety of functions. r 2006 Elsevier GmbH. All rights reserved. Keywords: Complement; C1q; Module; Globular domain; Immunity; Evolution Introduction Abbreviations: CBLN, cerebellin; CRP, C-reactive protein; EMILIN, elastin microfibril interface located protein; gC1q, globular domain of C1q; ghA, ghB and ghC, recombinant form of the carboxylterminal globular head modules of C1q A, B and C chains, respectively; PTX3, long prototypical pentraxin 3; THD, TNF homology domain; TNF, tumor necrosis factor Corresponding author. Tel.: +44 1895 266362; fax: +44 1895 274348. E-mail address: [email protected] (U. Kishore). 0171-2985/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.imbio.2006.11.001 C1q is the target recognition protein of the classical complement pathway that is crucial for the clearance of pathogens and apoptotic cells (Kishore and Reid, 2000). It is involved in a number of immunological processes such as phagocytosis of bacteria, neutralization of retroviruses, cell adhesion, modulation of dendritic cells (DC), B cells and fibroblasts, and maintenance of immune tolerance via clearance of apoptotic cells (Kishore et al., 2004b). In addition to binding ARTICLE IN PRESS 254 R. Ghai et al. / Immunobiology 212 (2007) 253–266 antigen-bound IgG and IgM, C1q can engage a variety of self and non-self ligands including envelope proteins of certain retroviruses, b-amyloid fibrils, lipopolysaccharides (LPS), porins from Gram-negative bacteria, phospholipids, and some acute phase reactants including pentraxins (Kishore et al., 2004a; Kishore et al., 2004b). However, how C1q manages to specifically bind to a large number of structurally diverse self and nonself targets continues to puzzle us. C1q has a hexameric structure that looks like a bouquet of tulips under electron microscopy. It contains an N-terminal collagen-like region and a C-terminal globular (gC1q) domain (Kishore and Reid, 1999). The gC1q domain, which is the main ligand recognition domain of C1q, has a heterotrimeric structure, which is composed of the C-terminal halves of the A (ghA), B (ghB) and C (ghC) chains (Kishore and Reid, 2000). Despite various attempts, the residues involved in gC1q–ligand interactions have not been defined due to technical difficulties in expressing the heterotrimeric gC1q domain in a recombinant form. However, the availability of recombinant forms of the individual modules (ghA, ghB, and ghC) for mutational studies and X-ray crystal structures for in silico analysis has taken us closer to identifying the binding surfaces and the specific residues involved. The expression and functional characterization of individual modules have revealed structural and functional autonomy within the gC1q domain in terms of preferentially binding a diverse range of C1q ligands (Kishore et al., 1998; Kishore et al., 2003). C1q interacts with charged polyampholytic proteins, such as IgG, C-reactive protein (CRP) and the gC1q receptor (gC1qR), as well as with hydrophobic ligands (b-amyloid peptide and fibrils, LPS and viral proteins such as gp41of HIV-1)(Kishore et al., 2004b). A charge pattern recognition mechanism has been proposed for C1q, but a discrete pattern has not been conclusively established. It is also not clear how many binding sites exist on the gC1q domain, whether they overlap, and if conformational changes are required for specific binding or activation. Recent studies involving structural aspects of the gC1q domain (Gaboriaud et al., 2004) and a series of mutational studies using recombinant forms of individual globular head modules have enhanced our understanding of the C1q–ligand interaction (Kojouharova et al., 2004; Roumenina et al., 2005, 2006; Zlatarova et al., 2006). The identification of a diverse range of proteins that contain a gC1q signature domain, belonging to the C1q family, has triggered a number of interesting studies involving the gC1q domain (Kishore et al., 2004a; Kishore et al., 2004b). This review is an update on the structure–function studies on human C1q following the last C1 Mainz meeting (2002), with additional information on the evergrowing C1q family. Modular organization of the gC1q domain Studies using recombinant ghA, ghB, and ghC modules have suggested that each of the three modules of the gC1q domain of C1q bind different ligands independently (Kishore et al., 1998, 2003; Kojouharova et al., 1998; Roumenina et al., 2006). The X-ray crystal structure, which has been of immense value in designing mutational studies, shows that the gC1q domain has a compact, spherical heterotrimeric organization with a non-crystallographic pseudo-three-fold symmetry (Fig. 1(A) and (B)). Each of the individual globular head modules, with their N- and C-termini emerging at the base of the trimer, has a jelly-roll topology consisting of a 10-stranded b-sandwich made up of two five-stranded anti-parallel b-sheets (Gaboriaud et al., 2003). The gC1q is held together predominantly by non-polar interactions, with contributions from a series of interactions along its three-fold axis that include hydrogen bonds, a well-exposed Ca2+ ion located near the apex and main-chain polar interactions. Additional lateral interactions, which are hydrophobic at the base, and polar and hydrophilic towards the apex, further stabilize the heterotrimeric assembly. The three modules within gC1q (A, B, and