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

• DOI: 10.1073/pnas.1304894110
• Publication Date: 2013-05-07T04:00:47Z
• Authors: Isabelle Bally; Sarah Ancelet; Christine Moriscot; Florence Gonnet; Alberto Mantovani; Régis Daniel; Guy Schoehn; Gérard J. Arlaud; Nicole M. Thielens
• Keywords: Tetramer; HEK 293 cells; Complement C1q

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