Jarkko Huuskonen

The impact of phospholipid transfer protein (PLTP) on HDL metabolism

High-density lipoproteins (HDL) play a major protective role against the development of coronary artery disease. Phospholipid transfer protein (PLTP) is a main factor regulating the size and composition of HDL in the circulation and plays an important role in controlling plasma HDL levels. This is achieved via both the phospholipid transfer activity of PLTP and its capability to cause HDL conversion. The present review focuses on the impact of PLTP on HDL metabolism. The basic characteristics and structure of the PLTP protein are described. The two main functions of PLTP, PLTP-mediated phospholipid transfer and HDL conversion are reviewed, and the mechanisms and control, as well as the physiological significance of these processes are discussed. The relationship between PLTP and the related cholesteryl ester transfer protein (CETP) is reviewed. Thereafter other functions of PLTP are recapitulated: the ability of PLTP to transfer cholesterol, alpha-tocopherol and lipopolysaccharide (LPS), and the suggested involvement of PLTP in cellular cholesterol traffic. The discussion on PLTP activity and mass in (patho)physiological settings includes new data on the presence of two forms of PLTP in the circulation, one catalytically active and the other inactive. Finally, future directions for PLTP research are outlined.

Mutation of the Cytoplasmic Domain of the Integrin Subunit DIFFERENTIAL EFFECTS ON CELL SPREADING, RECRUITMENT TO ADHESION PLAQUES, ENDOCYTOSIS, AND PHAGOCYTOSIS

The cytoplasmic domain of the β subunit of the αβ3integrin is required for cell spreading on fibrinogen. Here we report that deletion of six amino acids from the COOH terminus of the β3(ITYRGT) totally abolished cell spreading and formation of adhesion plaques, whereas retaining Ilepartially preserved these functions. We further found that substitution of Tyrwith Ala also abolished αβ3-mediated cell spreading. The effects of these and other mutations on additional functions of αβ3were also studied. Progressive truncations of β3, in which stop codons were inserted at amino acid positions 759-756, caused partial defects in the recruitment of αβ3to preestablished adhesion plaques and a gradual decrease in the ability of αβ3to mediate internalization of fibrinogen-coated particles. The Tyr Ala substitution mutant was almost totally inactive in both of these assays. Point mutations at Tyr, and at a conserved area close to the transmembrane domain of β3, decreased integrin recruitment to preestablished adhesion plaques but allowed αβ3-mediated formation of these structures and partial cell spreading. Deletion of the cytoplasmic domain of β3did not affect the constitutive endocytosis of αβ3.

Quantification of human plasma phospholipid transfer protein (PLTP): relationship between PLTP mass and phospholipid transfer activity

A sensitive sandwich-type enzyme-linked immunosorbent assay (ELISA) for human plasma phospholipid transfer protein (PLTP) has been developed using a monoclonal capture antibody and a polyclonal detection antibody. The ELISA allows for the accurate quantification of PLTP in the range of 25-250 ng PLTP/assay. Using the ELISA, the mean plasma PLTP concentration in a Finnish population sample (n = 159) was determined to be 15.6 +/- 5.1 mg/l, the values ranging from 2.30 to 33.4 mg/l. PLTP mass correlated positively with HDL-cholesterol (r = 0.36, P < 0.001), apoA-I (r = 0.37, P < 0.001), apoA-II (r = 0.20, P < 0.05), Lp(A-I) (r=0.26, P=0.001) and Lp(A-I/A-II) particles (r=0.34, P<0.001), and negatively with body mass index (BMI) (r = -0.28, P < 0.001) and serum triacylglycerol (TG) concentration (r = -0.34, P < 0.001). PLTP mass did not correlate with phospholipid transfer activity as measured with a radiometric assay. The specific activity of PLTP, i.e. phospholipid transfer activity divided by PLTP mass, correlated positively with plasma TG concentration (r=0.568, P<0.001), BMI (r=0.45, P<0.001), apoB (r = 0.45, P < 0.001). total cholesterol (r=0.42, P < 0.001), LDL-cholesterol (r = 0.34, P < 0.001) and age (r = 0.36, P < 0.001), and negatively with HDL-cholesterol (r= -0.33, P < 0.001), Lp(A-I) (r= -0.21, P < 0.01) as well as Lp(A-I/A-II) particles (r = -0.32, P < 0.001). When both PLTP mass and phospholipid transfer activity were adjusted for plasma TG concentration, a significant positive correlation was revealed (partial correlation, r = 0.31, P < 0.001). The results suggest that PLTP mass and phospholipid transfer activity are strongly modulated by plasma lipoprotein composition: PLTP mass correlates positively with parameters reflecting plasma high density lipoprotein (HDL) levels, but the protein appears to be most active in subjects displaying high TG concentration.

Phospholipid transfer protein in lipid metabolism

Phospholipid transfer protein (PLTP) is one of the main modulators of plasma HDL size and composition. The publications discussed in the present review have substantially increased our knowledge on the physiological importance of PLTP-mediated phospholipid transfer, especially between triglyceride-rich lipoproteins and HDL. Furthermore, novel data have provided clues about the transfer mechanism, and evidence for the direct involvement of PLTP in atheroprotection has recently been presented. The development of assays for PLTP mass determination has offered new tools for the elucidation of the physiological role of PLTP.

Acyl chain and headgroup specificity of human plasma phospholipid transfer protein

Phospholipid transfer protein (PLTP) is a plasma protein with two reported in vitro activities: transfer of phospholipids and modulation of HDL particle size. The mechanism of PLTP-mediated phospholipid transfer was studied by determining the acyl chain and headgroup specificity and comparing the results with those obtained with the non-specific lipid transfer protein (ns-LTP), a previously characterised intracellular transfer protein. To verify the results obtained with purified plasma PLTP, recombinant PLTP produced in COS-1 cells was used. The transfer rates were determined by monitoring the transfer of fluorescent, pyrene-labeled phospholipids from quenched donor phospholipid vesicles to HDL3 particles. When the length of the pyrene-labeled acyl chain was varied from 6 to 14 carbons, a fairly monotonous decrease in the transfer rate was observed. No difference in rate was observed for the isomers having the pyrene-labeled and unlabeled acyl chains in reversed positions. PLTP mediated equally the transfer of the various headgroup derivatives except phosphatidylethanolamine (PE), which was transferred 2-3-fold more slowly. In all experiments the plasma and recombinant PLTP behaved identically. The specificity patterns observed for PLTP and ns-LTP were very similar. No PLTP-phospholipid intermediate could be observed, indicating that PLTP, like ns-LTP, does not form a tight complex with the lipid substrate and may thus mediate the transfer of phospholipid via another, yet unspecified mechanism.

Oxidative modification of HDL3 in vitro and its effect on PLTP-mediated phospholipid transfer.

The oxidation of HDL3 by Cu(II) and its effect on the ability of these particles to act as phospholipid acceptors in human plasma phospholipid transfer protein (PLTP)-mediated lipid transfer were investigated. Oxidation of HDL3 was monitored by measuring the following parameters: (i) formation of conjugated dienes, (ii) production of thiobarbituric acid reactive substances (TBARS), (iii) decrease in reactive lysine and (iv) tryptophan residues, (v) change in particle charge and (vi) diameter, and (vii) oligomerisation of apoA-I and apoA-II. Formation of conjugated dienes was the parameter responding to the oxidative treatment with the fastest kinetics. The appearance of TBARS and modification of apolipoprotein tryptophan residues were detected simultaneously but required higher Cu(II) concentrations for maximal kinetics. Cross-linking of the major protein constituents of HDL3, apoA-I and apoA-II, represented later steps of the oxidation process. Further, the oxidative modification was accompanied by a progressive change in HDL3 particle charge and a minor increase in particle diameter. PLTP-mediated phospholipid transfer to the oxidized particles was investigated using an assay measuring the transfer of fluorescent, pyrene-labeled PC. The transfer was significantly inhibited, but only after extensive modification of the HDL proteins, suggesting that the HDL oxidative modifications occurring in vivo do not essentially impair its phospholipid acceptor function. A similar but less pronounced inhibition was observed when two other phospholipid transfer proteins, the nonspecific lipid transfer protein (ns-LTP) and the phosphatidylcholine transfer protein (PC-TP), were studied in parallel. This indicates that the inhibition was partly due to unspecific effects of the modification on acceptor particle surface properties, but included an aspect specific for PLTP.

Structure and phospholipid transfer activity of human PLTP: Analysis by molecular modelling and site-directed mutagenesis

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 HDL-binding, 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.