Characterization of a 95 kDa High Affinity Human High Density Lipoprotein-Binding Protein

Abstract

A new human 95 kDa high density lipoprotein (HDL)-binding protein (HBP) corresponding to a high affinity HDL-binding site with K(d) = 1.67 microg/mL and a capacity of 13.4 ng/mg was identified in human fetal hepatocytes. The HDL binding with the 95 kDa HBP plateaus at 2.5-5 microg/mL under reducing and nonreducing conditions. The association of HDL(3) with the 95 kDa HBP plateaued in 15-30 min while dissociation was complete in 30 min. HDL(3), apoA-I, and apoA-II were recognized by the 95 kDa HBP while low density lipoproteins (LDL) and tetranitromethane-modified HDL were not. The 95 kDa HBP predominantly resides on the surface of cells since trypsin treatment of HepG2 cells eliminated nearly 70% of HDL binding. All studied human cells and cell lines (HepG2, Caco-2, HeLa, fibroblasts, SKOV-3, PA-I) demonstrated the presence of the 95 kDa protein. Both RT-PCR and Western blotting for HB-2/ALCAM were negative in human fetal hepatocytes while Gp96/GRP94 was clearly differentiated from the 95 kDa HBP by two-dimensional electrophoretic mobility. Moreover, deglycosylation of HepG2 membrane preparations did not affect either HDL binding to the 95 kDa HBP or its size, while in contrast it affected the molecular weights of HB-2/ALCAM and SR-BI/CLA-1. We conclude that the 95 kDa HBP is a new HDL receptor candidate widely expressed in human cells and cell lines.

Apolipoprotein A-I induces translocation of protein kinase Cα to a cytosolic lipid-protein particle in astrocytes

“…The rapid reaction seems to involve phospholipase C␥ and PI turnover, so that it should be initiated by the interaction of apoA-I with a receptor-like signal-mediating membrane protein, whether directly or indirectly. Although many reports indicated the initiation of the signaling cascade by apoA-I or HDL, there is no clear indication of the signal-mediating membrane protein that may directly interact with apolipoprotein or HDL (35)(36)(37)(38)(39)(40). ABCA1 has been identified as a key protein for the generation of HDL by apolipoprotein from cellular lipid, but it is still unclear whether this protein interacts directly with apolipoprotein to generate HDL or plays an indirect role for the HDL assembly reaction (41)(42)(43)(44)(45).…”

Lipopolysaccharide Down Regulates Both Scavenger Receptor B1 and ATP Binding Cassette Transporter A1 in RAW Cells

“…125 I-HDL binding experiments were performed as previously described (3). RAW cells cultured in 12-well plates were incubated with 1 g of LPS per ml for 24 h. After three subsequent washes with PBS, the cells were chilled on ice and binding of 125 I-HDL (5 g/ml) was determined in the absence (total binding) or presence (nonspecific binding) of a 50-fold excess of unlabeled ligand.…”

Expression Cloning and Characterization of a Novel Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein, GPI-HBP1

“…Several HDL-binding proteins have been identified including class B type I scavenger receptor (SRBI) (2,3), two candidate hepatic HDL receptors designated HDL-binding proteins 1 and 2 (4,5), 80-and 130-kDa GPI-anchored HDL-binding proteins expressed in human macrophages (6), 110-kDa GPI-anchored HDL-binding protein expressed in HepG2 cells (7), and recently characterized 95-kDa HDL-binding protein (8). To date, only SRBI appears to be a physiological HDL receptor based on the selective uptake of cholesterol esters into cells and the efflux of cholesterol from cells to HDL mediated by SRBI (1).…”