C) show clear differences in their electrostatic surface potentials. The modules ghA and ghC both show a combination of basic and acidic residues scattered on their external face, whereas module ghB shows a predominance of positive charges, especially a continuous patch of arginine residues (ArgB101, ArgB114, and ArgB129), that have been implicated in the C1q–IgG interaction (Gaboriaud et al., 2003; Kojouharova et al., 2004). Molecular modeling, also based on the crystal structure of the gC1q of human C1q, has revealed that within the ghB module, which is the most accessible of the three modules due to its outward-facing equatorial position, ArgB129 and GluB162 seem central to the C1q–IgG interaction, with additional ionic interactions provided by ArgB114 and ArgB161 (Fig. 1C). The modeling has also proposed that TyrB175 and LysA200, located at the apex of the heterotrimer, can potentially form complementary CRP binding sites. Thus, the autonomous character of the individual modules, with different surface charge patterns and spatial orientations, gives flexibility and versatility of the ligand recognition to the gC1q domain (Kishore et al., 2004a, b). Flexibility within the gC1q modules during C1q–ligand recognition The structural modeling proposes a predominant role for the ghB module in the C1q–IgG interaction (Gaboriaud et al., 2003). It suggests that after the initial ARTICLE IN PRESS R. Ghai et al. / Immunobiology 212 (2007) 253–266 255 Fig. 1. The gC1q domain. Two different views of the compact, spherical heterotrimeric gC1q domain are shown (ghA blue, ghB brown, ghC green): side view (A), top view (B). The red sphere indicates the calcium ion located at the apex of the domain. Residues described in the text as considered important for the C1q–ligand interactions are explicitly labeled (C, D). Some residues are labeled with a yellow background for improved visibility only. The figure was generated using PyMOL. electrostatic attraction to the apex, the two molecules align allowing Asp270 and Lys322 of IgG to form salt bridges with Arg129 and Glu162 of the ghB, respectively, with Arg114 and ArgB161 stabilizing the complex. In this orientation, the Arg129 appears to act like a wedge between the CH2 and light chain constant domains. Therefore, the Fab/Fc orientation may be a critical factor in dictating access of the ghB module to the CH2 domain (Gaboriaud et al., 2003). Variations at this position in the different IgG subtypes may explain their differential ability to fix complement. Mutational studies have also identified a number of amino acid residues (ArgA162, ArgB114, HisB117, ArgB129, ArgB163, and ArgC156) on the gC1q domain as important for IgG binding (Fig. 1(C) and (D)) (Kojouharova et al., 2004). The flexibility and diversity of C1q binding is further highlighted by the interaction of the gC1q domain with CRP, a major acute phase reactant that binds, via the face of its pentameric ring, to the phosphocholine component of membrane phospholipid (Kishore et al., 2004b). The overall dimensions of the C1q and CRP molecules appear to suggest that only one gC1q can bind to each CRP pentamer through one of the five available binding sites on CRP. This interaction involves a series of residues including CRP Glu88, Asp112 and Tyr175, and Lys114 from a neighboring subunit (Agrawal et al., 2001). Molecular modeling has revealed a shape complementarity between the top of the gC1q and the central pore of the pentameric CRP ring, suggesting a central role for ghA, ghB, and CRP Asp112 and Tyr175 in this interaction (Gaboriaud et al., 2003). Therefore, the organization of the modules within the gC1q domain allows recognition of different ligands via different faces, by two or even three subunits, thus providing a structural basis for its versatile recognition properties. Mutagenesis data (Roumenina et al., 2006) revealed that residues both from the top of gC1q trimer (with participation of all three chains) as well as the equatorial surface of the B-chain are important for the interaction with CRP. ARTICLE IN PRESS 256 R. Ghai et al. / Immunobiology 212 (2007) 253–266 Identification of APO (without calcium) and HOLO (with calcium) planes within the gC1q structure signal transmission from the gC1q domain to the serine protease complex, located in the collagen-like region. Recently, we have found that the exposed Ca2+ within the gC1q heterotrimer primarily influences the target recognition properties of C1q towards IgG, IgM, CRP, and pentraxin 3 (PTX3) (Roumenina et al., 2005). In silico modeling of the gC1q domain at pH 7.4 indicates that the loss of Ca2+ leads to changes in the direction of the electric moment from a co-axial position in the calcium-saturated HOLO form (towards the gC1q apex) to one perpendicular to the molecular axis in the calcium-depleted APO form (towards equatorial side of the B chain). Thus, two planes – normal to the electric moment vectors in the HOLO form (the HOLO plane) and in the APO form (the APO plane) – can be defined with potential importance for target recognition (Fig. 2). An analysis of ghA, ghB, and ghC sequences individually, using a computer program called ConSurf (Glaser et al., 2003), has identified a number of functionally critical residues that are highly variable and map within the potential binding area on the gC1q crystal structure (Kishore