# STATE-OF-THE-ART RESEARCH ON C1Q AND THE CLASSICAL COMPLEMENT PATHWAY

EDITED BY: Uday Kishore, Nicole M. Thielens and Christine Gaboriaud PUBLISHED IN: Frontiers in Immunology

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ISSN 1664-8714 ISBN 978-2-88945-058-9 DOI 10.3389/978-2-88945-058-9

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# **STATE-OF-THE-ART RESEARCH ON C1Q AND THE CLASSICAL COMPLEMENT PATHWAY**

### Topic Editors:

**Uday Kishore,** Brunel University London, UK **Nicole M. Thielens,** Institut de Biologie Structurale, France **Christine Gaboriaud,** Institut de Biologie Structurale, France

Model of the human C1q molecule, illustrating its multi-chain organization. It is a surface pattern recognition protein including 6 chains of C1qA (colored in dark blue), 6 of C1qB (in green), 6 of C1qC (in cyan).

The structure of the heterotrimeric recognition globular domain has been solved experimentally by X-ray crystallography. The other collagen-like parts of the molecule have been modelled.

White colour at the middle of the collagen-like stems highlights the approximate location of the effector proteases binding site; magenta spots locate a recognition binding site.

Figure by Christine Gaboriaud

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 derived from self, non-self and altered self via its heterotrimeric globular (gC1q) domain and thus trigger the classical complement 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. C1q circulates in serum as part of the C1 complex, in association with a catalytic tetrameric assembly of two homologous yet distinct serine proteases, C1r and C1s. Binding of C1q to appropriate targets leads to sequential activation of C1r and C1s, the latter being able to cleave complement components C4 and C2 thereby triggering the complement cascade. Activation of the classical pathway plays an important role in innate immune protection against pathogens and damaged elements from self. However, its involvement has been shown in various pathologies including ischemia-reperfusion injury and hereditary angioedema. Unexpected roles for the classical pathway have also been discovered recently, linked to both physiological and pathological aspects of development, including brain and cancer cells. These new perspectives should arouse renewed interest in a search for specific inhibitors of the classical pathway. In addition, C1q has recently been shown to have a number of functions that are independent of the activation of the classical pathway.

This research topic is aimed at providing a state-of-the-art overview of the classical pathway, including, but not restricted to emerging functions of C1q and of the C1 complex, as well as pathological consequences of C1 activation or of the presence of anti-C1q autoantibodies. Contributions are included in the areas such as structural basis of C1q ligand recognition, C1q family proteins, inhibitors of the classical pathway identified in pathogens and improved derived inhibitors, structural determinants of the substrate specificities of C1r and C1s, elucidation of the architecture of C1, structural and functional homology of C1 with the initiating complexes of the lectin complement pathway, and novel involvement of C1q in processes such as ageing, cancer, synaptic pruning, and pregnancy.

**Citation:** Kishore, U., Thielens, N. M., Gaboriaud, C., eds. (2016). State-of-the-Art Research on C1q and the Classical Complement Pathway. Lausanne: Frontiers Media. doi: 10.3389/978-2-88945-058-9

# Table of Contents

*05 Editorial: State-of-the-Art Research on C1q and the Classical Complement Pathway* Uday Kishore, Nicole M. Thielens and Christine Gaboriaud

# **1. C1q and gC1q Containing Proteins: Structure and Emerging Functions**

*08 The Human C1q Globular Domain: Structure and Recognition of Non-Immune Self Ligands*

Christine Gaboriaud, Philippe Frachet, Nicole M. Thielens and Gérard J. Arlaud

# **2. C1q and the C1 Complex in Pregnancy and Autoimmunity**

*57 Transcriptional Factor PU.1 Regulates Decidual C1q Expression in Early Pregnancy in Human*

Shanmuga Priyaa Madhukaran, Uday Kishore, Kaiser Jamil, Boon Heng Dennis Teo, Mahesh Choolani and Jinhua Lu

Berhane Ghebrehiwet, Kinga K. Hosszu, Alisa Valentino, Yan Ji and Ellinor I. B. Peerschke

# **3. Classical Complement Pathway Activation and Inhibition**

Lakshmi Carmel Wijeyewickrema, Renee Charlene Duncan and Robert Neil Pike

*94 Deciphering the Fine Details of C1 Assembly and Activation Mechanisms: "Mission Impossible"?*

Christine Gaboriaud, Wai Li Ling, Nicole M. Thielens, Isabelle Bally and Véronique Rossi

# Editorial: State-of-the-Art Research on C1q and the Classical Complement Pathway

*Uday Kishore1 \*, Nicole M. Thielens2 \* and Christine Gaboriaud2*

*1College of Health and Life Sciences, Brunel University London, Uxbridge, UK, 2 Institut de Biologie Structurale (IBS), Université Grenoble Alpes, CEA, CNRS, Grenoble, France*

Keywords: complement, C1 complex, C1q, gC1q structure, C1s, classical pathway activation, autoimmunity, emerging functions

# **The Editorial on the Research Topic**

#### **State-of-the-Art Research on C1q and the Classical Complement Pathway**

Complement protein C1q is a fascinating innate immune molecule. C1q is the first subcomponent of the classical complement pathway. Its primary three-chain structure (A, B, and C chains), which is composed of a triple-helical collagen-like region and a C-terminal ligand-recognizing globular head (gC1q) domain, yields a tulip-like organization with six gC1q domains, each representing a heterotrimer (the C-terminal regions of the A, B, and C chains) (1). In addition to binding to immune complexes containing IgG and IgM, the gC1q domain also engages with a number of self and non-self ligands. A summary of the structural basis of C1q–ligand interactions has been elegantly presented in the review by Gaboriaud et al. For example, it shows that C1q can recognize multiple ligands on the apoptotic cell surface, which illustrates its versatile surface recognition properties. A common binding area for several of these non-immune ligands has been observed in the subunit C, which could play a role in restricting the activation of the classical complement pathway.

It has become evident over the course of the last two decades that the gC1q domain is widely conserved across a diverse range of vertebrate and invertebrate proteins (2). The functions of these proteins can range from being immunological to structural. Through a review article, Ghebrehiwet et al. have cited two examples of C1q functions that are distinct from its involvement in the classical pathway: its ability to induce apoptosis in prostate cancer cells, and to modulate vascularization for fetal–maternal interaction. Given the existence of a C1q–TNF superfamily based on the remarkable structural similarities between C1q and TNF family members (1), a good argument has been made for a cytokine-like property of C1q. The complexity and diversity related to the functions of C1q family have been greatly exemplified by a structure–function review article by Colombatti et al., who describe the functions associated with the gC1q domain of two C1q family members – such as EMILIN and multimerin. A slight structural variation from the prototypical structure, as revealed by the NMR solution structure, is insightful, purporting one residue in the interaction of gC1q domain with α4β1 and α9β1 integrins.

In recent years, a number of functions of C1q have emerged that are complement independent. This is reflected in the local synthesis of C1q by various immune and non-immune cells. The diversity of C1q functions includes its involvement in dendritic cell maturation, immune modulation, cell differentiation, cancer progression, neuronal synapse pruning, and pregnancy. This has been extensively summarized by Kouser et al. In the backdrop of a recent observation that C1q gene knockout mice show nearly all features of preeclampsia, the role of C1q in normal and complicated pregnancies

#### *Edited and Reviewed by:*

*Francesca Granucci, University of Milano-Bicocca, Italy*

#### *\*Correspondence:*

*Uday Kishore<EMAIL_ADDRESS><EMAIL_ADDRESS>Nicole M. Thielens<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

*Received: 26 August 2016 Accepted: 20 September 2016 Published: 04 October 2016*

#### *Citation:*

*Kishore U, Thielens NM and Gaboriaud C (2016) Editorial: State-of-the-Art Research on C1q and the Classical Complement Pathway. Front. Immunol. 7:398. doi: 10.3389/fimmu.2016.00398*

has become a burning issue in the reproductive immunology. In this issue, Madhukaran et al. have reported a link between the expression of the transcription factor PU.1 and C1q in human trophoblasts and stromal cells, similar to DCs and macrophages, using early decidual tissue.

Given its involvement in a wide range of homeostatic functions, it is not surprising that C1q is central to many human diseases. C1q, as a key molecule in self-tolerance mechanisms, is involved in clearing immune complexes and apoptotic/ necrotic cells. C1q-deficient mice have been shown to have lupus-like symptoms. However, this issue is further confounded by the fact that anti-C1q autoantibodies are found in a number of pathological situations, more so in systemic lupus erythematosus (SLE). These autoantibodies are certainly required for the development of lupus nephritis. Since C1q itself is an IgG-binding protein, identification and characterization of anti-C1q autoantibodies is a challenging task. Mahler et al. have addressed the technicalities of this endeavor, while reassessing the pathological consequences of such autoantibodies in a disease context. In line with the involvement of C1q in tolerance and SLE, Ghebrehiwet et al. have addressed the importance of monocyte surface expressed C1q in association with C1r and C1s. This review examines the role of cell-bound C1q in capturing and processing circulating immune complexes and pathogen-associated molecular patterns (PAMPs). The ability of C1q to modulate PAMP-recognizing receptors (PRRs) makes it a potent and versatile immune surveillance molecule of the innate immunity.

The final section of the issue is dedicated to the understanding of the mechanisms underlying activation of the classical pathway and their specific inhibition for therapeutic purposes.

In an original research article, Wijeyewickrema et al. aim at further deciphering the interaction of the C1s protease with its C4 protein substrate. Previous studies had provided evidence for homologous C4 interaction exosites in the CCP and serine protease domains of both C1s and MASP-2 (3). The authors focus here on the role of C1s Lys628 residue of the serine protease domain. Using site-directed mutagenesis and a peptide substrate library, they show that this residue plays a different role in cleaving peptide versus protein substrates by interacting with C4 in order to facilitate its cleavage. The architecture of the active site at this position is markedly different in C1s compared with MASP-2 (4), which might provide clues toward designing specific inhibitors of the classical pathway.

In a review article, Sharp et al. focus on complement inhibitors of the classical pathway for application in transfusion medicine. Uncontrolled antibody-initiated complement activation plays a central role in hemolytic diseases such as acute intravascular hemolytic transfusion reaction (AIHTR). The authors have

# REFERENCES

1. Kishore U, Gaboriaud C, Waters P, Shrive AK, Greenhough TJ, Reid KBM, et al. C1q and tumor necrosis factor superfamily: modularity and versatility. *Trends Immunol* (2004) 10:551–61. doi:10.1016/j.it.2004.08.006

identified peptide inhibitors of C1 (PIC1) derived from a region of the coat protein of astrovirus with homology to human neutrophil peptide 1 that inhibits complement activation by binding to the collagen-like regions of C1q (5). They have developed a simple rat model of AIHTR for future preclinical studies of PIC1. A review of complement inhibitors, marketed as well as currently under development, is presented, and their therapeutic potential in transfusion and blood disorders is discussed.

In their perspective article, Gaboriaud et al. provide an update on the current knowledge about the structural basis of the mechanisms involved in the assembly and activation of the C1 complex. The main protein players involved in C1 activation and its control are presented together with the molecular dissection strategy used to define structure–function relationships of C1 subcomponents and to decipher key protein–protein interactions. However, the conformational changes required for allowing C1 activation following target binding are still not elucidated, and this is one among 16 still unanswered questions identified by the authors. Hopefully, new technological developments in structural biology, such as a combination of X-ray crystallography and electron microscopy, will help fulfilling this "mission impossible."

This special topic issues clearly highlights the diversity and complexity associated with structures and functions of C1q and C1q family members. We have come a long way in understanding the structural basis of the gC1q interaction with self and non-self ligands. While additional members of C1q family are still being identified and characterized, the importance of the gC1q domain in the evolutionary history of animals is unique. From being a prototypical innate immune molecule, C1q is now regarded as an independent modulator of a diverse range of functions that are not dependent on its involvement in the complement activation. These complement-independent functions of C1q are likely to be the major thrust of research in coming years.

# AUTHOR CONTRIBUTIONS

All authors listed have made substantial, direct, and intellectual contribution to the work and approved it for publication.

# ACKNOWLEDGMENTS

We are grateful to all the authors who contributed to this special issue of Frontiers in Immunology. We are also indebted to all the reviewers who took special care and attention in ensuring that all the accepted manuscripts reached the high standard we set for ourselves.

serine protease domain of C1s. *J Immunol* (2012) 189:2365–73. doi:10.4049/ jimmunol.1201085

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2016 Kishore, Thielens and Gaboriaud. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# The human C1q globular domain: structure and recognition of non-immune self ligands

# *Christine Gaboriaud, Philippe Frachet, Nicole M. Thielens and Gérard J. Arlaud \**

Groupe IRPAS, Institut de Biologie Structurale, Grenoble, France

#### *Edited by:*

Uday Kishore, Brunel University, UK

#### *Reviewed by:*

Uday Kishore, Brunel University, UK Paul Quax, Leiden University Medical Center, Netherlands

#### *\*Correspondence:*

Gérard J. Arlaud, Commissariat à l'énergie atomique, CNRS, Institut de Biologie Structurale, Université Joseph Fourier-Grenoble 1, 41 rue Jules Horowitz, 38027 Grenoble Cedex 1, France. e-mail<EMAIL_ADDRESS>
C1q, the ligand-binding unit of the C1 complex of complement, is a pattern recognition molecule with the unique ability to sense an amazing variety of targets, including a number of altered structures from self, such as apoptotic cells. The three-dimensional structure of its C-terminal globular domain, responsible for its recognition function, has been solved by X-ray crystallography, revealing a tightly packed heterotrimeric assembly with marked differences in the surface patterns of the subunits, and yielding insights into its versatile binding properties. In conjunction with other approaches, this same technique has been used recently to decipher the mechanisms that allow this domain to interact with various non-immune self ligands, including molecules known to provide eat-me signals on apoptotic cells, such as phosphatidylserine and DNA. These investigations provide evidence for a common binding area for these ligands located in subunit C of the C1q globular domain, and suggest that ligand recognition through this area down-regulates C1 activation, hence contributing to the control of the inflammatory reaction. The purpose of this article is to give an overview of these advances which represent a first step toward understanding the recognition mechanisms of C1q and their biological implications.

