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.     

Role of A kinase anchor proteins in the tissue-specific regulation of lipoprotein lipase

Activation of protein kinase A by catecholamines inhibits lipoprotein lipase (LPL) activity through the elaboration of an RNA binding complex, which inhibits LPL translation by binding to the 3'-untranslated region of the LPL mRNA. To better define this process, we reconstituted the inhibitory RNA binding complex in vitro and demonstrated that the K homology (KH) domain of A kinase anchor protein (AKAP) 121/149 plays a vital role in the inhibition of LPL translation. Inhibition of LPL translation occurred in vitro only when the Calpha subunit, R subunit, and AKAP 149 were present. Using different glutathione-S-transferase fusion proteins of AKAP 149, sequences containing the KH domain were required for inhibition of LPL translation, and the inhibition of AKAP 121 expression in 3T3-F442A adipocytes with short interfering RNA resulted in loss of epinephrine-mediated translation inhibition. After epinephrine injection into mice, LPL activity was inhibited in white adipose tissue but not in brown adipose tissue (BAT) or muscle. LPL activity and synthetic rate were inhibited in vitro by the addition of epinephrine to 3T3-F442A adipocytes, but there was no effect in L6 muscle cells and cultures of brown adipocytes. Corresponding with these differences in LPL translation, AKAP 121 protein and mRNA were abundantly expressed in mouse white adipose tissue, but was either very low or undetectable in BAT and muscle. Thus, AKAP 121/149 contains a KH region that is essential to the translation inhibition of LPL in response to epinephrine. BAT and muscle do not express significant AKAP 121/149, and this likely explains some of the tissue-specific differences in LPL regulation.     

Role of protein kinase C in the translational regulation of lipoprotein lipase in adipocytes

The hypertriglyceridemia of diabetes is accompanied by decreased lipoprotein lipase (LPL) activity in adipocytes. Although the mechanism for decreased LPL is not known, elevated glucose is known to increase diacylglycerol, which activates protein kinase C (PKC). To determine whether PKC is involved in the regulation of LPL, we studied the effect of 12-O-tetradecanoyl phorbol 13-acetate (TPA) on adipocytes. LPL activity was inhibited when TPA was added to cultures of 3T3-F442A and rat primary adipocytes. The inhibitory effect of TPA on LPL activity was observed after 6 h of treatment, and was observed at a concentration of 6 nM. 100 nM TPA yielded maximal (80%) inhibition of LPL. No stimulation of LPL occurred after short term addition of TPA to cultures. To determine whether TPA treatment of adipocytes decreased LPL synthesis, cells were labeled with [35S]methionine and LPL protein was immunoprecipitated. LPL synthetic rate decreased after 6 h of TPA treatment. Western blot analysis of cell lysates indicated a decrease in LPL mass after TPA treatment. Despite this decrease in LPL synthesis, there was no change in LPL mRNA in the TPA-treated cells. Long term treatment of cells with TPA is known to down-regulate PKC. To assess the involvement of the different PKC isoforms, Western blotting was performed. TPA treatment of 3T3-F442A adipocytes decreased PKC alpha, beta, delta, and epsilon isoforms, whereas PKC lambda, theta, zeta, micro, iota, and gamma remained unchanged or decreased minimally. To directly assess the effect of PKC inhibition, PKC inhibitors (calphostin C and staurosporine) were added to cultures. The PKC inhibitors inhibited LPL activity rapidly (within 60 min). Thus, activation of PKC did not increase LPL, but inhibition of PKC resulted in decreased LPL synthesis by inhibition of translation, indicating a constitutive role of PKC in LPL gene expression.     

Chimeras of hepatic lipase and lipoprotein lipase. Domain localization of enzyme-specific properties.

Chimeric molecules between human lipoprotein lipase (LPL) and rat hepatic lipase (HL) were used to identify structural elements responsible for functional differences. Based on the close sequence homology with pancreatic lipase, both LPL and HL are believed to have a two-domain structure composed of an amino-terminal (NH2-terminal) domain containing the catalytic Ser-His-Asp triad and a smaller carboxyl-terminal (COOH-terminal) domain. Experiments with chimeric lipases containing the HL NH2-terminal domain and the LPL COOH-terminal domain (HL/LPL) or the reverse chimera (LPL/HL) showed that the NH2-terminal domain is responsible for the catalytic efficiency (Vmax/Km) of these enzymes. Furthermore, it was demonstrated that the stimulation of LPL activity by apolipoprotein C-II and the inhibition of activity by 1 M NaCl originate in structural features within the NH2-terminal domain. HL and LPL bind to vascular endothelium, presumably by interaction with cell surface heparan sulfate proteoglycans. However, the two enzymes differ significantly in their heparin affinity. Experiments with the chimeric lipases indicated that heparin binding avidity was primarily associated with the COOH-terminal domain. Specifically, both HL and the LPL/HL chimera were eluted from immobilized heparin by 0.75 M NaCl, whereas 1.1 M NaCl was required to elute LPL and the HL/LPL chimera. Finally, HL is more active than LPL in the hydrolysis of phospholipid substrates. However, the ratio of phospholipase to neutral lipase activity in both chimeric lipases was enhanced by the presence of the heterologous COOH-terminal domain, demonstrating that this domain strongly influences substrate specificity. The NH2-terminal domain thus controls the kinetic parameters of these lipases, whereas the COOH-terminal domain modulates substrate specificity and heparin binding.     

Epidermal growth factor receptor binding is affected by structural determinants in the toxin domain of transforming growth factor-alpha-Pseudomonas exotoxin fusion proteins.

TGF-alpha-PE40 is a hybrid protein composed of transforming growth factor-alpha (TGF-alpha) fused to a 40,000-dalton segment of Pseudomonas exotoxin A (PE40). This hybrid protein possesses the receptor-binding activity of TGF-alpha and the cell-killing properties of PE40. These properties enable TGF-alpha-PE40 to bind to and kill tumor cells that possess epidermal growth factor (EGF) receptors. Unexpectedly, TGF-alpha-PE40 binds approximately 100-fold less effectively to EGF receptors than does native TGF-alpha (receptor-binding inhibition IC50 = 540 and 5.5 nM, respectively). To understand the factors governing receptor binding, deletions and site-specific substitutions were introduced into the PE40 domain of TGF-alpha-PE40. Removal of the N-terminal 59 or 130 amino acids from the PE40 domain of TGF-alpha-PE40 improved receptor binding (IC50 = 340 and 180 nM, respectively) but decreased cell-killing activity. Substitution of alanines for cysteines at positions 265 and 287 within the PE40 domain dramatically improved receptor binding (IC50 = 37 nM) but also decreased cell-killing activity. Similar substitutions of alanines for cysteines at positions 372 and 379 within the PE40 domain did not significantly affect receptor-binding or cell-killing activities. These studies indicate that the PE40 domain of TGF-alpha-PE40 interferes with EGF receptor binding. The cysteine residues at positions 265 and 287 of PE40 are responsible for a major part of this interference.     

Insulin increases the synthetic rate and messenger RNA level of lipoprotein lipase in isolated rat adipocytes

Lipoprotein lipase (LPL) is the enzyme responsible for hydrolysis of circulating triglyceride-rich lipoproteins and is important for storage of adipocyte lipid. To study the regulation of LPL synthetic rate in adipose tissue, primary cultures of isolated rat adipocytes were pulse-labeled with [35S]methionine, and LPL was immunoprecipitated with an LPL-specific antibody. A pulse-chase experiment identified the cellular and secreted forms of LPL as a 55-57-kDa protein. In the presence of heparin, there was a large increase in secretion of newly synthesized LPL from the cells, although heparin did not stimulate cellular LPL synthetic rate. When cells were exposed to insulin for 2 h, pulse-labeling revealed that insulin stimulated a maximal dose-related increase in LPL synthetic rate of 300% of control. This increase in LPL synthetic rate was observed after an exposure to insulin for as little as 60 min and was accompanied by only a 10-25% increase in total protein synthesis. In addition, insulin had no effect on the turnover of intracellular LPL. Using a cDNA probe for LPL, insulin induced a 2-fold increase in the LPL mRNA. Thus, insulin stimulated an increase in specific LPL mRNA in isolated rat adipocytes. This increase in LPL mRNA then leads to an increase in the synthetic rate of the LPL protein.     