et al., 2004b). Several of these highly variable residues can also be mapped on these two planes. Strikingly, all residues that have been experimentally demonstrated to be important for the target-binding via side-directed mutagenesis (Kojouharova et al., 2004; Roumenina et al., 2005, 2006; Zlatarova et al., 2006), as well as proposed by molecular modeling (Gaboriaud et al., 2003) can be mapped on to these two planes. The calcium-controlled reorientation of the electric moment vector and the initial target recognition by the HOLO plane, followed by rotation towards the APO plane are proposed to be key steps in complement activation C1q–IgM interaction involves rotation around ArgB108–ArgB109 axis Mutational studies using recombinant ghA, ghB, and ghC modules and a comparison of their interactions with IgG and IgM have suggested that the IgG- and IgM-binding sites within the gC1q domain are different but may overlap (Zlatarova et al., 2006). Whereas ArgB108, Arg B109, and TyrB175 are the critical IgM binding site residues, ArgB114, ArgB129, ArgB163, and HisB117 have been shown to be central to IgG binding (Kojouharova et al., 2004), but are not important for the C1q–IgM interaction. Given the location of ArgB108, ArgB109, and TyrB175 in the gC1q crystal structure, it is likely that C1q interacts with IgM via the top of the gC1q domain (Fig. 1). Residues ArgB108 and ArgB109, which belong to both the APO and HOLO planes of the C1q molecule, are keen candidates for participation in the recognition of IgG, IgM and other targets. ArgB108 is more important for IgG binding, while the contributions of ArgB108 and ArgB109 to the C1q–IgM binding are comparable. Most likely these residues are important for the initial stage of IgG as well as IgM recognition. TyrB175 is most likely involved in the formation of the IgM binding site of C1q, while it contributes minimally to IgG binding. TyrB175 can participate in both hydrogen bonding via its hydroxyl group and hydrophobic/stacking interactions via its aromatic ring. It probably forms a hydrogen bond with a complementary partner from IgM (Zlatarova et al., 2006). Thus, the residues that have been implicated previously in IgG Fig. 2. Our recent model involving the APO and HOLO planes of the gC1q trimer at the centee of C1q–ligand interaction. C1q in serum is present in the Ca2+-bound (HOLO) form and its molecular electrical moment is directed toward the apex of the gC1q heterotrimer. In the initial phase of the C1q–target interaction, when bound to C1q, Ca2+ facilitates recognition of negatively charged molecules; the negative field of the target molecule accelerates calcium removal from C1q (APO); and this leads to rotation of the gC1q domain around the ArgB108–ArgB109–AsnB104 axis and a shift in the angle between adjacent collagen arms. The two APO and HOLO planes appear to contain most of the residues that have been shown to be crucial for C1q–ligand interaction via crystal structure, mutational, and computational analyses. ARTICLE IN PRESS R. Ghai et al. / Immunobiology 212 (2007) 253–266 binding (ArgB114, HisB117, ArgB129, and ArgB163) (Kojouharova et al., 2004) do not seem important for the C1q–IgM interaction. Therefore, the charged residues from the apex of the heterotrimeric gC1q (ArgB108, ArgB109, and TyrB175 participate in the recognition of both IgG and IgM, while ArgB114, ArgB129, ArgB163, and HisB117 are exclusively involved in IgG binding. TyrB175 is involved in the C1q–CRP interaction, and LysB136, which is located near the collagen arm, seems to be quite important for C1q binding to IgG, IgM, CRP, and PTX3 (Roumenina et al., 2005; Roumenina et al., 2006). In addition to the location of the residues thought to be important in binding IgM, its overall structure suggests that it is very unlikely that the top as well as the side surfaces of the gC1q domain is engaged in IgM binding simultaneously. It has been recently considered that at the very first stage of the recognition of IgG and IgM, C1q uses the apex, where ArgB108, ArgB109, and TyrB175 are located (Zlatarova et al., 2006). The differences in binding the two immunoglobulins appear later. Following the rotation of the gC1q due to Ca2+ release (Roumenina et al., 2005), Asp270 and Lys322 of IgG form salt bridges with ArgB129 and GluB162, respectively, with additional ionic interactions provided by ArgB114 and ArgB161. However, the geometry of IgM, which is very different from that of IgG, does not allow the involvement of residues from the side surface of the B chain during C1q–IgM interaction. Previously, Perkins et al. (1991) proposed a model to describe the C1q–IgM interaction. According to their model, two adjacent heads of C1q are involved in the interaction with two Cm3 domains on two adjacent subunits of IgM, when C1q is in an active ‘‘closed’’ conformation (angle between adjacent collagenous arms being 281). The open conformation of C1q, with angle of 40–451 is thought to be non-active, since the C1q heads are too far apart for two adjacent C1q heads to attach to the Cm3 domains on adjacent subunits in IgM. However, according to Gaboriaud et al., C1 in the closed conformation is non-active while it is complement activating in the open conformation (Gaboriaud et al., 2003; Gaboriaud et al., 2004). Our data and proposed model (Zlatarova et al., 2006) are consistent with