Structural and functional characterization of β2 -glycoprotein I domain 1 in anti-melanoma cell migration

Abstract

We previously found that circulating β₂ -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis by diverse mechanisms. In the present study, we investigated the antitumor activities of β₂ -glycoprotein I using structure-function analysis and mapped the critical region within the β₂ -glycoprotein I peptide sequence that mediates anticancer effects. We constructed recombinant cDNA and purified different β₂ -glycoprotein I polypeptide domains using a baculovirus expression system. We found that purified β₂ -glycoprotein I, as well as recombinant β₂ -glycoprotein I full-length (D12345), polypeptide domains I-IV (D1234), and polypeptide domain I (D1) significantly inhibited melanoma cell migration, proliferation and invasion. Western blot analyses were used to determine the dysregulated expression of proteins essential for intracellular signaling pathways in B16-F10 treated with β₂ -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide showed stronger potency in suppressing tumor growth. Structural analysis showed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecule for β₂ -glycoprotein I by immunofluorescence and coimmunoprecipitation assays. Interaction between specific amino acids of β₂ -glycoprotein I D1 and annexin A2 was later evaluated by the molecular docking approach. Moreover, five amino acid residues were selected from fragments A and B for functional evaluation using site-directed mutagenesis, and P11A, M42A, and I55P mutations were shown to disrupt the anti-melanoma cell migration ability of β₂ -glycoprotein I. This is the first study to show the therapeutic potential of β₂ -glycoprotein I D1 in the treatment of melanoma progression.

Keywords: anti-melanoma cell migration; melanoma growth; protein structure analysis; recombinant β2-glycoprotein I polypeptide domain; site-directed mutagenesis.

Figure 1

Purified β₂‐glycoprotein I (β₂‐GPI) and recombinant β₂‐GPI polypeptides contribute to anti‐cell migration. B16‐F10 melanoma cells were cultured in 6‐well plates at a density of 1.8 × 10⁵ cells/well and treated with the indicated concentrations of purified β₂‐GPI, recombinant D1, D4, D5, D1234, and D12345 polypeptides, Fc vector, and BSA (control) for 48 h. A, B16‐F10 cells were scraped using a pipette tip (white dotted lines indicate the scratched edges) and the migrating cells were assessed by wound‐healing assay at 0 and 24 h. Representative photographs are shown using the inverted phase microscope at 40× magnification. The wound areas were assessed between 0 and 24 h and are represented as the percentage of the control group (set as 100%). ***P < .001. B, The effects of purified β₂‐GPI and its recombinant polypeptides on anti‐melanoma cell migration were also determined by transwell migration assay. Graph shows changes in cell migration as a percentage of the control group. Data represent mean ± SEM of triplicates. **P < .01, ***P < .001

Figure 2

Role of recombinant β₂‐glycoprotein I (β₂‐GPI) D1 polypeptide in the inhibition of melanoma cell migration. B16‐F10 melanoma cells were cultured in 6‐well plates and treated with or without the indicated concentrations of purified β₂‐GPI, recombinant D1 polypeptide, Fc vector, and BSA (control) for 48 h. A, Cells were scraped with a pipette tip (white dotted lines indicate the scratched edges) and the migrating cells were assessed by wound‐healing assay. Representative photographs are shown using an inverted phase microscope at 40× magnification. The wound areas between 0 and 24 h were calculated and represented as a percentage of the control group (set as 100%). Graph shows the relative wound width. Data represent mean ± SEM (three individual experiments). **P < .01, ***P < .001. B, Transwell migration assays using B16‐F10 cells at different doses of recombinant β₂‐GPI D1 polypeptide are shown. Changes in cell migration were also represented as a percentage of the control group. Data represent mean ± SEM of triplicates. ***P < .001

Figure 3

Purified β₂‐glycoprotein I (β₂‐GPI) and recombinant β₂‐GPI D1 polypeptide contribute to anti‐cell proliferation. B16‐F10 melanoma cells were cultured and treated with purified β₂‐GPI, recombinant peptides, Fc vector, or BSA (control). A, Cell proliferation was determined using a cell counting assay and cell numbers were scored at 24, 36, or 48 h. B, Cell proliferation was assessed by BrdU incorporation at 24 h. C, B16‐F10 melanoma cells were cultured and treated with the indicated concentrations of purified β₂‐GPI, recombinant peptides, Fc vector, and BSA (control) for 24 h by invasion analysis. Experiments were repeated three times. Representative images from one experiment are shown (top) and cell invasion was quantified (bottom). D, Invasion assay using B16‐F10 cells at different doses of recombinant β₂‐GPI D1 polypeptide were assessed. Data represent mean ± SEM of three individual experiments. *P < .01, **P < .01, ***P < .001

Figure 4

Effects of β₂‐glycoprotein I (β₂‐GPI) and its recombinant polypeptides on protein expression in B16‐F10 cells treated with β₂‐GPI or recombinant polypeptides. Western blotting analysis for the expression of (A) p‐AKT, AKT, p‐ERK, ERK, p‐p38, p38, p‐JNK, and JNK and (B) p‐IKKα, IKKα, p‐IκBα, IκBα, and the nuclear levels of p50 and p65. β‐Actin and lamin A/C were used as the cytosolic and nuclear protein loading control, respectively. α‐Tubulin was used to rule out contamination of cytosolic protein in nuclear protein extraction. Representative gels are shown (left) and the intensity of protein bands normalized by the internal control was quantitated as relative protein expression using ImageQuant software (right). Data are presented as mean ± SEM of at least three independent experiments. *P < .05, **P < .01 vs control group

Figure 5

Role of purified β₂‐glycoprotein I (β₂‐GPI) and recombinant polypeptides in melanoma growth. A syngeneic murine melanoma model was established in C57BL6 mice by implanting 5 × 10⁶ B16‐F10 melanoma cells/250 μL PBS into the dorsal flanks of each mouse. When tumors reached 100 mm³, purified β₂‐GPI and recombinant polypeptides D12345 and D1 were injected s.c. beside the tumors and the mice were killed on day 9. A, Comparison of tumor growth following injection of purified β₂‐GPI, recombinant polypeptides D1 and D12345, and PBS (mock). Tumor volumes were measured using a Vernier caliper every 2 days and were calculated using the formula: length (mm) × width² (mm²) × 0.5. There were four mice per group and tumor volumes are plotted as means ± SEM. B, Mice were killed and the tumors were excised and photographed. Fc represents mice bearing tumors with empty vector. Tumor weights from mice injected with purified β₂‐GPI, recombinant polypeptides D12345 and D1, and Fc protein were determined at the end of the experiment. Data representing medium and interquartile range are shown as horizontal lines

Figure 6

Structural comparison of β₂‐glycoprotein I (β₂‐GPI) D1 polypeptide and other β₂‐GPI domains. A, Comparison of sequence conservation between D1 and D2/D3/D4 polypeptides was carried out using the multiple sequence alignment (MSA) tool, ClustalW. Bar chart represents the degree of conservation at each amino acid position. Higher conservation scores are represented by higher bars colored with yellow. The amino acids colored with green show completely conserved residues, yellow indicates identical residues, and blue highlights residues with similar physicochemical properties. The lower line of the alignment is the consensus sequence. Fragment A (amino acids 6‐21) and fragment B (amino acids 41‐59) are the peptide regions with lowest similarity between D1 and D2/D3/D4 polypeptides. B, Solvent accessible surface area (SASA) and secondary structure (SS) were calculated using Dictionary of Secondary Structure of Proteins (DSSP) program23 program to estimate the structural characterization of D1 (red), D2 (blue), D3 (green) and D4 (gray). In the line graph, the Y‐axis represents the relative area (%) of solvent accessibility and the X‐axis represents the position of the amino acid residues. C,D, Structure comparison of fragments A (red) and B (green) of D1 illustrated as superimposed on the corresponding moieties of other peptide regions (blue) in D2, D3, and D4. Root mean square deviation (RMSD) using PyMol software was carried out. Black arrows show the distinctive regions of fragments A and B compared with the corresponding region (blue line) in the D2, D3, and D4 structures

Figure 7

Hotspot detection in fragments A and B of β₂‐glycoprotein I (β₂‐GPI) D1 polypeptide. A, Five nonconservative amino acid residues (P11, P17, K19, M42, and I55) are represented in purple in the illustration based on the PyMOL visualization of PDB ID 1QUB. The view for visualizing P11, P17, and K19 residues in fragment A (orange) is rotated 180° around the vertical axis for visualizing M42 and I55 residues in fragment B (green). B, Evolutionary conservation analysis was done using CONSURF software based on the secondary structure of fragments A and B. The conservation scores are divided into nine grades for visualization, from the most variable positions colored cyanine, through the intermediately conserved positions colored white, to the most conserved positions colored purple. C, Surface information was incorporated into the 3‐D structure of fragments A and B, colored from cyanine (variable) to purple (conserved), based on the analysis by CONSURF software. D, Using the default parameters in the CONSURF package, electrostatic surface representation of fragments A and B, colored red to blue from −5.5 to +5.5 kT/e, was conducted to explore the electrostatic environment around the five amino acids on the surface area of D1 polypeptide

Figure 8

Molecular counterpart of β₂‐glycoprotein I (β₂‐GPI) and the functional importance of β₂‐GPI D1 hotspot. A, Representative immunofluorescence images of B16‐F10 cells stained for β₂‐GPI (green) and annexin A2 (red). Merged image of β₂‐GPI and annexin A2 staining cells is shown in yellow. Cell nuclei were stained with DAPI (blue). Scale bar, 50 μm. B, Interaction of β₂‐GPI and annexin A2 was confirmed by coimmunoprecipitation (co‐IP) experiment. Cells were lysed and membrane protein extracts were immunoprecipitated with anti‐β₂ GP1 (left) or anti‐annexin A2 (right) antibody, followed by immunoblotting with anti‐β₂ GP1 or anti‐annexin A2. C, Interactions between the amino acid residues of annexin A2 (blue) and β2‐GPI D1 (orange) are illustrated. The figures of the structures depicted were retrieved from Protein Data Bank and PyMol software. Hydrogen‐bond interactions between amino acids of D1 and annexin A2 are shown as black dashed lines. D, Close view of amino acid interaction between β2‐GPI D1 (cyan) and annexin A2 (gray) is analyzed by using the PIPER module of Schrödinger Suite ( https://www.schrodinger.com/). Structural changes in mutant‐type amino acids P11A, M42A, and I55P were compared with wild‐type amino acids. E, Residue mutations of β₂‐GPI cDNA were constructed by site‐directed mutagenesis. Then, B16‐F10 cells were scraped with a pipette tip and treated with D1, D1 P11A, D1 P17Y, D1 K19T, D1 M42A, or D1 I55P, and functional analysis of site mutation was detected by migration assay. Representative photographs are shown at 40× magnification. Scale bars represent 0.2 mm. Percentage of the migrating area was calculated as: 100% − (wound areas at 24 h/wound areas at 0 h) × 100% and represented as a percentage of the control. Data represent mean ± SEM of at least three independent experiments. **P < .01 compared with the control