## 2 Results
### Recombinant LAIR-1 Ig-Like Domain Interacts with Serum-Derived C1q
For that purpose, the C1 complex was reconstituted by incubation of sC1q with recombinant C1r2s2 at increasing molar ratios before injection over immobilized LAIR-1. C1q was no more able to interact with LAIR-1 when it is fully associated with C1r2s2 (above 1:3 tetramer versus C1q molar ratio), revealing that the LAIR-1 binding site on C1q CLR might be the same or located in close proximity of the C1r2s2 binding site. Of note, the need for a molar ratio higher than 1:1 to fully inhibit C1q interaction with LAIR-1 can be explained by the fact that the concentration used (1 nM) is almost 10 times lower than the previously determined K\({}_{\text{D}}\) of C1q interaction with C1r2s2 (around 13 nM; [PERSON] et al. 2013) [20]. Importantly, such a total inhibition of LAIR-1 interaction with C1q when associated with C1r2s2 suggests that no other LAIR-1 binding site, distant from the C1r2s2 binding region, is present on C1q CLR (Figure 2B). As mentioned above, the C1r2s2 tetramer is known to interact with the collagen-like regions of C1q and C1q lysine residues are involved in this binding [20]. To assess if LAIR-1 binding to C1q CLRs also requires these lysine residues, we used a recombinant C1q mutant with an alanine substitution of LysA59, LysB61 and LysC58 (rC1q ABC) with recombinant wild-type C1q (rC1q WT) as a control. LAIR-1 binding to these two rC1q variants was assessed using SPR. Although rC1q ABC does not interact with C1r2s2, this variant retains its interaction properties with LAIR-1 with a K\({}_{\text{D}}\) similar to that of rC1q WT and sC1q (Figure 3 and Table 1). This reveals that C1r2s2 and LAIR-1 interact with C1q through different binding sites. As LAIR-1 interaction with C1q involves its collagen-like regions, we used a new C1 qCLR_nc2 construct derived from the CLR_nc2 fusion protein recently developed in our laboratory to investigate the collagen-like functions of C1q [21]. We needed to delete the GPP triplets initially added between the CLR and nc2 sequences in order to avoid addition of artifactual LAIR-1 binding sites into the molecule (see Section 3). The ability of LAIR-1 Ig-like domain to interact with wild-type C1 qCLR_nc2 was assessed by SPR. As expected, LAIR-1 WT interaction with C1 qCLR_nc2 exhibited a K\({}_{\text{D}}\) value in the same range as with sC1q (Figure 4B and Table 1), therefore confirming that C1q interacts with LAIR-1 via its CLR. \begin{table}
\begin{tabular}{c c} \hline
**Imobilized** & **LAIR-1 WT** \\
**Ligands** & **K\({}_{\text{D}}\) (\(\mu\)M)** \\ \hline sC1q & 112.3 \(\pm\) 6.2 (n = 4) \\ rC1q WT & 99.5 \(\pm\) 26.6 (n = 2) \\ rC1q ABC & 131.8 \(\pm\) 23.5 (n = 2) \\ C1 qCLR\_nc2 & 179.3 \(\pm\) 4.8 (n = 3) \\ C1 qCLR\_nc2 AR72E & 295.0 \(\pm\) 10.6 (n = 3) \\ C1 qCLR\_nc2 BK65E & 188.8 \(\pm\) 5.3 (n = 3) \\ C1 qstem\_nc2 & 174.9 \(\pm\) 0.6 (n = 2) \\ \hline \end{tabular} Values are means \(\pm\) SD from separate experiments. The number of replicates (n) are indicated next to the KD values. The dissociation constant KD was determined by global fitting of the data using a steady-state binding model.  
\end{table} 
Table 1: Equilibrium dissociation constants for the binding of LAIR-1 WT to C1q and C1q variants. We went further in engineering the C1 qstem_nc2 fusion fragment, obtained by deleting the N-terminal part of C1 qCLR_nc2 until the CLR hinge region. This C1 qstem_nc2 truncated fragment contains a single heterotrimeric C1q collagen stem comprising the C1r\({}_{2}\)s\({}_{2}\) and LAIR-1 binding sites with one C-terminal nc2 domain (Figure 4A). The C1 qstem_nc2 construction was tested for its interaction with LAIR-1 Ig-like domain using SPR and the results indicated that it interacts in a dose-dependent manner (Figure 4C). Kinetic analysis of this interaction reveals a K\({}_{\text{D}}\) value similar to the one obtained with the full length C1 qCLR_nc2. This confirms that the LAIR-1 binding site might not be located in the N-terminal collagen bundle.