Gaboriaud et al. We propose that two or three nonadjacent heads, in the closed (non-active) conformation, have to be involved in binding Cm3 domains of IgM. The binding aids Ca2+ release, which causes a reorientation of the electric moment of gC1q from apical to equatorial. This electrical change induces a rotation of the gC1q heads and an increase in the angle between the collagen arms from 281 to 451. In order to shift towards the active (open) conformation, without disengaging from the target, the gC1q heads have to rotate on the ArgB108–ArgB109 axis. We believe that the changes brought about by this rotation activate complement. Thus, ArgB108 and ArgB109 are likely to be central to the 257 initial recognition process involving both IgG and IgM, and form an axis for the rotation of the gC1q domains while maintaining target interaction (Zlatarova et al., 2006). C1q as a prototypical molecule of a novel C1q family In the human genome, the 32 proteins that possess a gC1q domain (140 residues) constitute the novel C1q family (Tables 1 and 2) (Kishore et al., 2004a, b). Examples of members of this family, which has expanded through gene duplication, are depicted in Fig. 3 in order to highlight the multiplicity of functions and domains with which the gC1q domain has been associated. There is a wide range of sequence similarity between the gC1q domains of the members, ranging from a low 21% (between C1QA and Multimerin 1) to a high 96% (between C1QTNF9 and LOC387911). Nearly all gC1q domains contain eight invariant residues (Fig. 4). These residues are critical in maintaining the structural integrity of the gC1q domain. Phylogenetic analysis of protein sequences of the gC1q domain suggests two distinct families (Fig. 4). The larger family comprises two groups which are indicated as C1q-like (because its members include C1QA, C1QB, and C1QC) and cerebellin-like (because of the presence of CBLN1, CBLN2, CBLN3, and CBLN4). All collagens (COL8A1, COL8A2, and COL10A1) as well as adiponectin belong to the C1q-like subgroup. The C1QL1, C1QL2, C1QL3, and C1QL4 proteins also belong to this sub-group. The smaller family, including EMILINs and multimerins, is distinct from the rest. There are some interesting cases, for instance LOC387911 possesses a slightly truncated gC1q Cterminus while C1QTNF4 has two complete gC1q domains. The entire gC1q domain containing genes is listed in Table 1, while the known functions of family members are described in Table 2. The genes coding for the majority of the C1q family members do not form clusters and are predominantly scattered singly through out the genome. The phylogenetic tree shown in Fig. 5, however, provides an indication of the level of similarity between them. There are a couple of examples of gene clusters, which includes the genes C1QA, C1QB, and C1QC that are clustered together on the locus 1p36, and C1QTNF9 and LOC387911 at locus 13q12.12. The C1QTNF9 and LOC387911 genes are separated by just two genes and the gC1q domains of these proteins are almost identical to each other (Fig. 5). There are also examples of other proteins with almost identical gC1q domains that are not clustered. Interestingly, there is a pseudogene positioned adjacent to gene LOC650568 (similar to a1 type VIII collagen precursor) ARTICLE IN PRESS 258 Table 1. R. Ghai et al. / Immunobiology 212 (2007) 253–266 List of human C1q domain family members Gene id Official gene symbol Description Gene aliases Location RefSeq mRNA RefSeq protein Mouse homolog 9370 ADIPOQ ACDC; ACRP30 3q27 NM_004797 NP_004788 Adipoq 712 C1QA 1p36.12 NM_015991 NP_057075 C1qa 713 C1QB 1p36.12 NM_000491 NP_000482 C1qb 714 C1QC C1QG; C1q-C 1p36.11 NM_172369 NP_758957 C1qc 65,981 10,882 C1QDC1 C1QL1 EEG1; EEG-1 CRF; C1QRF 12p11 17q21 NM_001002259 NM_006688 NP_001002259 NP_006679 C1qdc1 C1ql1 165,257 C1QL2 2q14.2 NM_182528 NP_872334 C1ql2 389,941 C1QL3 C1ql; K100 10p13 NM_001010908 NP_001010908 C1ql3 338,761 C1QL4 MGC131708 12q13.12 NM_001008223 NP_001008224 C1ql4 114,897 C1QTNF1 GIP; CTRP1; 17q25.3 NM_030968 NP_112230 C1qtnf1 114,898 C1QTNF2 CTRP2; zacrp2 5q33.3 NM_031908 NP_114114 C1qtnf2 114,899 C1QTNF3 NM_030945 NP_112207 C1qtnf3 C1QTNF4 11q11 NM_031909 NP_114115 C1qtnf4 114,902 C1QTNF5 CTRP3; Cors; CORS26; Corcs CTRP4; ZACRP4 CTRP5; LORD 5p13 114,900 11q23.3 NM_015645 NP_056460 C1qtnf5 114,904 C1QTNF6 114,905 C1QTNF7 390,664 C1QTNF8 338,872 C1QTNF9 869 147,381 140,689 1300 CBLN1 CBLN2 CBLN4 COL10A1 1295 1296 COL8A1 COL8A2 Adiponectin, C1Q and collagen domain containing Complement component 1, q subcomponent, A chain Complement component 1, q subcomponent, B chain Complement component 1, q subcomponent, C chain C1q domain containing 1 Complement component 1, q subcomponent-like 1 Complement component 1, q subcomponent-like 2 Complement component 1, q subcomponent-like 3 Complement component 1, q subcomponent-like 4 C1q and tumor necrosis factor related protein 1 C1q and tumor necrosis factor related protein 2 C1q and tumor necrosis factor related protein 3 C1q and tumor necrosis factor related protein 4 C1q and tumor necrosis factor related protein 5 C1q and tumor necrosis factor related protein 6 C1q and tumor necrosis factor related protein 7 C1q and tumor necrosis factor related protein 8 C1q and tumor necrosis factor related protein 9 Cerebellin 1 precursor Cerebellin 2 precursor Cerebellin 4 