Cachectin/tumor necrosis factor decreases human adipose tissue lipoprotein lipase mRNA levels, synthesis, and activity

The effects of the cytokine cachectin/tumor necrosis factor (TNF) on human adipose tissue lipoprotein lipase (LPL) were studied. TNF is produced by activated macrophages and is thought to play a role in mediating hypertriglyceridemia and wasting of adipose tissue triglyceride stores (cachexia) that often accompany infection and malignancy. TNF effects were studied in human adipose tissue fragments maintained in organ culture in the presence of insulin and dexamethasone to induce high LPL activity. Addition of TNF to the culture medium for 20 h caused a dose-dependent inhibition of LPL activity to an average of 37% of controls at 50 U/ml TNF. This inhibition of LPL activity was explained by specific decreases in levels of LPL mRNA (to 40% of controls) and rates of LPL synthesis determined by biosynthetic labeling and immunoprecipitation (to 32% of controls). The decline in LPL synthesis was specific, as it occurred despite a small increase in overall protein synthesis in the presence of TNF. Comparable decreases in LPL activity were observed when TNF was added to adipose tissue cultured solely in the presence of insulin. Thus, similar to results in rodent models, TNF is a potent inhibitor of LPL gene expression in human adipose tissue. TNF may therefore play a role in the disorders of triglyceride catabolism and the pathogenesis of cachexia that occur with stimulation of the immune system in humans.     

The role of glucose and glycosylation in the regulation of lipoprotein lipase synthesis and secretion in rat adipocytes

Several studies have suggested that insulin and glucose increase adipose tissue lipoprotein lipase (LPL). To study the mechanism of the glucose-induced stimulation of LPL, the effects of glucose and glycosylation were examined in primary rat adipocyte cultures. In cells cultured in the presence of 1 mg/ml glucose, a 55-kDa LPL protein was synthesized and secreted into the medium, whereas cells cultured in glucose-free medium synthesized a 49-kDa form of LPL which was not secreted. The treatment of the mature 55-kDa form of LPL with peptide:N-glycosidase-F resulted in the formation of a 49-kDa form of LPL. When cells were cultured in the presence of tunicamycin, a 49-kDa form of LPL was synthesized by the cells but was not secreted. In addition, LPL activity was reduced by 90% when glycosylation was blocked by either tunicamycin or glucose deprivation. LPL synthetic rate was examined in cells cultured in a spectrum of glucose concentrations. LPL synthetic rate increased directly with medium glucose concentration and was decreased 80% in the absence of glucose compared to the synthetic rate in the presence of 1 mg/ml glucose. In addition, LPL synthetic rate in the presence of insulin was approximately 200% of the synthetic rate in untreated control cells at all glucose concentrations and even in the absence of glucose. In spite of the effect of glucose on LPL synthetic rate, glucose had no effect on the level of LPL mRNA. In contrast, the mRNA for the 78-kDa glucose-regulated protein (GRP78) was increased in adipocytes cultured in glucose-free medium. In summary, glucose was essential for glycosylation of LPL, and glycosylation was essential for LPL catalytic activity and secretion. In addition, glucose stimulated LPL synthetic rate and potentiated the stimulatory effects of insulin, but had no specific effect on LPL mRNA. Whereas insulin stimulates LPL by increasing the level of LPL mRNA, glucose stimulates LPL translation and post-translational processing.     

Improvement in Yield and Purity of a Recombinant Malaria Vaccine Candidate Based on the Receptor-Binding Domain of Plasmodium vivax Duffy Binding Protein by Codon Optimization

A recombinant blood-stage vaccine for Plasmodium vivax malaria based on the functional receptor-binding domain of PvDBP (PvRII) has been developed. A synthetic gene coding for PvRII was expressed in Escherichia coli using codon optimization. Expression level of recombinant PvRII was 10% of the total cellular proteins. Truncated PvRII products, seen when the native PvRII gene was expressed, were absent in case of synthetic gene.     

Apolipoproteins C-I and C-III Inhibit Lipoprotein Lipase Activity by Displacement of the Enzyme from Lipid Droplets

Apolipoproteins (apo) C-I and C-III are known to inhibit lipoprotein lipase (LPL) activity, but the molecular mechanisms for this remain obscure. We present evidence that either apoC-I or apoC-III, when bound to triglyceride-rich lipoproteins, prevent binding of LPL to the lipid/water interface. This results in decreased lipolytic activity of the enzyme. Site-directed mutagenesis revealed that hydrophobic amino acid residues centrally located in the apoC-III molecule are critical for attachment to lipid emulsion particles and consequently inhibition of LPL activity. Triglyceride-rich lipoproteins stabilize LPL and protect the enzyme from inactivating factors such as angiopoietin-like protein 4 (angptl4). The addition of either apoC-I or apoC-III to triglyceride-rich particles severely diminished their protective effect on LPL and rendered the enzyme more susceptible to inactivation by angptl4. These observations were seen using chylomicrons as well as the synthetic lipid emulsion Intralipid. In the presence of the LPL activator protein apoC-II, more of apoC-I or apoC-III was needed for displacement of LPL from the lipid/water interface. In conclusion, we show that apoC-I and apoC-III inhibit lipolysis by displacing LPL from lipid emulsion particles. We also propose a role for these apolipoproteins in the irreversible inactivation of LPL by factors such as angptl4.     

