Characterization of lactoferrin-binding proteins of human macrophage membrane: multiple species of lactoferrin-binding proteins with polylactosamine-binding ability.
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.
Eda
S
S
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Japan.
Kikugawa
K
K
Beppu
M
M
D000606:Amino Sugars / Q000378:metabolism*; D001457:Anion Exchange Protein 1, Erythrocyte / Q000378:metabolism; D001426:Bacterial Proteins; D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry; D002352:Carrier Proteins / Q000378:metabolism*; D002846:Chromatography, Affinity; D006801:Humans; D007781:Lactoferrin / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D014407:Tumor Cells, Cultured
D000606:Amino Sugars; D001457:Anion Exchange Protein 1, Erythrocyte; D001426:Bacterial Proteins; D002241:Carbohydrates; D002352:Carrier Proteins; D011134:Polysaccharides; C027768:lactoferrin binding proteins, bacterial; C066929:polylactosamine; D007781:Lactoferrin