Epidermal growth factor-like domain repeat of stabilin-2 recognizes phosphatidylserine during cell corpse clearance

Abstract

Exposure of phosphatidylserine (PS) on the cell surface occurs early during apoptosis and serves as a recognition signal for phagocytes. Clearance of apoptotic cells by a membrane PS receptor is one of the critical anti-inflammatory functions of macrophages. However, the PS binding receptors and their recognition mechanisms have not been fully investigated. Recently, we reported that stabilin-2 is a PS receptor that mediates the clearance of apoptotic cells, thus releasing the anti-inflammatory cytokine, transforming growth factor beta. In this study, we showed that epidermal growth factor (EGF)-like domain repeats (EGFrp) in stabilin-2 can directly and specifically recognize PS. The EGFrps also competitively impaired apoptotic cell uptake by macrophages in in vivo models. We also showed that calcium ions are required for stabilin-2 to mediate phagocytosis via EGFrp. Interestingly, at least four tandem repeats of EGF-like domains were required to recognize PS, and the second atypical EGF-like domain in EGFrp was critical for calcium-dependent PS recognition. Considering that PS itself is an important target molecule for both apoptotic cells and nonapoptotic cells during various cellular processes, our results should help elucidate the molecular mechanism by which apoptotic cell clearance in the human body occurs and also have implications for targeting PS externalization of nonapoptotic cells.

FIG. 1.

EGF-like domain repeats (EGFrp) of stabilin-2 competitively inhibit the uptake of aged RBCs and apoptotic cells. (A) Schematic diagram of stabilin-2 protein and deletion mutant> proteins. A Nus tag was fused to each recombinant protein to enhance the solubility of the proteins. Stab-U1, -U2, and -U3 are composed of one EGFrp and two FAS1 domains. Stab-U4 is composed of an EGFrp, a FAS1 domain, and a Link domain. (B) Effect of four repeated units on engulfment of aged RBCs by L/Stab-2 cells. Aged RBCs were preincubated with 10 μM concentrations of the indicated stabilin-2 proteins and then added to L/Stab-2 cells. After incubation for 1 h, the percentages of cells engulfing the aged RBCs were determined. **, P < 0.01 (ANOVA). (C) Representative images of stabilin-2-mediated phagocytosis in the presence of each repeated unit. Scale bar, 50 μm. (D) Effect of EGFrp-containing proteins on the engulfment of aged RBCs by L/Stab-2 cells. Aged RBCs were preincubated with three different concentrations (0.1, 1.0, or 10 μM) of the indicated stabilin-2 proteins and then added to L/Stab-2 cells. After incubation for 1 h, the percentages of cells engulfing the aged RBCs were determined. (E) Representative images of apoptotic cell engulfment by L/Stab-2 cells in the presence of recombinant stabilin-2 proteins. Apoptotic Jurkat T cells were preincubated with the indicated stabilin-2 proteins (10 μM) and then added to L/Stab-2 cells. After incubation for 1 h, the cells were extensively washed, stained using an in situ cell-death detection kit (Roche), and then observed under a fluorescence microscope. Scale bar, 100 μm. (F) Effect of EGFrps on the engulfment of aged RBCs by L/Stab-2 cells. Aged RBCs were preincubated with three different concentrations (0.1, 1.0, or 10 μM) of the indicated proteins and then added to L/Stab-2 cells. The percentages of cells engulfing the aged RBCs were determined. The results are expressed as the means ± the standard deviation (SD) from at least three experiments.

FIG. 2.

EGFrps directly and preferentially interact with PS. (A) Affinity of recombinant stabilin-2 proteins to PS. ELISA plates were coated with PS and then incubated with the indicated stabilin-2 proteins. After 1 h, proteins bound to the wells were quantified using anti-His antibody. (B) ELISA plates were coated with PS, PC, phosphatidylethanolamine (PE), or phosphatidylinositol (PI) and then incubated with the EGFrp protein (E3). After 1 h, proteins bound to the wells were quantified using anti-His antibody. The results are expressed as the means ± the SD from at least three experiments.