**Keywords: C1q, complement, innate immunity, ligand recognition, X-ray crystallography**

# **INTRODUCTION**

Triggering of the classical complement pathway is mediated by C1, a multimolecular complex resulting from the association of a recognition subunit, C1q, and a Ca2+-dependent tetramer comprising two copies of two serine proteases, C1r, and C1s (Cooper, 1985; Gaboriaud et al., 2004). C1q is a 460-kDa hexameric protein assembled from six heterotrimeric collagen-like fibers, each being prolonged by a C-terminal globular domain which mediates the recognition function of C1 (Gaboriaud et al., 2003, 2004; Kishore et al., 2004). A major characteristic of this domain lies in its ability to sense and engage an amazing variety of ligands (Cooper, 1985; Kishore et al., 2004). Thus, C1q is classically known for its ability to bind IgG- and IgM-containing immune complexes. In addition, it recognizes the lectin SIGN-R1, C-reactive protein, and other pentraxins bound to pathogens and other surfaces, as well as various molecular motifs on several Gram-negative bacteria and viruses (Cooper, 1985; Szalai et al., 1999; Thielens et al., 2002; Kishore et al., 2004; Kang et al., 2006). In most cases, recognition of these non-self ligands by C1q triggers activation of the classical complement pathway, thereby contributing to their elimination through enhanced phagocytosis, lysis, and inflammation.

This traditional view of the biological role of C1q should be reconsidered in light of recent studies providing evidence that C1q has the ability to sense many altered structures from self, including the pathological form of the prion protein (Klein et al., 2001; Erlich et al., 2010), β-amyloid fibrils (Tacnet-Delorme et al., 2001),modified forms of low-density lipoprotein (Biro et al., 2007, 2010), and apoptotic cells (Taylor et al., 2000; Navratil et al., 2001). Recognition by C1q triggers efficient clearance of apoptotic cells by phagocytes, but in this case lysis and inflammation are

both inhibited, thus contributing to the maintenance of immune tolerance (Nauta et al., 2004; Fraser et al., 2009). Recent investigations on the C1q targets at the apoptotic cell surface have revealed that three molecules known to provide "eat-me" signals, phosphatidylserine, DNA, and calreticulin, are recognized by C1q, suggesting a multiligand-binding process (Païdassi et al., 2008a,b, 2011).

The three-dimensional (3-D) structure of the heterotrimeric globular domain of C1q, responsible for the recognition function of this protein, has been solved by X-ray crystallography, giving insights into its versatile binding properties (Gaboriaud et al., 2003). The same technique, as well as other approaches, have been recently applied to decipher the interaction of this domain with various non-immune self ligands, including molecules acting as eat-me signals on apoptotic cells, providing evidence for a common binding area (Païdassi et al., 2008a; Garlatti et al., 2010). The purpose of this article is to give an overview of these advances which shed light on the recognition of self ligands by C1q and reveal possible implications in the regulation of C1 activation.

# **ARCHITECTURE OF THE HUMAN C1q GLOBULAR DOMAIN**

The 3-D structure of the C-terminal globular domain of human C1q, obtained after digestion of the collagenous moiety of the protein with collagenase, has been solved by X-ray crystallography to a resolution of 1.9 Å (Gaboriaud et al., 2003). The structure revealed a globular, almost spherical heterotrimeric assembly, with a diameter of about 50 Å, the N- and C-termini of each subunit emerging at the base of the trimer, in close vicinity to one another (**Figures 1A,B**). A major information from the structure

was that the gC1q subunits are arranged clockwise in the order A, B, C when the assembly is viewed from the top, this indication enabling us to derive a 3-D model of the collagen-like triple helix of C1q, and thereby to reconstruct the whole C1q molecule (Gaboriaud et al., 2003, 2004). In the resulting C1q model (**Figure 1C**), the B subunit of each globular domain lies on the outer part of the molecule, whereas A and C are positioned inside. As discussed later, it is very likely that this particular configuration has direct implications in terms of ligand recognition and C1 activation.

The three subunits each exhibit a 10-stranded β sandwich fold with a jelly roll topology homologous to the one described initially for tumor necrosis factor (Eck and Sprang, 1989; Jones et al., 1989) and subsequently for members of the gC1q family (Shapiro and Scherer, 1998; Bogin et al., 2002; Kvansakul et al., 2002), consisting of two five-stranded β-sheets (A- , A, H, C, F and B- , B, G, D, E), each made of anti-parallel strands (**Figure 1D**). Comparison of the three subunits indicates strong conservation of the β-strands and significant variability in the loops connecting them, a characteristic that also applies to other gC1q domains. In addition to a free cysteine conserved in all gC1q domains, which is essentially buried in the structure, each subunit of human C1q contains two other cysteines engaged in a disulfide bond (**Figure 1D**).

Assembly of the C1q heterotrimer involves a tight association of the subunits, as shown by a total buried surface of 5490 Å2 equally contributed by each subunit, this value being significantly less, however, than those in collagens VIII and X (Bogin et al., 2002). Trimerization involves a central interface as well as lateral interactions which in both cases are hydrophobic at the base of the trimer and become more polar when going to the apex. This results in the formation of a discontinuous central channel which is closed at both extremities. Interestingly, despite its heterotrimeric structure, the C1q globular domain assembles in the same way as its homotrimeric counterparts in ACRP30 and collagens VIII and X. Nevertheless, attempts to assemble gC1q subunits as homotrimers *in silico* were found to result in severe steric clashes, particularly at the level of lateral interactions, thus providing a structural basis for their natural propensity to associate only as heterotrimers (Gaboriaud et al., 2003). The structure of the C1q globular domain also reveals the presence of a Ca2<sup>+</sup> ion bound at the top of the assembly (**Figures 1A,B**). The binding site is asymmetrical relative to the trimer, considering that Ca2<sup>+</sup> is coordinated by oxygen ligands contributed by subunits A and B, but is not connected to subunit C. In contrast to the buried Ca2<sup>+</sup> cluster observed in collagen X (Bogin et al., 2002), the single Ca2<sup>+</sup> ion of C1q is exposed to the solvent and defines the upper end of the central channel. In addition to contributing to the stability of the heterotrimeric

assembly, the Ca2<sup>+</sup> ion may have a functional role. Thus, the loss of Ca2<sup>+</sup> has been postulated to modify the direction of the electric moment of the C1q globular domain and thereby to influence the recognition of immune targets such as C-reactive protein and IgG (Roumenina et al., 2005). The fact that the Ca2<sup>+</sup> ion is accessible to the solvent also opens the possibility of a direct implication in the recognition of certain charged targets (Gaboriaud et al., 2003).

# **THE HETEROTRIMERIC STRUCTURE OF THE C1q GLOBULAR DOMAIN AS THE KEY TO ITS BINDING VERSATILITY**

A striking feature of the structure of the C1q globular domain lies in the fact that the three subunits exhibit marked differences in their surface patterns, with respect to both charged and hydrophobic residues (**Figure 2**). Thus, subunit A mainly shows a combination of arginine and acidic residues scattered on its surface (**Figure 2A**). Subunit C also shows a combination of basic and acidic residues spread over the surface (**Figure 2C**). In contrast, positively charged residues are predominant on the surface of module B, with in particular a cluster of three arginines ArgB101, ArgB114, and ArgB129. The latter two residues, which have been proposed to be involved in the interaction with IgG (Marqués et al., 1993), markedly protrude outside the structure (**Figure 2B**). Several hydrophobic residues are exposed to the solvent on the external face of each subunit, the most striking example being the IleB103, ValB105, ProB106 cluster lying over the ArgB101, ArgB114, ArgB129 triad (**Figure 2B**). In contrast, the only accessible aromatic residues (TyrC155, TrpC190) are found on the equatorial area of subunit C (**Figure 2C**). The top of the heterotrimer (**Figure 2D**) shows a predominance of positive charges mainly contributed by lysine residues. In each subunit, several hydrophobic patches and aromatic residues are also exposed.

The heterotrimeric structure of the C1q globular domain is very likely a major determinant of its versatile recognition properties. Thus, because they display quite different surface patterns in terms of charged and hydrophobic residues, the three subunits are expected to mediate different individual binding properties. In addition, considering the tightly packed structure of the domain, it appears likely that recognition of certain ligands will involve residues contributed by several subunits, thereby considerably enlarging the spectrum of the C1q targets. This assumption appears consistent with the observation that many C1q ligands have been found to interact in a significant way with different subunits of its globular domain (Kishore et al., 2003). As discussed later, the diversity of the recognition modes of C1q is well illustrated by the models proposed for C-reactive protein and IgG1, where binding is thought to involve the apex of the C1q globular domain, and the equatorial region of subunit B, respectively (Gaboriaud et al., 2003).

# **C1q RECOGNIZES MULTIPLE LIGANDS AT THE APOPTOTIC CELL SURFACE**

Recent investigations on the targets recognized by C1q at the apoptotic cell surface have allowed us to identify several ligands (Païdassi et al., 2008a,b, 2011). In all cases, the C1q moiety responsible for binding has been shown to be the globular domain.

Phosphatidylserine is currently one of the best documented recognition signal required for apoptotic cell clearance. Its tethering is mediated directly by phagocyte receptors, with or without the help of soluble bridging molecules (Savill et al., 2002). We have recently shown that C1q is one of the molecules that bridge phosphatidylserine exposed at the surface of early apoptotic cells to the phagocyte (Païdassi et al., 2008a). The interaction is mediated by its globular domain, which recognizes the phosphoserine moiety of phosphatidylserine. Annexin V, which binds avidly to phosphatidylserine, inhibits the interaction, and C1q and its globular domain both co-localize with phosphatidylserine on the apoptotic cell surface. Surface plasmon resonance analysis of the interaction between the C1q globular domain and phosphatidylserine has revealed high-affinity (**Figure 3A**) and the phosphoserine binding site on C1q has been identified by X-ray crystallography (**Figure 4**).

Nuclear splitting and DNA fragmentation are well-established features of apoptosis, and the generation of auto-antibodies directed against chromatin components is a characteristic of autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis (Shoenfeld et al., 1987). It was recently demonstrated that nucleic acids are rapidly exposed during apoptosis and provide ligands for C1q binding (Elward et al., 2005). In an attempt to characterize C1q binding to DNA,we have shown for the first time that the globular region of C1q displays a lectin-like activity that contributes to DNA binding (Païdassi et al., 2008b). This observation is consistent with the observation that treatment of apoptotic cells with DNase decreases significantly binding to C1q. Studies at the molecular level have demonstrated that the C1q globular domain binds DNA through its pentose moiety, and that deoxy-d-ribose inhibits formation of complexes between DNA and the C1q globular domain (**Figure 3B**). The structure of the deoxy-d-ribose binding site on the C1q globular domain has been deciphered at high resolution by X-ray crystallography.

Calreticulin was first identified as a receptor for the collagenous moiety of C1q and the collectins at the surface of phagocytes. However, based on more recent studies, it is now widely accepted that calreticulin is a major recognition signal at the apoptotic cell surface, acting as an "eat-me" signal in the uptake of apoptotic cells by macrophages (Gardai et al., 2006; Obeid et al., 2007). This latter finding evokes a double play for this protein, as both a phagocyte receptor and an "eat-me" signal on the apoptotic cell surface, with the help of the bridging molecule C1q. In support of this hypothesis, we have recently demonstrated that the C1q globular domain, responsible for C1q binding to apoptotic cells, binds calreticulin with high-affinity and co-localizes with this protein on the apoptotic cell surface (**Figure 3C**; Païdassi et al., 2011).

# **A COMMON BINDING AREA FOR NON-IMMUNE SELF LIGANDS**

To decipher the recognition properties of the C1q globular domain, its X-ray structure has been solved in the presence of

three different small ligands corresponding to molecular determinants recognized by C1q in larger target molecules from self: (i) phosphoserine, the motif recognized in phosphatidylserine, (ii) deoxy-d-ribose, a specific determinant of DNA, and (iii) two repetitive units of heparan sulfate (Païdassi et al., 2008a; Garlatti et al., 2010). The three structures were solved using crystal soaking because attempts to generate co-crystals of the C1q globular domain in complex with a ligand were unsuccessful. The soaking technique introduces space constraints, and is more favorable to small ligands. The smallest ligand tested, deoxyd-ribose, was observed at the highest resolution (1.25 Å) in a convincing binding site located in subunit C of the C1q globular

domain (Garlatti et al., 2010). It is accommodated in a small pocket lying between ArgC98 and ArgC111 (**Figure 4B**). Two of its hydroxyl groups are stabilized by direct and water-mediated polar interactions with AsnC113, ArgC111, and ArgC118. The interaction is clearly specific to deoxy-d-ribose, since no ligand fixation could be detected when the same protocol was applied using d-ribose. Indeed, the additional hydroxyl group present in ribose would clash sterically with AsnC113 in the observed configuration, providing a structural basis for the strict specificity of C1q toward deoxy-d-ribose. The other two ligands investigated, phosphoserine and heparan sulfate, are both negatively charged, and interact approximately in the same area of the C subunit of the C1q globular domain (**Figure 4A**), although they differ in their precise interaction modes (Païdassi et al., 2008a; Garlatti et al., 2010). Thus, heparan sulfate is interacting with TyrC155, TrpC190, and LysC129 (**Figure 4C**), whereas phosphoserine binds ArgC111, SerC126, and ThrC127 (**Figure 4D**). The sulfate/phosphate binding propensity of ArgC98, ArgC111, TyrC155, and TrpC190 suggests that this area has the ability to bind larger polyanionic molecules. If this hypothesis is correct, then the larger polyanionic molecules DNA and heparin are expected to compete with each other because of the close proximity of their binding sites. This hypothesis has been tested using competition experiments, providing evidence that, indeed, DNA and heparan sulfate fragments each inhibit in a dosedependent manner C1q binding to a HS-coated surface, in complete agreement with X-ray crystallography analyses (Garlatti et al., 2010).

# **THE LOCATION OF A LIGAND-BINDING SITE ON THE C1q GLOBULAR DOMAIN CORRELATES WITH ITS C1 ACTIVATION POTENTIAL**

According to the C1q model derived from the X-ray structure of its globular domain (**Figure 1C**), the binding sites for phosphoserine, deoxy-d-ribose, and heparan sulfate, all located in subunit C, would be positioned on the inner face of the C1q cone, and oriented toward the target (**Figures 4A** and **5A**). In contrast, the IgG binding site has been proposed to lie on the equatorial region of subunit B, on the outer part of the C1q molecule (Gaboriaud et al., 2003; Garlatti et al., 2010). In light of our current knowledge of the structure and activation mechanism of the C1 complex (Budayova-Spano et al., 2002; Gaboriaud et al., 2003, 2004; Bally et al., 2009; Brier et al., 2010), these locations have direct functional implications in terms of C1 activation. Thus, the X-ray structure of the zymogen C1r catalytic domain has led to the conclusion that an outward movement of the C1q stems is necessary to disrupt the resting head-to-tail dimeric structure of C1r and thereby trigger C1 activation (**Figures 5B–D**). If this assumption is correct, any ligand recognized through the outer part of the C1q globular domain, as proposed for IgG (**Figure 5A**), is expected to trigger efficient C1 activation (**Figure 5F**). Conversely, given the inner positioning of their interaction sites, binding of ligands such as phosphoserine, deoxy-d-ribose, and heparan sulfate would be unable to generate the appropriate outward movement of the C1q stems, particularly if these ligands are clustered in dense surface patches, thus preventing or restraining activation of the C1 complex. In full agreement with this hypothesis, it has been shown that immune complexes trigger efficient activation of C1 in the presence of C1 inhibitor, in contrast to DNA and heparin (**Figure 5E**; Ziccardi, 1982; Garlatti et al., 2010).

# **CONCLUSION**

Uncontrolled activation of the classical complement pathway is involved in many inflammatory pathologies. Conversely, C1q deficiency is associated with major defaults in the uptake of apoptotic cells and correlates with autoimmune diseases such as systemic lupus erythematosus and glomerulonephritis, emphasizing the crucial role of C1q in the maintenance of immune tolerance (Botto and Walport, 2002). Whereas heparan sulfate and other sulfated molecules are known to inhibit complement activation, DNA, and phosphatidylserine are both eat-me signals involved in the removal of apoptotic cells by C1q. It is tempting to hypothesize that, because the binding sites for these ligands are all located within the same area of the C1q globular domain, on the inner edge of the C1q cone, their recognition by C1q only generates low C1 activation, hence contributing to the control of the inflammatory reaction, in addition to complement regulators (Zipfel and Skerka,

2009). Recently, another molecule from self, heme, was shown to bind C1q and inhibit C1 activation by immune complexes and C-reactive protein (Roumenina et al., 2011). Interestingly, one of the two heme binding sites postulated by these authors is located in subunit C of the C1q globular domain, in the vicinity of the sites identified for DNA, phosphatidylserine, and heparan sulfate, suggesting that heme-mediated inhibition of C1 activation could involve a similar mechanism.

It is clear, however, that this scheme does not apply to all known non-immune C1q self ligands. Thus, whereas the oxidized form of low-density lipoprotein (ox-LDL) does not trigger C1 activation despite its ability to bind C1q with high-affinity (Biro et al., 2007), the enzymatically modified species (E-LDL) is a potent C1 activator (Biro et al., 2007, 2010). In the same way, cardiolipin, β-amyloid fibrils, as well as different forms of the prion protein have been shown to bind C1q and activate C1 to various extents (Peitsch et al., 1988; Tacnet-Delorme et al., 2001; Dumestre-Pérard et al., 2007; Sim et al., 2007; Sjöberg et al., 2008). Further investigations at the molecular level will be necessary to generate a more detailed map of the ligand-binding sites of C1q and uncover all the secrets of this unique sensor molecule. Such advances would have important potential therapeutic applications, considering that ligand recognition by C1q may elicit both beneficial and deleterious effects, as exemplified in neurodegenerative diseases where both

# **REFERENCES**

required to trigger activation of the C1 complex. *EMBO J.* 21, 231–239.

types of effects have been reported (van Beek et al., 2003;Veerhuis et al., 2011). Ideally, the aim would be to modulate the classical complement pathway by preventing or limiting its noxious effects while preserving its protective role. In the long term, this goal should be achievable through the design of inhibitory molecules able to specifically target the strong C1-activating binding sites located on the outer part of the C1q globular domain, while preserving the functionality of the inner binding area allowing recognition and efficient clearance of altered self ligands.

## **ACKNOWLEDGMENTS**

The authors greatly acknowledge the contributions to the studies referred to in this review of all past and present members of the laboratory, with particular attention to Claudine Darnault, Virginie Garlatti, Jordi Juanhuix, Monique Lacroix, Helena Païdassi, and Pascale Tacnet-Delorme.

of complement protein C1q provides a basis for its versatile recognition properties. *J. Biol. Chem.* 278, 46974–46982.

human C1q A, B, and C chains. *J. Immunol.* 171, 812–820.

C4b-binding protein directly. *Mol. Immunol.* 45, 3213–3221.

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 25 October 2011; paper pending published: 09 December 2011; accepted: 21 December 2011; published online: 06 January 2012.*

*Citation: Gaboriaud C, Frachet P, Thielens NM and Arlaud GJ (2012) The human C1q globular domain: structure and recognition of non-immune self ligands. Front. Immun. 2:92. doi: 10.3389/fimmu.2011.00092*

*This article was submitted to Frontiers in Molecular Innate Immunity, a specialty of Frontiers in Immunology.*

*Copyright © 2012 Gaboriaud, Frachet, Thielens and Arlaud. This is an openaccess article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.*

# *Berhane Ghebrehiwet 1\*, Kinga K. Hosszu1, Alisa Valentino1 and Ellinor I. B. Peerschke2*

<sup>1</sup> The Department of Medicine, Stony Brook University, Stony Brook, NY, USA

<sup>2</sup> The Department of Laboratory Medicine, Memorial Sloan–Kettering Cancer Center, New York, NY, USA

#### *Edited by:*

Uday Kishore, Brunel University, UK

#### *Reviewed by:*

Uday Kishore, Brunel University, UK Annapurna Nayak, Brunel University, UK

#### *\*Correspondence:*

Berhane Ghebrehiwet, Health Sciences Center, Stony Brook University School of Medicine, T-16, Room 040, Stony Brook, New York, NY 11794-8161, USA. e-mail: berhane.ghebrehiwet@ stonybrook.edu

Research conducted over the past 20 years have helped us unravel not only the hidden structural and functional subtleties of human C1q, but also has catapulted the molecule from a mere recognition unit of the classical pathway to a well-recognized molecular sensor of damage-modified self or non-self antigens. Thus, C1q is involved in a rapidly expanding list of pathological disorders – including autoimmunity, trophoblast migration, preeclampsia, and cancer.The results of two recent reports are provided to underscore the critical role C1q plays in health and disease. First is the observation by Singh et al. (2011) showing that pregnant C1q−/− mice recapitulate the key features of human preeclampsia that correlate with increased fetal death. Treatment of the C1q−/− mice with pravastatin restored trophoblast invasiveness, placental blood flow, and angiogenic balance and, thus, prevented the onset of preeclampsia. Second is the report by Hong et al. (2009) which showed that C1q can induce apoptosis of prostate cancer cells by activating the tumor suppressor molecule WW-domain containing oxydoreductase (WWOX or WOX1) and destabilizing cell adhesion. Downregulation of C1q on the other hand, enhanced prostate hyperplasia and cancer formation due to failure of WOX1 activation. C1q belongs to a family of structurally and functionally related TNF-α-like family of proteins that may have arisen from a common ancestral gene. Therefore C1q not only shares the diverse functions with the tumor necrosis factor family of proteins, but also explains why C1q has retained some of its ancestral "cytokine-like" activities. This review is intended to highlight some of the structural and functional aspects of C1q by underscoring the growing list of its non-traditional functions.

**Keywords: role of C1q in inflammation and autoimmunity, C1q and cancer, emerging role of C1q, functions of C1q**

### **INTRODUCTION**

The first component of complement, C1, is a multimolecular complex comprising of C1q and a Ca2+-dependent tetramer, C1r2–C1s2. The role of C1q within this complex is to serve as a recognition signal – a signal that is readily translated into a highly specific and orderly intramolecular rearrangement resulting in a sequential proteolytic activation – first of C1r and then of C1s – thereby triggering activation of the classical pathway (Cooper, 1985; Schumaker et al., 1987; Arlaud et al., 2001). However, the role of C1q is not restricted to recognition of immune complexes or other molecules that activate the classical pathway. Evidence accumulated to date clearly demonstrates that C1q is a key player in a rapidly expanding list of pathophysiological conditions which include: trophoblast invasion and placental development (Agostinis et al., 2010), onset of preeclampsia (Singh et al., 2011), regulation of autoimmune diseases such systemic lupus erythematosus (SLE; Walport et al., 1998) and activation of the tumor suppressor molecule WW-domain containing oxydoreductase, which plays a

critical role in the pathogenesis of prostate cancer (Hong et al., 2009).

 The synthesized molecule in turn, is either transiently expressed as a cell membrane associated molecular ligand (mC1q; Loos, 1983), or secreted into the pericellular milieu where it can modulate cell-specific biological responses in a manner that involves an autocrine and/or paracrine signaling through cell surface receptors and their signaling partners (Menzies et al., 2008). The diversity of immunological functions mediated by C1q could perhaps be explained by having a unique structural feature: a collagen-like domain (cC1q) and a globular heads domain (gC1q) present in the same protein. This paper will shed some light into the evolution and structural

**Abbreviations:** cC1q, the collagen domain of C1q; cC1qR, receptor for cC1q; CRT (identical to cC1qR), calreticulin; gC1q, the globular heads of C1q; gC1qR, receptor for gC1q; ghA, ghB, and ghC, globular heads of the A, B, and C chains of C1q respectively.

basis of C1q function and highlight some of its emerging novel functions.

#### **STRUCTURE OF HUMAN C1q**

*Human C1q* (460 kDa) is a collagen-like, structurally complex, hexameric glycoprotein, which associates with the Ca2+ dependent C1r2–C1s2 tetramer (360 kDa) to form the pentameric C1, the first component of the serum complement system (Calcott and Müller-Eberhard, 1972; Reid and Thompson, 1983; Reid, 1989). It is composed of six globular "*heads*" linked via six collagen-like "*stalks*" to a fibril-like central region (Shelton et al., 1972; Svehag et al., 1972; Knobel et al., 1975; Brodsky-Doyle et al., 1976; Sellar et al., 1991). The C1q molecule (**Figure 1**), consists of 3 remarkably similar but distinct polypeptide chains, A, B, and C, each of which occurs 6 times in the molecule to give rise to a total of 18 chains (6A, 6B, 6C; Reid, 1989). The three chains are the product of three distinct genes, which are aligned 5- ⇒3- , in the same orientation, in the order A–C–B on a 24-kb stretch of DNA on chromosome 1p (Reid, 1985; Sellar et al., 1991). Each polypeptide chain (A = 28 kDa, B = 25 kDa, C = 24 kDa) consists of 81 amino acid-long collagen-like *N*-terminal "*stalk*" and a *C*terminal, globular "*head*" of 136 amino acids (Reid, 1989). The chains are arranged to form 6 triple helical strands, three peptide chains, A, B, and C, forming one strand (**Figure 1**). In each strand, the triple helix is formed between the *N*-terminal collagenlike sequences of the three chains while the globular "*head*" is formed from the *C*-terminal portion of these chains (Sellar et al., 1991; Kishore and Reid, 1999). Chains A and B are disulfidelinked within a given strand throughformation of disulfide bridges between the half-cysteine residues located at A-4 and B-4, whereas the C chain forms a disulfide bond with the C chain of the next strand through cysteine residues found at position C-4 (Reid, 1989). Thus, the two disulfide-linked strands form a doublet, and three doublets are linked by non-covalent forces to give C1q its signature electron microscopic structure resembling a bouquet of tulips (Shelton et al., 1972; Brodsky-Doyle et al., 1976; **Figure 1**). Approximately 8% (w/w) of human C1q is carbohydrate, which is in the form of glucosylgalactosyl disaccharide units linked to hydroxylysine residues in the cC1q domain, and six asparaginelinked sugar chains located in the gC1q domain (Yonemasu et al., 1971; Calcott and Müller-Eberhard, 1972; Reid et al., 1972). The two types of sugars in turn account for approximately 69 and 31% of the total carbohydrate respectively (Mizuochi et al., 1979; Shinkai and Yonemasu, 1979; Reid, 1989).

From a structure–function point of view, the C1q molecule contains two very well defined major functional domains through which it enters into a wide range of molecular interactions. These domains are known as the *collagen-like domain (cC1q),* and the *globular* "*heads*"*domain (gC1q).* Of these, the gC1q domain,which recognizes a wide range of molecular patterns including modifiedself and non-self antigens, is considered to be key to the versatility of C1q function (Gaboriaud et al., 2009). The two domains of C1q can be purified separately after the intact C1q molecule is first subjected to either peptic digestion at pH 4.45, or collagenase digestion

#### **FIGURE 1 | Schematic model of the subunit structure and assembly of intact of C1q.** Three similar chains – A, B, and C – are assembled to form disulfide-linked A–B and C–C dimers. One strand of the molecule consists of an A–B dimer non-covalently linked toaCchain forming (A–B–C). The C chain of one strand is then disulfide-linked to the C chain of a neighboring strand to

give an A–B C–C B–A doublet and three such doublets are linked by non-covalent forces giving C1q its final signature "bouquet-like" structure. The length of each chain as well as the length of the gC1q and cC1q domains (for brevity, g- or c-domain) is given in parenthesis. (The figure was adapted from Reid, 1989).

at near neutral pH 7.4 (Knobel et al., 1974). The respective purified heterotrimeric domains can then be used in functional studies, although the affinity of the individual trimeric gC1q domain is likely to be several-fold less than that of the intact C1q because of the multivalency of the latter (Tacnet et al., 2008). Evidence accumulated to date convincingly shows that the two domains of C1q participate in a multiplicity of immunological functions by serving as a binding sites for a long list of pathogen-associated molecular patterns (PAMPs) as well as danger associated molecular patterns (DAMPs).  The gC1q domain has a heterotrimeric structure composed of the C-terminal halves of each of the three chains A, B, and C, and each of these heterotrimeric structures are postulated to function independently (Kishore and Reid, 1999, 2000). Because of the availability of recombinant forms of each of the globular heads (gh) or modules, it has now been possible to purify the individual modules (ghA, ghB, and ghC) as recombinant proteins and study their individual roles in ligand binding and biological function (Kishore et al., 2003; Ghai et al., 2007). Indeed, mutational studies using ghA, ghB, and ghC modules have identified the gh residues involved in recognition of IgG and IgM (Kojouharova et al., 2004; Gadjeva et al., 2008). In addition to recognition of self and non-self antigens, C1q can enter into a vast array of interactions by binding to cell surface molecules either via its cC1q or gC1q domain. C1q receptors mediate a plethora of immunologic functions including phagocytosis, and clearance of apoptotic cells (Bobak et al., 1987; Nauta et al., 2002; Païdassi et al., 2011).

Although the list of cell surface or cell associated molecules recognized by C1q is increasing, there are at least two well characterized and ubiquitously distributed cell surface molecules – known respectively as cC1qR and gC1qR – that recognize the two domains of C1q (Ghebrehiwet and Peerschke, 2004a). Although each of these molecules lacks a consensus sequence motif for a traditional transmembrane segment, the lack of direct conduit to intracellular signaling elements is circumvented by the formation of a docking/signaling complex with transmembrane proteins. This partnership in turn is dictated by the specific biological response to be induced (Ghebrehiwet et al., 2001; Ghebrehiwet and Peerschke, 2004a). Each of these receptors is a multiligand binding protein capable of inducing a wide range of cellular functions (Ghebrehiwet and Peerschke, 2004a). More importantly, binding of C1q to cell surfaces via either receptor induces a plethora of biological functions that play a critical role in innate and adaptive immunity, inflammation, infection, and cancer (Ghebrehiwet et al., 2001).

# **THE C1q FAMILY OF PROTEINS**

From data available to date, it has become clear that the unusual feature of having a *collagen-like* and *globular region* in the same protein is not unique to C1q. Instead, the data show that C1q belongs to a long list of oligomeric proteins that are structurally and functionally similar. These proteins, whose members include: *mannan-binding lectin* (*MBL*), *lung surfactant protein A* (*SP-A*), SP-D, *collectin-43* (*CL-43*), collectin-46 (CL-46), and *bovine conglutinin* (Malhotra et al., 1992) are collectively known as collectins (*col*lagen containing *lectins*) by virtue of having collagen-like sequences contiguous with non-collagen-like stretches (Hoppe and Reid, 1994). Collectins are calcium dependent molecules and belong to a group III of the family of lectins containing C-type carbohydrate recognition domains (CRD; Lu et al., 1993; Hakansson and Reid, 2000). Their genes characteristically contain a small exon encoding a 36 amino acid-long neck region,which links the collagen domain with the CRD. The CRD in turn, is responsible for recognition of glycoconjugates containing mannose and fucose on microorganisms but *not* on self-proteins, resulting in enhanced clearance of pathogenic microorganisms (Hakansson and Reid, 2000). Unlike the collectins however, C1q does not possess a consensus domainfor CRD, but it shares another common feature with the collectins in that it possesses galactose– glucose disaccharides attached to the collagenous regions of each protein (Mizuochi et al., 1979; Reid, 1979; Shinkai and Yonemasu, 1979). Despite lacking a CRD domain however, recent data suggest that C1q also has lectin-like activity suggesting that carbohydrates – especially those on pathogens and apoptotic cells – may provide additional binding targets where the binding of C1q is critical for removal of self waste (Païdassi et al., 2008).

In addition to the collectins, several other molecules, which are structurally similar to C1q, have also been described. One of these is, adipocyte complement-related protein of 30 kDa (ACRP30) or adiponectin (Scherer et al., 1995; Hu et al., 1996; Berg et al., 2001; Wong et al., 2004). ACRP30 or adiponectin, is secreted exclusively from fat cells and implicated in energy homeostasis and obesity as well as enhancement of hepatic insulin action (Berg et al., 2001; Wong et al., 2004). It is a protein of 247 amino acids consisting of an *N*-terminal collagen-like domain and a *C*terminal globular domain (Scherer et al., 1995; Berg et al., 2001). The globular domain shares a significant homology to the globular domains of type VIII and X collagens as well as subunits of C1q. More intriguing however is the fact that the crystal structure of a homotrimeric fragment from ACRP30 revealed an unexpected homology to the tumor necrosis factor (TNF) family of proteins. Because of identical folding topologies, key residue conservations, and similarity of trimer interfaces and intron positions, these studies have firmly established an evolutionary link between the TNF-α and the C1q family of proteins suggesting that TNF arose by divergence from an ancestral recognition molecule of the innate immune system (Shapiro and Scherer, 1998). The evolutionary connection between C1q-like proteins and TNF-α, which control many aspects of *inflammation*, adaptive immunity, *apoptosis*, and energy homeostasis, not only sheds insight into the shared diverse functions of this family of proteins (Shapiro and Scherer, 1998), but also explains why C1q has retained some of its "*cytokine-like*" activities. Structurally, the C1q/TNF family of proteins contains the same compact "jellyroll" β fold, usually organized as trimers but with diversefunctions rangingfrom cytokine activity (TNF-α), regulation of metabolism (ACRP30), connective tissue organization (collagen type VIII and X), cellular activation (CD40L), and innate immunity (C1q; Kishore et al., 2002, 2004; Gaboriaud et al., 2007). More recently, the crystal structure of the gC1q domain of C1q was solved and the structure reveals a compact, almost spherical heterotrimeric assembly held together mainly by non-polar

interactions, with a Ca2<sup>+</sup> ion bond at the top of the molecule (Gaboriaud et al., 2009). More importantly, the structural studies revealed that the heterotrimeric assembly of the gC1q domain appears to have unique features that allow its versatility (Gaboriaud et al., 2009). Other molecules in which the "gC1q domains" are found at the C-terminal end include: precerebellin (Kavety et al., 1994), multimerin (Hayward et al., 1995), and types VIII and X collagens (Bogin et al., 2002). Therefore, despite its similarities with the collectins, the structural folds of the gC1q domain, which are more similar to the TNF than to the CRD region of the collectins – make C1q more related to the TNF family of proteins (Kishore et al., 2004).

The significance of a particular human protein is sometimes underscored by the fact that pathogenic microorganisms specifically mimic its functional domain or antigenic determinant for the purpose of immune evasion and thus enhance their survival. For example, the three-dimensional structure of the major surface protein of *Bacillus anthracis* spore surface protein, bacillus collagenlike protein of anthracis (BclA) reveals remarkable similarity to C1q – although there is only low sequence identity between the two proteins (Sylvestre et al., 2002; Rety et al., 2005). Furthermore, both BclA and C1q have been shown to bind to lung alveolar surfactant component-SP-C, suggesting that both recognize common targets in the alveolar component (Rety et al., 2005). This molecular mimicry in turn, underscores not only the functional relevance of the gC1q domain, but also pathogenic microorganisms mimic this signature domain in order to get access into host cells. The exosporium of *B. cereus,*which belongs to the same genus of sporeforming aerobic bacteria as *B. anthracis*, contains a binding site for gC1qR. Although the nature of the *B. cereus* molecule has not been as yet identified, the fact that it recognizes gC1qR on cells for the initial spore attachment and entry suggests that this molecule may also contain a gC1q-like domain (Ghebrehiwet et al., 2009).

# **THE DIVERSE BIOLOGICAL FUNCTIONS OF C1q**

Although for many years, the traditionally accepted role of C1q is the recognition of immune complexes and activation of the classical pathway, there is an ever-growing body of evidence, which suggests that C1q is a major sensor of DAMPs and PAMPS and plays a critical role in inflammation as well as innate and adaptive immunity (**Figure 2**) (Kojouharova et al., 2003; Sontheimer et al., 2005; Ghai et al., 2007; Gadjeva et al., 2008; Païdassi et al., 2008; Hosszu et al., 2010). In addition, C1q induces a plethora of immunologic functions by interaction with distinct cell surface molecules, which recognize either, its gC1q or cC1q domain (Ghebrehiwet and Peerschke, 2004a).  Interaction of C1q with cell surface molecules, induces a wide range of biological responses including stimulation of leukocyte oxidative response (Tenner and Cooper, 1982), phagocytosis (Bobak et al., 1987), suppression of B and T cell proliferation (Ghebrehiwet et al., 1990), fibroblast and endothelial cell adhesion (Bordin et al.,1990;Chen et al.,1994; Feng et al.,2002), trophoblast

pneumonia (Nishino et al., 1981; Komatsu et al., 1982) as well as autoimmune diseases, a prototype of which is SLE. In fact, deficiency of C1q is considered to be a strong susceptibility factor for SLE as evidenced by the fact that almost all (≥92%) of the known patients with C1q deficiency have developed the disease (Botto et al., 1998; Walport et al., 1998; Pickering et al., 2000). The significance of C1q in SLE is further substantiated by the finding that, peripheral blood monocytes from SLE donors, who are not C1q deficient, have a significantly impaired ability to upregulate C1q synthesis both at the mRNA and protein levels, when compared to their healthy counterparts (Moosig et al., 2006). Furthermore, previous studies have shown SLE patients to synthesize high levels of a non-functional, low molecular weight form of C1q, that run with an apparent molecular weight of 150 kDa by gel filtration (Hoekzema et al., 1985). This non-functional form of C1q

cell migration (Agostinis et al., 2010), regulation of dendritic cells (Castellano et al., 2004a,b; Hosszu et al., 2010), angiogenesis (Bossi et al., 2011), and chemotaxis of eosinophils,mast cells, neutrophils, and dendritic cells (Ghebrehiwet et al., 1995; Kuna et al., 1996; Leigh et al., 1998; Vegh et al., 2005). Another interesting aspect of the C1qRs, is that both are released into the pericellular milieu, by proliferating, activated, or proinflammatory cells. Because both are capable of activating the classical pathway, it is believed that the secreted molecules may exacerbate or trigger the local inflammatory process. The binding site for gC1qR on C1q has been shown to be on the A-chain of C1q (Ghebrehiwet et al., 2001), which is the same or overlapping site to a putative IgG site (Marques et al., 1993).

