Abstract
Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. PLTP is present in plasma in two forms, one with high and the other with low phospholipid transfer activity. We have used the human hepatoma cell line, HepG2, as a model to study PLTP secreted from hepatic cells. PLTP activity was secreted by the cells into serum-free culture medium as a function of time. However, modification of a previously established ELISA assay to include a denaturing sample pretreatment with the anionic detergent sodium dodecyl sulphate was required for the detection of the secreted PLTP protein. The HepG2 PLTP could be enriched by Heparin-Sepharose affinity chromatography and eluted in size-exclusion chromatography at a position corresponding to the size of 160 kDa. PLTP coeluted with apolipoprotein E (apoE) but not with apoB-100 or apoA-I. A portion of PLTP was retained by an anti-apoE immunoaffinity column together with apoE, suggesting an interaction between these two proteins. Furthermore, antibodies against apoE but not those against apoB-100 or apoA-I were capable of inhibiting PLTP activity. These results show that the HepG2-derived PLTP resembles in several aspects the high-activity form of PLTP found in human plasma.
Highlights
Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution
We report that PLTP secreted from HepG2 cells resembles in several aspects the high-activity PLTP form in human plasma: it is poorly immunodetectable in its native form, shows affinity for heparin, displays an apparent size of about 160 kDa, and cofractionates with apolipoprotein E (apoE)
PLTP secretion by HepG2 cells To study the secretion of PLTP from the liver, we used the human hepatoma cell line HepG2 as a model system
Summary
Plasma phospholipid transfer protein (PLTP) is an important regulator of plasma HDL levels and HDL particle distribution. It has been suggested by Murdoch et al that the immunoreactivity of the two forms of PLTP from plasma differ and that the use of monoclonal PLTP antibodies in ELISA mass assays may underestimate the mass of the active form [21] To overcome this discrepancy of antibody reactivity, to improve the mass determination of inactive and active PLTP, and to elucidate further the interaction of PLTP with apolipoproteins, we have used the human hepatoma cell line HepG2 as an in vitro model. We report that PLTP secreted from HepG2 cells resembles in several aspects the high-activity PLTP form in human plasma: it is poorly immunodetectable in its native form, shows affinity for heparin, displays an apparent size of about 160 kDa, and cofractionates with apoE. We show that by pretreating the HepG2-derived PLTP with a strong denaturing anionic detergent, sodium dodecyl sulfate (SDS), we can significantly improve its immunochemical mass quantitation
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