precursor Collagen, type X, alpha 1 (Schmid metaphyseal chondrodysplasia) Collagen, type VIII, alpha 1 Collagen, type VIII, alpha 2 11,117 84,034 EMILIN1 EMILIN2 131,149 387,911 LOC131149 LOC387911 650,568 LOC650568 22,915 MMRN1 79,812 MMRN2 643,866 UNQ755 CTRP6; ZACRP6 CTRP7; ZACRP7 UNQ5829 22q13.1 NM_031910 NP_114116 C1qtnf6 4p16-p15 NM_031911 NP_114117 C1qtnf7 16p13.3 NM_207419 NP_997302 None MGC48915 13q12.12 NM_178540 NP_848635 C1qtnf9 16q12.1 18q22.3 20q13.13-q13.33 6q21-q22 NM_004352 NM_182511 NM_080617 NM_000493 NP_004343 NP_872317 NP_542184 NP_000484 Cbln1 Cbln2 Cbln4 Col10a1 3q12.3 1p34.2 NM_001850 NM_005202 NP_001841 NP_005193 Col8a1 Col8a2 2p23.3-p23.2 18p11.3 NM_007046 NM_032048 NP_008977 NP_114437 Emilin1 Emilin2 3q26.1 13q12.12 XM_067228 NM_001007537 XP_067228 NP_001007538 Gm414 None 22q11.21 XM_939652 XP_944745 None 4q22 NM_007351 NP_031377 Mmrn1 10q23.2 NM_024756 NP_079032 Mmrn2 14q12 NM_001039771 NP_001034860 Cbln3 CBLNL4 MGC9568 FECD; PPCD; PPCD2 Elastin microfibril interfacer 1 gp115; EMILIN Elastin microfibril interfacer 2 FOAP-10; EMILIN-2 Similar to otolin-1 LOC131149 Similar to hypothetical LOC387911 protein MGC48915 Similar to alpha 1 type VIII LOC650568 collagen precursor Multimerin 1 ECM; MMRN; GPIa*; EMILIN4 Multimerin 2 EMILIN3; ENDOGLYX1 Cerebellin LOC643866, CBLN3 The table provides several useful standard database identifiers (Entrez GeneID, RefSeq mRNA, and RefSeq protein) that may be used to gather more information about these genes. The known mouse homolog of each gene is also indicated. There are three genes in the gC1q family that are not detected in the mouse (LOC387911, C1QTNF8, and LOC650568). The C1QDC1 in humans is the only gC1q domain family member without a predicted signal peptide. ARTICLE IN PRESS R. Ghai et al. / Immunobiology 212 (2007) 253–266 Table 2. 259 Functions of C1q domain family members Protein Tissues of origin and presentation Function References ADIPOQ (adiponectin) Serum protein produced exclusively by adipocytes: 5–10 mg/ml Berg et al. (2001, 2002); Scherer et al. (1995); Yokota et al. (2000) C1q (C1QA, C1QB, C1QC) Serum protein produced by liver: 70 mg/ml EMILIN-1 Connective tissue, blood vessels, skin, heart, lung, kidney, cornea, small intestine, aorta, uterus and appendix, fetal heart and lungs EMILIN-2 Peripheral leukocytes, placenta, spinal cord, fetal heart and lung, major component of cochlear basilar membrane in mouse Multimerin 1 Platelet a granules, endothelial cell Weibel–Palade bodies, placenta, lung and liver, platelets, megakaryocytes, endothelium and subendothelium of blood vessels Blood vessel endothelial cell surface including capillaries, veins, arterioles and muscular arteries Major component of the basement membrane of the corneal endothelium (Descemet’s membrane); optic nerve, aorta, umbilical cord and tissues undergoing remodeling; calvarium, eye and skin; produced by endothelial cells Anti-diabetic and anti-atherogenic adipokine; energy homeostasis, lipid and carbohydrate metabolism and sensitivity to insulin; levels are reduced in obesity, insulin resistance and type II diabetes; suppression of inflammation, angiogenesis and fibrosis First subcomponent of classical pathway; immune tolerance; microbial clearance; cell adhesion; linking innate and acquired immunity Extracellular matrix glycoprotein of elastic fiber; supports adhesion of smooth muscle to elastic fibers, formation of elastic fibers, and regulation of vessel assembly; involved in b1-integrin-dependent cell adhesion Development of cochlear basilar membrane and heart chambers; matrix for assembly of other components of basement membrane; differentiation of chicken embryo aorta cells in culture Possible role in platelet factor V storage and stability. Supports adhesion of various cell lines via an RGDS motif Multimerin 2 Collagen alpha VIII (COL8A1, COL8A2) Collagen X (COL10A1) Hypertrophic chondrocytes in the epiphyseal growth plate of long bones, ribs, and vertebrae during endochondral bone formation, bone fracture callus, and osteoarthritic cartilage Gaboriaud et al. (2004); Kishore et al. (2004b); Kishore and Reid (2000) Doliana et al. (1999); Spessotto et al. (2003) Doliana et al. (2001) Hayward et al. (1995) Possible role in vasculogenesis, angiogenesis, hemostasis and cell–matrix adhesion Christian et al. (2001) Component of subepithelial and subendothelial extracellular matrices; required for normal anterior eye development; maintenance of integrity of blood vessels, vascular tissue development and remodeling; cell migration, plaque stability and thrombus organization; generation of a peri- or subcellular matrix environment that stimulates cell proliferation 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 Greenhill et al. (2000); Kvansakul et al. (2003); Muragaki et al. (1991); Plenz et al. (2003); Yamaguchi et al. (1989) Bogin et al. (2002); Dublet et al. (1999); Zhang and Chen (1999) ARTICLE IN PRESS 260 R. Ghai et al. / Immunobiology 212 (2007) 253–266 Table 2. (continued ) Protein Tissues of origin and presentation Function References Cerebellin 1 precursor Highly enriched in postsynaptic structures of cerebellar Purkinje cells in cartwheel neurons of the dorsal cochlear nucleus Albertin et al. (2000); Hirai et al. (2005); Kavety et al. (1994); Mazzocchi et al. (1999); Pang et al. (2000); Urade et al. (1991) C1QDC1 