The significance of C1q in health and disease is further underscored by the fact that deficiency in C1q leads to a plethora of diseases including recurrent infections, otitis media, meningitis,

# **C1q DEFICIENCY AND AUTOIMMUNITY**

is characterized by having an abnormal A–C disulfide bonding instead of the normal pattern, which is A–B and C–C (Hoekzema et al., 1985, 1987, 1989). Although this form of C1q retains affinity for the C1r2C1s2-complex, it does not bind to the Fc in IgG aggregates (Martin and Loos, 1988). Because cultured monocytes from normal individuals can synthesize both the dysfunctional as well as the normal forms of C1q, it is hypothesized that in disease states such as SLE, a condition may exist which favors a disproportionate concentration of dysfunctional C1q, especially since the normal C1q would be depleted due to complement activation (Hoekzema et al., 1985). Although the molecular underpinnings are still being worked out, two hypotheses have been put forth to explain the role of C1q in SLE. The *first* deals with the role of C1q in removal of apoptotic waste. Removal of apoptotic cells and cellular debris is essential for maintenance of tissue homeostasis, organogenesis, remodeling, development, and maintenance of the immune system, protection against neoplasia, and resolution of inflammation. The mechanism of this removal involves the recognition of the apoptotic cell surface and initiation of phagocytic uptake by macrophages and dendritic cells. C1q, has been shown to play a major role both in the recognition and removal of apoptotic cells (Casciola-Rosen et al., 1994; Korb and Ahearn, 1997) through interaction with C1q receptors expressed both on the phagocytic cell (e.g., cC1qR/CD91; Ogden et al., 2001; Vandivier et al., 2002), and the apoptotic cell (phosphatidylserine, DNA, and possibly gC1qR; Païdassi et al., 2008). This in turn implies that defective clearance of immune complexes and apoptotic cells, leads to immune recognition of hidden epitopes – a critical immunopathogenic event leading to the development of SLE, the prototype autoimmune disease. Indeed, C1q directly and specifically binds to antigens expressed early on the surface of apoptotic cells through its gC1q domain (Korb and Ahearn, 1997; Païdassi et al., 2008) and the common autoantigens targeted in SLE, such as phosphatidylserine and DNA are found in high concentrations on the surface of apoptotic cells (Casciola-Rosen et al., 1994). However, while its role in removal of apoptotic debris is clearly important, there are a number of redundant pathways involved in the removal of self waste, thus making the role of C1q not as critical (Ghebrehiwet and Peerschke, 2004b). The *second* hypothesis stipulates that autoimmunity arising from the absence or inefficient C1q function, especially at local sites, reflects an important role in maintaining tolerance (Ghebrehiwet and Peerschke, 2004b; Hosszu et al., 2008, 2010). Thus, while C1q could provide active protection from autoimmunity by silencing or regulating autoreactive immune cells, its absence or defective secretion could lead to a loss of peripheral tolerance as a sum total of impaired apoptotic cell clearance and negative signaling (Hosszu et al., 2010). Inherent in this postulate is the notion that the unique structure of C1q, which allows it to interact with its receptors via either its cC1q or gC1q domain, may control the transition from the monocyte state (innate immunity) toward the professional antigen presenting cell (APCs) state (adaptive immunity). Therefore the recent observation showing that C1q functions as a "*molecular switch*" during the narrow window of monocyte to DC transition (Hosszu et al., 2008, 2010), not only explains why C1q is primarily synthesized by potent APCs, but also underscores why its absence can impair antigen uptake and tolerance.

# **LOCAL SYNTHESIS AND FUNCTION OF C1q**

As described above, most inflammatory cells such as those found in atherosclerotic plaques (Peerschke et al., 2004) or cells that undergo phenotypic differentiation or proliferation are known to express and/or secrete C1q (Vegh et al., 2003; Bulla et al., 2008; Hosszu et al., 2008). The emerging postulate is therefore, that locally secreted C1q modulates cellular function (s) by an autocrine and/or paracrine signaling mechanism. For example, fetal trophoblast cells invading the decidua in the early stages of pregnancy not only actively synthesize and express C1q on their surface, but this C1q is actively involved in promoting trophoblast invasion of the decidua (Agostinis et al., 2010). This in turn is mediated by two cell surface receptors – gC1qR and α4β<sup>1</sup> integrin, which promote trophoblast adhesion and migration through the activation of ERK1/2 MAPKs (Agostinis et al., 2010). In support of this is the finding that C1q−/− mice manifested increased frequency of fetal resorption, reduced fetal weight, and smaller litter size when compared to their wild-type counterparts suggesting that defective local production of C1q may be involved in pregnancy disorders such as preeclampsia (Agostinis et al., 2010). The significance of C1q in pregnancy disorders is further supported by an independent study, which showed that pregnant C1q−/− mice express the key features of human preeclampsia that correlate with increased fetal death (Singh et al., 2011). Treatment of C1q−/− mice with pravastatin restored trophoblast invasiveness, placental blood flow, and angiogenic balance and, thus, prevented the onset of preeclampsia (Singh et al., 2011). It is also important to note within this context, the results of recent studies, which showed that *in vivo*, C1q induces angiogenesis and promotes wound healing. This in turn identifies not only a novel role but also identifies C1q as a potential pro-angiogenic molecule for the treatment of wounds and chronic ulcers (Bossi et al., 2011).

# **THE EMERGING ROLE OF C1q IN CARCINOGENESIS**

Another emerging role of C1q is in the regulation of cancer cell survival and progression. Recent studies have shown that C1q, which is expressed on basal and epithelial cells of prostate tissues, sustains the activation of the tumor suppressor WOX1 (WWdomain containing oxydoreductase), which is needed for blocking cancer cell proliferation. This suggests that C1q may induce apoptosis of prostate cancer cells by activating WOX1 and destabilizing cell adhesion. Conversely, downregulation of C1q was found to enhance prostate hyperplasia and cancerous formation due to failure of WOX1 activation (Hong et al., 2009). More importantly, although C1q is expressed in the normal prostate, it is significantly reduced in benign prostatic hyperplasia and prostate cancer tissues (Hong et al., 2009). Therefore, the C1q/WOX1-induced death of cancer cells could have a universal implication in cancer, as long as C1q and WOX1 are present in both the extracellular and intracellular milieu, and are sufficient in activating the apoptotic cascade (Hong et al., 2009). However, since the autocrine or paracrine signaling effect of locally expressed C1q is most likely going to be in concert with its cell surface receptors and signaling partners, it is important to note that both cC1qR/calreticulin (cC1qR/CRT) and gC1qR have also been implicated in carcinogenesis, albeit their roles seem to be rather divergent. Whereas cC1qR/CRT is reported to have tumor suppressive effect, gC1qR appears to be

Ghebrehiwet et al. C1q protein family

requisite for tumor cell progression and metastasis. For example, when Epstein–Barr virus (EBV)-immortalized B lymphocytes were injected into nude mice, they were found to reproducibly inhibit tumor growth (Tosato et al., 2003). This anti-tumor activity, which is released by the lymphoblastic cells, was later identified to be a fragment of cC1qR/CRT corresponding to the N-terminal residues 1–180. Subsequently, recombinant protein corresponding to this N-terminal fragment was found to inhibit endothelial cell proliferation *in vitro* and angiogenesis *in vivo* (Tosato et al., 2003). The inhibition of endothelial cell proliferation was due to direct binding of the cC1qR/CRT fragment to the extracellular matrix protein, laminin thereby interfering with endothelial cell attachment. Further studies showed that subcutaneous inoculation of recombinant CRT fragment into nude mice not only prevented the development of subcutaneous tumors, but also inhibited the growth of established ones (Tosato et al., 2003). Since the collagen tail of C1q (cC1q) binds to the N-terminal half of cC1qR/CRT, which contains several short (7–10 amino acids) CH2-like motifs (ExKxKx) similar to the C1q binding motif found in the CH2 domain of IgG (Sontheimer et al.,2003), these C1q binding regions of cC1qR/CRT have the potential to be developed into therapeutic modalities.

The receptor for the ghs of C1q, gC1qR, on the other hand, may have functions that enhance tumor growth and tumor progression. First, histochemical staining with anti gC1qR mAb revealed marked differential expression of gC1qR in thyroid, colon, pancreatic, gastric, esophageal, and lung adenocarcinomas compared to the non-malignant histologic counterparts. In contrast, differential expression was not observed in endometrial, renal, and prostate carcinomas (Rubinstein et al., 2004; Peerschke et al., 2011). Recent studies not only corroborated these findings, but additionally showed that cell surface expressed gC1qR is a receptor for the tumor homing peptide, LyP-1 (Fogal et al., 2008). Moreover, recent studies using human lung carcinoma A549 cells have shown the cell surface gC1qR to be a key regulator for lamellipodia formation and cancer metastasis via receptor tyrosine kinase activation. Furthermore, the growth factor-induced lamellipodia formation

### **REFERENCES**

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and cell migration were significantly decreased in gC1qR-depleted cells, with a concomitant blunt activation of the focal adhesion kinase and the respective receptor tyrosine kinases (Kim et al., 2001). These studies collectively suggest that gC1qR, which is highly upregulated on the surface of tumor cells and inflammatory cell scan serve not only as a tumor cell marker, but also can be a target for therapeutic intervention (Peerschke et al., 2011).

### **CONCLUDING REMARKS**

Because of the incredible number of reports that have come out in the past 20 years, it is not possible to list all the functions in which C1q is involved. Therefore, while touching on some of the well known functions of C1q, this paper underscores the role of C1q in novel and emerging areas of diseases (e.g., preeclampsia, cancer, and wound healing) in which either the soluble form or the membrane associated form of C1q appear to be intimately involved. The chimeric structure of C1q – containing two functional domains – together with the fact that it is synthesized by a wide range of proinflammatory cell types makes it likely that it is a potent orchestrator of molecular pathways involved not only in innate and adaptive immunity, but also in a wide range of inflammatory diseases including cancer. The heterotrimeric gC1q domain of C1q is also revealing itself as a powerful pattern recognition domain with the ability to sense a broad range of ligands – including a number of altered self-motifs and pathogenassociated molecular ligands – each of which will likely trigger a multiplicity of immunologic functions, which are either beneficial or deleterious to the host. More than ever, the availability of recombinant forms of the heterotrimeric trimeric gC1q domains will undoubtedly help us gain insight into the mechanism of interaction between C1q and the vast array of molecular patterns. The best is yet to come.

#### **ACKNOWLEDGMENTS**

This work was supported in part by grants from the National Institutes of Health (R01 AI 060866 and R01 AI-084178).

macrophages. *J. Immunol.* 138, 1150–1156.

Petry, F., Loos, M., Pandolfi, P. P., and Walport, M. J. (1998). Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. *Nat. Genet.* 19, 56–59.

and Arlaud, G. (2009). The crystal structure of the globular head of complement protein C1q provides a basis for its versatile recognition properties. *J. Biol. Chem.* 278, 46974–46982.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Received: 02 February 2012; accepted: 01 March 2012; published online: 05 April 2012.*

*Citation: Ghebrehiwet B, Hosszu KK, Valentino A and Peerschke EIB (2012) The C1q family of proteins: insights into the emerging non-traditional functions. Front. Immun. 3:52. doi: 10.3389/fimmu.2012.00052*

*This article was submitted to Frontiers in Molecular Innate Immunity, a specialty of Frontiers in Immunology.*

*Copyright © 2012 Ghebrehiwet, Hosszu, Valentino and Peerschke. This is an openaccess article distributed under the terms of the Creative Commons Attribution Non Commercial License, which permits non-commercial use, distribution, and reproduction in other forums, provided the original authors and source are credited.*

# The EMILIN/multimerin family

# *Alfonso Colombatti 1,2,3\*, Paola Spessotto1, Roberto Doliana1, Maurizio Mongiat 1, Giorgio Maria Bressan4 and Gennaro Esposito2,3*

<sup>1</sup> Experimental Oncology 2, Centro di Riferimento Oncologico, Istituto di Ricerca e Cura a Carattere Scientifico, Aviano, Italy

<sup>2</sup> Department of Biomedical Science and Technology, University of Udine, Udine, Italy

<sup>3</sup> Microgravity, Ageing, Training, Immobility Excellence Center, University of Udine, Udine, Italy

<sup>4</sup> Department of Histology Microbiology and Medical Biotechnologies, University of Padova, Padova, Italy

#### *Edited by:*

Uday Kishore, Brunel University, UK

#### *Reviewed by:*

Uday Kishore, Brunel University, UK Kenneth Reid, Green Templeton College University of Oxford, UK

#### *\*Correspondence:*

Alfonso Colombatti, Division of Experimental Oncology 2, Centro di Riferimento Oncologico, Istituto di Ricerca e Cura a Carattere Scientifico, 33081 Aviano, Italy. e-mail<EMAIL_ADDRESS>
Elastin microfibrillar interface proteins (EMILINs) and Multimerins (EMILIN1, EMILIN2, Multimerin1, and Multimerin2) constitute a four member family that in addition to the shared C-terminus gC1q domain typical of the gC1q/TNF superfamily members contain a N-terminus unique cysteine-rich EMI domain. These glycoproteins are homotrimeric and assemble into high molecular weight multimers. They are predominantly expressed in the extracellular matrix and contribute to several cellular functions in part associated with the gC1q domain and in part not yet assigned nor linked to other specific regions of the sequence. Among the latter is the control of arterial blood pressure, the inhibition of Bacillus anthracis cell cytotoxicity, the promotion of cell death, the proangiogenic function, and a role in platelet hemostasis. The focus of this review is to highlight the multiplicity of functions and domains of the EMILIN/Multimerin family with a particular emphasis on the regulatory role played by the ligand–receptor interactions of the gC1q domain. EMILIN1 is the most extensively studied member both from the structural and functional point of view. The structure of the gC1q of EMILIN1 solved by NMR highlights unique characteristics compared to other gC1q domains: it shows a marked decrease of the contact surface of the trimeric assembly and while conserving the jelly-roll topology with two β-sheets of antiparallel strands it presents a nine-stranded β-sandwich fold instead of the usual 10 stranded fold. This is likely due to the insertion of nine residues that disrupt the ordered strand organization and forma a highly dynamic protruding loop. In this loop the residue E933 is the site of interaction between gC1q and the α4β1 and α9β1 integrins, and contrary to integrin occupancy that usually upregulates cell growth, when gC1q is ligated by the integrin the cells reduce their proliferative activity.

**Keywords: α4β1 integrin, gC1q-dependent cell adhesion, cell migration, gC1q NMR solution structure, EMI domain, extracellular matrix, skin homeostasis**

# **INTRODUCTION**

The elastin microfibrillar interface protein (EMILIN) and Multimerin family of glycoproteins is part of the large superfamily of collagenous and non-collagenous proteins containing the gC1q signature (Shapiro and Scherer, 1998; Doliana et al., 1999; Kishore et al., 2004). A phylogenetic analysis of gC1q sequences of the 32 proteins present in the human genome carrying this domain (Tom Tang et al., 2005; Ghai et al., 2007), show that EMILINs/Multimerins form a small family well distinct from the rest of the superfamily. Comparison of the coding sequences of EMILIN/Multimerin family components against *Drosophila melanogaster* and other invertebrate genome database gives no matches, indicating that this gene family emerged during vertebrate evolution, first appearing in lower cordate, where orthologs of EMILIN1, EMILIN2, and MMRN2 have been identified (Mei and Gui, 2008; Milanetto et al., 2008). MMRN1 is present starting from birds, indicating that this member of the family originated much later during vertebrate evolution. The members of the EMILIN/Multimerin family exert a diverse range of functions directly linked to their gC1q domains although several important

functions depend on interactions of soluble proteins and/or cellular receptors with other domains. Structure, function, and physiological and pathological consequences of the activity and/or lack of activity of family members will be summarized.