Inhibition of lipoprotein lipase by alkanesulfonyl fluorides

A number of alkanesulfonyl halides (chlorides and fluorides) and esters were synthesized and their effect on the activity of lipoprotein lipase (LPL) was studied. Sulfonyl fluorides proved to be efficient inhibitors of LPL when the enzyme was incubated with a 10-fold molar excess of the inhibitors in a buffer containing bile salts (deoxycholate). Hexadecane- and dodecanesulfonyl fluorides caused 50% inhibition of LPL activity at concentrations of 10 to 20 μM.     

Evidence for Two Distinct Binding Sites for Lipoprotein Lipase on Glycosylphosphatidylinositol-anchored High Density Lipoprotein-binding Protein 1 (GPIHBP1)

GPIHBP1 is an endothelial membrane protein that transports lipoprotein lipase (LPL) from the subendothelial space to the luminal side of the capillary endothelium. Here, we provide evidence that two regions of GPIHBP1, the acidic N-terminal domain and the central Ly6 domain, interact with LPL as two distinct binding sites. This conclusion is based on comparative binding studies performed with a peptide corresponding to the N-terminal domain of GPIHBP1, the Ly6 domain of GPIHBP1, wild type GPIHBP1, and the Ly6 domain mutant GPIHBP1 Q114P. Although LPL and the N-terminal domain formed a tight but short lived complex, characterized by fast on- and off-rates, the complex between LPL and the Ly6 domain formed more slowly and persisted for a longer time. Unlike the interaction of LPL with the Ly6 domain, the interaction of LPL with the N-terminal domain was significantly weakened by salt. The Q114P mutant bound LPL similarly to the N-terminal domain of GPIHBP1. Heparin dissociated LPL from the N-terminal domain, and partially from wild type GPIHBP1, but was unable to elute the enzyme from the Ly6 domain. When LPL was in complex with the acidic peptide corresponding to the N-terminal domain of GPIHBP1, the enzyme retained its affinity for the Ly6 domain. Furthermore, LPL that was bound to the N-terminal domain interacted with lipoproteins, whereas LPL bound to the Ly6 domain did not. In summary, our data suggest that the two domains of GPIHBP1 interact independently with LPL and that the functionality of LPL depends on its localization on GPIHBP1.     

Contribution of the carboxy-terminal domain of lipoprotein lipase to interaction with heparin and lipoproteins

The C-terminal domain of lipoprotein lipase (LPL) is involved in several important interactions. To assess its contribution to the binding ability of full-length LPL we have determined kinetic constants using biosensor technique. The affinity of the C-terminal domain for heparin was about 500-fold lower than that of full-length LPL (K(d) = 1.3 microM compared to 3.1 nM). Replacement of Lys403, Arg405 and Lys407 by Ala abolished the heparin affinity, whereas replacement of Arg420 and Lys422 had little effect. The C-terminal domain increased binding of chylomicrons and VLDL to immobilized heparin relatively well, but was less than 10% efficient in binding of LDL compared to full-length LPL. Deletion of residues 390-393 (WSDW) did not change the affinity to heparin and only slightly decreased the affinity to lipoproteins. We conclude that the C-terminal folding domain contributes only moderately to the heparin affinity of full-length LPL, whereas the domain appears important for tethering triglyceride-rich lipoproteins to heparin-bound LPL.     