Heart, brain, placenta, lung, liver, muscle, kidney, pancreas, spleen, lymph nodes, thymus, tonsil, PBMCs, bone marrow, fetal liver Brain areas involved in motor function (Purkinje cells of the cerebellum, the accessory olivary nucleus, the pons and the red nucleus), also in several other brain regions Highly expressed in heart; also expressed in placenta, liver, skeletal muscle Precursor for several truncated derivatives, including the hexadecapeptide cerebellin. Role in development of Purkinje cell synapse. Enhances in vitro secretory activity of adrenal; involvement in synapse integrity and plasticity in the cerebellum Inhibitor of growth; cancerprotective role in final stages of erythropoesis Potential role in coordination and regulation of motor control, and/or a basic housekeeping role in neurons Berube et al. (1999) Interacts directly with vasopressin receptor V2; blocks vWF binding to collagen and thus inhibits collageninduced platelet aggregation 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 development of arthritis Formation of an extracellular hexagonal lattice between retinal pigment epithelium and Bruch’s membrane in the retinal epithelium Innamorati et al. (2002); Lasser et al. (2006) C1QL1 C1QTNF1 C1QTNF3 C1QTNF5 Prechondrocytes in developing cartilage; colon, small intestine, placenta, fibroblasts, white adipose tissue, blood and kidney; osteosarcoma, chondroblastoma, and giant cell tumors Retinal epithelium, lung placenta, cerebrum, and liver at chromosome locus 22q11.21, which is annotated as being similar to C1QTNF5. It contains the coding sequence for a gC1q domain. Structural aspects of the gC1q domains Four structures of gC1q domains have been solved by X-ray crystallography: mouse ACRP30 (Shapiro and Scherer, 1998), human collagen X (Bogin et al., 2002), mouse collagen a1 (VIII) (Kvansakul et al., 2003), and human C1q (Gaboriaud et al., 2003) (reviewed in Kishore et al., 2004a, b). Each gC1q domain exhibits a 10-stranded b-sandwich fold with a jelly-roll topology, consisting of two five-stranded b-sheets, each made of anti-parallel strands (Fig. 1(A) and (B)). Each of the conserved residues within C1q family proteins belongs Aerbajinai et al. (2004) Maeda et al. (2001); Schaffler et al. (2003) Hayward et al. (2003) to the hydrophobic core of the gC1q domain. The bstrands are strongly conserved in the different gC1q domains, both with respect to their relative orientation and size. All structures reveal similar tight assemblies with pseudo-three-fold symmetry, resulting in globular, ( in diameter). The Nalmost spherical domains (45–55 A and C-termini of the three subunits emerge on the same side of the trimer and are adjacent to one another. Structural homology is very high between collagen VIII and X, but much lower between distantly related collagen VIII and ACRP30. Trimerization involves a very tight association of the subunits, as judged from total buried surface (Bogin et al., 2002). Near the top of the solvent channel, the collagen X structure has a buried cluster of three Ca2+ ions surrounding the one located on the three-fold symmetry axis of the trimer. It is coordinated by acidic residues and other residues, which together form an intricate ARTICLE IN PRESS R. Ghai et al. / Immunobiology 212 (2007) 253–266 261 Fig. 3. Cartoon depicting the different domains present in gC1q containing proteins in the human genome. Most gC1q domain containing proteins have a collagenous region (typical Gly-X-Y repeats) just before the start of the gC1q domain. The proteins form either homotrimeric or heterotrimeric structures. The presence of such regions possibly helps in assembling the trimeric structure. CBLN1, 2, and 4, C1QDC1 and Multimerin1 however lack any collagenous repeats in their complete sequences. Fig. 4. Multiple alignment of human gC1q domain family members. An alignment of all the 32 human gC1q domains is shown. Eight residues that are completely conserved in all proteins are marked with an * above the alignment. The consensus sequence is shown at the bottom of the alignment, and a conservation graph is also provided. network of ionic bonds that probably contribute to the high stability of the collagen X gC1q trimer (absent from the collagen VIII). Ca2+ appears to have a stabilizing effect in the ACRP30 since the Ca2+-free structure is partly disordered. A single Ca2+ ion, also located at the upper end of the central channel, is present in the C1q structure. In contrast with the Ca2+ cluster of collagen X, the Ca2+ ion of C1q is well exposed to the solvent, and plays a crucial role in ligand recognition and the activation of complement (Roume- nina et al., 2005). The surfaces of the collagen VIII and X structures have three strips, each containing eight aromatic residues partially exposed to the solvent, extending across the shallow grooves between the subunits. In both cases, a detergent molecule is bound near the top of the grooves, suggesting the hydrophobic nature of the strips, which probably initiate supramolecular assembly. The ACRP30 gC1q structure also revealed that the gC1q topology is similar to the conserved C-terminal ARTICLE IN PRESS 262 R. Ghai et al. / Immunobiology 212 (2007) 253–266 Phylogenetic tree of Human C1q domain family members 950 840 Emilins and Multimerins 1000 970 1000 1000 396 495 491 984 Cerebellin-like 432 838 