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

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

X-Ray Structure of the Human Calreticulin Globular Domain Reveals a Peptide-Binding Area and Suggests a Multi-Molecular Mechanism

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca2+-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism

L'integrine plaquettaire GPIIb IIIa : clonage, sequencage et expression des ADN complementaires : etude realisee a partir de megacaryocytes humains

SIGLECNRS T Bordereau / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Reconnaissance et phagocytose des cellules apoptotiques "Rôle de C1q et de la calréticuline"

La mort cellulaire par apoptose est un processus biologique fondamental, nécessitant des interactions fines avec le système immunitaire pour une reconnaissance et une élimination efficace des cellules mortes. C'est ainsi que C1q, une molécule du complément, essentielle dans le système immunitaire inné, a été mise en évidence comme fortement impliquée dans le mécanisme de reconnaissance et d'élimination des cellules apoptotiques, notamment via sa région globulaire (C1qGR). Récemment, la translocation de la calréticuline (CRT) au niveau externe de la membrane plasmique des cellules cancéreuses a été identifiée comme un signal eat-me pouvant être immunogène. Initialement, l'interaction entre la CRT et C1q a été caractérisée à la surface des phagocytes et la CRT a été considérée comme un récepteur pour les queues collagènes de C1q (C1qCLF). L'ensemble de ces observations est en faveur d'un double rôle de la CRT, à la surface des phagocytes et des cellules apoptotiques. Dans un premier temps, l'élaboration d'une stratégie de FRET a permis de détecter une interaction directe entre la CRT et C1qGR à la surface de la cellule HeLa en apoptose précoce. Dans un second temps, la mise en place de tests de phagocytose a permis de montrer que la calréticuline exposée à la surface des cellules apoptotiques peut moduler la phagocytose : des effets opposés ont été observés selon le modèle cellulaire utilisé (HeLa traitées par des ARNi ou MEF CRT-/-) et dans certaines conditions, une modulation combinée de la calréticuline et de C1q a été observée sur la réponse inflammatoire (production de cytokines).Cell death by apoptosis is a fundamental biological process, requiring fine interactions with the immune system for the effective recognition and removal of the apoptotic cells. C1q, a complement molecule, essential in the innate immune system, has been shown to be strongly involved in the mechanism of recognition and elimination of apoptotic cells, mainly through its globular region (C1qGR). Recently, the translocation of calreticulin (CRT) at the surface of cancer cells has been identified as an eat-me signal, which can be immunogenic. Initially, the interaction between CRT and C1q was characterized on phagocytes surface and CRT was described as a receptor for the collagenous tails of C1q (C1qCLF). All these observations support a dual role of CRT at the surface of phagocytes and their targets. At first, using a FRET strategy we achieve to detect a direct interaction between CRT and C1qGR at the surface of early apoptotic HeLa cells. Second, the establishment of phagocytosis assays showed that calreticulin exposed at the surface of apoptotic cells could modulate phagocytosis: opposite effects were observed depending on the cellular model used (RNAi treated HeLa or MEF CRT-/-) and under certain conditions, a combined modulation of calreticulin and C1q was observed on the inflammatory response (cytokine production).SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Calreticulin Release at an Early Stage of Death Modulates the Clearance by Macrophages of Apoptotic Cells

International audienceCalreticulin (CRT) is a well-known "eat-me" signal harbored by dying cells participating in their recognition by phagocytes. CRT is also recognized to deeply impact the immune response to altered self-cells. In this study, we focus on the role of the newly exposed CRT following cell death induction. We show that if CRT increases at the outer face of the plasma membrane and is well recognized by C1q even when phosphatidylserine is not yet detected, CRT is also released in the surrounding milieu and is able to interact with phagocytes. We observed that exogenous CRT is endocytosed by THP1 macrophages through macropinocytosis and that internalization is associated with a particular phenotype characterized by an increase of cell spreading and migration, an upregulation of CD14, an increase of interleukin-8 release, and a decrease of early apoptotic cell uptake. Importantly, CRT-induced pro-inflammatory phenotype was confirmed on human monocytes-derived macrophages by the overexpression of CD40 and CD274, and we found that monocyte-derived macrophages exposed to CRT display a peculiar polarization notably associated with a downregulation of the histocompatibility complex of class II molecules hampering its description through the classical M1/M2 dichotomy. Altogether our results highlight the role of soluble CRT with strong possible consequences on the macrophage-mediated immune response to dying cell

