Activation of lipoprotein lipase by native and acylated peptides of apolipoprotein C-II.

Apolipoprotein C-II, a protein found associated with all major classes of plasma lipoproteins, is a potent activator of the enzyme lipoprotein lipase. We have prepared the maleyl, citraconyl and succinyl derivatives of apolipoprotein C-II, and compared the capacities of the intact and tryptically cleaved proteins to activate lipoprotein lipase. The NH2-terminal 50 residue peptide proved virtually inactive, even after removal of the masking groups from the citraconyl derivative. The COOH-terminal 29 residue peptides of maleyl and citraconyl apolipoprotein C-II were more active than the corresponding succinylated peptide. After deacylation of the citraconyl derivative, the COOH-terminal peptide had maximal activity as great as apolipoprotein C-II, although the profile of activation remained dissimilar at low activator concentrations.     

Activation of lipoprotein lipase by N-alpha-palmitoyl (56-79) fragment of apolipoprotein C-II

The effect of apolipoprotein C-II (apoC-II) and a synthetic fragment of apoC-II corresponding to residues 56-79 on the lipoprotein lipase (LpL) catalyzed hydrolysis of trioleoylglycerol in a monolayer of egg phosphatidylcholine and of dipalmitoylphosphatidylcholine vesicles was examined. Synthetic peptide 56-79, which does not associate with lipid, did not activate LpL at surface pressures greater than 30 mN/m; apoC-II is active up to 34 mN/m. However, acylation of the NH2-terminus of peptide 56-79 with palmitoyl chloride gave nearly identical LpL activating properties as compared to apoC-II. We conclude that at high surface pressures the lipid-binding region of apoC-II (residues 44-55) plays an essential role in LpL activation.     

Inhibition of lipoprotein lipase activity by synthetic peptides of apolipoprotein C-III.

In this study we have examined effects of synthetic polypeptide fragments of apoC-III on the kinetic properties of lipoprotein lipase (LPL) activity. Based on the loss of 79% of LPL-inhibitory activity after CNBr cleavage at the N-terminal portion of apoC-III and a systematic search for synthetic peptides with LPL-inhibitory activity spanning the apoC-III sequence, we concluded that the N-terminal domain is the most important in the modulation of LPL activity. In addition, there are multiple attachment sites in apoC-III for its interaction with LPL and these sites reside in the hydrophilic sequences of apoC-III. Probably for this reason the intact apo-CIII exhibited higher inhibitory potential than its peptide components. Based on the deduced inhibition constants derived for the synthetic apoC-III1-79 we concluded that apoC-III is likely to exhibit a physiological role in regulating LPL activity since the derived dissociation constants for the LPL-apoC-III interaction are within the physiological concentration range of plasma apoC-III. In addition, as the synthetic apoC-III1-79 lacks the carbohydrate moiety, we also concluded that the presence of the oligosaccharide in native apoC-III is not essential for its inhibitory activity on LPL. The fact that the I50 (concentration for inhibition of LPL at 50% activity) decreases for apoC-III-1 when assayed in the presence of apoC-II indicated that the activator actually caused an increased affinity between LPL and apoC-III and demonstrated that apoC-III does not compete for the activator site of apoC-II.     

Kinetics of product inhibition and mechanisms of lipoprotein lipase activation by apolipoprotein C-II

