Binding of phospholipid transfer protein (PLTP) to apolipoproteins A-I and A-II: location of a PLTP binding domain in the amino terminal region of apoA-I

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Plasma phospholipid transfer protein (PLTP) plays an important role in the maintenance of plasma highdensity lipoprotein (HDL) content and remodeling of HDL in the circulation. In the present study we have used different fractionation methods to investigate the distribution of PLTP in human plasma. A novel enzyme-linked immunosorbent assay developed during the study allowed for simultaneous assessment of both PLTP mass and activity in the fractions obtained. Size-exclusion chromatography and plasma fractionation by nondenaturing polyacrylamide gel electrophoresis (PAGE) yielded similar results demonstrating that PLTP associates in native plasma with two distinct particle populations, while ultracentrifugation with high salt leads to detachment of PLTP from lipoprotein particles and loss of a majority of its phospholipid transfer activity. Interestingly, analysis of the size-exclusion chromatography fractions demonstrated that PLTP exists in the circulation as an active population that elutes in the position of HDL corresponding to an average molecular mass of 160 ؎ 40 kDa and an inactive form with an average mass of 520 ؎ 120 kDa. The inactive fraction containing approximately 70% of the total PLTP protein eluted between HDL and low density lipoprotein (LDL). Thus, the two PLTP pools are associated with different types of lipoprotein particles, suggesting that the PLTP activity in circulation is modulated by the plasma lipoprotein profile and lipid composition. -

Section: Discussionsupporting

confidence: 73%

Distribution of phospholipid transfer protein in human plasma: presence of two forms of phospholipid transfer protein, one catalytically active and the other inactive

Oka¹,

Kujiraoka²,

Ito³

et al. 2000

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“…phospholipid transfer protein (pltp). PLTP binds both purified apoA-I and apoA-II (62). The PTLP effect on HDL size conversion was seen both in LpA-I and in LpA-I/ A-II (63).…”

Section: Roles In Hdl Structure-function and Metabolismmentioning

confidence: 96%

Role of apoA-II in lipid metabolism and atherosclerosis: advances in the study of an enigmatic protein

Blanco‐Vaca

Escolà‐Gil

Martín‐Campos

et al. 2001

122

Our understanding of apolipoprotein A-II (apoA-II) physiology is much more limited than that of apoA-I. However, important and rather surprising advances have been produced, mainly through analysis of genetically modified mice. These results reveal a positive association of apoA-II with FFA and VLDL triglyceride plasma concentrations; however, whether this is due to increased VLDL synthesis or to decreased VLDL catabolism remains a matter of controversy. As apoA-II-deficient mice present a phenotype of insulin hypersensitivity, a function of apoA-II in regulating FFA metabolism seems likely. Studies of human beings have shown the apoA-II locus to be a determinant of FFA plasma levels, and several genome-wide searches of different populations with type 2 diabetes have found linkage to an apoA-II intragenic marker, making apoA-II an attractive candidate gene for this disease. The increased concentration of apoBcontaining lipoproteins present in apoA-II transgenic mice explains, in part, why these animals present increased atherosclerosis susceptibility. In addition, apoA-II transgenic mice also present impairment of two major HDL antiatherogenic functions: reverse cholesterol transport and protection of LDL oxidative modification. The apoA-II locus has also been suggested as an important genetic determinant of HDL cholesterol concentration, even though there is a major species-specific difference between the effects of mouse and human apoA-II. As antagonizing apoA-I antiatherogenic actions can hardly be considered the apoA-II function in HDL, this remains a topic for future investigations. We suggest that the existence of apoA-II or apoA-I in HDL could be an important signal for specific interaction with HDL receptors such as cubilin or heat shock protein 60. -Blanco-Vaca, F., J. C. Escolà-Gil, J. M. Martín-Campos, and J. Julve. Role of apoA-II in lipid metabolism and atherosclerosis: advances in the study of an enigmatic protein. J.

“…It is possible that the bound lipids play a merely structural role. Inhibition of lipid insertion into the pockets could cause minor changes in the fold of the protein or its molecular interactions with HDL apolipoproteins (38) or phospholipids (15), causing the observed differences in phospholipid transfer activity. In this model PLTP would facilitate the transfer of phospholipids from the donor to the acceptor by bringing these particles in close contact.…”

Section: Discussionmentioning

confidence: 99%

Structure and phospholipid transfer activity of human PLTP: analysis by molecular modeling and site-directed mutagenesis

Huuskonen

Wohlfahrt

Jauhiainen

et al. 1999

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The plasma phospholipid transfer protein (PLTP) is an important regulator of high density lipoprotein (HDL) metabolism. We have here, based on sequence alignments of the plasma LPS-binding/lipid transfer protein family and the X-ray structure of the bactericidal/permeability increasing protein (BPI), modeled the structure of PLTP. The model predicts a two-domain architecture with conserved lipid-binding pockets consisting of apolar residues in each domain. By site-directed mutagenesis of selected amino acid residues and transient expression of the protein variants in HeLa cells, the pockets are shown to be essential for PLTP-mediated phospholipid transfer. A solid phase ligand binding assay was used to determine the HDL-binding ability of the mutants. The results suggest that the observed decreases in phospholipid transfer activity of the N-terminal pocket mutants cannot be attributed to altered HDLbinding, but the C-terminal lipid-binding pocket may be involved in the association of PLTP with HDL. Further, the essential structural role of a disulfide bridge between cysteine residues 146 and 185 is demonstrated. The structural model and the mutants characterized here provide powerful tools for the detailed analysis of the mechanisms of PLTP function. -Huuskonen, J., G. Wohlfahrt, M. Jauhiainen, C. Ehnholm, O. Teleman, and V. M. Olkkonen. Structure and phospholipid transfer activity of human PLTP: analysis by molecular modeling and site-directed mutagenesis.