The identification of specific high density lipoprotein3 binding sites on human blood monocytes using fluorescence-labeled ligand.
We previously reported the identity and purification of two HDL3-binding proteins 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 corresponding proteins that are present in human tissue. This report describes the identification of HDL3-binding sites on human monocytes with the use of 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 fluorescent grains on monocytes, but not lymphocytes. A significant increase in FITC intensity on monocytes, but not lymphocytes, was observed by flow cytometry analysis, and the interaction between FITC-HDL3 and human monocytes was concentration-dependent. Although very low density (VLDL) and low density lipoprotein (LDL) were ineffective competitors and HDL2 only partially competed 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 FITC-HDL3 remained bound to the cell surface. Two major HDL-binding proteins were identified in CHAPS-solubilized human mononuclear cells by ligand blotting, using HDL3 as the ligand. Both showed similar binding parameters, specificity, and molecular weight identical to HB1 and HB2 from rat liver plasma membrane. We conclude that corresponding candidate HDL receptors or a similar receptor complex also exist on human blood monocytes.
Hidaka
H
H
Department of Laboratory Medicine, Shinshu University School of Medicine, Matsumoto, Japan.
Hidaka
E
E
Tozuka
M
M
Nakayama
J
J
Katsuyama
T
T
Fidge
N
N
D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins; D002793:Cholic Acids; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006801:Humans; D008075:Lipoproteins, HDL / Q000378:metabolism*; D053440:Lipoproteins, HDL3; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D009000:Monocytes / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins; D018110:Receptors, Lipoprotein / Q000032:analysis; D014357:Trypsin / Q000494:pharmacology
D002352:Carrier Proteins; D002793:Cholic Acids; D005456:Fluorescent Dyes; D008075:Lipoproteins, HDL; D053440:Lipoproteins, HDL3; D016601:RNA-Binding Proteins; D018110:Receptors, Lipoprotein; C031616:high density lipoprotein receptors; C075197:high density lipoprotein binding protein; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate