Philippe Frachet

C1Q Binds Phosphatidylserine and Likely Acts as a Multiligand-Bridging Molecule in Apoptotic Cell Recognition.

Efficient apoptotic cell clearance is critical for maintenance of tissue homeostasis, and to control the immune responses mediated by phagocytes. Little is known about the molecules that contribute “eat me” signals on the apoptotic cell surface. C1q, the recognition unit of the C1 complex of complement, also senses altered structures from self and is a major actor of immune tolerance. HeLa cells were rendered apoptotic by UV-B treatment and a variety of cellular and molecular approaches were used to investigate the nature of the target(s) recognized by C1q. Using surface plasmon resonance, C1q binding was shown to occur at early stages of apoptosis and to involve recognition of a cell membrane component. C1q binding and phosphatidylserine (PS) exposure, as measured by annexin V labeling, proceeded concomitantly, and annexin V inhibited C1q binding in a dose-dependent manner. As shown by cosedimentation, surface plasmon resonance, and x-ray crystallographic analyses, C1q recognized PS specifically and avidly (KD = 3.7–7 × 10−8 M), through multiple interactions between its globular domain and the phosphoserine group of PS. Confocal microscopy revealed that the majority of the C1q molecules were distributed in membrane patches where they colocalized with PS. In summary, PS is one of the C1q ligands on apoptotic cells, and C1q-PS interaction takes place at early stages of apoptosis, in newly organized membrane patches. Given its versatile recognition properties, these data suggest that C1q has the unique ability to sense different markers which collectively would provide strong eat me signals, thereby allowing efficient apoptotic cell removal.

Investigations on the C1q-calreticulin-phosphatidylserine interactions yield new insights into apoptotic cell recognition.

Both C1q and calreticulin (CRT) are involved in the recognition of apoptotic cells. CRT was initially characterized as a receptor for the C1q collagen-like fragment (CLF), whereas C1q was shown to bind apoptotic cells through its globular region (GR). Using purified CRT and recombinant CRT domains, we now provide unambiguous experimental evidence that, in addition to its CLF, the C1q GR also binds CRT and that both types of interactions are mediated by the CRT globular domain. Surface plasmon resonance analyses revealed that the C1q CLF and GR domains each bind individually to immobilized CRT and its globular domain with K(D) values of (2.6-8.3) × 10(-7) M. Further evidence that CRT binds to the C1q GR was obtained by electron microscopy. The role of CRT in the recognition of apoptotic HeLa cells by C1q was analyzed. The C1q GR partially colocalized with CRT on the surface of early apoptotic cells, and siRNA (small interfering RNA)-induced CRT deficiency resulted in increased apoptotic cell binding to C1q. The interaction between CRT and phosphatidylserine (PS), a known C1q ligand on apoptotic cells, was also investigated. The polar head of PS was shown to bind to CRT with a 10-fold higher affinity (K(D)=1.5 × 10(-5) M) than that determined for C1q, and, accordingly, the C1q GR-PS interaction was impaired in the presence of CRT. Together, these observations indicate that CRT, C1q, and PS are all closely involved in the uptake of apoptotic cells and strongly suggest a combinatorial role of these three molecules in the recognition step.

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.

Proteins of the Innate Immune System Crystallize on Carbon Nanotubes but Are Not Activated

The classical pathway of complement is an essential component of the human innate immune system involved in the defense against pathogens as well as in the clearance of altered self-components. Activation of this pathway is triggered by C1, a multimolecular complex comprising a recognition protein C1q associated with a catalytic subunit C1s-C1r-C1r-C1s. We report here the direct observation of organized binding of C1 components C1q and C1s-C1r-C1r-C1s on carbon nanotubes, an ubiquitous component in nanotechnology research. Electron microscopy imaging showed individual multiwalled carbon nanotubes with protein molecules organized along the length of the sidewalls, often over 1 μm long. Less well-organized protein attachment was also observed on double-walled carbon nanotubes. Protein-solubilized nanotubes continued to attract protein molecules after their surface was fully covered. Despite the C1q binding properties, none of the nanotubes activated the C1 complex. We discuss these results on the adsorption mechanisms of macromolecules on carbon nanotubes and the possibility of using carbon nanotubes for structural studies of macromolecules. Importantly, the observations suggest that carbon nanotubes may interfere with the human immune system when entering the bloodstream. Our results raise caution in the applications of carbon nanotubes in biomedicine but may also open possibilities of novel applications concerning the many biochemical processes involving the versatile C1 macromolecule.

The lectin-like activity of human C1q and its implication in DNA and apoptotic cell recognition

C1q, the binding subunit of the C1 complex of complement, is an archetypal pattern recognition molecule known for its striking ability to recognize a wide variety of targets, ranging from pathogenic non self to altered self. DNA is one of the C1q ligands, but the precise region of C1q and the DNA motifs that support interaction have not been characterized yet. Here, we report for the first time that the peripheral globular region of the C1q molecule displays a lectin‐like activity, which contributes to DNA binding through interaction with its deoxy‐d‐ribose moiety and may participate in apoptotic cell recognition.

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.

Direct interaction between CD91 and C1q.

C1q‐mediated removal of immune complexes and apoptotic cells plays an important role in tissue homeostasis and the prevention of autoimmune conditions. It has been suggested that C1q mediates phagocytosis of apoptotic cells through a receptor complex assembled from CD91 (α‐2‐ macroglobulin receptor, or low‐density lipoprotein receptor‐related protein) and calreticulin, with CD91 being the transmembrane part and calreticulin acting as the C1q‐binding molecule. In the present study, we observe that C1q binds cells from a CD91 expressing monocytic cell line as well as monocytes from human blood. C1q binding to monocytes was shown to be correlated with CD91 expression and could be inhibited by the CD91 chaperone, receptor‐associated protein. We also report data showing a direct interaction between CD91 and C1q. The interaction was investigated using various protein interaction assays. A direct interaction between purified C1q and CD91 was observed both by ELISA and a surface plasmon resonance assay, with either C1q or CD91 immobilized. The interaction showed characteristics of specificity because it was time‐dependent, saturable and could be inhibited by known ligands of both CD91 and C1q. The results obtained show for the first time that CD91 recognizes C1q directly. On the basis of these findings, we propose that CD91 is a receptor for C1q and that this multifunctional scavenger receptor uses a subset of its ligand‐binding sites for clearance of C1q and C1q bound material.

Relative Contribution of C1q and Apoptotic Cell-Surface Calreticulin to Macrophage Phagocytosis

C1q 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 Is a Phosphatidylserine-binding Protein That Affects the Production and Function of Microvesicles.

Proteinase 3 (PR3), the autoantigen in granulomatosis with polyangiitis, is expressed at the plasma membrane of resting neutrophils, and this membrane expression increases during both activation and apoptosis. Using surface plasmon resonance and protein-lipid overlay assays, this study demonstrates that PR3 is a phosphatidylserine-binding protein and this interaction is dependent on the hydrophobic patch responsible for membrane anchorage. Molecular simulations suggest that PR3 interacts with phosphatidylserine via a small number of amino acids, which engage in long lasting interactions with the lipid heads. As phosphatidylserine is a major component of microvesicles (MVs), this study also examined the consequences of this interaction on MV production and function. PR3-expressing cells produced significantly fewer MVs during both activation and apoptosis, and this reduction was dependent on the ability of PR3 to associate with the membrane as mutating the hydrophobic patch restored MV production. Functionally, activation-evoked MVs from PR3-expressing cells induced a significantly larger respiratory burst in human neutrophils compared with control MVs. Conversely, MVs generated during apoptosis inhibited the basal respiratory burst in human neutrophils, and those generated from PR3-expressing cells hampered this inhibition. Given that membrane expression of PR3 is increased in patients with granulomatosis with polyangiitis, MVs generated from neutrophils expressing membrane PR3 may potentiate oxidative damage of endothelial cells and promote the systemic inflammation observed in this disease.

Cytoplasmic proliferating cell nuclear antigen connects glycolysis and cell survival in acute myeloid leukemia.

Cytosolic proliferating cell nuclear antigen (PCNA), a scaffolding protein involved in DNA replication, has been described as a key element in survival of mature neutrophil granulocytes, which are non-proliferating cells. Herein, we demonstrated an active export of PCNA involved in cell survival and chemotherapy resistance. Notably, daunorubicin-resistant HL-60 cells (HL-60R) have a prominent cytosolic PCNA localization due to increased nuclear export compared to daunorubicin-sensitive HL-60 cells (HL-60S). By interacting with nicotinamide phosphoribosyltransferase (NAMPT), a protein involved in NAD biosynthesis, PCNA coordinates glycolysis and survival, especially in HL-60R cells. These cells showed a dramatic increase in intracellular NAD+ concentration as well as glycolysis including increased expression and activity of hexokinase 1 and increased lactate production. Furthermore, this functional activity of cytoplasmic PCNA was also demonstrated in patients with acute myeloid leukemia (AML). Our data uncover a novel pathway of nuclear export of PCNA that drives cell survival by increasing metabolism flux.