The kinetics of product inhibition of bovine milk lipoprotein lipase (LPL) were studied in a system of emulsified trioleoylglycerol (TG) at different fixed initial concentrations of oleic acid [( OA]0) without a fatty acid (FA) acceptor. In the absence of apolipoprotein C-II (C-II), the apparent Vmax and the nH(TG) (the slope of the corresponding Hill plot for TG) of 1.82 decreased by about 52% and [TG]0.5 increased 13-fold by raising the [OA]0 to 0.3 mM. At low [OA]0, product inhibition was competitive with respect to TG: the nH(OA) averaged 1.1, and [OA]0.5 was increased about 2-fold by TG. At the higher [OA]0, nH(OA) was 3.5, and TG had no effect on [OA]0.5. In the presence of 3 micrograms/mL C-II, the apparent Vmax was 4.3-7.1-fold higher than in its absence, and the nH(TG) was 2.45. Both parameters decreased by only 20-25%, and [TG]0.5 increased only 3-fold at an [OA]0 of 0.3 mM. Conversely, nH(OA) decreased by 35% and [OA]0.5 increased 6-fold by increasing TG concentrations. Similar kinetics were observed with very low density lipoproteins (VLDL). At saturating TG and varying C-II concentrations, nH(C-II) was 1.78, and product inhibition was found to be competitive with respect to C-II. At the [OA]0 employed, the FA had no effect on enzyme binding to TG emulsions, and there was no evidence that LPL catalyzes the reverse reaction. It is concluded that (a) the LPL kinetics are those of a multisite enzyme that probably has three high-affinity binding sites for TG, two for C-II, and four for OA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of bovine milk lipoprotein lipase and the mechanism of enzyme activation by apolipoprotein C-II

The kinetics of bovine milk lipoprotein lipase (LPL) were studied in order to determine the reaction mechanism of this enzyme. Reaction velocities were determined at varying concentrations of emulsified trioleoylglycerol (TG) and different fixed concentrations of apolipoprotein C-II (C-II) or at varying C-II concentrations and different fixed concentrations of TG. Neither the apparent Km(TG) nor the apparent Km(C-II) was affected by varying the concentrations of C-II or TG, respectively. However, C-II increased the apparent Vmax for the enzyme about 20-fold. The following kinetic parameters were calculated from Lineweaver-Burk plots: Km(C-II) = 2.5 X 10(-8) M and Km (TG) = 2.5 X 10(-3) M. The dissociation constant (KS) of the enzyme-TG binary complex was determined from Scatchard plots to be 7.6 X 10(-8) M. Heparin was found to be a competitive dead-end inhibitor against both TG and C-II. Tricapryloylglycerol represented a competitive inhibitor against TG but a noncompetitive inhibitor against C-II. C-II was shown to interact with dansylated bovine milk LPL, increasing its fluorescent emission by inducing a conformational change in the enzyme. Based on these studies, it was concluded that the LPL-catalyzed reaction follows a random, bireactant, rapid-equilibrium mechanism and the role of C-II in the activation process involves an increase in the catalytic rate constant (Kp) resulting from conformational changes of LPL induced by C-II.     

Chain length dependence of phosphatidylcholine hydrolysis catalyzed by lipoprotein lipase. Effect of apolipoprotein C-II

The effect of apolipoprotein C-II (apoC-II) on the bovine milk lipoprotein lipase (LpL)-catalyzed hydrolysis of a homologous series of saturated phosphatidylcholines was examined with respect to the fatty acyl chain length of the substrates. Dilauryl-, dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine solubilized by Triton X-100 and sonicated vesicles of dimyristoylphosphatidylcholine were used as substrates. The maximal rate of the LpL-catalyzed hydrolysis of each of these lipids was determined in the absence and presence of apoC-II. The activation factor (the ratio of enzyme activity with apoC-II to that without the activator protein) increased with increasing mol ratios of apoC-II to LpL and was maximal at a ratio of approximately 50. At all apoC-II/LpL mole ratios tested, the activation factor increased as a function of fatty acyl chain length. A quantitative relationship between fatty acyl chain length and the extent of maximal activation of LpL by apoC-II was observed: the logarithm of the activation factor is a linear function of the number of carbon atoms of a single fatty acyl chain of the substrates.     

Hydrolysis of chylomicron triacylglycerol by endothelium-bound lipoprotein lipase. Effect of decreased apoprotein C-II/C-III ratio.

Chylomicrons with a decreased ratio of C-II/C-III apoproteins on their surface produced by the addition of apoproteins C-III-0 or C-III-3 to intact rat lymph chylomicrons. These chylomicrons inhibited the activity of soluble lipoprotein lipase in vitro, but had no effect on the activity of the endothelium-bound enzyme in the perfused heart.     

