Abstract
We have previously reported that propolypeptide of von Willebrand factor (pp-vWF) promotes melanoma cell adhesion in a beta1 integrin-dependent manner. In this report, we identified the alpha subunit of the cell adhesion receptor for pp-vWF as alpha4. Human leukemia cell lines that express alpha4beta1 integrin (very late antigen-4, VLA-4), but not cell lines which lack VLA-4, attached well to pp-vWF substrate and these adhesions were completely inhibited by anti-alpha4 integrin monoclonal antibody HP2/1. Adhesion of mouse melanoma expressing alpha4 integrin was also inhibited by anti-mouse alpha4 mAb PS/2. Furthermore, transfection of human alpha4 cDNA into alpha4(-) Chinese hamster ovary cells resulted in an acquisition of adhesive activity to pp-vWF, indicating that pp-vWF is a ligand for VLA-4 integrin. Using a recombinant fragment of pp-vWF, the cell attachment site was shown to be located within amino acid residues 376-455 of pp-vWF. A series of synthetic peptides covering this region were tested for the ability to promote cell attachment and a 15-residue peptide designated T2-15 (DCQDHSFSIVIETVQ, residues numbered 395-409) promoted VLA-4 dependent cell adhesion. The peptide was also capable of inhibiting cell adhesion to pp-vWF, suggesting that this sequence represents the cell attachment site. By affinity chromatography using peptide T2-15-Sepharose, it was found that alpha4beta1 integrin complex from extracts of surface iodinated B16 cells specifically bound to the peptide. These results strongly suggest that pp-vWF is a novel physiological ligand for VLA-4.
Highlights
Propolypeptide of von Willebrand factor,1 which is called von Willebrand antigen II [1], is an unusually large propolypeptide (ϳ100 kDa) produced only in endothelial cells and megakaryocytes together with blood coagulation protein von Willebrand factor [2]
We report that the receptor responsible for cell adhesion to propolypeptide of von Willebrand factor (pp-vWF) is VLA-4 (␣41 integrin)
At first we tested more than 20 cell lines, which grow on culture dishes, for their ability to adhere to pp-vWF and found that only the cultured tumor cell lines with melanoma origin, including mouse melanoma B16, human melanoma G-361 and MeWo, and hamster melanoma RPMI 1846, adhered on pp-vWF substrate
Summary
Materials—Monoclonal antibody (mAb) 4B4 recognizing human 1 integrin was a gift from Dr C. Polyclonal antibody against C terminus of the ␣7 subunit was prepared by immunizing a synthetic peptide having sequence of cytoplasmic tail of human ␣7B subunit (DAHPILAADWHPELG) in our laboratory These polyclonal antisera all recognized respective subunits from mouse integrin because of the high interspecies conservation of the sequence in the cytoplasmic region. After blocking nonspecific protein-binding sites by incubation with 10 mM Tris-HCl, 150 mM NaCl, pH 7.4, containing 1% bovine serum albumin and 100 g/ml mouse IgG at room temperature for 1 h, the chips were placed at the bottom of 48-well tissue culture dishes (Costar 3548) and overlaid with 2–5 ϫ 105 cells in 50 l of serum-free medium. The samples were boiled in reducing Laemmli sample buffer and analyzed by SDS-PAGE on a 7.5% polyacrylamide gel as described above
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