Figure 4.

LYVE-1 binds both immobilized and soluble HA. LYVE-1, expressed as a soluble IgFc fusion protein in transiently transfected human COS fibroblasts, was compared with CD44 for binding to HA and other glycosaminoglycans. A shows LYVE-1 and CD44H Fc fusion proteins isolated from the supernatants of [³⁵S] methionine/cysteine-labeled transfectants and electrophoresed on a 7.5% polyacrylamide SDS-PAGE gel. Samples were the protein A–Sepharose adsorbed proteins from control untransfected cells (lane 1), CD44H Fc transfected cells (lane 2), and LYVE-1 transfected cells (lane 3). The LYVE-1 fusion protein comprises residues 1–232 of the extracellular domain fused to the hinge (H), CH2, and CH3 domains of human IgG₁. Details of the CD44H Fc protein, which includes residues 1–200 of the extracellular domain, have been published previously (1). For ligand binding assays, LYVE-1 Fc was compared with CD44H Fc and the negative control fusion proteins CD33 Fc and ICAM-2 Fc for adhesion to immobilized and soluble HA in 96-well microtiter plates (see Materials and Methods). B shows binding of the fusion proteins to immobilized HA, in the absence of competing glycosaminoglycans; C shows binding (LYVE-1 Fc only) in the presence of free chondroitin-4-SO₄, chondroitin-6-SO₄, or heparin; and D shows binding to soluble biotinylated HA. Detection of bound fusion protein and biotinylated HA was carried out using peroxidase-conjugated anti–human IgFc antibody and peroxidase-conjugated streptavidin, respectively. Values are the mean ± SEM of at least three replicates.