Kinetics of human and bovine milk lipoprotein lipase and the mechanism of enzyme activation by apolipoprotein C-II

The kinetics of human and bovine milk lipoprotein lipase (HM-LPL and BM-LPL, respectively) were compared by varying apolipoprotein C-II (C-II) or triacylglycerol (TG) concentrations. The apparent Km (TG) and Km (C-II) for HM-LPL were 2.2 and 6.7-fold higher than for BM-LPL. Plots of 1/v vs 1/[TG] or 1/[C-II] intercepted the respective abscissas at the same points: C-II had no effect on Km (TG) and TG had no effect on Km (C-II). Replots of slope 1/s vs 1/[C-II] gave straight lines which yielded KA values identical to Km (C-II). It is concluded that the HM-LPL system follows a random, bireactant, rapid equilibrium mechanism as shown previously for BM-LPL.     

Interaction of synthetic peptides of apolipoprotein C-II and lipoprotein lipase at monomolecular lipid films

The triacylglycerol hydrolyase and phospholipase A1 activities of bovine milk lipoprotein lipase toward long-chain fatty acyl ester substrates were investigated with monomolecular lipid films containing trioleoylglycerol and phosphatidylcholine. In a monolayer of egg phosphatidylcholine containing 3 mol% [14C]trioleoylglycerol, and in the presence of apolipoprotein C-II, a 79 amino acid activator protein for lipoprotein lipase, enzyme activity was maximal at a surface pressure of 21-22 mN X m-1 (37 mumol oleic acid released/h per mg enzyme); enzyme activity was enhanced 9-fold by apolipoprotein C-II. At surface pressures between 22 and 30 mN X m-1, lipoprotein lipase activity decreased over a broad range and was nearly zero at 30 mN X m-1. Apolipoprotein C-II and the synthetic fragments of the activator protein containing residues 56-79, 51-79 and 44-79 were equally effective at 20 mN X m-1 in enhancing lipoprotein lipase catalysis. However, at surface pressures between 25 and 29 mN X m-1, only apolipoprotein C-II and the phospholipid-associating fragment containing residues 44-79 enhanced enzyme catalysis. The effect of apolipoprotein C-II and synthetic peptides on the phospholipase A1 activity of lipoprotein lipase was examined in sphingomyelin:cholesterol (2:1) monolayers containing 5 mol% di[14C]myristoylphosphatidylcholine. At 22 mN X m-1, apolipoprotein C-II and the synthetic fragments containing residues 44-79 or 56-79 enhanced lipoprotein lipase activity (70-80 nmol/h per mg enzyme). In contrast to trioleoylglycerol hydrolysis, the synthetic fragments were not as effective as apolipoprotein C-II enhancing enzyme activity towards di[14C]myristoylphosphatidylcholine at higher surface pressures. We conclude that the minimal amino acid sequence of apolipoprotein C-II required for activation of lipoprotein lipase is dependent both on the lipid substrate and the packing density of the monolayer.     

Mechanism of action of lipoprotein lipase on triolein particles: effect of apolipoprotein C-II

Triolein particles stabilized by a phosphatidylcholine monolayer were used to study the lipoprotein lipase (LpL) reaction. They were prepared in two different sizes and with triolein and phosphatidylcholine in the molar ratios of 0.9-1.2 : 1 (small particles) and 8-17 : 1 (large particles). The rate of hydrolysis by LpL of phosphatidylcholine on the surface of both lipid particles was only 1/20 as much as that of triolein, even if it was activated to the maximum by apolipoprotein C-II (apoC-II). Thus, the phospholipase activity of LpL was low enough to measure the initial rate of hydrolysis of triolein without causing a gross change of the surface of the lipid particle. When the hydrolysis of triolein by LpL was monitored, fatty acid was released at a constant rate until all of the triolein molecules were hydrolyzed. The enzyme required 220 +/- 17 and 66 +/- 9 nM apoC-II for its half-maximal activity (Km (apoC-II] with small and large particles as a substrate (1.15 mM triolein for small and 2.13 mM triolein for large particles), respectively, using various concentrations of LpL. The Km(apoC-II) values for these two substrates became similar when LpL activity was analyzed with respect to the density of apoC-II on the phosphatidylcholine monolayer at the surface of the particles (bound apoC-II/phosphatidylcholine). The concentration of substrate particles did not affect the Km(apoC-II) values. The presence of an adequate amount of apoC-II increased the maximal activity of LpL (Vmax(triolein)) from 0.48 +/- 0.21 to 6.81 +/- 0.45 and from 0.32 +/- 0.04 to 7.13 +/- 0.64 mmol/h/mg with a slight decrease in the apparent Michaelis constant (Km(triolein)) for small (from 90 to 54 microM triolein) and large (from 1.00 to 0.65 mM triolein) particles, respectively. Although the apparent Km for triolein in large particles was about ten times greater than that in small particles, the values became similar when they were corrected for the concentration of phosphatidylcholine (50-100 microM phosphatidylcholine), which corresponded to the surface area of the substrate particles. It was suggested that bound apoC-II molecules were transferred relatively slowly to other lipid particles while LpL molecules moved rapidly among the lipid particles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reduction of plasma triglycerides in apolipoprotein C-II transgenic mice overexpressing lipoprotein lipase in muscle