1000 1000 833 518 310 840 1000 434 961 738 998 595 922 937 C1q-like 458 449 574 1000 MMRN2 MMRN1 EMILIN1 EMILIN2 CBLN1 CBLN2 CBLN4 UNQ755-CBLN3 C1QDC1 C1QTNF3 C1QTNF4-domain2 C1QTNF4-domain1 C1QTNF1 C1QTNF6 C1QTNF8 C1QTNF9 LOC387911 ADIPOQ LOC131149 C1QTNF2 C1QTNF7 COL8A2 COL8A1 COL10A1 C1QTNF5 C1QC C1QB C1QA C1QL1 C1QL3 C1QL4 C1QL2 Fig. 5. Phylogenetic tree of human C1q family members. An alignment of all complete 32 gC1q domains found in the human genome was performed using ClustalW. C1QTNF4 is the only protein with two gC1q domains (indicated as domain 1 and 2). The tree shown was built by neighbor-joining with 1000 bootstraps. The numbers shown next to the nodes show the number of times (out of 1000) that the two branches occur together during the bootstrap process. tumor necrosis factor (TNF) homology domain (THD) within the TNF ligand family proteins (Kishore et al., 2004a; Shapiro and Scherer, 1998). Each of the 10 b-strands of ACRP30 can be superimposed with the 10 strands of TNF-a, TNF-b, and the CD40 ligand. The relative positions and lengths of these b-strands are almost identical between ACRP30 and the TNF ligands. The C1q and TNF family proteins, which establish a C1q and TNF superfamily, also have similar gene structures: their gC1q or THD domains are each encoded within one exon, while introns in both families are restricted to respective Nterminal collagen or stalk regions, suggesting divergence from a common precursor molecule of the innate immune system. Domain architecture and evolutionary spread of the C1q family proteins The gC1q domain has an ancient evolutionary history. During the course of time, it has entrenched itself into various functional roles by variation of critical amino acids as well as associating with other domains. However, the distribution of the gC1q domain shows some clear preferences for some taxonomic groups. Notably, the gC1q domain is restricted to metazoans, echinoderms molluscs, and for some unexplained reasons, to some Bacillus species. It is completely absent (or as yet undetected) in plants and fungi. Following the comparative taxonomic occurrence of gC1q and TNF domains (Fig. 6), the simplest domain architectures are ARTICLE IN PRESS R. Ghai et al. / Immunobiology 212 (2007) 253–266 263 Fig. 6. Domain architecture and evolutionary comparisons of gC1q and TNF domain. The distribution and representative domain architectures of the gC1q and TNF domain across various evolutionary taxa have been compared via presence (black) or absence (white) of the domain architecture. TM: transmembrane region; EGF: epidermal growth factor domain; EMI: EMILIN domain, a protein–protein interaction motif which may have a role in the oligomerization of EMILIN-1; RING (Really Interesting New Gene) finger domain probably involved in mediating protein–protein interactions, and has a general E3-ubiquitin ligase activity; BBOX (Bbox type zinc finger domain) domain is generally associated with the RING domain; PRY (associated with SPRY (SPla/RYanodine receptor) domain), PRY, BBOX and RING domains are frequently associated with the pyrin domain (an immunoglobulin like domain), which is a protein–protein interaction motif, belongs to a family comprising death, death effector, and caspase recruitment domains; LRR: Leucine-Rich Repeat domain; CLECT: C-type lectin domain. (Collagen)n: One or more instances of the collagen domain may be present; (CLECT)2: exactly two CLECT domains are present adjacent to each other; 7tm_1: G-protein-coupled receptor domain; Pkinase: serine threonine-type protein kinase. the single gC1q domain itself, or its duplicated version (gC1q+gC1q). The simplest gC1q version is seen in all but Ascidians (e.g., Ciona intestinalis). The most prosperous domain pairing of the gC1q domain has been with the collagen domain, as several instances of such proteins are present across almost all taxa shown. The gC1q domain is also paired to a transmembrane region (with or without collagen), which is similar with the extensive pairing of the related THD with a transmembrane domain. Indeed, this domain architecture (transmembrane+THD) seems to be the most widespread for the THD, but less so for the gC1q domain. The pairing of the EGF domain with the gC1q domain seems to be restricted to birds and mammals only. The most varied domain architectures for the gC1q domain are seen in actinopterygii (ray-finned fishes) and mammals (seven different types, four common to both). While in mammals, the gC1q domain associates with domains like EMI and EGF, which are important for protein–protein interactions in the extracellular milieu; in actinopterygii it is primarily associated with domains important in innate immunity. In birds, the gC1q domain pairs with collagen and the Peptidase_M3 domain, a large family of mammalian and bacterial metallopeptidases that cleave mediumsized peptides. In Bacillus sp., the gC1q domain occurs singly (Rety et al., 2005). The gC1q and TNF domains have been utilized by many different taxa in order to carry out varied functions, highlighting their importance, and that of the C1q and TNF superfamily. However, the gC1q domain has been more promiscuous than the TNF domain when domain association diversity is taken into account. Apart from itself and a transmembrane region, the TNF domain is found, throughout taxa, paired only with a