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.

Keywords: C1 complex; innate immunity; protein engineering.

Fig. 1.

SDS/PAGE and MALDI-TOF mass spectrometry analyses of serum and WT rC1q. (A) SDS/PAGE analysis: lane 1, unreduced serum C1q; lane 2, unreduced WT rC1q; lane 3, reduced serum C1q; lane 4, reduced WT rC1q. The molecular weights of unreduced and reduced markers indicated in kDa are shown on the left and right sides, respectively. (B and C) MALDI-TOF mass spectra of serum C1q (B) and WT rC1q (C). Analyses were performed on reduced samples as described under Materials and Methods. A representative spectrum of four measurements is shown in the case of WT rC1q, and, therefore, the mass values indicated are slightly different from those stated in the text, which represent mean values ±SD of the different measurements.

Fig. 2.

Negative-staining electron microscopy of WT rC1q (A and B) and serum C1q (C and D). (A and C) Overall views. (B and D) Selected images of individual molecules showing top views (upper row) and side views (lower row). Samples were stained with sodium silicotungstate as described under Materials and Methods.

Fig. 3.

SPR analyses of the binding of C1s-C1r-C1r-C1s (A) and MASP-3 (B) to immobilized serum C1q, WT rC1q, and rC1q variants. Analyses were performed as described in SI Materials and Methods. The C1s-C1r-C1r-C1s and MASP-3 concentration was 18 nM. In A, the curves obtained for the LysB61Ala and LysC58Ala mutants are virtually identical and cannot be distinguished.

Fig. 4.

C1 activation by serum C1q and rC1q variants. (A) Spontaneous activation. Complexes (0.25 µM) reconstituted from the different C1q samples and proenzyme C1s-C1r-C1r-C1s were incubated for various periods at 37 °C. ▼, LysB61Ala mutant; ■, LysC58Ala mutant; □, LysA59Ala, LysB61Ala, LysC58Ala triple mutant. (B) Activation by IgG–ovalbumin immune aggregates. The reconstituted complexes were incubated for 1 h at 37 °C in the presence of 0.2 mg/mL IgG–ovalbumin aggregates and 1 µM C1 inhibitor. The C1 activation extent was measured in each case by SDS/PAGE, followed by Western blot analysis using an anti-C1s antibody (21). In A, the background activation level measured when incubating C1s-C1r-C1r-C1s alone for 1 h is indicated by +. Results in B are expressed as means ±SD of two independent experiments.