LPL and its specific physiological activator, apolipoprotein C-II (apoC-II), regulate the hydrolysis of triglycerides (TGs) from circulating TG-rich lipoproteins. Previously, we developed a skeletal muscle-specific LPL transgenic mouse that had lower plasma TG levels. ApoC-II transgenic mice develop hypertriglyceridemia attributed to delayed clearance. To investigate whether overexpression of LPL could correct this apoC-II-induced hypertriglyceridemia, mice with overexpression of human apoC-II (CII) were cross-bred with mice with two levels of muscle-specific human LPL overexpression (LPL-L or LPL-H). Plasma TG levels were 319 +/- 39 mg/dl in CII mice and 39 +/- 5 mg/dl in wild-type mice. Compared with CII mice, apoC-II transgenic mice with the higher level of LPL overexpression (CIILPL-H) had a 50% reduction in plasma TG levels (P = 0.013). Heart LPL activity was reduced by approximately 30% in mice with the human apoC-II transgene, which accompanied a more modest 10% decrease in total LPL protein. Overexpression of human LPL in skeletal muscle resulted in dose-dependent reduction of plasma TGs in apoC-II transgenic mice. Along with plasma apoC-II concentrations, heart and skeletal muscle LPL activities were predictors of plasma TGs. These data suggest that mice with the human apoC-II transgene may have alterations in the expression/activity of endogenous LPL in the heart. Furthermore, the decrease of LPL activity in the heart, along with the inhibitory effects of excess apoC-II, may contribute to the hypertriglyceridemia observed in apoC-II transgenic mice.     

No evidence for linkage between familial hypertriglyceridemia and apolipoprotein B,apolipoprotein C-III or lipoprotein lipase genes

Familial hypertriglyceridemia has been suggested to be an autosomal dominant condition with age-dependent penetrance, but so far the underlying defective gene has not been elucidated. We examined the possible role of three candidate gene loci by linkage analysis in six Finnish families with familial clustering of hypertriglyceridemia. The probands were initially recruited from a group of hyperlipidemic outpatients after measurement of serum triglyceride concentrations exceeding 2.00 mmol/l on two occasions. Altogether, 71 subjects were included in the linkage analyses. Bior multiallelic DNA polymorphisms were used as markers for the apolipoprotein B gene (chromosome 2), lipoprotein lipase gene (chromosome 8), and apolipoprotein A-I/C-III/A-IV gene cluster (chromosome 11). Linkage analysis was performed by applying two alternative phenotyping models, one adopting quantitative serum triglyceride concentrations and another using qualitative classification of the subjects into hypertriglyceridemic, normotriglyceridemic, and borderline hypertriglyceridemic groups. Using either approach, the cumulative lod scores of each of the three candidate genes in the six families were less than -2.0 at the recombination fraction 0.0. These results suggest that none of the candidate genes investigated is involved in familial clustering of hypertriglyceridemia in our study.     

Triglyceridase and phospholipase A1 activities of rat-heart lipoprotein lipase. Influence of apolipoproteins C-II and C-III.

The influence of purified human apolipoprotein C-II on phospholipase A1 and triglyceridase activities of lipoprotein lipase were compared. Lipoprotein lipase was obtained from rat hearts by perfusion with a medium containing heparin and purified on a heparin Sepharose 4-B column. Using phosphatidyl-ethanolamine-coated triglyceride particles as substrate it was found that the phospholipase A1 and triglyceridase activities of lipoprotein lipase similarly depend on the presence of apolipoprotein C-II. Apolipoprotein C-III cannot replace apolipoprotein C-II. However, addition of apolipoprotein C-III in the presence of C-II affects both lipase activities. While strong inhibition of triglyceridase activity was observed under these conditions, phospholipase A1 activity was slightly stimulated. On the basis of these findings a model was constructed for the role of apolipoprotein C-II in lipoprotein lipase action.     

Phospholipase activity of bovine milk lipoprotein lipase on phospholipid vesicles: influence of apolipoproteins C-II and C-III.

The effect of human plasma apolipoproteins C-II and C-III on the hydrolytic activity of lipoprotein lipase from bovine milk was determined using dimyristoyl phosphatidylcholine (DMPC) vesicles as substrate. In the absence of apoC-II or C-III, lipoprotein lipase has limited phospholipase activity. When the vesicles were preincubated with apoC-II and then phospholipase activity determined, there was a time dependent release of lysolecithin; activity was dependent upon both apoC-II and lipoprotein lipase concentrations. The addition of apoC-III to DMPC did not stimulate phospholipase activity. We conclude that apoC-II has an activator effect on the phospholipase activity of lipoprotein lipase and that the mechanism is beyond that of simply altering the lateral compressibility of the lipid.     

Molecular basis of familial chylomicronemia: mutations in the lipoprotein lipase and apolipoprotein C-II genes.

The molecular basis of familial chylomicronemia (type I hyperlipoproteinemia), a rare autosomal recessive trait, was investigated in six unrelated individuals (five of Spanish descent and one of Northern European extraction). DNA amplification by polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) analysis allowed rapid identification of the underlying mutations. Six different mutant alleles (three of which are previously undescribed) of the gene encoding lipoprotein lipase (LPL) were discovered in the five LPL-deficient patients. These included an 11 bp deletion in exon 2, and five missense mutations: Trp 86 Arg (exon 3), His 136 Arg (exon 4), Gly 188 Glu (exon 5), Ile 194 Thr (exon 5), and Ile 205 Ser (exon 5). The Trp 86 Arg mutation is the only known missense mutation in exon 3. The other missense mutations lie in the highly conserved "central homology region" in close proximity with the catalytic site of LPL. These and other previously reported missense mutations provide insight into structure/function relationships in the lipase family. The missense mutations point to the important role of particular highly conserved helices and beta-strands in proper folding of the LPL molecule, and of certain connecting loops in the catalytic process. A nonsense mutation (Arg 19 Term) in the gene encoding apolipoprotein C-II (apoC-II), the cofactor of LPL, was found to underlie chylomicronemia in the sixth patient who had normal LPL but was apoC-II-deficient.     

Association of lipoprotein lipase and apolipoprotein C-III genes polymorphism with acute myocardial infarction in diabetic patients

Lipoprotein lipase (LPL) and Apolipoprotein C-III (APOC-III) play an important role in lipid metabolism. The aim of this study was to explore the possible associations of the gene polymorphisms (LPL HindIII, LPL Ser(447)-Ter and APOC3 SstI), diabetes mellitus, and plasma lipids with myocardial infarction. The polymorphisms were assessed by restriction assay in 200 Egyptian MI patients (100 diabetic and 100 non-diabetic) and 100 healthy controls. This study demonstrated that individuals with the H2H2 genotype or S2 allele have more than three times higher relative risk of suffering from MI than those carrying the H1H1 or S1S1. Type 2 DM mainly lowers HDL-C levels in MI patients who carry H2H2 or S2S2 genotype and increases TC, TG, and LDL levels in MI patients carrying H2H2 or S2S2 genotype compared with non-diabetic MI patients carrying the same genotypes. In S447X polymorphism, it was observed that DM led to loss of the protective lipid profile in MI patients carrying 447XX genotype. These findings suggest that H2H2 or S2S2 genotypes are associated with dyslipidemia and increased risk of myocardial infarction. The S447X polymorphism is associated with a favorable lipid profile. However, the association of diabetes mellitus with these polymorphisms leads to unfavorable lipid profile.     

Characterization of the lipid-binding properties and lipoprotein lipase inhibition of a novel apolipoprotein C-III variant Ala23Thr

We have identified a G-to-A transition in exon 3 of the APOC3 gene resulting in a novel Ala23Thr apolipoprotein (apo) C-III variant, associated with apoC-III deficiency in three unrelated Yucatan Indians. The Ala23Thr substitution modifies the hydrophobic/hydrophilic repartition of the helical N-terminal peptide and hence could disturb the lipid association. In vitro expression in Escherichia coli of wild-type and mutant apoC-III enabled the characterization of the variant. Compared with wild-type apoC-III-Ala23, the mutant apoC-III-Thr23 showed reduced affinity for dimyristoylphosphatidylcholine (DMPC) multilamellar vesicles with higher amounts of free apoC-III. Displacement of apoE from discoidal apoE:dipalmitoylphosphatidycholine (DPPC) complex by apoC-III-Thr23 was comparable to wild type but the less efficient binding of the apoC-III-Thr23 to the discoidal complex resulted in a higher apoE/apoC-III (mol/mol) ratio (34%) than with wild-type/apoE:DPPC mixtures. The inhibition of lipoprotein lipase (LPL) by apoC-III-Thr23 was comparable to that of wild type, and therefore effects on LPL activity could not explain the lower triglyceride (Tg) levels in Thr-23 carriers. Thus, these in vitro results suggest that in vivo the less efficient lipid binding of apoC-III-Thr23 might lead to a faster catabolism of free apoC-III, reflected in the reduced plasma apoC-III levels identified in Thr-23 carriers, and poorer competition with apoE, which might enhance clearance of Tg-rich lipoproteins and lower plasma Tg levels seen in Thr-23 carriers.     

Activation of lipoprotein lipase by blood platelets

When an homogenate of human blood platelets is added to preparations of partially purified adipose tissue lipoprotein lipase, the lipase activity is markedly enhanced. The degree of activation is related to the amount of platelet homogenate in the reaction mixture. The lipoprotein lipase activator in platelets is heat labile, nondialyzable, partially resistant to papain, and present mostly in the particulate fraction of platelet homogenates.     

Interaction of lipoprotein lipase and apolipoprotein C-II with sonicated vesicles of 1,2-ditetradecylphosphatidylcholine: comparison of binding constants

The interaction of lipoprotein lipase (LpL) and its activator protein, apolipoprotein C-II (apoC-II), with a nonhydrolyzable phosphatidylcholine, 1,2-ditetradecyl-rac-glycero-3-phosphocholine (C14-ether-PC), was studied by fluorescence spectroscopy. A complex of 320 molecules of C14-ether-PC per LpL was isolated by density gradient ultracentrifugation in KBr. The intrinsic tryptophan fluorescence emission spectrum of LpL was shifted from 336 nm in the absence of lipid to 330 nm in the LpL-lipid complex; the shift was associated with a 40% increase in fluorescence intensity. Addition of C14-ether-PC vesicles to apoC-II caused a 2.5-fold increase in intrinsic tryptophan fluorescence and a shift in emission maximum from 340 to 317 nm. LpL and apoC-II/C14-ether-PC stoichiometries and binding constants were determined by measuring the increase in the intrinsic tryptophan fluorescence as a function of lipid and protein concentrations; for LpL the rate and magnitude of the fluorescence increases were relatively independent of temperature in the range 4-37 degrees C. A stoichiometry of 270 PC per LpL for the LpL-lipid complex compares favorably with the value obtained in the isolated complex. The dissociation constant (Kd) of the complex is 4.3 X 10(-8) M. For apoC-II, the stoichiometry of the complex is 18 PC per apoprotein, and the Kd is 3.0 X 10(-6) M. These data suggest that LpL binds more strongly than apoC-II to phosphatidylcholine interfaces.