# **FUNDAMENTAL NOTIONS ABOUT ECM AND INTEGRIN RECEPTORS**

The cells of the microenvironment are embedded in a supporting network of extracellular matrix (ECM) constituents that include collagens, elastin, proteoglycans, and glycoproteins (Li et al., 2007; Zhang and Huang, 2011). The ECM does not constitute a mere structural scaffold but it elicits profound influences on cell behavior and affects cell growth, differentiation, motility, and viability (Bissell et al., 2005; Marastoni et al., 2008; Hynes, 2009;Cukierman and Bassi, 2010). The complexity of the microenvironment is further magnified by the property of ECM molecules to function as reservoirs of growth factors, cytokines, matrix metalloproteinases (MMPs), and processing enzymes (Sternlicht and Werb, 2001). The relative availability of these elements may be affected when the ECM rearranges during wound healing or tumor progression.

The cells sense their microenvironment through interactions with specific receptors, the integrins. Integrins, a large family of αβ heterodimeric cell membrane receptors, are key sensory molecules that translate chemical and physical cues from the dynamic constituents of ECM into biochemical signals that regulate many interrelated cellular processes (Legate et al., 2009). Integrins function as mechanotransducers transforming mechanical forces created by the ECM or the cytoskeleton into cell movement (Chen et al., 2004; Hynes, 2009; Schwartz, 2010). Eighteen α and eight β subunits have been so far discovered in mammals and at least 24 different combinations of α/β subunits have been characterized. Integrins display overlapping ligand specificity and many cells express multiple integrin receptors for the same ligand. Ligation of different integrin receptors by a certain ligand may induce different cellular effects in response to ECM ligation. Following integrin ligation a variety of downstream signaling events ensue: for instance, integrins activate survival pathways via the PI3K and MAPK pathways and act as essential cofactors for growth factors stimulation. Elevated integrin expression in the absence of appropriate ligands, or in the presence of natural or synthetic antagonists, promotes apoptosis under otherwise permissive growth conditions. Moreover, integrins coordinate survival or death responses or cell behavior as a function not only of ECM composition but also of its relative stiffness (Levental et al., 2009; Provenzano and Keely, 2011). This dual function provides an elegant mechanism through which tissue-remodeling events may regulate cell death or survival in a temporal, ECM-governed manner (Dupont et al., 2011). Thus, growth factor shortage and deficiency of pro-survival stimuli or absence of integrin activation leads to intracellular signals, mainly Akt down-regulation, which in turn activate a peculiar type of apoptosis in substrate-adherent cells, anoikis (Gilmore, 2005). Similar balances are at work if one considers the other cellular processes regulated by the many integrin–ECM relationships.

### **THE EMILIN/MULTIMERIN FAMILY OF PROTEINS**

Proteins containing the EMI domain, a cysteine-rich sequence of approximately 80 amino acids, define the EMI domain endowed (EDEN) superfamily in mammals (Braghetta et al., 2004). This superfamily comprises seven genes, which can be subdivided into three families on the basis of the major protein domains. The first family is formed by Emu1 and Emu2 genes (Leimeister et al., 2002) that except for the presence of an EMI domain do not share structural similarities with the other EDEN members. The second family comprises only one gene EMILIN-truncated (EMILIN-T) with a structure similar to the third larger family, but lacking the C-terminal globular domain of C1q (gC1q). The third is the EMILIN/Multimerin family. It is characterized by the N-terminal EMI domain, a central part of the molecule formed by a long (approximately 700 amino acids) region with high probability for coiled-coil structures, and a region homologous to the gC1q domain (**Figure 1**). The family includes EMILIN1 (Colombatti et al., 1985; Bressan et al., 1993; Doliana et al., 1999), EMILIN2 (Doliana et al., 2001), Multimerin1 (MMRN1; Hayward et al., 1991, 1995), and Multimerin2 (MMRN2; Sanz-Moncasi et al., 1994; Christian et al., 2001). Apart from MMRN1 that is deposited in the ECM but for the most part is sequestered in megakaryocytes, endothelial cells (ECs), and platelets granules (Adam et al., 2005), the members of the EMILIN family are constituents of the ECM Under normal conditions the expression of each individual gene overlaps with those of some other members of the family; the only exception is apparently the central nervous system, where only EMILIN2 is detected outside blood vessels, at least at the mRNA level (Braghetta et al., 2004). Moreover, EMILIN2 is specifically and abundantly expressed in mouse cochlear basilar membrane (Amma et al., 2003).

#### **DOMAIN ORGANIZATION**

The EMI domain (Doliana et al., 2000a) is located at the Nterminus in all the proteins in which it is expressed and displays a high sequence homology rangingfrom 60 to 70% between thefamily members. Cysteine-rich repeat modules are present in various ECM proteins, including several constituents or proteins associated with the elastic fiber (Pereira et al., 1993; Sinha et al., 1998; Doliana et al., 2000a). The EMI domain is rather unique: in fact, while most of the cysteine-rich domains described to date contain either six or eight cysteine residues, it has seven cysteine residues located at regular positions, with the exception of MMRN1 in which the second cysteine residue is missing. The specific spacing of cysteines and a number of strongly conserved aromatic and hydrophobic residues represent a diagnostic feature of this sequence. The consensus WRCCPG(Y/F)xGxxC toward the Cterminus of the domain is highly preserved and apparently unique of the EMI domain. Together with the invariable N-terminal position in all proteins discovered to date it clearly distinguishes the EMI domain from other cysteine-rich domain as, for example, the EGF-like that are often embedded within the protein sequence. In the last decade on the basis of reiterated sequence homology searches other proteins including *Drosophila* CED-1, fasciclin, and periostin have been proposed to harbor an EMI domain (Callebaut et al., 2003). However, after careful comparison refined by eye, it appears that these cysteine-rich domains very scarcely conform to the EMI domain consensus motif and should be classified as novel entities.

A few ECM proteins present long domains in which the 3–4–3– 4 spacing of hydrophobic residues (heptad repeats) predicts that they form α-helical coiled-coils which result in a rigid rod (Berger et al., 1995; Wolf et al., 1997). The large central regions of all the EMILIN/Multimerin family members shows a very low or undetectable homology at the primary sequence level; however, when analyzed by specific algorithms it reveals a common structural homology as all the members contain several regions in which the probability for coiled-coil formation is very high.

Between the long central coiled-coil part of the molecules and the gC1q C-terminal domain each component of the family presents different sequences that could confer specific structural and/or functional properties. EMILIN1 presents a 90 residues long sequence, including two sequences corresponding to structures referred to as "leucine zipper." Although its precise function has not been determined yet it might contribute to the coiled-coil association in the C- to N-terminal direction for the assembly of a proper EMILIN1 trimer (see below). Right downstream the leucine zipper there is a short collagenic region formed by 17 GXY triplets organized in a trypsin-resistant triple helix (Mongiat et al., 2000). In place of the leucine zipper motif EMILIN2

harbors a unique proline-rich motif of 53 residues that is highly hydrophilic and exposed to the solvent and thus available for potential interactions with other ligands or provide some flexibility that is not present in EMILIN1. Just upstream of the gC1q domain EMILIN2 also presents a collagenous sequence consisting of 17 triplets like EMILIN1, but with four interruptions, which makes it very unlikely that this sequence could form a stable triple helix. MMRN1 and MMRN2 do not harbor any collagenous sequence. On the contrary, in the corresponding position MMRN1 presents an EGF-like domain and MMRN2 an arginine rich sequence, whose functions are still undisclosed.

The gC1q domain lengths vary between 131 (C1q-C chain of C1q) and 151 (EMILIN1) residues with a high level of conservation of several hydrophobic and uncharged residues. Long before the first crystal structure was resolved Fourier-transform infrared spectroscopy and structure prediction of 15 gC1q sequences suggested a β-sheet secondary structure arrangement for this domain (Smith et al., 1994). The structure was then formally demonstrated by the analysis of the ACRP30/AdipoQ C1q crystal (Shapiro and Scherer, 1998) followed by the solution of the crystal structures of the gC1q of human collagen X (Bogin et al., 2002), mouse collagen VIII (Kvansakul et al., 2003), and human complement gC1q (Gaboriaud et al., 2003). All these crystals show similar quaternary structures obtained by the non-covalent association of three polypeptides whose tertiary structure adopts invariability a beta sandwich topology formed by 10 beta strands organized in two beta sheets. We were then the first to determine the three dimensional NMR solution structure of the human EMILIN1 gC1q homotrimer (Verdone et al., 2004, 2008, 2009). This domain exhibits a striking homology to the gC1q domains of several other members of the C1q/TNF superfamily but, at the same time, displays very peculiar characteristics (**Figure 2**). The quaternary structure of the complex is formed by three mostly identical subunits, apart from very minor differences inherently associated

**FIGURE 2 | Top: NMR solution structure of the homotrimeric EMILIN1 gC1q domain.** The structure was downloaded from database of protein structures maintained at NCBI site (http://www.ncbi.nlm.nih.gov/Structure/ mmdb/mmdbsrv.cgi?uid = 68072). Ribbon representation of the assembly, as side view, is presented in **(A)** the three protomers in the trimer are shown in different colors (pink, blue, and brown). Each monomer has a nine-stranded folding topology, with strands labeled according to the gC1q/tumor necrosis factor superfamily nomenclature. **(B)** The residues highly conserved in all the members of the gC1q superfamily and essential for a correct domain folding are shown in yellow only in one monomer for clarity. **(C)** Top view of EMILIN1 gC1q domain. The yellow bar highlights the solvent exposed position of the unstructured segment Tyr927–Gly945 **(D)** the X-ray structure of type VIII collagen gC1q, http://www.ncbi.nlm.nih. gov/Structure/mmdb/mmdbsrv.cgi?uid = 25284, showing the buried position of strand F. The same position of strand F is present in the X-ray solution structures of ACRP, type X collagen and Complement gC1q domains. Bottom: Sequence alignment between strand F (residues in bold) of representative members of the C1q superfamily and EMILINs gC1q. Asterisks indicate proteins for which the structure has been solved. Similar residues that are conserved in all proteins are shaded in gray, and the glutamic acid residue interacting whit α4β1 is in yellow.

with the molecular dynamic simulations (Verdone et al., 2008, 2009). Each protomer conserves the typical jelly-roll topology of two β-sheets of antiparallel strands. However, in contrast to all the gC1q structures solved to date, it presents a nine-stranded

β-sandwich fold instead of the 10-stranded fold. By sequence homology alignment of C1q family members, the lack of the F beta strand in EMILIN1 gC1q may be due to the insertion of nine residues in the middle of strand F that disrupt its secondary structure. As an important consequence, the highly ordered and rigid strand is substituted by a 19-residues long segment spanning from Y927 to G945. This sequence is unfolded, highly dynamic and highly accessible to solvent, with 10–11 residues protruding from the main globular structure making this region a good candidate for hosting an interaction site. Indeed, by site-directed mutagenesis experiments focused on this segment, we mapped the acidic residue E933 as the site of interaction between gC1q and the α4β1 integrin. This loop is not present in any of the already determined gC1q structures, and seems the only region of gC1q where single mutation or even deletions do not alter the overall domain structure. Interestingly, the quaternary structure of EMILIN1 gC1q assembly locates the unstructured loop at the apex of the homotrimer (**Figure 2D**). Another important information derived from the NMR solution structure of the EMILIN1 gC1q is a marked decrease of the monomer contact surface in the trimeric assembly (3,000 Å2), compared to ACRP (5,324 Å2), complement (5,490 Å2), Type VIII (6,150 Å2), and type X collagen (7,360 Å2) gC1q domains. Since the conserved residues are nearly identical in the different gC1q domains, it seems reasonable that the lower thermal stability detected in some of the domains, namely ACRP30 and EMILIN1 depends on the partly different neighboring residues among the various domains. Moreover, the decrease of the buried surface may in part be due to the missing contribution of unstructured element, or even to a contact disturbing activity, as suggested by the decrease of trimer thermal stability observed in some mutants (Doliana et al., unpublished data). Despite various attempts, we were unable to express a soluble form of EMILIN2 gC1q domain and hence to determine its structure, but, based on the sequence homology and functional data, it is conceivable that also the gC1q of EMILIN2 presents the same unstructured loop able to interact with the integrin receptor (see below). None of the other gC1q of the superfamily possess the described unstructured insertion and there are no data on a possible interaction with the α4β1 integrin. It is of note that the E993 residue is the only site of interaction with an integrin localized to date in a gC1q domain.

