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. 2016 May 1;196(9):3896-3909.
doi: 10.4049/jimmunol.1502122. Epub 2016 Apr 1.

The C-Terminal Acidic Region of Calreticulin Mediates Phosphatidylserine Binding and Apoptotic Cell Phagocytosis

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The C-Terminal Acidic Region of Calreticulin Mediates Phosphatidylserine Binding and Apoptotic Cell Phagocytosis

Sanjeeva Joseph Wijeyesakere et al. J Immunol. .
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Calreticulin is a calcium-binding chaperone that is normally localized in the endoplasmic reticulum. Calreticulin is detectable on the surface of apoptotic cells under some apoptosis-inducing conditions, where it promotes the phagocytosis and immunogenicity of dying cells. However, the precise mechanism by which calreticulin, a soluble protein, localizes to the outer surface of the plasma membrane of dying cells is unknown, as are the molecular mechanisms that are relevant to calreticulin-induced cellular phagocytosis. Calreticulin comprises three distinct structural domains: a globular domain, an extended arm-like P-domain, and a C-terminal acidic region containing multiple low-affinity calcium binding sites. We show that calreticulin, via its C-terminal acidic region, preferentially interacts with phosphatidylserine (PS) compared with other phospholipids and that this interaction is calcium dependent. Additionally, exogenous calreticulin binds apoptotic cells via a higher-affinity calcium-dependent mode that is acidic region dependent. Exogenous calreticulin also binds live cells, including macrophages, via a second, lower-affinity P-domain and globular domain-dependent, but calcium-independent binding mode that likely involves its generic polypeptide binding site. Truncation constructs lacking the acidic region or arm-like P-domain of calreticulin are impaired in their abilities to induce apoptotic cell phagocytosis by murine peritoneal macrophages. Taken together, the results of this investigation provide the first molecular insights into the phospholipid binding site of calreticulin as a key anchor point for the cell surface expression of calreticulin on apoptotic cells. These findings also support a role for calreticulin as a PS-bridging molecule that cooperates with other PS-binding factors to promote the phagocytosis of apoptotic cells.


Figure 1
Figure 1. Expression and characterization of calreticulin truncation constructs
(A) (Upper left panel) Model for the structure of mCRT(1-351) based on published structures of the globular and P-domains of calreticulin and calnexin (10-12, 51) (Upper right panel) Representative SDS-PAGE gel depicting the migration positions of purified forms of various mCRT constructs used in this investigation. (Lower panel) Clustal-W2 alignments of the amino acid sequences of the murine calreticulin (mCRT) constructs used in this study. The constructs are: mCRT(WT), wild type mCRT; mCRT(ΔC), mCRT lacking the C-terminal acidic domain (residues 340-399); mCRT(ΔP), mCRT lacking the arm-like P-domain (residues 187-283); mCRT(1-351), mCRT lacking the C-terminus of the acidic C-domain (residues 352-399); mCRT(1-351 ΔP), mCRT containing the globular domain alone (lacking both the P-domain (residues 187-283) as well as the C-terminal region of the acidic C-domain (residues 352-399); GB1-mCRT(P), mCRT containing the P-domain alone (residues 187-283) expressed as a fusion with the B1 domain of protein G (GB1); GB1-mCRT(C), mCRT containing the acidic C domain alone (residues 340-399) expressed as a fusion with GB1. (B) Representative bio-layer interferometry analysis (from 2 independent experiments) depicting the binding of GB1-mCRT(P) to varying ERp57 concentrations. The calculated steady-state binding affinities (KD) for GB1-mCRT(P) and mCRT(WT) are 0.101 ± 0.008 and 0.282 ± 0.038 μM respectively (C) Representative far-UV CD spectra (out of 2 independent experiments) of GB1-mCRT(C) or GB1 in the presence or absence of calcium. (D) Representative ITC thermograms (from 2 independent experiments) showing binding of GB1-mCRT(C) or GB1 to calcium. Plots show the raw titration curves (above) and corresponding curve fits (below). Calculated thermodynamic parameters are shown in Table I.
Figure 2
Figure 2. Calreticulin binds lipids in a calcium- and C-terminal acidic region-dependent manner
Figure shows representative ITC thermograms (from of 3-5 independent experiments) showing the interaction of the indicated calreticulin constructs with liposomes containing POPC or POPS in the presence or absence of 5 mM CaCl2. Plots show the raw titration curves (above) and corresponding curve fits (below). Unless indicated otherwise, mCRT-lipid interactions were measured in the presence of 5 mM CaCl2. Calculated thermodynamic parameters are described in Table I.
Figure 3
Figure 3. Calcium-bridged interactions with the acidic C-terminal region of calreticulin mediates mCRT binding to apoptotic, but not live cells
Flow cytometric analysis of the binding of FITC-labeled GB1-mCRT(C) (A and B), mCRT(WT) (C and F), mCRT(ΔP) (D) and mCRT(ΔC) (E) to apoptotic (A, C-E) or live (B and F) mouse embryonic fibroblasts (MEFs) in the presence or absence of calcium as indicated. Figures show representative plots from 3 (A), 2 (B), 5 (C), 2 (D and E) and 4 (F) independent experiments. In all analyses, binding was measured in the 7AAD or DAPI populations by flow cytometry.
Figure 4
Figure 4. Distinct modes of calreticulin binding to apoptotic cells compared to live cells or macrophages
Inhibition assays (panels A, B and D) or direct binding assays (panel C) are shown. Panels A, B and D show representative IC50 plots for the dose-dependent inhibition of the binding of FITC-labeled mCRT(WT) to apoptotic MEFs (A), live MEFs (B) or live macrophages (D) by the indicated mCRT domains. Calculated inhibition constants and data replicates are described in Table II. (C) Flow cytometric analysis of the dose-dependent binding of FITC-mCRT(WT) (shown as filled circles) or GB1-mCRT(C) (shown as filled squares) to macrophages in the presence or absence of calcium as indicated. Panel shows representative plots from 6 (mCRT(WT) in 5 mM Ca2+) or 2 (mCRT(WT) in 0 mM Ca2+ and GB1-mCRT(C)) independent experiments. In all analyses, binding was measured in the 7AAD or DAPI populations by flow cytometry.
Figure 5
Figure 5. Calreticulin surface exposure in nocoadazole-treated MEFs and calreticulin-mediated phagocytosis induction in vitro and in vivo
(A) CRT−/− (K42) MEFs or K42 cells reconstituted with mCRT(WT) were treated with 300 μM oxaliplatin (OXP) (for 16 hours), 150 μM cisplatin (CDDP) (for 16 hours) or 1 μM nocodazole (NOCO) (for 48 hours) and analyzed for surface calreticulin expression by flow cytometry. Panels depict representative histograms (from 3 independent experiments) showing cell-surface calreticulin expression with the analyses gated on the apoptotic (Annexin-V+/7AAD) population. (B) Upper left panel shows representative flow cytometry plots depicting the externalization of calreticulin and PS (detected via Annexin V staining) in nocodazole-treated (top row) or untreated MEFs (bottom row), with cells gated on the 7AAD population. There is low level of non-specific binding of the anti-CRT antibody to a cell-surface factor that is upregulated following drug treatment. Upper middle panel shows the quantification of the percentage of CRT+ MEFs within the Annexin V+ population. Upper right panel shows the percentage of total Annexin V+ cells following nocodazole-treatment of CRT−/− MEFs or those reconstituted with mCRT(WT). Averaged data ± SEM from 4 independent experiments are shown in the middle and right panels, with statistical significance assessed via paired t-tests and statistically significant differences indicated (*). Lower panel shows representative fluorescence microscopy images (original magnification ×40) (from 2 independent experiments) depicting the co-localization of mCRT(WT) and Annexin V (to mark PS) on the surface of nocodazole-treated MEFs. (C and D) Phagocytosis of nocodazole-treated MEFs by murine peritoneal macrophages as assessed in vitro via fluorescence microscopy (C) or in vivo via flow cytometry (D). In D, CMFDA-labeled, nocodazole-treated apoptotic CRT−/− cells or those reconstituted with mCRT(WT) were injected i.p. and macrophages were harvested by peritoneal lavage 60 minutes post-injection. The percentages of macrophages (CD11b+ cells) positive for the target cell marker (CMFDA) are shown. ‘PBS’ represents a control where mice were injected with sterile PBS alone in the absence of apoptotic target cells. Left-hand panels in C and D show representative fluorescence images or flow cytometry data used to measure the uptake of nocodazole-treated CMFDA-labeled mCRT(WT)-expressing MEFs by murine peritoneal macrophages (CD11b+ cells) (C). Right-hand panels in C and D show the mean phagocytic uptake ± SEM from 4 independent experiments, with 2 mice per data point shown for the in vivo experiments (panel D). Statistical significance was assessed via one-tailed paired t-tests and statistically significant differences indicated (*).
Figure 6
Figure 6. Roles for calreticulin domains in the phagocytic uptake of apoptotic cells and in binding to apoptotic cells
(A-C) Phagocytosis by murine peritoneal macrophages of apoptotic (UV-treated) MEFs coated with indicated calreticulin constructs, assessed by flow cytometry (A, C) or microscopy (B). Phagocytosis measurements were done at 37 °C, with 4 °C incubations serving as adhesion controls. In all cases, phagocytosis was assessed as the %CMFDA+ cells within the macrophage (CD11b+) population. The left-hand panel in A shows a representative flow cytometric analysis. The left-hand panel in B shows representative fluorescence images (original magnification ×40) of the uptake of CMFDA-labeled mCRT(WT)-coated apoptotic MEFs by murine peritoneal macrophages. Bar graphs (right panels) in panels A and B show the mean phagocytic uptake ± SEM from 5 (A) or 7 (B) independent experiments. Statistical significance within each temperature group was assessed via repeated measures one-way ANOVA followed by a Dunnett’s post-hoc test (panel A) or paired t-tests (panel B) and statistically-significant differences are indicated (*). Panel C shows a representative EC50 plot (from 3 independent experiments) depicting dose-dependent changes in the efferocytosis of apoptotic MEFs coated with 0-40 μM mCRT(WT) by murine peritoneal macrophages as assessed via flow cytometry. (D) Assessment of sequestration of externalized PS by calreticulin. Left panel shows representative flow-cytometry plots (measured within the 7AAD population) showing the binding of the indicated mCRT-FITC constructs (24 μM) followed by Annexin V-PE (to mark externalized PS) to apoptotic cells. Other panels depict quantifications of the percentage (± SEM) of total Annexin V-PE+ cells (second panel), total CRT+ cells (third panel) and CRT+ cells within the Annexin V+ population (fourth panel) from 3 independent experiments, with statistical significance assessed via paired t-tests and statistically significant differences indicated (*).
Figure 7
Figure 7. Proposed model for the pro-phagocytic role of calreticulin
(A and D) The acidic C-terminal regions of mCRT(WT) anchors it to exposed phosphatidylserine on the surface of the apoptotic target cell via calcium-bridged interactions. The arm-like P-domain of calreticulin interacts with receptors on the phagocyte (such as LRP-1), allowing calreticulin to function as a pro-phagocytic signal and promote uptake of the dying cell. Phagocytosis models in which the globular domain engages the phagocyte receptor (A) or apoptotic cell co-receptor (D) are both consistent with the data. (B and C) Proposed models for the PS-independent (B) and dependent modes (C) of interaction between apoptotic target cells and mCRT(ΔC) and mCRT(ΔP) respectively.

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