The role of plasma phospholipid transfer protein (PLTP) in HDL remodeling in acute-phase patients.

During reverse cholesterol transport plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and pre-beta-HDL. The acute-phase response is accompanied with dramatic changes in lipid metabolism including alterations in HDL concentration, composition, and thereby its function as a substrate for HDL remodeling proteins in circulation. To evaluate how acute-phase HDL (AP-HDL) functions in PLTP-mediated HDL conversion, we collected plasma samples from patients with severe acute-phase response (n=17), and from healthy controls (n=30). Subsequently, total HDL (1.063

Dynamic changes in mouse lipoproteins induced by transiently expressed human phospholipid transfer protein (PLTP): importance of PLTP in prebeta-HDL generation.

The plasma phospholipid transfer protein (PLTP) plays an important role in the regulation of plasma high density lipoprotein (HDL) levels and governs the distribution of HDL sub-populations. In the present study, adenovirus mediated overexpression of human PLTP in mice was employed to investigate the distribution of PLTP in serum and its effect on plasma lipoproteins. Gel filtration experiments showed that the distributions of PLTP activity and mass in serum are different, suggesting that human PLTP circulated in mouse plasma as two distinct forms, one with high and the other with low specific activity. Our study further demonstrates that overexpression of PLTP leads to depletion of HDL and that, as PLTP activity declines, replenishment of the HDL fraction occurs. During this process, the lipoprotein profile displays transient particle populations, including apoA-IV and apoE-rich particles in the LDL size range and small particles containing apoA-II only. The possible role of these particles in HDL reassembly is discussed. The increased PLTP activity enhanced the ability of mouse sera to produce pre(beta)-HDL. The present results provide novel evidence that PLTP is an important regulator of HDL metabolism and plays a central role in the reverse cholesterol transport (RCT) process.

Acute-phase HDL in phospholipid transfer protein (PLTP)-mediated HDL conversion.

In reverse cholesterol transport, plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and prebeta-HDL. During the acute-phase reaction (APR), serum amyloid A (SAA) becomes the predominant apolipoprotein on HDL. Displacement of apo A-I by SAA and subsequent remodeling of HDL during the APR impairs cholesterol efflux from peripheral tissues, and might thereby change substrate properties of HDL for lipid transfer proteins. Therefore, the aim of this work was to study the properties of SAA-containing HDL in PLTP-mediated conversion. Enrichment of HDL by SAA was performed in vitro and in vivo and the SAA content in HDL varied between 32 and 58 mass%. These HDLs were incubated with PLTP, and the conversion products were analyzed for their size, composition, mobility in agarose gels, and apo A-I degradation. Despite decreased apo A-I concentrations, PLTP facilitated the conversion of acute-phase HDL (AP-HDL) more effectively than the conversion of native HDL(3), and large fusion particles with diameters of 10.5, 12.0, and 13.8 nm were generated. The ability of PLTP to release prebeta from AP-HDL was more profound than from native HDL(3). Prebeta-HDL formed contained fragmented apo A-I with a molecular mass of about 23 kDa. The present findings suggest that PLTP-mediated conversion of AP-HDL is not impaired, indicating that the production of prebeta-HDL is functional during the ARP. However, PLTP-mediated in vitro degradation of apo A-I in AP-HDL was more effective than that of native HDL, which may be associated with a faster catabolism of inflammatory HDL.

Phospholipid transfer is a prerequisite for PLTP-mediated HDL conversion.

