Fig 1: ?-Carboxylation is required for growth arrest-specific 6 (Gas6) activity. (A) HEK293 cells were transfected with pSecTaq-hGas6 plasmid (10) to collect Gas6-containing media (CM). Serum-free culture media was supplemented with either 2 µM warfarin (W) or 4.4 µM vitamin K (K) to abolish or induce ?-carboxylation of Gas6. The conditioned media was subjected to SDS-PAGE gel to assess total Gas6 and ?-carboxylation level by Gas6 and ?-carboxylglutamic acid immunoblotting. (B) hAxl/IFN?R1 reporter cells were stimulated by warfarin-treated Gas6-CM (W) or vitamin K-treated Gas6-CM (K). The activation of Axl receptor was assessed by pSTAT1 immunoblotting. (C) hAxl/IFN?R1 cells were incubated with warfarin-treated Gas6 (Gas6-W, red) conditioned media or vitamin K-treated Gas6 (Gas6-K, black) conditioned media. The binding of Gas6 to hAxl/IFN?R1 receptors was assessed in flow cytometry by using anti-Myc-PE antibody (left), with geometric MFI quantification from independent experiments (n = 3) in the right panel. Error bar, SEM. n.s., not significant. (D) Schematic structure of Myc-tagged mutant Gas6 proteins. WT, wild-type Gas6; ?G, Gas6 that lacks Gla domain; ?E, Gas6 that lacks EGF repeats; ?GE, Gas6 that lacks both Gla domain and EGF repeats. (E) HEK293 cells were transfected with the pUcD2SRa-rGas6-Myc plasmid (32) in the presence or absence of vitamin K. The expression level of the Gas6 proteins in the conditioned media was assessed by Myc-immunoblotting. (F) The ?-carboxylation level of the Gas6 proteins was assessed by ?-carboxylglutamic acid immunoblotting. (G) Human TAM/IFN?R1 reporter cells were stimulated by wild-type or mutant Gas6-CM, and the activations of TAM receptors were assessed by pSTAT1 immunoblotting. (H) The binding of different (warfarin or vitamin K-treated wild-type, or mutant) Gas6 proteins to hAxl/IFN?R1 cells was assessed by flow cytometry using anti-Myc-PE antibody (left), with MFI quantification from independent experiments (n = 3) in the right panel. Error bar, SEM, n.s., not significant. (I) Warfarin-treated (W) or mutant Gas6 was added to hAxl/IFN?R1 cells in culture dish for 30 min. The cells were then washed by PBS and wild-type Gas6 with fully ?-carboxylation was added to the cells. The activation of Axl was assessed by pSTAT1 immunoblotting. The Western blotting results are representative results from at least three independent experiments.
Fig 2: Homology modeling for the association between Gla domain of growth arrest-specific 6 (Gas6) and phosphatidylserine (PS). (A) The sequences of Gla domain of Gas6 proteins between species and (B) the sequences of Gla domain of different human vitamin K-dependent proteins were pairwise aligned with conserved glutamic acids highlighted in orange. (C) Homology model of Gla domain of Gas6 is based on Gla domain of bovine prothrombin PDB: 1NL2. The amino acids that may provide binding sites for PS were pointed out by green arrows. In human Gas6, two PS molecules (PS1 and PS2) are predicted to be associated with Gla domain from two symmetrical interfaces of the protein; those amino acids were pointed out by green and blue arrows separately. (D) Molecular surface of Gla domain of Gas6 was shown in pink, and the ?-carboxylated E residues shown in green, associated with calcium ions as brown balls, two PS molecules are shown in blue and POC lipids as gray wire models. (E) Possible contacts between PS and E residues are highlighted with magenta clouds. (F) Point mutations were induced to switch 54 and 55 glutamic acids (E) to aspartic acids (D). HEK293 cells were transfected with the mutant Gas6 plasmid, and the ?-carboxylation and expression of wild-type or E54 and 55D Gas6 proteins were assessed by ?-carboxylglutamic acid or Gas6 immunoblotting (upper panel). hAxl/IFN?R1 reporter cells were stimulated by wild-type or E54 and 55D Gas6, and the activation of Axl was assessed by pSTAT1 immunoblotting (lower panel). The Western Blotting results are representative results from at least three independent experiments.
Fig 3: Immunohistochemical staining of growth-arrest specific gene 6 (Gas6) protein in triple negative breast cancer (TNBC) tissues (200×). (a) Gas6 positive staining. (b) Gas6 negative staining.
Fig 4: Phosphatidylserine (PS)-positive apoptotic and calcium-stressed cells differentially affect Tyro3, Axl, and Mertk (TAMs) activation by growth arrest-specific 6 (Gas6) or protein S (Pros1). (A) Jurkat, H1299, and MDA-MB231 cells (top to bottom) were treated with calcium ionophore A23187 (10 µM) for 15 min in 37°C and then stained with FITC-Annexin V and PI to assess PS externalization. (B) Jurkat, H1299, and MDA-MB231 cells (top to bottom) were treated with UV radiation and then stained with FITC-Annexin V and PI to assess PS externalization. (C) Percentage (left) and MFI (right) quantification of the Annexin V+/PI- cells that were induced by calcium stress or UV radiation from independent experiments (n = 3). Error bar, SEM, **p < 0.005, ****p < 0.0001. (D) Recombinant Gas6 protein at concentration of 50 nM was added to 1.0 × 106 apoptotic or calcium-stressed Jurkat cells and subsequently stained with anti-hGas6 primary and antirabbit Alexa Fluor 647 secondary antibody to assess Gas6-cell surface association by flow cytometry. Background (gray): Jurkat cells without treatment were stained with primary and secondary antibodies; Mock + Gas6 (blue): Jurkat cells without treatment were incubated with 50 nM Gas6 and then stained with primary and secondary antibodies. (E) Three modes (resting, calcium-stressed and apoptotic) of Jurkat, H1299 and MDA-MB231 cells were mixed with Gas6-containing media (Gas6-CM, Gas6 concentration estimated as 250 nM) or Pros1 (100 nM) to stimulate hTAM/IFN?R1 reporter cells. The activation of TAM receptors was assessed by pSTAT1 immunoblotting. The Western blotting results are representative results from at least three independent experiments.
Fig 5: LC-MS/MS analysis of ?-carboxylation sites in Gla domain of growth arrest-specific 6 (Gas6). (A) Gas6-containing media (Gas6-CM) was subjected to SDS-PAGE electrophoresis, and the band at 72 kDa was excised and subjected to LC-MS/MS analysis. (B) In the Gla domain of Gas6, glutamic acids 54, 55, 64, 73, 74, 77, 80, and 84 were identified as ?-carboxylated by LC-MS/MS. Among them, three independent carboxylated peptides, 49AFQVFEEAK57, 73EEAR76, and 77EVFENDPETDYFYPR91, were identified with glutamic acid residues shown in red. Each peptide was found in three forms: non-?-carboxylated (N), single carboxylated at one of the E residues (M), or dually carboxylated at both E residues (D). The frequencies of each form of the peptides were listed. (C) Representative MS/MS spectrum of a doubly-charged ion (m/z 578.76) is corresponding to the peptide sequence of 49AFQVFEEAK57 with carboxylation modification at E54 and E55. The observed y- and b-ion series confirmed the peptide sequence and modification.
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