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

Abstract

Complement 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.

Structural basis of the C1q/C1s interaction and its central role in assembly of the C1 complex of complement activation

“…Recent mutagenesis data show that LysB61 and LysC58 are essential for binding, whereas LysA59 is less important, so it may not bind to C1r or C1s (16). From the structure, it is not possible to determine whether the B or C chains of C1q contribute the lysine side chain, because the register of the collagenous domain of C1q is not known (i.e., which of the A, B, and C chains is leading, middle, and trailing).…”

A C1q domain containing protein from Crassostrea gigas serves as pattern recognition receptor and opsonin with high binding affinity to LPS

“…The structure differences suggest the different functions of C1qDCs in vertebrates and invertebrates. In human, the collagen domain forms tripolymer and higher-order tertiary structure to activate the downstream protease of complement pathway [6,11]. CgC1qDC-1 in the present study was consisted of eight b-propeller strands, which formed the globular structure, similar to the globular domain of C1q from H. sapiens.…”

Heteromeric Complexes of Native Collectin Kidney 1 and Collectin Liver 1 Are Found in the Circulation with MASPs and Activate the Complement System

“…Based on the structure of C1q, proteins with collagen-like regions may form heteromeric complexes via coordinated interactions in the collagen-like region and other regions (30,31). The same ability most likely applies for CL-L1 and CL-K1, which are highly similar to each other with respect to domain organization and an identical number of residues in both the collagen-like region and the a-helical coiled-coil neck region (Fig.…”