G-protein-coupled receptor domain (7tm_1) or a serine threonine protein kinase domain. Perspectives and concluding remarks It is largely considered that C1q–ligand interactions are ionic in nature; however, the complementary ligandbinding sites on C1q and the mechanisms of interactions are still unclear. Progress has been made in the last few years aided by the solution of the X-ray crystal structure of gC1q domain of human C1q and the availability of the recombinant forms of ghA, ghB, and ghC. Mutational studies have identified a number of residues important for C1q–target interactions that have been corroborated by chemical modification, molecular modeling, bioinformatics, and in silico theoretical calculations of electric moment vectors. Charged residues belonging to the apex of the gC1q heterotrimer (with participation of all three chains) as well as the side ARTICLE IN PRESS 264 R. Ghai et al. / Immunobiology 212 (2007) 253–266 of the ghB seem crucial for C1q binding to ligands, and their contribution to each interaction is different. It is likely that a set of charged residues from the gC1q surface participate via different ionic and hydrogen bonds with corresponding residues from the ligand, instead of forming separate binding sites. C1q is a rigid molecule formed by distinctive independent modules composed mainly of b-sheets. Therefore, great mobility of the individual modules within such a structure is not anticipated. The change in the relative position of the collagen stalk and the gC1q domain, coupled with the loss of the Ca2+ ion, has suggested a possible mechanism of C1 activation. There are sufficient data to suggest that the ligand binding sites can be found on different faces of gC1q that correspond to the specific features of different ligands. It is also becoming clear that there are two common important binding surfaces on the gC1q domain rather than discrete binding sites: the apex of the heterotrimer with the participation of all three chains and the equatorial outward-facing surface of the B chain. We envisage that a set of charged residues from these APO and HOLO planes within the gC1q heterotrimer form different ionic and hydrogen bonds with complementary residues in a manner similar to polyspecific antibodies. The highly variable residues identified by ConSurf that are mapped on to the APO and HOLO planes may allow different members of the C1q family to carry out a diverse range of functions while having a similar overall fold. It is worth speculating, however, that for any one ligand, such as IgM or IgG, several binding mechanisms, probably of varying affinities may exist. The fact that ghA, ghB, and ghC can all bind with apparent varying affinity to several ligands (Kishore et al., 2003) strongly suggests that in the C1q heads, there is more than one mode of binding to several classes of ligands. This may also reflect partially that for complex ligands like IgG, more than one type of binding site for C1q may exist (in different, or perhaps even in the same molecule of IgG). In IgG, the binding site identified by Duncan and Winter (1988) (residues Glu318, Lys320, and Lys322, identified from a murine IgG2b background, but present in all human IgG) is not the C1q binding site in Rituximab, a monoclonal antibody with human IgG1 background, in which positions Asp270, Lys322, Pro329, and Pro331 form the main C1q binding site (Idusogie et al., 2000). Modeling studies can probably predict the best fit binding sites for various ligands, but secondary (not-quite-the-best-fit) sites should perhaps not be ignored. The unfolding of the structural and functional aspects of the gC1q domain of other members of this family appears to highlight the pathological importance of the C1q family. For instance, C1q deficiency is a likely cause of SLE due to an impaired clearance of apoptotic cells (Botto et al., 1998). Adiponectin has been shown to reverse insulin resistance associated with obesity by decreasing triglyceride content in the muscle and liver of obese mice, thus being central to insulin-resistant diabetes (Berg et al., 2001). A mild autosomal disorder associated with growth plate abnormalities, called ‘Schimid’s metaphyseal chondrodysplasia’, has been associated with missense mutations in the gC1q domain of collagen X, which disrupts the hydrophobic core and perturbs trimer assembly (Bogin et al., 2002). A Ser163Arg mutation in the gC1q domain of another C1q family member, CTRP5, has recently been associated with late-onset retinal degeneration (Hayward et al., 2003). Further studies, such as those by Wong et al. (2004) are likely to dissect the clinical importance of the members of C1q family that will lead to a better understanding of the gC1q domains. Acknowledgments The work described herein was made possible by grants from the Deutsche Forschungsgemeinshaft through the Graduate College of Biochemistry of Nucleoprotein Complexes (GK370) at Justus Liebig University, Giessen, Germany and the German National Genome Network (NGFN-2). UK acknowledges financial support from the European Commission, Alexander von Humboldt Foundation and Brunel University. UK is gratetful to KBR for introducing him to the field of Clq and classical pathway. References Aerbajinai, W., Lee, Y.T., Wojda, U., Barr, V.A., Miller, J.L., 2004. 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