Interactions of ferritin with scavenger receptor class A members

Abstract

Scavenger receptors are a superfamily of membrane-bound receptors that recognize both self and nonself targets. Scavenger receptor class A (SR-A) has five known members (SCARA1 to -5 or SR-A1 to -A5), which are type II transmembrane proteins that form homotrimers on the cell surface. SR-A members recognize various ligands and are involved in multiple biological pathways. Among them, SCARA5 can function as a ferritin receptor; however, the interaction between SCARA5 and ferritin has not been fully characterized. Here, we determine the crystal structures of the C-terminal scavenger receptor cysteine-rich (SRCR) domain of both human and mouse SCARA5 at 1.7 and 2.5 Å resolution, respectively, revealing three Ca²⁺-binding sites on the surface. Using biochemical assays, we show that the SRCR domain of SCARA5 recognizes ferritin in a Ca²⁺-dependent manner, and both L- and H-ferritin can be recognized by SCARA5 through the SRCR domain. Furthermore, the potential binding region of SCARA5 on the surface of ferritin is explored by mutagenesis studies. We also examine the interactions of ferritin with other SR-A members and find that SCARA1 (SR-A1, CD204) and MARCO (SR-A2, SCARA2), which are highly expressed on macrophages, also interact with ferritin. By contrast, SCARA3 and SCARA4, the two SR-A members without the SRCR domain, have no detectable binding with ferritin. Overall, these results provide a mechanistic view regarding the interactions between the SR-A members and ferritin that may help to understand the regulation of ferritin homeostasis by scavenger receptors.

Keywords: SCARA5; SR class A; SRCR domain; X-ray crystallography; cell surface receptor; ferritin; protein structure; scavenger receptor.

Figure 1.

SCARA5 interacts with L-ferritin via the SRCR domain. A, schematic representation of the full-length SCARA5 (isoform 1), SCARA5 (isoform 4), and SCARA5ΔSRCR. B, FACS data showed that the FITC-labeled L-ferritin bound to the HEK293 cells transfected with hSCARA5 (isoform 1) or hSCARA5 (isoform 4) but did not bind to the SCARA5ΔSRCR-transfected cells. C, SDS-PAGE of the pulldown assays with FLAG tag showed that the ectodomain (ECTO) and the SRCR domain of SCARA5 could interact with L-ferritin, but the ectodomain without the SRCR domain (ECTOΔSRCR) had no interaction with L-ferritin. Empty beads had no interaction with L-ferritin either. D, fluorescent images showed that the hSCARA5 (isoform 1)– or the hSCARA5 (isoform 4)–transfected cells could internalize the FITC-labeled L-ferritin, but the SCARA5ΔSRCR-transfected cells had no binding to L-ferritin (bar, 25 μm).

Figure 2.

Crystal structures of the SRCR domain of SCARA5. A, SEC profiles of the CL-SRCR fragments of mSCARA1 and hSCARA5 and the SDS-PAGE of the CL-SRCR fragment of hSCARA5 from the SEC elution peak (fractions 1–5), showing the CL-SRCR fragment (orange arrow) as well as the SRCR fragment resulting from degradation (green arrow). B, SEC profile and SDS-PAGE of the CL-SRCR fragment of hSCARA5 after 36 h of degradation, resulting in an SRCR fragment for crystallization (green arrow). C, superposition of the crystal structures of human (orange) and mouse (blue) SRCR domain of SCARA5. Three Ca²⁺-binding sites (dashed red circles) are zoomed in at insets (Ca²⁺ are shown as green spheres; water molecules are shown as small red spheres), respectively. The glycans (N-acetylglucosamine, NAG) are colored in yellow. The surface electrostatic potential of the SRCR domain of hSCARA5 is shown (bottom left) with the same orientation as the crystal structure; the positively charged region is indicated by the dashed red oval.

Figure 3.

Mutagenesis studies of the interaction between the SRCR domain of SCARA5 and L-ferritin. A, residues around the Ca²⁺-binding sites and the loop region (yellow) involved in the mutagenesis studies on the SRCR domain of hSCARA5. B, ELISA data showed that the ectodomain of hSCARA5 (ECTO) and the SRCR domains of human and mouse SCARA5 bound to L-ferritin in the presence of Ca²⁺ (5 mm), and the binding affinities (K_D) were calculated based on the curve fitting, respectively. By contrast, Mg²⁺ reduced the binding affinity with L-ferritin, and EDTA could eliminate the binding completely. C, ELISA data showed that all of the mutants except D426A could reduce the binding of the SRCR domain with L-ferritin. D, FACS data showed all of the mutants of SCARA5 except D426A could reduce the binding of L-ferritin with the mutant-transfected cells. Mutants D458A/D459A and N481A from the Ca²⁺-binding site 2 retained some binding affinities with L-ferritin. Mock, nontransfected cells. E, fluorescent images showed that all of the mutants of SCARA5 except D426A could reduce the internalization of L-ferritin with the mutant-transfected cells (bar, 25 μm). Error bars, S.D.

Figure 4.

SCARA5 recognizes both L-ferritin and H-ferritin. A, FACS data showed that both FITC-labeled L-ferritin and H-ferritin bound to the hSCARA5 transfected cells, but not the hSCARA5 (E486A)-transfected cells. B, fluorescent images showed that both L-ferritin and H-ferritin could be internalized by the hSCARA5-transfected cells, but not the hSCARA5 (E486A)-transfected cells (bar, 25 μm). C, ELISA data showed that both L-ferritin and H-ferritin could bind to the SRCR domain of hSCARA5, but not to the SRCR (E486A) mutant. D, FLAG tag pulldown assays showed that both L-ferritin and H-ferritin could interact with the SRCR domain of hSCARA5. E, FACS data showed that the unlabeled L-ferritin could block the binding of the FITC-labeled H-ferritin by the SCARA5-transfected cells almost completely. F, FACS data showed that unlabeled H-ferritin could partially block the binding of the FITC-labeled L-ferritin by the SCARA5-transfected cells. Error bars, S.D.

Figure 5.

Mutagenesis studies of the SCARA5 binding region on ferritin. A, residues on the surface of L-ferritin (left; Protein Data Bank entry 2FFX) involved in the mutagenesis studies are labeled in the green rectangles. The potential binding region of SCARA5 on ferritin is indicated by the red rectangles. B, FACS data showed that mutants S19A, Q26A, Q80A/D81A, K83A, K106A, and D113A of L-ferritin reduced binding affinities with SCARA5. C, FACS data showed that mutants K84A, E87A, D88A/E89A, K92A/D95A, and K98A of L-ferritin retained similar binding affinities with SCARA5 as the WT. D, ELISA data showed that mutants S19A, Q26A, Q80A/D81A, K83A, K106A, and D113A of L-ferritin reduced the binding affinities with the SRCR domain SCARA5. E, ELISA data showed that mutants K84A, E87A, D88A/E89A, K92A/D95A, and K98A of L-ferritin retained similar binding affinities with the SRCR domain SCARA5. F, FACS data showed that the mutant K87A of H-ferritin reduced the binding with SCARA5 significantly. G, ELISA data showed that the mutant K87A of H-ferritin reduced the binding with the SRCR domain of SCARA5 significantly. Error bars, S.D.

Figure 6.

Interactions of L-ferritin with the SR-A members. A, FACS data (left) and fluorescent images (right) showed that human SCARA1, MARCO, and SCARA5 could bind to L-ferritin, whereas SCARA3, SCARA4, SCARA1ΔSRCR, SCARA1 (D376A/D377A), and CD163 had no binding to L-ferritin (bar, 25 μm). B, ELISA data showed that the SRCR domain of human SCARA5 had higher binding affinity with L-ferritin than the SRCR domains of human SCARA1 and MARCO. C, schematic model for the recognition of ferritin by SCARA5 on the cell surface. Error bars, S.D.