Role of C1q in Efferocytosis and Self-Tolerance — Links With Autoimmunity

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Nanoscale imaging of CD47 informs how plasma membrane modifications shape apoptotic cell recognition

International audienceCD47 recognized by its macrophage receptor SIRPα serves as a “don’t eat-me” signal protecting viable cells from phagocytosis. How this is abrogated by apoptosis-induced changes in the plasma membrane, concomitantly with exposure of phosphatidylserine and calreticulin “eat-me“ signals, is not well understood. Using STORM imaging and single-particle tracking, we interrogate how the distribution of these molecules on the cell surface correlates with plasma membrane alteration, SIRPα binding, and cell engulfment by macrophages. Apoptosis induces calreticulin clustering into blebs and CD47 mobility. Modulation of integrin affinity impacts CD47 mobility on the plasma membrane but not the SIRPα binding, whereas CD47/SIRPα interaction is suppressed by cholesterol destabilization. SIRPα no longer recognizes CD47 localized on apoptotic blebs. Overall, the data suggest that disorganization of the lipid bilayer at the plasma membrane, by inducing inaccessibility of CD47 possibly due to a conformational change, is central to the phagocytosis process

Relative contribution of c1q and apoptotic cell-surface calreticulin to macrophage phagocytosis.

International audienceC1q has been shown to recognize apoptotic cells, to enhance their uptake and to modulate cytokine release by phagocytes and thus promote immune tolerance. Surface-exposed calreticulin (CRT), known as a C1q receptor, is also considered to be an early eat-me signal that enhances the phagocytosis of apoptotic cells and is capable of eliciting an immunogenic response. However, the molecular mechanisms that trigger these functions are not clear. We hypothesized that CRT and C1q might act together in these processes. We first showed, by means of fluorescence resonance energy transfer (FRET), that CRT interacts with the C1q globular region at the surface of early apoptotic cells. Next, we pointed out that knockdown of CRT on early apoptotic HeLa cells impairs the enhancement effect of C1q on their uptake by THP-1 monocyte-derived macrophages. Furthermore, a deficiency of CRT induces contrasting effects on cytokine release by THP-1 macrophages, increasing interleukin (IL)-6 and monocyte chemotactic protein 1/CCL2 and decreasing IL-8. Remarkably, these effects were greatly reduced when apoptotic cells were opsonized by C1q, which counterbalanced the effect of the CRT deficiency. These results demonstrate that CRT-C1q interaction is involved in the C1q bridging function and they highlight the particular ability of C1q to control the phagocyte inflammatory status, i.e. by integrating the molecular changes that could occur at the surface of dying cells

Proteinase 3 Interferes With C1q-Mediated Clearance of Apoptotic Cells

International audienceProteinase 3 (PR3) is the autoantigen in granulomatosis with polyangiitis, an autoimmune necrotizing vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCAs). Moreover, PR3 is a serine protease whose membrane expression can potentiate inflammatory diseases such as ANCA-associated vasculitis and rheumatoid arthritis. During apoptosis, PR3 is co-externalized with phosphatidylserine (PS) and is known to modulate the clearance of apoptotic cells through a calreticulin (CRT)-dependent mechanism. The complement protein C1q is one mediator of efferocytosis, the clearance of altered self-cells, particularly apoptotic cells. Since PR3 and C1q are both involved in the clearance of apoptotic cells and immune response modulation and share certain common ligands (i.e., CRT and PS), we examined their possible interaction. We demonstrated that C1q binding was increased on apoptotic rat basophilic leukemia (RBL) cells that expressed PR3, and we demonstrated the direct interaction between purified C1q and PR3 molecules as shown by surface plasmon resonance. To better understand the functional consequence of this partnership, we tested C1q-dependent phagocytosis of the RBL cell line expressing PR3 and showed that PR3 impaired C1q enhancement of apoptotic cell uptake. These findings shed new light on the respective roles of C1q and PR3 in the elimination of apoptotic cells and suggest a novel potential axis to explore in autoimmune diseases characterized by a defect in apoptotic cell clearance and in the resolution of inflammation