Effects of protein and gene transfer of the angiopoietin-1 fibrinogen-like receptor-binding domain on endothelial and vessel organization

The vessel-stabilizing effect of angiopoietin-1 (Ang1)/Tie2 receptor signaling is a potential target for pro-angiogenic therapies as well as anti-angiogenic inhibition of tumor growth. We explored the endothelial and vascular specific activities of the Ang1 monomer, i.e. dissociated from its state as an oligomer. A truncated monomeric Ang1 variant (i.e. DeltaAng1) containing the isolated fibrinogen-like receptor-binding domain of Ang1 was created and recombinantly produced in insect cells. DeltaAng1 ligated the Tie2 receptor without triggering its phosphorylation. Moreover, monomeric DeltaAng1 was observed to bind alpha(5)beta(1) integrin with similar affinity compared with Tie2. Unexpectedly, in vitro treatment of endothelial cells with DeltaAng1 showed some of the known effects of full-length Ang1, including inhibition of basal endothelial cell permeability and stimulation of cell adhesion as well as activation of MAPKs. Local treatment of the microvasculature of the developing chicken chorioallantoic membrane with the DeltaAng1 protein led to profound reduction of the mean vascular length density, thinning of vessels, and reduction of the number of vessel branching points. Similar effects were observed in side-by-side experiments with the recombinant full-length Ang1 protein. These effects of simplification of the vessel branching pattern were confirmed through local gene transfer with lentiviral particles encoding DeltaAng1 or full-length Ang1. Together, our findings suggest a potential use for exogenous Ang1 in reducing rather than increasing vascular density. Furthermore, we show that the isolated receptor-binding domain of Ang1 is capable of mediating some effects of full-length Ang1 independently of Tie2 phosphorylation, possibly through integrin ligation.     

Role of leucine residues in the C-terminal region of human interleukin-6 in the biological activity.

Site-directed mutagenesis of two sets of three periodic leucine residues which appear at every seventh position in the C-terminal region of human interleukin-6 (IL-6) was performed. Both receptor-binding and immunoglobulin (Ig)-induction activities of a triple mutant Leu168,175,182-->Val were only 1% compared with those of wild-type IL-6. However, the mutant Leu152,159,166-->Val had 13% receptor-binding and 2% Ig-induction activities of those of wild-type IL-6. In order to obtain more direct information on the receptor-binding region, we prepared two synthetic peptides. A significant binding activity was observed for the peptide Leu168-Met185, but not for the peptide Leu152-Arg169. These results indicate that leucine residues in the C-terminal region, especially Leu168, Leu175, and Leu182, play an important role in the receptor-binding and Ig-induction activities.     

1,1'-bis(anilino)-4-,4'-bis(naphtalene)-8,8'-disulfonate acts as an inhibitor of lipoprotein lipase and competes for binding with apolipoprotein CII

Lipoprotein lipase (LPL) is dependent on apolipoprotein CII (apoCII), a component of plasma lipoproteins, for function in vivo. The hydrophobic fluorescent probe 1,1'-bis(anilino)-4,4'-bis(naphthalene)-8,8'-disulfonate (bis-ANS) was found to be a potent inhibitor of LPL. ApoCII prevented the inhibition by bis-ANS, and was also able to restore the activity of inhibited LPL in a competitive manner, but only with triacylglycerols with acyl chains longer than three carbons. Studies of fluorescence and surface plasmon resonance indicated that LPL has an exposed hydrophobic site for binding of bis-ANS. The high affinity interaction was characterized by an equilibrium constant Kd of 0.10-0.26 microm and by a relatively high on rate constant kass = 2.0 x 10(4) m(-1) s(-1) and a slow off-rate with a dissociation rate constant kdiss = 1.2 x 10(-4) s(-1). The high affinity binding of bis-ANS did not influence interaction of LPL with heparin or with lipid/water interfaces and did not dissociate the active LPL dimer into monomers. Analysis of fragments of LPL after photoincorporation of bis-ANS indicated that the high affinity binding site was located in the middle part of the N-terminal folding domain. We propose that bis-ANS binds to an exposed hydrophobic area that is located close to the active site. This area may be the binding site for individual substrate molecules and also for apoCII.     

Atorvastatin decreases lipoprotein lipase and endothelial lipase expression in human THP-1 macrophages

Macrophage-derived lipases are associated with atherosclerosis in human and animal studies. Despite numerous non-lipid-lowering effects of statins, their effect on macrophage LPL and endothelial lipase (EL) expression has not been investigated. In the present study, atorvastatin and simvastatin dose-dependently decreased LPL and EL expression as well as Rho, liver X receptor alpha (LXRalpha), and nuclear factor kappaB (NF-kappaB) activation in THP-1 macrophages. Atorvastatin-reduced LPL and EL expression was only partially recovered by mevalonate cotreatment, indicating that mechanisms independent of reductase inhibition may be present. By contrast, Rho activation by lysophosphatidyl acid further decreased LPL and EL expression in the presence or absence of atorvastatin. