Abstract
Formation of the haptoglobin (Hp)-hemoglobin (Hb) complex in human plasma leads to a high affinity recognition by the endocytic macrophage receptor CD163. A fast segregation of Hp-Hb from CD163 occurs at endosomal conditions (pH <6.5). The ligand binding site of CD163 has previously been shown to involve the scavenger receptor cysteine-rich (SRCR) domain 3. This domain and the adjacent SRCR domain 2 of CD163 contain a consensus motif for a calcium-coordinated acidic amino acid triad cluster as originally identified in the SRCR domain of the scavenger receptor MARCO. Here we show that site-directed mutagenesis in each of these acidic triads of SRCR domains 2 and 3 abrogates the high affinity binding of recombinant CD163 to Hp-Hb. In the ligand, Hp Arg-252 and Lys-262, both present in a previously identified CD163 binding loop of Hp, were revealed as essential residues for the high affinity receptor binding. These findings are in accordance with pairing of the calcium-coordinated acidic clusters in SRCR domains 2 and 3 with the two basic Arg/Lys residues in the Hp loop. Such a two-point electrostatic pairing is mechanistically similar to the pH-sensitive pairings disclosed in crystal structures of ligands in complex with tandem LDL receptor repeats or tandem CUB domains in other endocytic receptors.
Highlights
CD163 mediates endocytosis of haptoglobin-hemoglobin complexes formed upon intravascular hemolysis
The resulting data showed that the scavenger receptor cysteine-rich (SRCR) domain 2 mutant protein (D185, D186A,E252A) has a significant reduction in affinity compared with the WT counterpart, and that effects of the single (E359A) and double mutations (D292A, D293A) in SRCR domain 3 are dramatic (Fig. 2D)
The present mutagenesis study reveals the essential receptor and ligand residues involved in high affinity binding of the Hp-Hb complex to CD163 in humans
Summary
CD163 mediates endocytosis of haptoglobin-hemoglobin complexes formed upon intravascular hemolysis. Conclusion: A two-point electrostatic pairing between Ca2ϩ-coordinated acidic clusters in receptors and basic ligand residues explains high-affinity CD163-(haptoglobin-hemoglobin) binding. Hp Arg-252 and Lys-262, both present in a previously identified CD163 binding loop of Hp, were revealed as essential residues for the high affinity receptor binding These findings are in accordance with pairing of the calcium-coordinated acidic clusters in SRCR domains 2 and 3 with the two basic Arg/Lys residues in the Hp loop. Such a two-point electrostatic pairing is mechanistically similar to the pH-sensitive pairings disclosed in crystal structures of ligands in complex with tandem LDL receptor repeats or tandem CUB domains in other endocytic receptors. Based on a series of binding experiments with mutated variants of both CD163 and Hp, we here present data pinpointing the contact region between CD163 and its high affinity ligand Hp-Hb and propose a common model for Ca2ϩ-dependent coupling and uncoupling of ligand
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