## 2 Results  
### Recombinant LAIR-1 Ig-Like Domain Interacts with Serum-Derived C1q  
The recombinant soluble Ig-like domain of wild-type LAIR-1 (WT) and all the variants used in the present study were expressed in bacteria and purified as described in Section 4. LAIR-1 extracellular domain contains one N-glycosylation site at the asparagine residue 69. For that reason, LAIR-1 extracellular region was also produced in HEK293F cells to assess its binding and the importance of the LAIR-1 glycosylation. LAIR-1 Ig-like domain produced in E. coli or HEK293F cells showed similar levels of interaction with C1q (Figure S1) but with slight differences in the binding curves possibly accounting for posttranslational modifications. Consequently, the results presented in this study were obtained using LAIR-1 produced in bacteria. The quality of each LAIR-1 variant was analyzed by ESI-MS and SDS-PAGE (Figure 1). As illustrated in Figure 1A, each LAIR-1 Ig-like domain variant exhibited an ESI-measured mass in agreement with the theoretical mass calculated from the amino acid sequence. Of note, a difference of 2 Da is observed between the theoretical and measured masses accounting for the presence of an intact disulfide bond, thus supporting the correct folding of the Ig-like domain. As expected, SDS-PAGE analysis of LAIR-1 WT and mutated samples reveals a unique protein band with an apparent molecular weight of about 15 kDa, consistent with masses determined by mass spectrometry (Figure 1B). We first assessed the interaction of the recombinant LAIR-1 Ig-like domain with C1q by surface plasmon resonance (SPR). LAIR-1 interacts with serum-derived C1q (sC1q) in two SPR configurations, i.e., soluble LAIR-1 on immobilized sC1q or soluble sC1q on immobilized LAIR-1 (red curves in Figure 2A,B, respectively). However, the difference in the curve shapes of the two configurations suggests avidity effects in the interaction of C1q with immobilized LAIR-1, which is absent in the other configuration, when C1q is immobilized. This likely reflects the presence of multiple LAIR-1 binding sites on the sC1q molecule. In order to avoid avidity constraints in SPR analyses, we decided to perform all SPR experiments using immobilized C1q fragments, with the exception of the C1r\({}_{2}\)s\({}_{2}\) competition assay for which the injection of soluble sC1q is required. Figure 1: Quality control of the LAIR-1 Ig-like domain variants. (**A**) Summary table of the theoretical masses calculated from the amino acid sequences and the measured masses by ESI-MS experiments of the LAIR-1 variants produced. (**B**) SDS-PAGE analysis of the LAIR-1 variants on a 14% polyacrylamide gel under reducing condition. Figure 2: Identification of the C1q functional region interacting with LAIR-1 and competition assay with the C1r\({}_{2}\)s\({}_{2}\) tetramer. (**A**) LAIR-1 (10 \(\upmu\)M) was injected over immobilized serum-derived C1q (sC1q, red curve), sCLR (green curve) or sGR (blue curve). (**B**) sC1q (1 nM) was preincubated with C1r\({}_{2}\)s\({}_{2}\) at increasing molar ratios (1–5) and injected over immobilized LAIR-1. The grey dashed line represents the C1r\({}_{2}\)s\({}_{2}\) tetramer injection at a concentration of 10 nM as a control. LAIR-1 Interacts with C1q Collagen Stems Through a Site in Close Proximity but Different from the C1r2s2 Tetramer Binding Site  
In order to identify which C1q functional region is involved in LAIR-1 interaction, soluble LAIR-1 Ig-like domain was injected over immobilized serum-derived GR or CLR. As illustrated in Figure 2A, LAIR-1 interacts with both sC1q and sCLR but no interaction signal was detected on immobilized sGR. This result reveals that LAIR-1 Ig-like domain interacts with C1q exclusively through its collagen-like regions. With a view to locate more precisely the LAIR-1 binding site in the C1q collagen-like regions, a competition experiment was then performed with a C1q ligand known to interact with the CLR within the C1 complex: the C1r2s2 tetramer.