Article Figures & Data
Figures
- Figure 1. The lectin domain bound to soluble integrins αvβ3 or αIIbβ3 in ELISA-type binding assays.
(A) The CD62P lectin and the EGF domains in the crystal structure of CD62P. (B) Binding of soluble integrins to the immobilized lectin domain of CD62P. Wells of the 96-well microtiter plate were coated with the CD62P lectin domain and the remaining protein-binding sites were blocked with BSA. Wells were incubated with soluble integrin αvβ3 or αIIbβ3 (1 μg/ml) for 1 h in 1 mM Mn2+ and bound integrins were quantified using anti-β3 mAb and anti-mouse IgG conjugated with HRP. (C) The CD62P lectin domain binds better to soluble integrins than the combined lectin and EGF domains (at a coating conc. of 50 μg/ml). (D) The disintegrin domain of ADAM15, another ligand for αvβ3 or αIIbβ3 suppresses the binding of soluble integrins to the lectin domain. (E, F) The binding of soluble integrins to the immobilized CD62P lectin domain (coating concentration at 50 μg/ml) in 1 mM different cations. Data are shown as means ± SD (n = 3). Statistical analysis was performed by ANOVA in Prism 7.
- Figure S1. Supplementary information.
(A) SDS–PAGE of the CD62P lectin domain and the combined CD62P lectin and EGF-like domain. The molecular weights predicted from amino acid sequences are 21,172 and 25,771, respectively. (B) Effect of integrin antagonists on the CD62P lectin domain binding to soluble αvβ3. ELISA-type binding assays were performed as described in the Materials and Methods section. Wells of the 96-well microtiter plate were coated with the CD62P lectin domain (50 μg/ml) and incubated with soluble integrins (1 μg/ml) in the presence of cyclic RGDfV (10 μg/ml, specific to αvβ3), mAb 7 × 103 (10 μg/ml, specific to β3). Data are shown as means ± SD (n = 3). (C, D, E) Effect of PSGL-1-Fc (C), P8G6 (D) or KF38789 (E) on the CD62P lectin domain binding to soluble integrins. ELISA-type binding assays were performed as described in the Materials and Methods section. Wells of 96-well microtiter plate were coated with the CD62P lectin domain (50 μg/ml) and incubated with soluble integrins (1 μg/ml) in the presence of PSGL-1Fc (10 μg/ml), P8G6 (10 μg/ml) or KF38789 (up to 10 μg/ml). Data are shown as means ± SD (n = 3). (F, G) Effect of integrin antagonists on the lectin binding to soluble α4β1. ELISA-type binding assays were performed as described in the Materials and Methods section. Wells of the 96-well microtiter plate were coated with the CD62P lectin domain (50 μg/ml) and incubated with soluble integrins (1 μg/ml) in the presence Bio1211 (1 μM, specific to α4β1) or the fibronectin fragment H120 (100 μg/ml) fused to GST. Data are shown as means ± SD (n = 3).
- Figure 2. The CD62P lectin domain binding to integrin and PSGL-1.
(A) The CD62P-αvβ3 docking model was superposed with the crystal structure of the CD62P–PSGL1 peptide complex (1G1S.pdb). The superposed model predicts that integrin-binding site and PSGL-1-binding site are distinct. R16/K17 of the CD62P lectin domain is close to integrin αvβ3 and E88 of the lectin domain is close to PSGL-1 peptide and glycan. (B) Docking simulation of the interaction between open/active αvβ3 (1L5G.pdb) and the CD62P lectin domain (1G1Q.pdb) was performed using Autodock3. The specificity loop (residues 177–184 of β3) is next to site 1. This loop is located between site 1 and site 2 (Fig 4A). The amino acid residues selected for mutagenesis are shown. (C) Alignment of P-, L-, and E-selectins. R16/K17 and E88 are conserved in selectins. (D, E) The binding of the CD62P lectin domain mutants to soluble integrins αvβ3 or αIIbβ3. Data are shown as means ± SD (n = 3). Statistical analysis was done by ANOVA in Prism7.
- Figure 3. Adhesion of CHO cells (PSGL-1 negative) to the CD62P lectin domain.
PSGL-1 is expressed in leukocytes, but not in CHO cells. (A, B) Effect of cations on cell adhesion to the CD62P lectin domain. The wells were coated with WT CD62P lectin domain and the remaining protein-binding sites were blocked with BSA. The wells were then incubated with CHO or β3-CHO cells for 1 h at 37°C in DMEM or Tyrode–HEPES buffer with 1 mM Mg2+ and bound cells were quantified after brief rinsing using endogenous phosphatase activity. (C, D) Time-course of cell adhesion to the CD62P lectin domain. Wells were coated with the lectin domain (50 μg/ml) and remaining protein binding sites were blocked with BSA. The wells were incubated with CHO or β3-CHO cells for 30 or 60 min in 1 mM Mg2+ and bound cells were quantified as described above. (E, F) Effect of the lectin domain mutants on adhesion to the CD62P lectin domain. Wells of 96-well microtiter plate were coated with the CD62P lectin domain (WT or mutants, coating concentration at 50 μg/ml) and the remaining protein-binding sites were blocked with BSA. Wells were incubated with CHO cells or β3-CHO cells in Tyrode–HEPES/1 mM Mg2+ or DMEM. The E88D mutant is defective in binding to the glycan ligand and the R16E/K17E mutant is defective in integrin binding. Data are shown as means ± SD (n = 3). Statistical analysis was done by ANOVA in Prism7.
- Figure 4. Activation of integrin αvβ3 by the CD62P lectin domain.
(A) A docking model of the CD62P lectin domain binding to site 2 of αvβ3. Docking simulation was performed as described in the Materials and Methods section. The specificity loop (residues 177–184 of β3) located between site 1 and site 2 is shown. Site 1 is on the opposite side of site 2 (Fig 2B). (B, C) Activation of soluble integrins αvβ3 (B) and αIIbβ3 (C) by the CD62P lectin domain in ELISA-type activation assays. Wells of 96-well microtiter plate were coated with ligands (γC399tr for αvβ3 at 50 μg/ml, and γC390-411 for αIIbβ3 at 20 μg/ml) and the remaining protein-binding sites were blocked with BSA. Wells were incubated with soluble integrins (1 μg/ml) and the CD62P lectin domain (0–50 μg/ml) in Tyrode–HEPES buffer with 1 mM Ca2+ for 1 h, and bound integrins were quantified using anti-β3 mAbs and HRP-conjugated anti-mouse IgG. Data are shown as means ± SD (n = 3). Statistical analysis was done by ANOVA in Prism7. (D, E) Effect of mutations in the predicted site 2-binding interface (Table 2) on activation of αvβ3 (D) or αIIbβ3 (E). Because site 1-binding (Table 1) and site 2-binding (Table 2) interfaces in the CD62P lectin domain overlap, we used the same mutations that affect binding to site 1 for activation assays. Data are shown as means ± SD (n = 3). Statistical analysis was done by ANOVA in Prism7.
- Figure 5. Activation of cell-surface integrins αvβ3 and αIIbβ3 by the CD62P lectin domain.
(A) Activation of cell-surface αvβ3 on β3-CHO cells by the CD62P lectin domain (flow cytometry). Cells were incubated with FITC-labeled γC399tr in the presence of the CD62P lectin domain (100 μg/ml) and the binding of γC399tr was measured in flow cytometry. (B) Median fluorescent intensity (MFI) of the binding of FITC-labeled γC399tr to β3-CHO cells. MFI in flow cytometry was calculated. Data are shown as means ± SD (n = 3). Statistical analysis was done by ANOVA in Prism7. (C) Activation of cell-surface αIIbβ3 on αIIbβ3-CHO cells by the CD62P lectin domain (flow cytometry). Cells were incubated with FITC-labeled γC390-411 in the presence of the CD62P lectin domain (100 μg/ml) and the binding of FITC-labeled γC390-411 was measured in flow cytometry. (D) MFI of the binding of FITC-labeled γC390-411 to αIIbβ3-CHO cells. MFI in flow cytometry was calculated. Data are shown as means ± SD (n = 3). Statistical analysis was done by ANOVA in Prism7. (E) Position of the cyclic site 2 peptide of β3 (residues 260–288 of β3). (F) Binding of site 2 peptides from β1 and β3 to the CD62P lectin domain. Wells of the 96-well microtiter plate were coated with the CD62P lectin domain (20 μg/ml) and remaining protein-binding sites were blocked with BSA. Wells were incubated with site 2 peptides fused to GST (100 μg/ml) and bound GST was quantified using HRP-conjugated anti-GST.
