Characterization of a 95 kDa high affinity human high density lipoprotein-binding protein.
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.
Bocharov
A V
AV
Center for Biologics Evaluation and Research, Division of Cellular and Gene Therapy, Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892, USA. bucharov@cber.fda.gov
Vishnyakova
T G
TG
Baranova
I N
IN
Patterson
A P
AP
Eggerman
T L
TL
D020410:Activated-Leukocyte Cell Adhesion Molecule / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000378:metabolism; D018955:CD36 Antigens / Q000378:metabolism; D018938:Caco-2 Cells; D002352:Carrier Proteins; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008075:Lipoproteins, HDL / Q000378:metabolism*; D053440:Lipoproteins, HDL3; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism*; D008970:Molecular Weight; D016877:Oxidants / Q000494:pharmacology; D011485:Protein Binding; D016601:RNA-Binding Proteins; D011869:Radioligand Assay; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein / Q000096:biosynthesis / Q000378:metabolism; D051116:Receptors, Scavenger; D019163:Reducing Agents / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051122:Scavenger Receptors, Class B; D013774:Tetranitromethane / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D020410:Activated-Leukocyte Cell Adhesion Molecule; D000951:Antigens, Neoplasm; D018955:CD36 Antigens; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C087410:Hdlbp protein, rat; D008024:Ligands; D008075:Lipoproteins, HDL; D053440:Lipoproteins, HDL3; D008565:Membrane Proteins; D016877:Oxidants; D016601:RNA-Binding Proteins; D011971:Receptors, Immunologic; D018110:Receptors, Lipoprotein; D051116:Receptors, Scavenger; D019163:Reducing Agents; C454739:Scarb1 protein, mouse; D051122:Scavenger Receptors, Class B; C031616:high density lipoprotein receptors; C052646:sarcoma glycoprotein gp96 rejection antigens; C075197:high density lipoprotein binding protein; D013774:Tetranitromethane