## 3 Discussion  
Besides its major role in the initiation of the classical pathway of the complement cascade, C1q achieves many complement-independent functions such as the opsonization of apoptotic cells and pathogens or maintenance of immune tolerance. Therefore, C1q interaction network on immune cells surface is highly complex and the underlying molecular mechanisms need to be explored. In this study, we investigated the molecular basis of C1q interaction with LAIR-1, an inhibitory immune receptor present on various immune cells. Molecular dissection of C1q unambiguously showed that LAIR-1 Ig-like domain interacts with the C1q collagen-like regions on a binding site located close to the one of C1r\({}_{2}\)s\({}_{2}\) as shown by SPR competition assays. In addition, the interactions of LAIR-1 with recombinant C1q with a wild-type sequence or mutated at the C1r\({}_{2}\)s\({}_{2}\) binding site were similar, revealing that LAIR-1 binding does not engage the same C1 qCLR residues as C1r\({}_{2}\)s\({}_{2}\). Interestingly, we showed that LAIR-1 interaction with C1q is only possible when it is devoid of its associated proteases. This result is in line with previous studies on C1q receptors showing that CR1 and LRP1 can interact with C1q but not with the C1 complex (i.e., C1q in complex with the C1r\({}_{2}\)s\({}_{2}\) tetramer), showing that these three receptors share the same conditional rule for C1q binding and might share a common binding region on the C1q collagen stems [31; 32]. This highlights the dual role of C1q in complement related function when associated with C1r\({}_{2}\)s\({}_{2}\) and complement independent functions when devoid of its cognate proteases, through the interaction with cellular receptors. Even if serum C1q is almost exclusively found associated with C1r\({}_{2}\)s\({}_{2}\) within the C1 complex, it is not unusual to find C1q alone in physiological conditions. Namely, single C1q can be locally expressed by several cell types or remain after C1-inhibitor mediated C1 complex dissociation [33; 34]. C1q production by tissue-resident monocyte-derived cells is a process tightly controlled by numerous stimuli. For instance, C1q synthesis by macrophages was shown to be increased in response to tissue injury and constitutes a key process for resolution of inflammation [35]. Our study used C1 qCLR_nc2, a variant of the CLR_nc2 recombinant fusion protein designed in our lab, previously reported to retain the C1r\({}_{2}\)s\({}_{2}\) interaction properties of C1 qCLR [21]. In the particular case of LAIR-1 study, we had to remove the three additional GPP collagen triplets initially introduced between the CLR and non-collagenous domain to improve the C1 qCLR_nc2 folding. This GPP removal was done in order to avoid introducing artificial interaction sites for LAIR-1. It is likely that during processing and maturation, these additional GPP triplets undergo proline hydroxylation as post-translational modification, resulting in GPO collagen triplets. It is well established that GPO triplets are high-affinity ligands of LAIR-1 and such additional putative binding sites would have introduced nonspecific signal of LAIR-1 binding to CLR_nc2 [12; 13]. Indeed, kinetics SPR experiments using the previously published CLR_nc2 protein containing the additional GPP triplets displayed a lower \(K_{\text{D}}\) value and a higher surface binding capacity (Rmax) substantiating the presence of additional binding sites and an increased avidity (Figure S4). Therefore, all experiments presented in this study were done using the C1 qCLR_nc2 devoid of additional GPP triplets. We report here that LAIR-1 interacts similarly with C1 qCLR_nc2 and sC1q, thus validating the use of C1 qCLR_nc2 in the context of studies on C1q collagen-like regions'receptors. By deleting the N-terminal portion of each C1q collagen chain until the hinge region, we managed to produce one isolated heterotrimeric C1q collagen stem (called C1 qstem_nc2).