FIG. 3.

Binding of aged RBCs by L cells expressing a third EGFrp of stabilin-2 (L/EGF3). (A) Schematic representation of mutant stabilin-2 protein (Stab-E3-C and Stab-U4-C). (B) Surface expression of EGFrp on L cells expressing a third EGFrp of stabilin-2 (L/EGF3). Isotype-matched control antibody was used as a control for anti-stabilin-2 antibody (5G3). A representative result of three independent experiments is shown. (C) Dose-dependent binding of PS liposome to the surface of L/EGF3 cells. L/EGF3 cells were incubated with two different concentrations (10 or 100 μM) of fluorescence-labeled PS or PC liposomes for 1 h at 4°C. After extensive washing, the amount of fluorescence-labeled liposome associated with the cells was determined via flow cytometry. (D) Adhesion of L/EGF3 cells to PS. ELISA plates were coated with PS (right) or PC (left) and then incubated with L/Mock, L/Stab-2, L/EGF3, or L/FAS7 cells at 37°C for 1 h. After extensive washing, cells attached to the surfaces were quantified by hexosaminidase assay. (E) Representative images of aged RBC binding in L/EGF3 (right) and L/Mock cells (left). Scale bar, 50 μm. (F) Microscopic quantification of the binding and engulfment of aged RBCs in L/Mock, L/Stab-2, and L/EGF3 cells. The results are expressed as the means ± the SD from at least three experiments. **, P < 0.01 (ANOVA). (G) Binding of the aged RBCs by L/EGF3 cells in the presence of liposomes containing PC or PS (10 μM), anti-stabilin-2 antibody, or isotype-matched control antibody. L/EGF3 cells were preincubated with liposomes containing PC or PS (10 μM), anti-stabilin-2 antibody, or isotype-matched control antibody, and then aged RBCs were added. After incubation for 1 h, the percentages of cells binding the aged RBCs were determined. The results are expressed as the means ± the SD from at least three experiments. **, P < 0.01 (ANOVA). (H) Binding and engulfment of aged RBCs in L/Mock, L/Stab-2, and L/U4 cells. The results are expressed as the means ± the SD from at least three experiments. **, P < 0.01 (ANOVA).

FIG. 4.

EGFrp inhibits the phagocytosis of apoptotic cells in vitro and in vivo. (A) Effect of EGFrp on the engulfment of aged RBCs by HMDMs. Aged RBCs were preincubated with 10 μM EGFrp and then added to HMDMs. The percentages of cells engulfing apoptotic cells were then determined. The results are expressed as the means ± the SD from at least three experiments. **, P < 0.01 (ANOVA). (B) EGFrp (E3) inhibits the phagocytic clearance of apoptotic thymocytes during sterile peritonitis. A graph shows the percentages of inflammatory macrophages from C57BL/6 mice engulfing apoptotic thymocytes at different time points after injection. Apoptotic thymocytes were preincubated with a 1 μM concentration of the third EGFrp (E3, ▪) or control Nus protein (□) prior to injection. The results are expressed as the means ± the SD from at least three experiments. (C) FACS analysis of the phagocytic uptake of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled apoptotic cells by Mac-1⁺ peritoneal macrophages 30 min after the injection of apoptotic cells in C57BL/c mice. A representative result of three independent experiments is shown.

FIG. 5.

Stabilin-2 mediated phagocytosis via calcium-dependent PS recognition. (A) Effect of calcium ions on stabilin-2-mediated phagocytosis. L/Stab-2 cells were incubated with aged RBCs in the presence of the indicated concentrations of calcium ion under serum-free conditions. After incubation for 1 h, the percentages of cells engulfing the aged RBCs were determined. The results are expressed as the means ± the SD from at least three experiments. (B) Dependence of NBD-PC or NBD-PS fluorescence quenching on the concentration of EGFrp (E3). EGFrp protein was added to NBD-liposome containing binding buffer. Quenching of the NBD-liposome fluorescence was then monitored by using a fluorescence spectrometer. The extent of quenching is plotted versus the inverse protein concentration as previously described (5). The results are expressed as the means ± the SD from at least three experiments.

FIG. 6.

Protein structure of stabilin-2 and alignments of the EGF-like domains. (A) Domain structure of stabilin-2. Stabiln-2 contains four EGF-like domain repeats (EGRrps, E1 to E4). Each EGFrp consists of two atypical EGF-like domains and four standard EGF-like domains (the fourth EGFrp [E4] has three EGF-like domains). (B) Alignment of the EGF-like domains in stabilin-2. The consensus sequences of atypical and standard EGF-like domains are derived from previous studies (6, 22, 39).

FIG. 7.

Characterization of bacterial EGFrp. (A) Gel filtration chromatography of bacterial EGFrp. Fast-performance liquid chromatography-gel filtration was carried out by using a Superdex 75 HR 10/30 column that had been calibrated with molecular mass standards. The molecular mass standards were indicated in the chromatogram (arrowheads indicate 67, 43, 25, and 13.7 kDa). Arrow, monomeric fraction. (B) SDS-PAGE of the purified monomer of EGFrp. Lane 1, molecular weight markers (kDa); lane 2, purified EGFrp monomer with DTT; lane 3, purified EGFrp monomer without DTT. (C and D) MALDI-TOF MS analysis of purified EGFrp monomer under nonreducing (DTT−, C) or reducing (DTT+, D) conditions. Arrows, monomeric EGFrp. (E) E3-Fc, E3-ori, or E3-16 was added to NBD-liposome containing binding buffer. Quenching of NBD-liposome fluorescence was then monitored by using a fluorescence spectrometer. The binding affinities of EGFrps (E3-Fc, E3-Ori, and E3-16) were determined by using the Kapp (apparent association constant, which is given by 1/K, using the Stern-Volmer law, which is as follows: Fo/(Fo − F) = 1/fa + 1/(faK[Mt]). The quenching constant, K, after buffer normalization is directly related to the binding affinity of the complex, which can be determined by plotting the quenching extent [Fo/(Fo − F)] versus the inverse protein concentration (1/[protein]).

FIG. 8.

EGFrps containing atypical EGF-like domains mediate the recognition of PS. (A) Schematic representation of deletion mutant stabilin-2 proteins. (B and C) NBD-PS-labeled PS liposomes were successively incubated with C-terminal deletion (B) or N-terminal deletion (C) mutants of EGFrp, and the proteins bound to the liposomes were quantified by fluorescence quenching assays. The extent of quenching is plotted versus the inverse protein concentration. (D and E) Aged RBCs were preincubated with C-terminal deletion (D) or N-terminal deletion (E) mutants of stabilin-2 and then added to L/Stab-2 cells. After incubation for 1 h, the percentages of cells engulfing aged RBCs were determined. The results are expressed as the means ± the SD from at least three experiments. *, P < 0.05; **, P < 0.01 (ANOVA).

FIG. 9.

The presence of a second atypical EGF-like domain is a key role in calcium-dependent PS recognition. (A) Schematic representation of EGFrp deletion mutants containing four EGF-like domains. (B) EGFrp deletion mutants were added to NBD-PS liposome containing binding buffer. Quenching of the NBD-PS liposome fluorescence was then monitored by using a fluorescence spectrometer. The extent of quenching is plotted versus the inverse protein concentration. (C) The binding affinities of EGFrp mutants were determined by using the Kapp [apparent association constant, which is given by 1/K, using the Stern-Volmer law, which is as follows: Fo/(Fo − F) = 1/fa + 1/(faK[Mt])]. The quenching constant, K, after buffer normalization is directly related to the binding affinity of the complex, which can be determined by plotting the quenching extent [Fo/(Fo − F)] versus the inverse protein concentration (1/[protein]). (D) The inhibitory effects of each EGFrp mutant were tested by using a phagocytosis assay as described in Materials and Methods, and the percentages of cells engulfing aged RBCs were determined. The results are expressed as the means ± the SD from at least three experiments. *, P < 0.05 (ANOVA).