Deletion or replacement of the second EGF-like domain of protein S results in loss of APC cofactor activity

Mille-Baker, Blandine; Rezende, Suely Meireles; Simmonds, Rachel E.; Mason, Philip J.; Lane, David A.; Laffan, Michael

doi:10.1182/blood-2002-08-2353

Cited by 29 publications

(47 citation statements)

References 25 publications

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“…In addition, EGFrps composed of only standard EGF-like domains showed only minimal PS binding activity, which is consistent with the previous observation that, although protein S contained four tandem repeats of the EGFlike domain, its N-terminal Gla domain is responsible for binding to the appropriate phospholipid membranes in the presence of Ca 2ϩ ions (27). However, a recent study showed that EGF-like domains are also important for protein S activity and suggested that EGF modules may be able to constrain the protein S molecule in an appropriate configuration (21). This is consistent with our observation that at least four tandem repeats of the EGF-like domain were required for binding to PS, which suggests that at least four repeats of the EGF-like domains may be necessary to form a conformation that presents the second atypical EGF-like domain in the proper orientation to target PS.…”

Section: Discussionsupporting

confidence: 79%

Epidermal Growth Factor-Like Domain Repeat of Stabilin-2 Recognizes Phosphatidylserine during Cell Corpse Clearance

Park

Kim

Jung

et al. 2008

Molecular and Cellular Biology

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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 ␤. 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.

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Section: Discussionsupporting

confidence: 79%

Epidermal Growth Factor-Like Domain Repeat of Stabilin-2 Recognizes Phosphatidylserine during Cell Corpse Clearance

Park

Kim

Jung

et al. 2008

Molecular and Cellular Biology

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“…A recent report has suggested a role for EGF2, since APC cofactor function was lost upon its replacement or deletion. 45 This suggestion is supported by the association of qualitative PS deficiency with naturally occurring mutations in EGF2 (Lys155Glu 46 and Cys134Phe 47 ). EGF4 of PS has also been suggested to be important to keep EGF1 in optimal alignment for interaction with APC, 39 although a recombinant EGF4 alone has failed to exhibit APC cofactor activity in a clotting assay.…”

Section: Apc-dependent Anticoagulant Functions Of Psmentioning

confidence: 94%

Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S–C4b binding protein complex

Rezende

Simmonds

Lane

2004

Blood

193

171

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“…Despite numerous studies describing the important protein S domains/residues for protein S cofactor function with APC (22,26,28,37), the mapping of APC residues involved in mediating the interaction with protein S has been limited. One study, however, broadly implicated residues 22-45 of the APC Gla domain in enabling protein S to act as an APC cofactor (18).…”

Section: Discussionmentioning

confidence: 99%

Multifunctional Specificity of the Protein C/Activated Protein C Gla Domain

Preston

Ajzner

Razzari

et al. 2006

Journal of Biological Chemistry

Self Cite

103

164

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Activated protein C (APC) has potent anticoagulant and antiinflammatory properties that are mediated in part by its interactions with its cofactor protein S and the endothelial cell protein C receptor (EPCR). The protein C/APC Gla domain is implicated in both interactions. We sought to identify how the protein C Gla domain enables specific protein-protein interactions in addition to its conserved role in phospholipid binding. The human prothrombin Gla domain, which cannot bind EPCR or support protein S cofactor activity, has 22/45 residues that are not shared with the human protein C Gla domain. We hypothesized that the unique protein C/APC Gla domain residues were responsible for mediating the specific interactions. To assess this, we generated 13 recombinant protein C/APC variants incorporating the prothrombin residue substitutions. Despite anticoagulant activity similar to wild-type APC in the absence of protein S, APC variants APC(PT33-39) (N33S/V34S/D35T/ D36A/L38D/A39V) and APC(PT36/38/39) (D36A/L38D/A39V) were not stimulated by protein S, whereas APC(PT35/36) (D35T/D36A) exhibited reduced protein S sensitivity. Moreover, PC(PT8/10) (L8V/H10K) displayed negligible EPCR affinity, despite normal binding to anionic phospholipid vesicles and factor Va proteolysis in the presence and absence of protein S. A single residue variant, PC(PT8), also failed to bind EPCR. Factor VIIa, which also possesses Leu-8, bound soluble EPCR with similar affinity to wild-type protein C, collectively confirming Leu-8 as the critical residue for EPCR recognition. These results reveal the specific Gla domain residues responsible for mediating protein C/APC molecular recognition with both its cofactor and receptor and further illustrate the multifunctional potential of Gla domains.

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Deletion or replacement of the second EGF-like domain of protein S results in loss of APC cofactor activity

Cited by 29 publications

References 25 publications

Epidermal Growth Factor-Like Domain Repeat of Stabilin-2 Recognizes Phosphatidylserine during Cell Corpse Clearance

Epidermal Growth Factor-Like Domain Repeat of Stabilin-2 Recognizes Phosphatidylserine during Cell Corpse Clearance

Coagulation, inflammation, and apoptosis: different roles for protein S and the protein S–C4b binding protein complex

Multifunctional Specificity of the Protein C/Activated Protein C Gla Domain

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