Deciphering complement receptor type 1 interactions with recognition proteins of the lectin complement pathway.

International audienceComplement receptor type 1 (CR1) is a membrane receptor expressed on a wide range of cells. It is involved in immune complex clearance, phagocytosis, and complement regulation. Its ectodomain is composed of 30 complement control protein (CCP) modules, organized into four long homologous repeats (A-D). In addition to its main ligands C3b and C4b, CR1 was reported to interact with C1q and mannan-binding lectin (MBL) likely through its C-terminal region (CCP22-30). To decipher the interaction of human CR1 with the recognition proteins of the lectin complement pathway, a recombinant fragment encompassing CCP22-30 was expressed in eukaryotic cells, and its interaction with human MBL and ficolins was investigated using surface plasmon resonance spectroscopy. MBL and L-ficolin were shown to interact with immobilized soluble CR1 and CR1 CCP22-30 with apparent dissociation constants in the nanomolar range, indicative of high affinity. The binding site for CR1 was located at or near the MBL-associated serine protease (MASP) binding site in the collagen stalks of MBL and L-ficolin, as shown by competition experiments with MASP-3. Accordingly, the mutation of an MBL conserved lysine residue essential for MASP binding (K55) abolished binding to soluble CR1 and CCP22-30. The CR1 binding site for MBL/ficolins was mapped to CCP24-25 of long homologous repeat D using deletion mutants. In conclusion, we show that ficolins are new CR1 ligands and propose that MBL/L-ficolin binding involves major ionic interactions between conserved lysine residues of their collagen stalks and surface exposed acidic residues located in CR1 CCP24 and/or CCP25

Functional recombinant human complement C1q with different affinity tags

International audienceComplement C1q is a multifunctional protein able to sense pathogens and immune molecules such as immunoglobulins and pentraxins, and to trigger the classical complement pathway through activation of its two associated proteases, C1r and C1s. C1q is a multimeric protein composed of three homologous yet distinct polypeptide chains A, B, and C, each composed of an N-terminal collagen-like sequence and a C-terminal globular gC1q module, that assemble into six heterotrimeric (A-B-C) subunits. This hexameric structure exhibits the characteristic shape of a bouquet of flowers, comprising six collagen-like triple helices, each terminating in a trimeric C-terminal globular head. We have produced previously functional recombinant full-length C1q in stably transfected HEK 293-F cells, with a FLAG tag inserted at the C-terminal end of C1qC chain. We report here the generation of additional recombinant C1q proteins, with a FLAG tag fused to the C-terminus of C1qA or C1qB chains, or to the N-terminus of the C1qC chain. Two other variants harboring a Myc or a 6-His tag at the C-terminal end of C1qC were also produced. We show that all C1q variants, except for the His-tagged protein, can be produced at comparable yields and are able to bind with similar affinities to either IgM, a ligand of the globular regions, or to the C1r2-C1s2 tetramer, and to trigger IgM-mediated serum complement activation. These new recombinant C1q variants provide additional tools to investigate the multiple functions of C1q

Impact of the surface charge of polydiacetylene micelles on their interaction with human innate immune protein C1q and the complement system

International audiencePolydiacetylene (pDA) micelles have been demonstrated to be effective drug carriers for cancer therapy in mouse model. However, little is known about their interaction with the human complement system, which constitutes an important part of the innate immune system and can cause severe hypersensitivity reactions. Herein, we investigate the influence of micelle surface charge on the binding of complement protein C1q, the target recognition unit that activates the classical complement pathway and performs a range of other important physiological functions. Besides the classical pathway, we also investigate the surface charge effect on complement activities through the other activation pathways, namely, the MBL-dependent lectin pathway and the alternative pathway. We synthesized three samples of pDA micelles bearing neutral, anionic, and cationic surface charge motifs, respectively. Surface plasmon resonance showed that none of these micelles interacted with C1q. Results from serum complement activation assays indicated that all micelles were inert to complement, except for the anionic pDA micelles, which activated the alternative pathway

Production of functional full-length recombinant human C1q

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Mode of PEG Coverage on Carbon Nanotubes Affects Binding of Innate Immune Protein C1q

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Expression of recombinant human complement C1q allows identification of the C1r/C1s-binding sites.

International audienceComplement C1q is a hexameric molecule assembled from 18 polypeptide chains of three different types encoded by three genes. This versatile recognition protein senses a wide variety of immune and nonimmune ligands, including pathogens and altered self components, and triggers the classical complement pathway through activation of its associated proteases C1r and C1s. We report a method for expression of recombinant full-length human C1q involving stable transfection of HEK 293-F mammalian cells and fusion of an affinity tag to the C-terminal end of the C chain. The resulting recombinant (r) C1q molecule is similar to serum C1q as judged from biochemical and structural analyses and exhibits the characteristic shape of a bunch of flowers. Analysis of its interaction properties by surface plasmon resonance shows that rC1q retains the ability of serum C1q to associate with the C1s-C1r-C1r-C1s tetramer, to recognize physiological C1q ligands such as IgG and pentraxin 3, and to trigger C1r and C1s activation. Functional analysis of rC1q variants carrying mutations of LysA59, LysB61, and/or LysC58, in the collagen-like stems, demonstrates that LysB61 and LysC58 each play a key role in the interaction with C1s-C1r-C1r-C1s, with LysA59 being involved to a lesser degree. We propose that LysB61 and LysC58 both form salt bridges with outer acidic Ca(2+) ligands of the C1r and C1s CUB (complement C1r/C1s, Uegf, bone morphogenetic protein) domains. The expression method reported here opens the way for deciphering the molecular basis of the unusual binding versatility of C1q by mapping the residues involved in the sensing of its targets and the binding of its receptors

Functional characterization of the recombinant human C1 inhibitor serpin domain: insights into heparin binding.

International audienceVariants of the human C1 inhibitor serpin domain containing three N-linked carbohydrates at positions 216, 231, and 330 (C1inhDelta97), a single carbohydrate at position 330 (C1inhDelta97DM), or no carbohydrate were produced in a baculovirus/insect cells system. An N-terminally His-tagged C1inhDelta97 variant was also produced. Removal of the oligosaccharide at position 330 dramatically decreased expression, precluding further analysis. All other variants were characterized chemically and shown to inhibit C1s activity and C1 activation in the same way as native C1 inhibitor. Likewise, they formed covalent complexes with C1s as shown by SDS-PAGE analysis. C1 inhibitor and its variants inhibited the ability of C1r-like protease to activate C1s, but did not form covalent complexes with this protease. The interaction of C1 inhibitor and its variants with heparin was investigated by surface plasmon resonance, yielding K(D) values of 16.7 x 10(-8) M (C1 inhibitor), 2.3 x 10(-8) M (C1inhDelta97), and 3.6 x 10(-8) M (C1inhDelta97DM). C1s also bound to heparin, with lower affinity (K(D) = 108 x 10(-8) M). Using the same technique, 50% inhibition of the binding of C1 inhibitor and C1s to heparin was achieved using heparin oligomers containing eight and six saccharide units, respectively. These values roughly correlate with the size of 10 saccharide units yielding half-maximal potentiation of the inhibition of C1s activity by C1 inhibitor, consistent with a "sandwich" mechanism. Using a thermal shift assay, heparin was shown to interact with the C1s serine protease domain and the C1 inhibitor serpin domain, increasing and decreasing their thermal stability, respectively

Recognition protein C1q of innate immunity agglutinates nanodiamonds without activating complement

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Reactive oxygen species levels control NF-κB activation by low dose deferasirox in erythroid progenitors of low risk myelodysplastic syndromes

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