Structural and functional characterization of β 2 -glycoprotein I domain 1 in anti-melanoma cell migration.

We previously found that circulating β2 -glycoprotein I inhibits human endothelial cell migration, proliferation, and angiogenesis via 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 B16F10 treated with β2 -glycoprotein I and variant recombinant polypeptides. Using a melanoma mouse model, we found that D1 polypeptide exhibited stronger potency in suppressing tumor growth. Structural analysis revealed that fragments A and B within domain I would be the critical regions responsible for antitumor activity. Annexin A2 was identified as the counterpart molecular for β2 -glycoprotein I by immunofluoresence and co-immunoprecipation 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 treatment of melanoma progression. This article is protected by copyright. All rights reserved.