The identification of specific high density lipoprotein(3) binding sites on human blood monocytes using fluorescence-labeled ligand

We previously reported the identity and purification of two HDL3-binding pr oteins in rat liver plasma membranes, As these proteins are candidate high density lipoprotein (HDL) receptors and probably multifunctional, including a role in HDL metabolism, we have considerable interest in identifying cor responding proteins that are present in human tissue. This report describes the identification of HDL3-binding sites on human monocytes with the use o f fluorescence microscopy and flow cytometry assay. After the incubation of mononuclear cells from human blood with fluorescein isothiocyanate (FITC)- labeled human HDL3, fluorescence micrographs showed dense signals of fluore scent grains on monocytes, but not lymphocytes. A significant increase in F ITC intensity on monocytes, but not lymphocytes, was observed by Bow cytome try analysis, and the interaction between FITC-HDL3 and human monocytes was concentration-dependent, Although very lo tv density (VLDL) and low densit y lipoprotein (LDL) were ineffective competitors and HDL2 only partially co mpeted for binding, a 50-fold concentration of HDL3 did compete effectively for binding of FITC-HDL3 to human monocytes. Trypsin treatment reduced the FITC intensity of monocytes, showing that a portion of cell-associated FIT C-HDL3 remained bound to the cell surface, Two major HDL-binding proteins w ere identified in CHAPS-solubilized human mononuclear cells by ligand blott ing, using HDL3 as the ligand. Both showed similar binding parameters, spec ificity, and molecular weight identical to HB1 and HB2 from rat liver plasm a membrane. We conclude that corresponding candidate HDL receptors or a sim ilar receptor complex also exist on human blood monocytes., Identification of specific high density lipoproteins binding sites on human blood monocyte s using fluorescence-labeled ligand.