#### **GENOMIC ORGANIZATION**

The EMI domain of EMILIN1 is contained in exons 2 and 3 (Doliana et al., 2000b) and the organization of this domain is remarkably similar to that of EMILIN2 (Doliana et al., 2001). Exon 4 (almost 2000 bp in length) encodes for four coiled-coil structures and two leucine zippers which may represent a sort of functional unit. The short collagenous region of EMILIN1 is encoded by two very short exons (5, 6) and part of exon 7. Finally, the gC1q domain is encoded by part of exon 7 and exon 8. Similarly to EMILIN1 also the EMILIN2 gene consists of eight exons and seven introns. However, whereas the exon structure is remarkably similar between the two genes, several introns of EMILIN2 are much larger than those of EMILIN1 resulting in an overall gene size of about 40 kb as opposed to the remarkably compact EMILIN1 gene (8 kb). Nevertheless, the exon size pattern and location of introns in the coding sequence are very well conserved between the two genes likely indicating that they evolved from a common ancestor. Whereas most of gC1q domains are encoded within one exon, with the notable exception of cerebellin family where the coding sequence is splitted in three exons, those of EMILIN1 and EMILIN2 are encoded by two exons. MMRN1 and MMRN2 genes have a similar organization with EMILINs genes only limited to the EMI domain and the large exon coding for the coiled-coil structures. At variance, MMRNs present two additional short exons interposed between the EMI domain coding exons and the coiled-coil coding exon. In both genes the gC1q domain is encoded within a single exon.

Before describing in more detail the functions of EMILIN1/Multimerin family formally associated with the gC1q domain, a short summary of the various functions of EMILIN/Multimerin proteins not directly dependent gC1q will follow (**Figure 3**).

# **gC1q-INDEPENDENT FUNCTIONS**

### **EMILIN1 AND BLOOD PRESSURE CONTROL**

Arterial blood pressure is a function of the vasculature resistance and of the cardiac output, i.e., the amount of blood pumped out by the heart. The integrity and elasticity of the vessels and the modulation of blood pressure are determined by smooth muscle cells and ECs lining the vascular walls and by their relationship with ECM. EMILIN1 is intimately associated with elastic fibers and microfibrils in blood vessels (Colombatti et al., 1985, 2000). EMILIN1 is implicated in elastogenesis and in maintenance of blood vascular cell morphology (Zanetti et al., 2004). *Emilin*1−/<sup>−</sup> mice display elevated systemic blood pressure independent of cardiac output without defects in vascular contractility or mechanical properties. They have significantly narrower arteries causing the hypertensive phenotype. EMILIN1 binds to proTGF-β1 prior to the cleavage of LAP and upstream of the furin convertases and prevents its processing (Zacchigna et al., 2006). The increased TGF-β in the absence of EMILIN1 as it occurs in *Emilin*1−/<sup>−</sup> mice leads to reduced vascular cell proliferation and results in narrower blood vessels and increased peripheral resistance, thereby causing hypertension. Also *Emilin*2−/<sup>−</sup> and *MMRN*2−/<sup>−</sup> mice mice display hypertensive phenotypes but the underlying mechanisms are different and do not affect proTGF-β1 processing (G. M. Bressan et al., unpublished data). While the pathogenetic mechanism of blood hypertension is largely explained by the interaction between the EMI domain and proTGF-β, EMILIN1 may also be involved in the hypertensive phenotype with other domains; for instance, in anchoring smooth muscle cells to elastic fibers or in the process of vessel assembly. In fact, in the media of arteries there are specific sites of interaction between smooth muscle cells and elastic fibers (Daga-Gordini et al., 1987). At these sites the cell basement membranes are interrupted and the cell surface is in direct contact with the periphery of the amorphous elastin core, a region enriched in fibrillin (Sakai et al.,1986) and EMILIN1 (Bressan et al.,1993). The finding that EMILIN1 gC1q is an adhesive substrate (see below) and provides anchorage and/or homeostatic control of cell proliferation (see below) suggests that it might also contribute to affect the cell number and the size of smooth muscle cells in arterial walls.

#### **DEFENSE FUNCTION OF EMILIN/MULTIMERIN FAMILY MEMBERS**

The informational spectrum method (ISM), a virtual spectroscopy approach, is a fast and simple structure analysis of proteins and their functionally important domains (Veljkovic and Metlas, 1988; Shepherd et al., 2003; Wen et al., 2005). By applying the ISM to the human sequence database all EMILIN/Multimerin family members were predicted to interact with anthrax protective antigen (PA). The interaction with PA was then formally proven for EMILIN1 and EMILIN2 by solid phase assays using recombinant proteins (Doliana et al., 2008). The interacting part of EMILIN2 resides in the first N-terminal 108 residues. Residue D425 of PA is important in mediating *Bacillus anthracis* cell toxicity (Ahuia et al., 2003) and its deletion results in a PA variant which still assembles but forms non-functional complexes leading to a complete inhibition of the channel forming ability of PA (Sellman et al., 2001). The deletion mutant (del425) of PA binds to EMILIN1 with very low efficiency suggesting that EMILIN1 interacts with this residue to prevent cell toxicity. The interaction is independent of the presence of divalent cations and does not involve PA aspartic residue at 683, a critical residue in cell surface receptor binding. The functional consequences of the specific interaction of PA *in vitro* and the inhibition of cell cytotoxicity by the LF–PA complex in the presence of EMILIN1 suggests that EMILINs/Multimerins, along with the LDL receptor-related protein LRP6 (Mayer et al., 2001), represent additional targets and/or binding proteins potentially useful for countermeasures against *B. anthracis* toxin lethality.

### **EMILIN2 AND CELL DEATH PROMOTION**

Among the few ECM molecules that impair cell viability (Shichiri and Hirata, 2001; Todorovicç et al., 2005; Seidler et al., 2006; Ramakrishnan et al., 2007; Tai and Tang, 2008; Juric et al., 2009) EMILIN2 adopts an entirely different mechanism, i.e., the activation of the extrinsic apoptotic pathway through a direct binding to death receptor 4 (DR4) and 5 (DR5; Pan et al., 1997; LeBlanc and Ashkenazi, 2003). This binding, in analogy with the downstream events that occur after ligation of the death ligand TRAIL to DR4 or DR5, is followed by receptor clustering, co-localization with lipid rafts, DISC assembly, and caspase activation (Mongiat et al., 2007). Thus, EMILIN2 mimics the activity of the known death receptor ligands (Walczak et al., 1997; Ashkenazi, 2008; Johnstone et al.,2008). This pro-apoptoticfunction resides in a homotrimeric 90 residues long region toward the N-terminus of the molecule, positioned right after the EMI domain in the coiled-coil domain (Mongiat et al., 2010). Since normal cells do not express DR4 or DR5 it can be speculated that EMILIN2 or specific peptides encompassing the pro-apoptotic region might be used as antitumor agents in combination with anti-angiogenic drugs such as bevacizumab (Mongiat et al., 2010) and in cases where TRAIL resistance is involved (Hengartner, 2000; Fulda, 2009).

### **MMRN2, EMILIN2, AND ANGIOGENESIS**

MMRN2 is found along the blood vessels in close contact with ECs in normal and tumor vasculature (Sanz-Moncasi et al., 1994; Christian et al., 2001; Koperek et al., 2007) and contributes to the maintenance of blood vessels' homeostasis by affecting the VEGF-A/VEGFR2 axis (Lorenzon et al., 2011). Neither proliferation nor apoptotic rate are affected. By preventing the interaction

of VEGF-A with VEGFR1 and VEGFR2, MMRN2 reduces their activation and impaires the non-integrin dependent motility of ECs leading to a decreased intra-tumoral angiogenesis and consequent impaired tumor growth. It is conceivable that MMRN2 attains a high EC pericellular concentration and thus competes for the binding of VEGF-A to its receptors. MMRN2 may thus represent a novel key homeostatic molecule indispensable for the maintenance of blood vessel integrity.

Despite the strong reduction of tumor growth induced by the pro-apoptotic function of EMILIN2 (Mongiat et al., 2007, 2010), EMILIN2 also increases the overall intratumor blood vessel density. As a consequence, added bevacizumab is able to further inhibit tumor growth (Mongiat et al., 2010).

#### **MMRN1 AND PLATELET FUNCTION**

MMRN1 is a soluble S–S linked homopolymer stored in platelets, megakaryocytes, and ECs and deposited in ECM (Hayward, 1997; Adam et al., 2005). It supports the adhesion of platelets, neutrophils, and ECs via integrin αvβ3 and αIIbβ3 (Adam et al., 2005). It binds to collagen and it is able to enhance von Willebrand factor-dependent platelet adhesion to collagen thus supporting thrombus formation. MMRN1 has a high affinity for factor V (Jeimi et al., 2008) and this facilitates the co-storage in platelet αgranules. When MMRN1 is released from platelets during platelet activation it regulates thrombin formation limiting thrombus formation. This protein is playing an important homeostatic control in platelets aggregation and its consequences. Unfortunately, all these multiple functions have not been assigned to any of the domains of MMRN1 apart from cell adhesion that depends on an RGD peptide at the N-terminus (Adam et al., 2005).

# **gC1q-DEPENDENT FUNCTIONS HOMOTRIMER ASSOCIATION**

Several members of the C1q/TNF superfamily associate into trimers. The chain composition of these different superfamily members varies from heterotrimers formed of three distinct chains as in C1q, or of homotrimers as in collagen typesVIII (Rosenblum, 1996; Illidge et al., 1998; Greenhill et al., 2000), and X (Thomas et al.,1991;Reichenberger et al.,1992;Chan et al.,1996; Frischholtz et al., 1998), ACRP30 (Hu et al., 1996; Shapiro and Scherer, 1998), MMRN1 (Hayward et al., 1995). The potential self-interaction of the EMILIN1 gC1q domain was formally demonstrated with the use of two-hybrid system approaches, yeast mating and cotransformation that both confirm the self-association of the single protomers (Mongiat et al., 2000).

### **SUPRAMOLECULAR ASSEMBLY**

The smallest EMILIN1 protomer under reducing conditions is a trimer held together by strong non-covalent bonds. Likely the formation of the gC1q homotrimer acts as a nucleation in a C- to N-terminal direction, facilitating the formation of the triple helix. Several of the gC1q domain-containing molecules assemble to high order S–S bonded structures composed of 9–12 polypeptides multimers, as in ACRP (Scherer et al., 1995), 18 polypeptides as in C1q (Reid, 1983; Kishore and Reid, 2000), collagens type X (Kwan et al., 1991), and probably also type VIII (Sawada et al., 1990). The sizes of MMRN1 multimers are well above 24 polypeptides (Hayward et al., 1991). EMILIN1 multimers can reach sizes of several million daltons, corresponding to S–S bonded complexes composed of several dozens of polypeptides (Mongiat et al., 2000). We have preliminary evidence that secreted recombinant EMILIN2 is assembled in smaller multimers compared to EMILIN1.

At present, the mechanistic basis for the formation of EMILINs supramolecular aggregates is unknown. While covalent S–S bonds are involved in the association it is possible that also non-covalent interactions participate to allow supramolecular assembly. In addition, it is not clear whether each EMILIN exclusively forms homotypic macro-aggregates consisting of only one member or also heterotypic macro-aggregates consisting of more members. This latter possibility is supported, at least for EMILIN1 and EMILIN2, by the partial overlapping gene expression pattern *in vivo* in many tissues (Braghetta et al., 2002), and by the codistribution of the secreted deposited proteins in *in vitro* cell cultures (Doliana et al., 2001). In situations where both EMILINs are expressed simultaneously, it is theoretically possible that each EMILIN forms either separate homotypic or that they may form heterotypic assemblies. The isolation of EMILIN2 from expression libraries using the gC1q domain of EMILIN1 as a bite proved that gC1q domains from distinct but similar molecules could interact at least in the two-hybrid system (Doliana et al., 2001). Then, also homo- and hetero-interactions between gC1q and EMI domains were determined by qualitative and quantitative two-hybrid assays and by immunochemical evidence of co-expression in yeast human cell extracts (Bot et al., in preparation). It is possible, at least *in vitro*, that gC1q and EMI domains from the same or different EMILINs interact in a head-to-tail fashion. The question remains whether there is the need for adapter molecules that favor this interaction or whether this interaction is direct and EMILIN1 and EMILIN2 can co-polymerize head-to-tail to form heterotypic non-covalent multimers *in vivo*.

# **INTERACTION OF gC1q WITH α4β1 INTEGRIN**

α4β1 integrin binds to the cell surface vascular cell adhesion molecule-1 (VCAM-1) on activated endothelium (Hamann et al., 1994) and to the ECM molecule fibronectin (FN; Humphries et al., 1986; Komoriya et al., 1991). The key VCAM-1 binding site is the tripeptide motif sequence IDS. This sequence is homologous to the LDV active motif on the CS-1 peptide of FN, suggesting that α4β1 may interact with FN and VCAM-1 through a similar mechanism. EMILIN1 is an adhesive ligand for α4β1 (Spessotto et al., 2003, 2006;Verdone et al., 2008; Danussi et al., 2011) and this function is fully accounted for by the gC1q domain (Spessotto et al., 2003). All polypeptides with integrin binding capability display exposed aspartic or glutamic acid residues located in mobile loops protruding from the main core of the ligand (Leahy et al., 1996; Casasnovas et al., 1998). These residues are critical for integrin recognition. The three dimensional conformation of the active site on ligand–receptor binding is extensively demonstrated (in particular for RGD containing ligands; Humphries, 1990; Leahy et al., 1996;Arnaout et al., 2007; Barczyk et al., 2010). The asparticacid-based sequences (e.g., RGD, LDV, KGD, RTD, and KQAGD) bind to the majority of integrins (Yamada, 1991) and mutations around the aspartic acid residue, i.e., G in RGD or L in LDV, greatly affect integrin recognition (Pierschbacher and Ruoslahti, 1984; Komoriya et al., 1991; Cherny et al., 1993). Other integrins

interact with ligands that contain glutamic-acid-based sequences (Michishita et al.,1993). However,it is likely that the scenario in the EMILIN1–α4β1 interaction is more complex than in conventional α4β1 binding to short linear peptide consensus sequences, representing the common feature of ECM integrin ligands (Humphries, 1990; Humphries et al., 2006). A gC1q domain in which the glutamic acid at position 933 (E933) was substituted with an alanine residue is no longer functional in cell adhesion assays and it is not recognized by α4β1 integrin suggesting that this residue plays a major role in this interaction. We exclude that the loss of activity of the E933A mutant or of deletion mutants encompassing this residue is due to a global folding change of the protein (Verdone et al., 2008). Thus, the gC1q–α4β1 interaction provides a further example of the functional relevance of disordered regions in proteins (Radivojac et al., 2007).

