Interaction of C1q With Pentraxin 3 and IgM Revisited: Mutational Studies With Recombinant C1q Variants

Bally, Isabelle; Inforzato, Antonio; Dalonneau, Fabien; Stravalaci, Matteo; Bottazzi, Barbara; Gaboriaud, Christine; Thielens, Nicole M.

doi:10.3389/fimmu.2019.00461

Cited by 27 publications

(59 citation statements)

References 36 publications

(57 reference statements)

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“…A hydrophobic patch on CDR3 formed by residues Pro102, Pro103, Trp105, Pro106 protrudes from the β-sandwich immunoglobulin fold and is inserted in a C1q cavity delimited by the C1q-B chain loop carrying Glu209B, and the loop carrying Thr102B ( Figure 4E). The latter residue is close in sequence to Arg108B and Arg109B, known to mediate interactions with pentraxin 3 and IgM (18). The cavity is likely shielded from the solvent due to proximity of C1qNb75 Arg108 to Glu209B on one side, and the presence of gC1q Arg156C on the other side ( Figure 4E).…”

Section: C1qnb75 Sterically Prevents the Interaction Between C1q And mentioning

confidence: 96%

Functional and Structural Characterization of a Potent C1q Inhibitor Targeting the Classical Pathway of the Complement System

et al. 2020

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The classical pathway of complement is important for protection against pathogens and in maintaining tissue homeostasis, but excessive or aberrant activation is directly linked to numerous pathologies. We describe the development and in vitro characterization of C1qNb75, a single domain antibody (nanobody) specific for C1q, the pattern recognition molecule of the classical pathway. C1qNb75 binds to the globular head modules of human C1q with sub-nanomolar affinity and impedes classical pathway mediated hemolysis by IgG and IgM. Crystal structure analysis revealed that C1qNb75 recognizes an epitope primarily located in the C1q B-chain that overlaps with the binding sites of IgG and IgM. Thus, C1qNb75 competitively prevents C1q from binding to IgG and IgM causing blockade of complement activation by the classical pathway. Overall, C1qNb75 represents a high-affinity nanobody-based inhibitor of IgG-and IgM-mediated activation of the classical pathway and may serve as a valuable reagent in mechanistic and functional studies of complement, and as an efficient inhibitor of complement under conditions of excessive CP activation.

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Section: C1qnb75 Sterically Prevents the Interaction Between C1q And mentioning

confidence: 96%

Functional and Structural Characterization of a Potent C1q Inhibitor Targeting the Classical Pathway of the Complement System

et al. 2020

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“…C1q collagen stalks (CLR) and C1q globular heads (GR) were prepared according to Tacnet-Delorme et al (13). The recombinant protease tetramer C1r2s2 was produced and purified according to Bally et al (14). Full-length LRP1 (soluble LRP1) was produced and purified according to De Nardis et al (15).…”

Section: Proteins Cells and Reagentsmentioning

confidence: 99%

Complement C1q Interacts With LRP1 Clusters II and IV Through a Site Close but Different From the Binding Site of Its C1r and C1s-Associated Proteases

et al. 2020

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Unless otherwise stated, all reagents are from Gibco ®. CHO-null and Jurkat cells were respectively cultured in DMEM-F12 (Dulbecco's modified Eagle's medium), or in RPMI (Roswell Park Memorial Institute medium), supplemented with 10% (v/v) Fetal Bovine Serum (FBS) at 37°C with an humidified atmosphere and 5% CO 2. For CHO clones expressing LRP1 receptors (full-length or mini IV), the media were supplemented with G418 (geneticin sulfate) at 400 µg/ml.

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“…pcDNA3.1/Zeo C1sWT was obtained by insertion of a PCR amplicon of pFastBac1/C1s (11) in pcDNA 3.1/Zeo between NheI and EcoRI. For purification purposes a FLAG-Tag was also inserted on the 3'-end (12). Variants pcDNA3.1/Zeo C1sV316del, pcDNA3.1/Zeo C1sC294R, and pcDNA3.1/Zeo C1s Fg40 fragment were engineered by site directed mutagenesis.…”

Section: Production Of C1s Variants In Mammalian Cells and Purificationmentioning

confidence: 99%

“…The pcDNA3.1 C1s constructs were used for stable transfection of mammalian HEK293F cells and secreted proteins were then purified from cell culture supernatants on a 2.5 mL ANTI-FLAG M1 agarose column as described for the recombinant C1s-C1r-C1r-C1s tetramer (12). The concentration of the purified C1s proteins was estimated using the absorption coefficient A 1%,1cm at 280 nm calculated using the PROTPARAM program on the Expasy Server (http:// web.expasy.org/protparam/), and the experimental molecular mass.…”

Section: Production Of C1s Variants In Mammalian Cells and Purificationmentioning

confidence: 99%

Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes

et al. 2019

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Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorders characterized by soft connective tissue alteration like joint hypermobility and skin hyper-extensibility. We previously identified heterozygous missense mutations in the C1R and C1S genes, coding for the complement C1 proteases, in patients affected by periodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leading to premature loss of teeth and connective tissue alterations. Up to now, there is no clear molecular link relating the nominal role of the C1r and C1s proteases, which is to activate the classical complement pathway, to these heterogeneous symptoms of periodontal EDS syndrome. We aim therefore to elucidate the functional effect of these mutations, at the molecular and enzymatic levels. To explore the molecular consequences, a set of cell transfection experiments, recombinant protein purification, mass spectroscopy and N-terminal analyses have been performed. Focusing on the results obtained on two different C1S variants, namely p.Val316del and p.Cys294Arg, we show that HEK293-F cells stably transfected with the corresponding C1s variant plasmids, unexpectedly, do not secrete the full-length mutated C1s, but only a truncated Fg40 fragment of 40 kDa, produced at very low levels. Detailed analyses of the Fg40 fragments purified for the two C1s variants show that they are identical, which was also unexpected. This suggests that local misfolding of the CCP1 module containing the patient mutation exposes a novel cleavage site, between Lys353 and Cys354, which is not normally accessible. The mutation-induced Fg40 fragment contains the intact C-terminal serine protease domain but not the N-terminal domain mediating C1s interaction with the other C1 subunits, C1r, and C1q. Thus, Fg40 enzymatic activity escapes the normal physiological control of C1s activity within C1, potentially providing a loss-of-control. Comparative enzymatic analyses show that Fg40 retains the native esterolytic activity of C1s, as well as its cleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereas the nominal complement C4 activation cleavage is impaired. These new results open the way to further molecular explorations possibly involving subsidiary C1s targets.

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Interaction of C1q With Pentraxin 3 and IgM Revisited: Mutational Studies With Recombinant C1q Variants

Cited by 27 publications

References 36 publications

Functional and Structural Characterization of a Potent C1q Inhibitor Targeting the Classical Pathway of the Complement System

Functional and Structural Characterization of a Potent C1q Inhibitor Targeting the Classical Pathway of the Complement System

Complement C1q Interacts With LRP1 Clusters II and IV Through a Site Close but Different From the Binding Site of Its C1r and C1s-Associated Proteases

Two Different Missense C1S Mutations, Associated to Periodontal Ehlers-Danlos Syndrome, Lead to Identical Molecular Outcomes

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