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Characterization of a 95 kDa high affinity human high density lipoprotein-binding protein
A V Bocharov1, T G Vishnyakova, I N Baranova
1Center 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
Biochemistry
|April 4, 2001
Summary
Researchers discovered a new 95 kDa high-density lipoprotein (HDL)-binding protein (HBP) in human cells. This protein acts as a potential HDL receptor, binding HDL(3), apoA-I, and apoA-II, and is widely expressed across various human cell types.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Medicine
Background:
- High-density lipoprotein (HDL) plays a crucial role in reverse cholesterol transport.
- Identification of specific HDL receptors is vital for understanding HDL metabolism and its therapeutic potential.
- Previous research has identified several HDL-binding proteins, but a comprehensive understanding of all receptors is ongoing.
Purpose of the Study:
- To identify and characterize a novel high-density lipoprotein (HDL)-binding protein (HBP) in human fetal hepatocytes.
- To determine the binding characteristics and specificity of the identified HBP.
- To investigate the cellular localization and expression pattern of the 95 kDa HBP.
Main Methods:
- High-affinity HDL-binding assay using human fetal hepatocytes to identify and characterize the 95 kDa HBP.
- Analysis of HDL binding kinetics, including association and dissociation rates.
- Cell surface protein analysis using trypsin treatment and Western blotting.
- RT-PCR and two-dimensional gel electrophoresis to differentiate the 95 kDa HBP from other known proteins.
- Deglycosylation studies to assess the protein's glycosylation status and its effect on HDL binding.
Main Results:
- A novel 95 kDa HDL-binding protein (HBP) was identified with high affinity (K(d) = 1.67 microg/mL) and capacity (13.4 ng/mg) in human fetal hepatocytes.
- The 95 kDa HBP specifically binds HDL(3), apoA-I, and apoA-II, but not LDL or modified HDL.
- The protein is predominantly located on the cell surface, as indicated by trypsinization experiments, and is widely expressed in various human cell lines.
- The 95 kDa HBP was differentiated from HB-2/ALCAM and Gp96/GRP94, and its binding and size were unaffected by deglycosylation, unlike HB-2/ALCAM and SR-BI/CLA-1.
Conclusions:
- The 95 kDa HBP represents a newly discovered HDL receptor candidate.
- This protein is widely expressed across different human cell types, suggesting a significant role in HDL interactions.
- Further research into the 95 kDa HBP could elucidate novel mechanisms in HDL metabolism and cholesterol regulation.