Fig. 5: Multimerisation of CD163 allows uptake of lower-avidity ligands.

a A close-up of the interface between CD163_A and CD163_B, showing the location of N807, which becomes modified with an N-linked glycan in the R809T mutant. b SEC-MALLS analysis of wild-type (WT) CD163 ectodomain (blue) and the ectodomain of the R809T mutant (green) with the left y-axis showing the differential refractive index, while the right y-axis displays the molecular weight. This is representative of two repeats at a concentration of 24 μM. c Assessment of the binding of CD163-R809T ectodomain, immobilised through a C-terminal biotin, to Hp(1-1)Hb, Hp(2-2)Hb and HpSPHb by SPR analysis. In the presence of Ca²⁺, the sensorgrams represent twofold dilution series from a maximum concentration of 5 nM for Hp(1-1)Hb, 2.5 nM for Hp(2-2)Hb and 80 nM for HpSPHb. In the absence of Ca²⁺, one injection of Hp(1-1)Hb at 10 nM is displayed. Data were shown as black lines, while fitting to a one-to-one binding model is depicted as dashed red lines. These are representative of n = 3. d Assessment of the binding of Hp (green) and Hb (orange) to CD163-R809T ectodomain using MST. Each point represents the mean of three replicates, and the error bars are the standard deviation. e Measurement of the ability of different ligands to compete for the uptake of fluorescently labelled Hp(2-2)Hb into HEK293 cells transfected with CD163-R809T. BSA is included as a control for CD163-independent effects. Each point depicts the mean of three replicates, and the error bars are standard deviations. f A model for the CD163-mediated uptake of HpHb. At the cell surface, calcium mediates multimerisation of CD163. Two states exist in equilibrium, with the arms either free to bind to ligand or forming arm-arm interactions which occlude ligand-binding sites. On addition of ligand, the free arms of CD163 mould around the ligand to form a complex. On internalisation into the endosome, a drop in calcium concentration and pH results in monomerisation of CD163 and ligand release. CD163 can then be recycled back to the cell surface while HpHb is degraded and detoxified.