- Figure 6. The CD62P lectin domain binds to and activate integrins α5β1 and α4β1.
(A) The binding of biotinylated α5β1 to immobilized CD62P lectin domain was determined in 1 mM Mn2+ as described in Fig 1 except that bound integrin was quantified using streptavidin conjugated with HRP. (B) Activation of cell-surface α5β1 on CHO cells by the CD62P lectin domain (flow cytometry). Cells were incubated with FITC-labeled fibronectin cell-binding domain (FN8-11) in the presence of the CD62P lectin domain (50 μg/ml) and the binding of FN8-11 was measured in flow cytometry. (C) Median fluorescent intensity (MFI). MFI in flow cytometry was calculated. (D) The binding of the CD62P lectin domain to integrins α4β1. The binding of biotinylated α4β1 to immobilized CD62P lectin domain was determined in 1 mM Mn2+ as described in Fig 1 except that bound integrin was quantified using streptavidin conjugated with HRP. (E) Activation of cell-surface α4β1 on CHO cells by the CD62P lectin domain (flow cytometry). Cells were incubated with FITC-labeled fibronectin α4β1-binding fragment (H120) in the presence of the CD62P lectin domain (50 μg/ml) and the binding of H120 was measured in flow cytometry. (F) MFI in flow cytometry was calculated.
Tables
- Table 1.
Amino acid residues in interaction between the CD62P lectin domain (1G1Q.pdb) and αvβ3 (1L5G.pdb) as predicted by the docking simulation.
CD62P lectin domain αv β3 Asn13, Ile14, Arg16, Lys17, Tyr18, Gln20, Asn21, Thr24, Asp25, Lys55, Asn56, Lys58, Thr61, Val63, Gly64, Thr65, Lys66, Lys67, Ala68, Asn83, Lys 84, Arg85, Asn86, Asn87, Asp89, His108, Leu110, Ala120, Cys122, Gln123, Asp124, Met125, Lys129, Glu132, Cys133, Glu135, Tyr145 Ala149, Asp150, Phe177,Tyr178, Gln180, Arg211, Thr212, Ala213, Gln214, Ala215, Ile216, Asp218, Arg218, Tyr122, Ser123, Met124, Lys125, Asp126, Asp127, Trp129, Ser130, Gln132, Asn133, Lys137, Asp179, Met180, Lys181, Thr182, Arg214, Asp251, Ser334, Met335, Asp336, Ser337, Asn339, Val340, Leu341, Gln342 Amino acid residues within 0.6 nm between the CD62P lectin domain and αvβ3 were selected using pdb viewer (version 4.1). Amino acid residues in the CD62P lectin domain selected for mutagenesis are shown in bold.
- Table 2.
Amino acid residues in interaction between the CD62P lectin domain (1G1Q.pdb) and αvβ3 (1JV2.pdb) as predicted by the docking simulation.
CD62P lectin domain αv β3 Thr7, Lys8, Trp12, Asn13, Ile14, Arg16, Lys17, Tyr18, Gln20, Asn21, Arg22, Tyr23, Thr24, Asp25, Arg54, Lys55, Asn56, Asn57, Lys58, Thr59, Val63, Gly64, Thr65, Lys66, Lys67, Ala68, Asn86, Asn87, Asp89, Leu110, Tyr118, Thr119, Ala120, Ser121, Cys122, Gln123, Asp124, Met125, Glu135 Glu15, Asn44, Thr45, Thr46, Pro48, Gly49, Ile50, Val51, Glu52, Gly76, Asn77, Asp79, Asp83, Asp84, Pro85, Phe88, His91, Gln120, Arg122 Lys159, Pro160, Val161, Met165, Ile167, Ser168, Glu171, Ala172, Glu174, Asn175, Pro176, Cys177, Tyr178, Asp179, Met180, Lys181, Thr183, Cys184, Pro186, Met187, Phe188, Val275, Gly276, Ser277, Asp278, His280, Tyr281, Ser282, Ser284, Thr285, Thr286, Amino acid residues within 0.6 nm between the CD62P lectin domain and αvβ3 were selected using pdb viewer (version 4.1). Amino acid residues in β3 site 2 peptides are shown in bold. Amino acid residues in the CD62P lectin domain selected for mutagenesis are shown in bold.
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