Characterization of lactoferrin-binding proteins of human macrophage membrane: multiple species of...

Human lactoferrin (LF) specifically binds to human monocytic leukemia cell line THP-1 cells differentiated into macrophages, and it has been suggested that the poly-N-acetyllactosaminyl saccharide chains of LF are involved. We partially purified and characterized LF-binding proteins with affinity for polylactosamines from THP-1 cells. LF-binding activity was solubilized by nonionic detergent Triton X-100 from THP-1 cell membrane, and subjected to affinity chromatography using an LF-Sepharose column. LF-binding activity, detected by ligand blotting assay, was eluted and further fractionated by affinity chromatography using a Sepharose column coupled with band 3, a polylactosaminyl chain-containing glycoprotein of human erythrocyte membrane. LF-binding activity was separated into three fractions (frs. B1, B2, and B3). These fractions exhibited band 3-binding activity as detected by ligand blotting assay. Dodecylsulfate-polyacrylamide gel electrophoresis of frs. B1, B2, and B3, followed by detection of LF-binding activity on Western blots, indicated that frs. B1, B2, and B3 contained LF-binding proteins with a molecular mass of 35, 50 and 80, and 35-37 kDa, respectively. Binding of LF to each of the fractions on the dot blots was partially inhibited by LF oligosaccharides, band 3 oligosaccharides and lacto-N-neotetraose, each containing di-N-acetyllactosaminyl or analogous structure, Gal beta 1-->4GlcNAc beta 1-->3Gal beta 1-->4GlcNAc (or Glc). These results suggest that the 35, 50 and/or 80, and 35-37 kDa proteins on THP-1 cells are LF-binding proteins with polylactosamine-binding ability.