How would an homotrimeric molecule such as gC1q mechanistically interact with the α4β1 integrin? The explanation that a linear tripeptide from one single monomer as in CS-1 peptide of FN could represent the α4β1 ligand binding site on gC1q is apparently excluded by the finding that the L932A mutant does not loose its cell adhesion binding property. Several mutant residues around E933 are still functional and this indicates that the α4β1 integrin binding site involves only E933. Thus, this residue plays an absolutely central role in α4 integrin mediated interaction, whereas several residues close to E933 do not seem to participate in this interaction (Verdone et al., 2008). The NMR solved structure locates residue E933 in the flexible loop at the apex of the EMILIN1 gC1q domain assembled into a symmetric trimer. The stoichiometry of three integrin molecules each recognizing one E933 residue on the unstructured loops of the gC1q trimer is ruled out for steric reasons. Thus, the interaction pattern with α4β1 might well entail the binding of the three E933 carboxylate groups to a single integrin molecule. An example of an integrin–ligand interaction that requires two structural elements residing on different polypeptide chains has been already reported: to be recognized by αvβ3 integrin the LG1–3 globular domain of laminin α chain requires the intact heterotrimeric C-terminal portion of the coiled-coil domain, including the most distant C-terminal region of the β and γ chains of laminin (Künneken et al., 2004). In that case it has been hypothesized that the C-terminus of the γ chain is assembled together with a specific region of LG1–3, leading to the formation of an interchain functional ligand tripeptide. The gC1q–α4 interaction could represent the first example of an integrin binding site located on a homotrimeric assembly that employs a different mode of integrin engagement and prospects a new model for the interaction geometry recognition between an integrin binding site and a functional ligand located on a homotrimeric assembly. In this scenario we hypothesize the involvement of two/three E933 residues for a single integrin molecule engagement for a proper interaction able to fit the α4 pocket in the βMIDAS region. One possibility to tackle this question is the use of bicistronic vectors coding for wild type and non-functional mutant E933A gC1q in order to obtain trimers with a single copy or two copies of the wild type sequence on average in the homotrimer. In this systems the mutant and wild type monomers are randomly assembled into trimers to form a heterogeneous population and can be isolated by affinity chromatography. Preliminary data suggests that

cell adhesion to heterogeneous gC1q trimers composed by both wild type and E933A mutants, is negatively affected implying the need of all three wild type monomers to achieve a proper integrin ligation with the homotrimer (Capuano et al., in preparation).

# **FUNCTIONAL CONSEQUENCES OF THE gC1q/INTEGRIN LIGATION**

#### **CELL ADHESION**

It is known that the adhesion of α5β1 or αvβ3 integrins to FN and vitronectin (VN) promotes a profound cytoskeletal reorganization (Ruoslahti and Pierschbacher, 1987; Defilippi et al., 1999; Ruoslahti, 1999; Zamir and Geiger, 2001; Campbell, 2008). On the contrary, the α4β1-dependent interactions extensively studied in hematopoietic cells (Morimoto et al., 1998; Steeber et al., 2005; Rose et al., 2007) as well as in transfected adherent cells (Pinco et al., 2002) prompt only the initial and intermediate stages of cell adhesion, i.e., attachment and spreading, whereas focal adhesion and stress fiber formation, characterizing strong cell adhesion, are rarely if ever observed (Iida et al., 1995).

Contrary to common beliefs that consider α4β1 an exclusive leukocyte integrin, there is a wealth of data available that demonstrates that this integrin is widely expressed, although at lower levels, in normal tissues including brain, heart, kidney, lung, muscle,liver prostate, skin, and ovary as well as their tumoral counterparts; α4β1 is also expressed in colon, bladder, breast, cervix, and melanoma tumor cells (Holzmann et al., 1998). In addition, its expression is upregulated by inflammatory cytokines (Weber et al., 1996; Milner and Campbell, 2003). This data makes α4β1 an integrin which is potentially involved in multiple and extensive interactions under several biological conditions including tumor spread.

Cells attached to EMILIN1 display a pattern of actin and paxillin distribution with accumulation of ruffles-inducing signals and lack of polarization and of stress fiber formation (Spessotto et al., 2003). Recognition of the gC1q domain does not require exogenous activation of α4β1 nor the addition of Mn2<sup>+</sup> nor anti-β1-activating mAbs; instead the presence of Ca2<sup>+</sup> or Mg2<sup>+</sup> is necessary since EDTA treatment fully abolishes cell adhesion (Spessotto et al., 2003). Similar results are observed upon binding to EMILIN2 (Spessotto et al., unpublished observations). On the contrary, MMRN1 that possesses a RGD site binds to αvβ3 and αIIbβ3 integrins on platelets (Adam et al., 2005).

## **CELL MIGRATION**

Among the numerous cell types that migrate on EMILIN1 trophoblasts are particularly interesting. The finding that trophoblast cells could attach to and very efficiently migrate and haptoractically move toward EMILIN1 using the α4β1 integrin without any prior artificial cellular activation was rather unexpected (Spessotto et al., 2006) because directional haptotaxis was much more efficient toward EMILIN1 compared with FN. In addition, under several conditions the cooperation of MMPs with integrins plays a contributing role in order to better direct cellular migration (Deryugina et al., 2001). Accordingly, MT1-MMP is strongly upregulated when trophoblasts cells are co-cultured with EMILIN1-producing decidual stromal cells. The finding that the general MMP inhibitor GM6001 is more effective and affects cell

migration toward EMILIN1 at 1-h migration, whereas at 4 h the inhibitory effect is stronger when cells move in an α5β1 integrin dependent mode toward FN (Spessotto et al., 2006), suggests that both ligands might be involved in the uterine wall invasion process but with a sequential role. Deposited EMILIN1 in the uterine tissue could thus favor the initial α4β1 dependent invasion by trophoblast cells to be assisted at later times by other ECM constituents such as FN and different integrins.

# **CELL PROLIFERATION**

It is generally known that integrin engagement positively regulates cell growth (Clark and Brugge, 1995; Walker and Assoian, 2005; Gilcrease, 2007; Streuli, 2009). The finding that EMILIN1 by the direct engagement of the gC1q domain modulates skin cell proliferation points out a novel function of α4β1 as well as the structurally and functionally similar α9β1 integrin (Danussi et al., 2011). The lack of integrin occupancy by EMILIN1, as it occurs in *Emilin*1−/<sup>−</sup> mice, results in increased Ki67 positive cells in epidermis and dermis. This is accompanied by reduction of PTEN phosphatase and strong upregulation of pErk1/2. Accordingly, when HT1080 and CaCo-2 cells expressing α4β1 or α9β1 integrins, respectively, adhere to gC1q PTEN levels increase. Even the addition of soluble gC1q to cells attached to plastic upregulates PTEN and the addition of TGF-β further increases PTEN levels. This is accompanied by decreased pErk1/2 levels and suggests a link between PTEN and α4/α9 integrin engagement: when α4/α9 integrins are not ligated by gC1q PTEN is down-regulated determining the activation of proliferative pathways such as pAkt and pErk1/2. Mechanistically, the increased pErk1/2 reduces TGF-β signaling by the phosphorylation of Smad2 at inhibitory Ser245/250/255 residues. It seems likely that these downstream changes concur *in vivo* to the decreased proliferation of basal keratinocytes and dermal fibroblasts (**Figure 4**).

# **ROLE OF EMILIN1 gC1q IN PHYSIOLOGICAL AND PATHOLOGICAL CONDITIONS**

Targeted inactivation of the *Emilin*1 gene in the mouse induces multiple phenotypes characterized by: decreased diameter of arterial vessels and systemic hypertension (Zacchigna et al., 2006); a lymphatic phenotype with a significant reduction of anchoring filaments (AFs) and enlargement of lymphatic vessels leading to a mild lymphatic dysfunction (Danussi et al., 2008); dermal and epidermal hyperproliferation (Danussi et al., 2011). Only the last phenotype is strictly dependent on the lack of gC1q.

### **EMILIN1 IN LYMPHATIC VESSEL FUNCTION**

The connections between ECM and lymphatic endothelial cells (LECs) that critically affect lymphatic vasculature function are mediated by AFs (Swartz and Skobe, 2001). These fibrillin rich structures, which are part of a hierarchical fibrillar elastic apparatus (Gerli et al., 2000), are highly sensitive to interstitial stresses (Swartz and Skobe, 2001) and exert a significant tension on LECs: their main function is to widen the capillary lumen and open the overlapping cell junctions facilitating lymph formation (Cueni and Detmar, 2006). Abnormalities of AFs may reduce adsorption from interstitium and propulsion of lymph and cells and promote pathological conditions such as lymphedema or diseases related to

impaired immune responses (De Cock et al., 2006). Dysfunctions may also be induced by extensive and chronic degradation of the ECM that renders lymphatic vessels non-responsive to the changes in the interstitium (Pelosi et al., 2007).

Consistent with the absence of a basement membrane *in vivo* (Pepper and Skobe, 2003), LECs secrete very little ECM (Farnsworth et al., 2006). EMILIN1 is produced by LECs *in vitro* and is present at the abluminal side of lymphatic vessels. It forms fibers directed from LECs to the surrounding ECM. Therefore, the detection of abundant EMILIN1 suggested the involvement of this protein in the structure and function of lymphatic vessels (Danussi et al., 2008). In fact, a comparative study between wild type and *Emilin*1−/<sup>−</sup> mice highlighted that *Emilin*1−/<sup>−</sup> mice display hyperplasia and enlargement of lymphatic vessels compared to wt mice. Morphological alterations of the lymphatic vessels of *Emilin*1−/<sup>−</sup> mice also are evident at the ultrastructural level. Structural abnormalities such as the reduction in the number of AFs and the formation of extensive multiple overlapping junctions of LECs are among the most evident alterations. LECs in *Emilin*1−/<sup>−</sup> mice frequently appear detached from the underlying ECM and from each other and result in the formation of irregular and multiple intraluminal flaps suggesting that EMILIN1 is involved in the process of lymphatic remodeling observed in these mice (Danussi et al., 2008). These alterations are associated with inefficient lymph drainage, enhanced lymph leakage, and lymphedema. However, in contrast to other recently described lymphatic-lineage gene targeting in mouse models, that in most cases result embryonic or perinatal lethal (Tammela et al., 2005), homozygous disruption of *Emilin*1 gene induces only a mild phenotype. Notably, this

is the first abnormal lymphatic phenotype associated with the deficiency of an ECM protein. Finally, the valve defects observed in collecting vessels are grossly similar to those of α9 integrin null mice (Bazigou et al., 2009), suggesting that the interaction of EMILIN1 and α9 integrin demonstrated with basal skin keratinocytes (Danussi et al., 2011) may also be important in valve morphogenesis in LECs.

### **EMILIN1 IN SKIN HOMEOSTASIS**

The phenotype of the *Emilin*1−/<sup>−</sup> mouse skin revealed increased thickness of epidermis and dermis.

Elastin microfibrillar interface protein 1 interacts with α4β1 (expressed on fibroblasts) and the closely related α9β1 (expressed on keratinocytes) to provide an important external cue for the maintenance of a correct homeostasis between proliferation and differentiation (Danussi et al., 2011). In this context, signals emanating from EMILIN1-ligated α4/α9 integrins are antiproliferative (Danussi et al., 2008, 2011). But how does EMILIN1 brings about the control of cell proliferation? EMILIN1 is produced only by skin fibroblasts and it is deposited in the dermis where it is abundantly expressed. However, EMILIN1-positive fibrils depart from near the BM and reach the basal keratinocyte layer, thus connecting the epidermis to the underlying dermal layer. *In vitro* co-cultures of EMILIN1-producing fibroblasts and keratinocytes provided the formal demonstration that the contact between deposited EMILIN1 and α4/α9 integrins directly regulates cell proliferation: only "contact" and not "transwell" (so that the two cell types are physically separated) co-cultures of keratinocytes with wild type EMILIN1-producing fibroblasts

inhibits proliferation. Consequently, silencing of EMILIN1 stimulates the proliferation of co-cultured keratinocytes. Proliferative and anti-proliferative signals occur simultaneously and the latter, EMILIN1/gC1q ligation in this case, can override the proproliferative signals only when they reach a certain threshold (Müller et al., 2008). It is likely that under certain circumstances the pericellular concentration of EMILIN1 in basal keratinocytes (and fibroblasts) exceeds this threshold and stops cell proliferation. Our findings open new perspectives in the molecular mechanisms of quiescence versus proliferation in basal keratinocytes. In addition, given the high expression of α9β1 integrin on these cells, the demonstration that EMILIN1 is a novel ligand for this integrin (Danussi et al., 2011) supports a scenario in which one of the functional consequences is the integration of EMILIN1 into the complex connections of basal keratinocyte turn over and the cross talks between basal keratinocytes, underlying ECM and stromal cells.

#### **EMILIN1 IN CANCER DEVELOPMENT**

In *Emilin*1−/<sup>−</sup> mice subjected to a skin carcinogenesis protocol tumor appearance was significantly accelerated and the number and size of tumors compared to wild type mice increased (Danussi et al., submitted). It seems likely that the aberrant skin homeostasis generated by EMILIN1 deficiency in these mice (Danussi et al.,

#### **REFERENCES**