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Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells

Nature Immunology volume 4, pages 87–91 (2003)Cite this article

Abstract

Rapid phagocytosis of apoptotic cells is thought to limit the development of inflammation and autoimmune disease. Serum enhances macrophage phagocytosis of apoptotic cells. Here we identified protein S as the factor responsible for serum-stimulated phagocytosis of apoptotic cells. Protein S is best known for its anti-thrombotic activity, serving as a cofactor for protein C. Purified protein S was equivalent to serum in its ability to stimulate macrophage phagocytosis of apoptotic lymphoma cells, and immunodepletion of protein S eliminated the prophagocytic activity of serum. Protein S acted by binding to phosphatidylserine expressed on the apoptotic cell surface. Protein S is thus a multifunctional protein that can facilitate clearance of early apoptotic cells in addition to regulating blood coagulation.

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Figure 1: FCS stimulates the ability of human macrophages to phagocytose apoptotic cells.
Figure 2: Protein S stimulates phagocytosis of apoptotic BL-41 cells by macrophages.
Figure 3: Immunodepletion of protein S from serum removes prophagocytic activity from serum.
Figure 4: Protein S binds to apoptotic cells.
Figure 5: Protein S binds to phosphatidylserine on the surface of apoptotic cells.

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Acknowledgements

We thank V. Calvert for supplying human monocytes; E. Shores and G. Tosato for critical reading of the manuscript; and E. Petricoin and Y. M. Zhao for expert technical advice.

Author information

Author notes

  1. Howard A. Anderson and Caroline A. Maylock: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Therapeutic Proteins, Laboratory of Biochemistry, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, 20892, MD, USA

    Howard A. Anderson & Emily Shacter

  2. Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, 20815, MD, USA

    Caroline A. Maylock

  3. Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, 20892, MD, USA

    Joy A. Williams

  4. Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, 20892, MD, USA

    Cloud P. Paweletz

  5. Department of Pharmacology, University of Texas, Southwestern Medical Center, Dallas, 75390, TX, USA

    Hongjun Shu

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  1. Howard A. Anderson
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Corresponding author

Correspondence to Emily Shacter.

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Supplementary information

Web Fig. 1.

Identification of protein S in the prophagocytic serum fraction. (a) Two-dimensional gel of the purified fraction stained with Coomassie blue. Proteins that were identified by tandem mass spectrometry (MS) are indicated (Pr.S, protein S; Kin., kininogen, BSA, bovine serum albumin). (b) MS spectra of tryptic digests of the Coomassie blue-stained protein S spot. CID represents collision-induced dissociation of peptide analyzed in c. (c) MS/MS profile analysis of ion 908.50 identifying protein S from the unique peptide sequence VYFAGVPR. Peptides from kininogen (YSIVFIAR) and BSA (KVPQVSTPTLVEVSR) were analyzed similarly (data not shown). (PDF 162 kb)

Web Fig. 2.

Immunoanalysis of protein S in enriched phagocytosis fraction. Proteins from serum (25 μg), the purified prophagocytic fraction from serum (5 μg) and human protein S (5 μg) were resolved by SDS-PAGE and stained for protein with Sypro Ruby Protein Gel Stain (Molecular Probes). Immunoblotting of proteins transferred to PVDF membranes was performed using a rabbit anti-protein S IgG primary antibody followed by HRP-conjugated goat anti-rabbit IgG. The three polypeptides seen in the commercial human protein S preparation and the differences in molecular weight observed between bovine and human protein S are most likely attributed to the numerous post-translational modifications that are known to occur to this molecule (for example, proteolysis, glycosylation, γ-glutamyl carboxylation). (PDF 195 kb)

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Anderson, H., Maylock, C., Williams, J. et al. Serum-derived protein S binds to phosphatidylserine and stimulates the phagocytosis of apoptotic cells. Nat Immunol 4, 87–91 (2003). https://doi.org/10.1038/ni871

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