Phospholipid transfer protein (PLTP) is an important regulator of high-density lipoprotein (HDL) metabolism. The two main functions of PLTP are transfer of phospholipids between lipoprotein particles and modulation of HDL size and composition in a process called HDL conversion. These PLTP-mediated processes are physiologically important in the transfer of surface remnants from lipolyzed triglyceride-rich lipoproteins to nascent HDL particles and in the generation of prebeta-HDL, the initial acceptor of excess peripheral cell cholesterol. The aim of the study presented here was to investigate the interrelationship between the two functions of PLTP. Plasma PLTP was chemically modified using diethylpyrocarbonate or ethylmercurithiosalicylate. The modified proteins displayed a dose-dependent decrease in phospholipid transfer activity and a parallel decrease in the ability to cause HDL conversion. Two recombinant PLTP mutant proteins, defective in phospholipid transfer activity due to a mutation in the N-terminal lipid-binding pocket, were produced, isolated, and incubated together with radioactively labeled HDL(3). HDL conversion was analyzed using three methods: native gradient gel electrophoresis, ultracentrifugation, and crossed immunoelectrophoresis. The results demonstrate that the mutant proteins (i) are able to induce only a modest increase in HDL particle size compared to the wild-type protein, (ii) are unable to release apoA-I from HDL(3), and (iii) do not generate prebeta-mobile particles following incubation with HDL(3). These data suggest that phospholipid transfer is a prerequisite for HDL conversion and demonstrate the close interrelationship between the two main activities of PLTP.

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

Plasma phospholipid transfer protein (PLTP) plays an important role in the maintenance of plasma high-density 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.

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 (PLTP) causes proteolytic cleavage of apolipoprotein A-I.

Plasma phospholipid transfer protein (PLTP) is a factor that plays an important role in HDL metabolism. In this study we present data suggesting that PLTP has an inherent protease activity. After incubation of HDL3 in the presence of purified plasma PLTP, the d < 1.25 g/ml particles (fusion particles) contained intact 28.2 kDa apoA-I while the d > 1.25 g/ml fraction (apoA-I-PL complexes) contained, in addition to intact apoA-I, a cleaved 23 kDa form of apoA-I. Purified apoA-I was also cleaved by PLTP and produced a similar 23 kDa apoA-I fragment. The cleavage of apoA-I increased as a function of incubation time and the amount of PLTP added. The process displayed typically an 8-10 h lag or induction period, after which the cleavage proceeded in a time-dependent manner. This lag-phase was necessary for the development of the cleavage activity during incubation at 37 degrees C. The specific apoA-I cleavage activity of different PLTP preparations varied between 0.4-0.8 microg apoA-I degraded/h per 1000 nmol per h of PLTP activity. The 23 kDa apoA-I fragment reacted with monoclonal antibodies specific for the N-terminal part of apoA-I, indicating that the apoA-I cleavage occurred in the C-terminal portion. The apoA-I cleavage products were further characterized by mass spectrometry. The 23 kDa fragment yielded a mass of 22.924 kDa, demonstrating that the cleavage occurs in the C-terminal portion of apoA-I between amino acid residues 196 (alanine) and 197 (threonine). The intact apoA-I and the 23 kDa fragment revealed identical N-terminal amino acid sequences. The cleavage of apoA-I could be inhibited with APMSF and chymostatin, suggesting that it is due to a serine esterase-type of proteolytic activity. Recombinant PLTP produced in CHO cells or using the baculovirus-insect cell system caused an apoA-I cleavage pattern identical to that obtained with plasma PLTP. The present results raise the question of whether PLTP-mediated proteolytic cleavage of apoA-I might affect plasma HDL metabolism by generating a novel kinetic compartment of apoA-I with an increased turnover rate.

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.

The interaction of plasma phospholipid transfer protein (PLTP) with HDL has not been characterized in detail, although we have reported that the apoA-I/apoA-II molar ratio in the HDL particle influences PLTP-mediated HDL conversion, but not phospholipid transfer. The aim of this study was to examine whether PLTP binds apoA-I or apoA-II, and if this occurs, then determine the PLTP-binding domain of the apoA-I molecule. To study the PLTP/apolipoprotein interaction we used a solid phase ligand binding assay, the ELISA technique, and apoA-I and apoA-II affinity chromatography. PLTP bound to both apoA-I and apoA-II affinity columns, a finding subsequently utilized in the purification of PLTP. PLTP also bound to both apoA-I and apoA-II on ELISA plates in a concentration-dependent manner, and the binding could be displaced by preincubating the PLTP sample with purified apolipoproteins. To determine which portion of apoA-I is recognized by PLTP, we coated ELISA plates with either recombinant full-length apoA-I or three shortened apoA-I forms sequentially truncated from the C-terminus. To characterize the PLTP binding ability of the C-terminal region of apoA-I, we used both C-terminal CNBr-fragment and a synthetic C-terminal peptide of apoA-I. To further confirm the identity of the binding region, we probed the interaction with a polyclonal and several monoclonal anti-apoA-I antibodies. The antibodies that inhibited the interaction between PLTP and apoA-I were directed towards apoA-I epitopes localized between amino acids 27-141. The polyclonal antibody, R33, and the monoclonal antibody A-I-1 (epitope between amino acids 27-48) were most effective and reduced PLTP binding by 70%. These results show that PLTP binds to both apoA-I and apoA-II, and that the PLTP binding domain of apoA-I resides in the amino terminal region.

Transformation of high density lipoprotein 2 particles by hepatic lipase and phospholipid transfer protein.

High density lipoprotein 2 (HDL2) was incubated with phospholipid transfer protein (PLTP) or with hepatic lipase (H-TGL), and the incubation products were separated into a d < 1.22 g/ml and a d > 1.22 g/ml fractions. The d < 1.22 g/ml fraction produced by PLTP was larger, had lower apolipoprotein A-I and higher lipid and apolipoprotein A-II content than native HDL2. The d > 1.22 g/ml fraction represented 30% of the initial HDL2 protein and consisted of small, apolipoprotein A-I and phospholipid-rich particles, with a high sphingomyelin:phosphatidylcholine ratio. Incubation with H-TGL led to a d < 1.22 g/ml fraction which was comparable to native HDL2 regarding size and chemical composition. The d > 1.22 g/ml particles represented only 5% of the initial HDL2 protein and had slightly higher diameter and sphingomyelin:phosphatidylcholine ratio than those produced by PLTP. Enrichment of HDL2 with triglyceride prior to incubation increased the amount of protein released into the d > 1.22 g/ml fraction (20%) but had no effect on size and chemical composition of the particles. We conclude that PLTP and H-TGL promote the formation of small, pre-beta-like HDL particles from HDL2.

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.

Phospholipid transfer protein mediated conversion of high density lipoproteins generates pre beta 1-HDL.

High density lipoproteins (HDL) subclasses can be differentiated by two-dimensional non-denaturing polyacrylamide gradient gel electrophoresis (2D-PAGGE) and subsequent immunoblotting. The quantitatively minor HDL-subclasses pre beta 1-LpA-I and gamma-LpE are initial acceptors of cell-derived cholesterol into the plasma compartment. In this study we analysed the effect of phospholipid transfer protein (PLTP) on the electrophoretic distribution of HDL-subclasses in plasma as well as the ability of plasma, pre beta 1-LpA-I, and gamma-LpE to take up [3H]cholesterol from labeled fibroblasts. Pre beta 1-LpA-I but not gamma-LpE disappeared during a 16 hours incubation in the absence of PLTP. During a one minute incubation pre beta 1-LpA-I of pre-incubated plasma released 75% less [3H]cholesterol from radiolabeled fibroblasts than pre beta 1-LpA-I of control plasma. Pre-incubation of plasma reduced the uptake of [3H]cholesterol by gamma-LpE by 40%. Totally, the cholesterol efflux capacity of plasma decreased by 10% compared to the original sample. The amount of immunodetectable pre beta 1-LpA-I increased when plasma was incubated in the presence of PLTP while the amount of immunodetectable gamma-LpE did not change. After one minute incubation of PLTP-conditioned plasma with [3H]cholesterol-labeled fibroblasts, the amount of radioactive cholesterol taken up by pre beta 1-LpA-I was twice as high as in control plasma whereas the amount of [3H]cholesterol taken up by gamma-LpE remained unchanged. As a net result, treatment with PLTP increased the cholesterol efflux into total plasma by 40%. Together with results of previous studies our data suggest that the conversion of alpha-LpA-I3 into alpha-LpA-I2 by PLTP generates pre beta 1-LpA-I but not gamma-LpE. PLTP helps to enhance the uptake of cell-derived cholesterol by pre beta 1-LpA-I and, thereby, the cholesterol efflux capacity of normal plasma.

The role of protein disulphide isomerase in the microsomal triacylglycerol transfer protein does not reside in its isomerase activity.

The microsomal triacylglycerol transfer protein (MTP), an alpha beta dimer, is obligatory for the assembly of apoB-containing lipoproteins in liver and intestinal cells. The beta subunit is identical with protein disulphide isomerase, a 58 kDa endoplasmic reticulum luminal protein involved in ensuring correct disulphide bond formation of newly synthesized proteins. We report here the expression of the human MTP subunits in Spodoptera frugiperda cells. When the alpha subunit was expressed alone, the polypeptide formed insoluble aggregates that were devoid of triacylglycerol transfer activity. In contrast, when the alpha and beta subunits were co-expressed, soluble alpha beta dimers were formed with significant triacylglycerol transfer activity. Expression of the alpha subunit with a mutant protein disulphide isomerase polypeptide in which both -CGHC- catalytic sites had been inactivated also yielded alpha beta dimers that had comparable levels of lipid transfer activity relative to wild-type dimers. The results indicate that the role of the beta subunit in MTP seems to be to keep the alpha subunit in a catalytically active, non-aggregated conformation and that disulphide isomerase activity of the beta subunit is not required for this function.

The mechanism of human plasma phospholipid transfer protein-induced enlargement of high-density lipoprotein particles: evidence for particle fusion.

1. Phospholipid transfer protein (PLTP) mediates conversion of high-density lipoprotein (HDL3) to large particles, with concomitant release of apolipoprotein A-I (apoA-I). To study the mechanisms involved in this conversion, reconstituted HDL (rHDL) particles containing either fluorescent pyrenylacyl cholesterol ester (PyrCE) in their core (PyrCE-rHDL) or pyrenylacyl phosphatidylcholine (PysPC) in their surface lipid layer (PyrPC-rHDL) were prepared. Upon incubation with PLTP they behaved as native HDL3, in that their size increased considerably. 2. When PyrPC-rHDL was incubated with HDL3 in the presence of PLTP, a rapid decline of the pyrene excimer/monomer fluorescence ratio (E/M) occurred, demonstrating that PLTP induced mixing of the surface lipids of PyrPC-rHDL and HDL3. As this mixing was almost complete before any significant increase in HDL particle size was observed, it represents PLTP-mediated phospholipid transfer or exchange that is not directly coupled to the formation of large HDL particles. 3. When core-labelled PyrCE-rHDL was incubated in the presence of PLTP, a much slower, time-dependent decrease of E/M was observed, demonstrating that PLTP also promotes mixing of the core lipids. The rate and extent of mixing of core lipids correlated with the amount of PLTP added and with the increase in particle size. The enlarged particles formed could be visualized as discrete, non-aggregated particles by electron microscopy. Concomitantly with the appearance of enlarged particles, lipid-poor apoA-I molecules were released. These data, together with the fact that PLTP has been shown not to mediate transfer of cholesterol esters, strongly suggest that particle fusion rather than (net) lipid transfer or particle aggregation is responsible for the enlargement of HDL particles observed upon incubation with PLTP.4.ApoA-I rHDL, but not apoA-II rHDL, were converted into large particles, suggesting that the presence of apoA-I is required for PLTP-mediated HDL fusion. A model for PLTP-mediated enlargement of HDL particles is presented.

Pig plasma phospholipid transfer protein facilitates HDL interconversion.

Phospholipid transfer protein (PLTP) from pig plasma was purified to homogeneity using ultracentrifugation and a combination of hydrophobic-, heparin-Sepharose-, and anti-pig-PLTP-affinity chromatography techniques. The molecular weight of PLTP is 78,000 as estimated by SDS-PAGE and by gel filtration. The effect of pig plasma PLTP on the particle size distribution of either human or pig high density lipoprotein (HDL) was studied by incubating HDL with PLTP. Incubation of human HDL3 or pig HDL with the highly purified preparations of PLTP induced a conversion of the homogeneous HDL into two main populations of particles. The conversion products were isolated by ultracentrifugation at density 1.21 g/ml. The size changes were evident as analyzed by native gradient gel electrophoresis and by high resolution gel filtration. The diameters of the large and small particles formed were 10.8 nm and 7.6-7.9 nm, respectively. In addition, pig HDL conversion products included a third population of particles with a diameter of 11.5 nm. The degree of conversion was dependent on time and PLTP activity. Neither cholesteryl ester transfer protein nor lecithin:cholesterol acyltransferase activity could be detected in the PLTP preparations. The present study demonstrates that purified pig PLTP can act as a conversion factor: it has the ability to convert HDL into populations of large and small particles. The release of apoA-I is an essential part of the conversion process.

Human plasma phospholipid transfer protein causes high density lipoprotein conversion.

The effect of human plasma phospholipid transfer protein (PLTP) on the particle size distribution of human high density lipoprotein (HDL) was studied by incubating human HDL3 (particle diameter, 8.7 nm) together with PLTP in vitro. Incubation of HDL3 with highly purified preparations of PLTP, devoid of cholesterol ester transfer protein (CETP), induced a conversion of the homogenous population of HDL particles into two main populations of particles, one larger, particle diameter 10.9 nm, and one smaller, particle diameter 7.8 nm, than the original HDL3. These size changes were evident as analyzed by gradient gel electrophoresis and by high resolution gel filtration. The degree of the conversion was dependent on the amount of PLTP added to the incubation and on incubation time. An inhibitory monoclonal antibody (TP-1) directed against CETP had no effect on the HDL conversion. The PLTP used was purified to homogeneity from human plasma using ultracentrifugation and a combination of hydrophobic, cation-exchange, heparin-Sepharose-, anion-exchange, and gel filtration chromatographies. The monoclonal anti-CETP antibody (TP-1), which inhibits lipid transfer catalyzed by CETP, did not react with PLTP or inhibit its activity. The estimated molecular weight of PLTP is 75,000. The present study demonstrates that PLTP can act like the putative conversion factor and has the ability to convert HDL3 into populations of larger and smaller HDL particles. The mechanism(s) involved in this process and its physiological relevance remain to be established.

A novel polymorphism of apolipoprotein A-IV is the result of an asparagine to serine substitution at residue 127.

We have identified a hitherto unknown genetic polymorphism of apolipoprotein A-IV (apoA-IV). The molecular basis for this polymorphism is an A to G substitution at nucleotide 1687 resulting in an Asn to Ser change of amino acid 127. The frequencies of the two apoA-IV alleles (designated apoA-IV127Asn and apoA-IV127Ser), determined by Hin c II restriction analysis of PCR amplified exon three of the apoA-IV gene, were 0.788 and 0.212, respectively, in a Finnish population sample. Allele frequencies of another polymorphism due to a Thr to Ser substitution at amino acid 347 were determined using Hinf I restriction analysis. The allele frequencies were 0.823 for apoA-IV347Thr and 0.177 for apoA-IV347Ser. None of the apoA-IV polymorphisms (apoA-IV127:Asn----Ser, apoA-IV347:Thr----Ser and apoA-IV360:Gln----His) had any effect on plasma lipid and lipoprotein concentrations in cohorts of dyslipidemic men and in a population sample of normolipidemic controls. There was also no association between the history of previous myocardial infarction and any of the apoA-IV alleles.

The apolipoprotein A-I binding protein of placenta and the SP-40,40 protein of human blood are different proteins which both bind to apolipoprotein A-I.

A complement-associated protein SP-40,40, which is a normal constituent of human blood, binds to the main apoprotein, apoA-I, of high density lipoprotein (HDL). This protein, which is identical to apolipoprotein J, was compared to another apoA-I binding protein purified from human placenta. Immunologically the two apoA-I binding proteins are different.

The mutation causing the common apolipoprotein A-IV polymorphism is a glutamine to histidine substitution of amino acid 360.

Apolipoprotein (apo) A-IV is a protein involved in the metabolism of chylomicrons and high density lipoproteins. This protein displays genetic polymorphism due to two main codominant alleles, A-IV1 and A-IV2. We have identified the mutation that leads to this polymorphism. It is caused by a single-base substitution of guanine for thymine in the third base of codon 360. This substitution leads to a glutamine to histidine change. Direct sequencing of amplified DNA from eight subjects in a three-generation pedigree has demonstrated that the guanine to thymine substitution can explain the apo A-IV polymorphism. In 32 unrelated individuals, a correspondence between apo A-IV phenotype determined by isoelectric focusing and genotype determined with Fnu4HI digestion of amplified DNA could be demonstrated. The enzyme lecithin:cholesteryl acyltransferase (LCAT) is activated by apo A-IV. Under our in vitro conditions, the isoprotein apo A-IV 1-1 is a better LCAT activator than is the isoprotein apo A-IV 2-2. A knowledge of the molecular mechanism underlying the apo A-IV polymorphism will help to elucidate the mechanisms involved in LCAT activation.

Characterization of the enzyme activity of human plasma lipoprotein (a) using synthetic peptide substrates.

The plasma concentration of lipoprotein (a) [Lp(a)] is correlated with the risk of atherosclerosis. It is a lipoprotein particle consisting of apoprotein (a) [Lp(a)] is correlated with the risk of atherosclerosis. It is a lipoprotein particle consisting of apoprotein (a) [apo(a)], a protein showing considerable amino acid sequence identity with plasminogen. bound to low-density lipoprotein. The apo(a) portion of Lp(a) was recently shown to have serine-proteinase-type amidolytic activity and to be able to degrade the adhesive glycoprotein fibronectin. To characterize this enzyme activity further, we used chromogenic peptide substrates and inhibitors. Of the substrates tested, those with arginine at the scissile bond [N-alpha-benzoyl-L-Arg p-nitroanilide (pNA), N-alpha-benzoyl-Ile-Glu-Gly-Arg-pNA, N-alpha-benzyloxycarbonyl-Arg-Gly-Arg-pNA] gave the highest hydrolysis rates. Synthetic substrates with plasmin specificity (Val-Leu-L-Lys-pNA and Val-Phe-L-Lys-pNA) were not hydrolysed by Lp(a). Neither tissue plasminogen activator nor urokinase had any effect on the enzyme activity. The addition of antibodies to these plasminogen activators did not inhibit the enzyme activity of Lp(a). Inhibition experiments with phenylmethanesulphonyl fluoride, carbodi-imide, dichloroisocoumarin and competitive peptide inhibitors demonstrated that Lp(a) has enzyme activity that closely resembles that of serine proteinases. Whether this serine-proteinase activity of Lp(a) plays any role in the genesis of atherosclerosis remains to be established.

Interaction of lipoprotein(a) with fibronectin and its potential role in atherogenesis.

The plasma concentration of lipoprotein (a) (Lp(a] varies widely in humans, and elevated concentrations of this lipoprotein are correlated with progression of atherosclerosis. Structural studies of Lp(a) have revealed that it is a low density lipoprotein (LDL)-like particle containing a unique glycoprotein, apo(a), which shares extensive homology with plasminogen. The apo(a) portion of Lp(a) binds to the carboxy-terminal heparin-binding domain of fibronectin. Incubation of Lp(a) or isolated apo(a) with fibronectin results in proteolytic cleavage of fibronectin which is, as visualized by gel electrophoresis and immunoblotting, distinct from that caused by plasmin or kallikrein. The proteolytic activity of apo(a) is of serine proteinase-type and displays specificity for arginine rather than lysine bonds. The molecular mechanism(s) underlying the association between Lp(a) and atherosclerosis remains an enigma.