Enzymatically Modified Low-Density Lipoprotein Is Recognized by C1q and Activates the Classical Complement Pathway

Several studies suggest that the complement system is involved in atherogenesis. To further investigate this question, we have studied the ability of native and modified forms of LDL to bind and activate C1, the complex protease that triggers the classical pathway of complement. Unlike native LDL, oxidized (oxLDL) and enzymatically modified (E-LDL) derivatives were both recognized by the C1q subunit of C1, but only E-LDL particles, obtained by sequential treatment with a protease and then with cholesterol esterase, had the ability to trigger C1 activation. Further investigations revealed that C1q recognizes a lipid component of E-LDL. Several approaches, including reconstitution of model lipid vesicles, cosedimentation, and electron microscopy analyses, provided evidence that C1 binding to E-LDL particles is mediated by the C1q globular domain, which senses unesterified fatty acids generated by cholesterol esterase. The potential implications of these findings in atherogenesis are discussed

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

Neutron scattering study of the (γ-B) catalytic domains of complement proteases Cl̄r and Cl̄s

AbstractThe catalytic domains of Cl̄r and Cl̄s, comprising the C-tenninal region of the A chain (γ), disulphide-linked to the B chain, were obtained by limited proteolysis of the native proteases with chymotrypsin and plasmin, respectively, and studied by small angle neutron scattering. For CIs (γ-B), a molar mass of 45 000 ± 5000 gmol, and a relatively large radius of gyration (Rg) of 28 ± 1 Å were determined, excluding a single globular domain. The corresponding values for Cl̄r (γ-B), (90,000 gmol, Rg, = 34 ± 1 Å) are consistent with a dimer involving the loose packing of two (γ-B) subunits. Various models of the dimer are discussed in the light of neutron scattering and other data

The serine protease domain of MASP-3: enzymatic properties and crystal structure in complex with ecotin.

International audienceMannan-binding lectin (MBL), ficolins and collectin-11 are known to associate with three homologous modular proteases, the MBL-Associated Serine Proteases (MASPs). The crystal structures of the catalytic domains of MASP-1 and MASP-2 have been solved, but the structure of the corresponding domain of MASP-3 remains unknown. A link between mutations in the MASP1/3 gene and the rare autosomal recessive 3MC (Mingarelli, Malpuech, Michels and Carnevale,) syndrome, characterized by various developmental disorders, was discovered recently, revealing an unexpected important role of MASP-3 in early developmental processes. To gain a first insight into the enzymatic and structural properties of MASP-3, a recombinant form of its serine protease (SP) domain was produced and characterized. The amidolytic activity of this domain on fluorescent peptidyl-aminomethylcoumarin substrates was shown to be considerably lower than that of other members of the C1r/C1s/MASP family. The E. coli protease inhibitor ecotin bound to the SP domains of MASP-3 and MASP-2, whereas no significant interaction was detected with MASP-1, C1r and C1s. A tetrameric complex comprising an ecotin dimer and two MASP-3 SP domains was isolated and its crystal structure was solved and refined to 3.2 Å. Analysis of the ecotin/MASP-3 interfaces allows a better understanding of the differential reactivity of the C1r/C1s/MASP protease family members towards ecotin, and comparison of the MASP-3 SP domain structure with those of other trypsin-like proteases yields novel hypotheses accounting for its zymogen-like properties in vitro

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

Complement Protein C1q Recognizes Enzymatically Modified Low-Density Lipoprotein through Unesterified Fatty Acids Generated by Cholesterol Esterase

International audienceWe previously reported that enzymatically modified low-density lipoprotein (E-LDL) particles obtained by LDL treatment with trypsin and then cholesterol esterase are recognized by C1q and activate the C1 complex of complement. The objective of this study was to identify the E-LDL component(s) recognized by C1q. In addition to trypsin, plasmin, thrombin, tryptase, and matrix metalloprotease-2 each yielded E-LDL particles with high C1-activating efficiency, and the C1 activation extent was strictly dependent on cholesterol esterase treatment in all cases. When incorporated into vesicles, the lipid fraction of E-LDL, but not of native LDL, triggered C1 activation, and activation correlated with the amount of unesterified cholesterol generated by cholesterol esterase. Whereas treatment of E-LDL particles with human serum albumin reduced their fatty acid content, both cholesterol and unesterified fatty acids were decreased by methyl-beta-cyclodextrin, both treatments resulting in dose-dependent inhibition of the C1-activating ability of the particles. Incorporation of linoleic acid into phosphatidylcholine-containing model vesicles enabled them to interact with the C1q globular domain and to trigger C1 activation, and cholesterol enhanced both processes by facilitating incorporation of the fatty acid into the vesicles. Direct evidence that C1q binds E-LDL through its globular domains was obtained by electron microscopy. This study demonstrates that C1 binding to E-LDL particles involves recognition by the C1q globular domain of the unesterified fatty acids generated by cholesterol esterase. The potential implications of these findings in atherogenesis are discussed

Enzymatically modified low-density lipoprotein is recognized by c1q and activates the classical complement pathway

Several studies suggest that the complement system is involved in atherogenesis. To further investigate this question, we have studied the ability of native and modified forms of LDL to bind and activate C1, the complex protease that triggers the classical pathway of complement. Unlike native LDL, oxidized (oxLDL) and enzymatically modified (E-LDL) derivatives were both recognized by the C1q subunit of C1, but only E-LDL particles, obtained by sequential treatment with a protease and then with cholesterol esterase, had the ability to trigger C1 activation. Further investigations revealed that C1q recognizes a lipid component of E-LDL. Several approaches, including reconstitution of model lipid vesicles, cosedimentation, and electron microscopy analyses, provided evidence that C1 binding to E-LDL particles is mediated by the C1q globular domain, which senses unesterified fatty acids generated by cholesterol esterase. The potential implications of these findings in atherogenesis are discussed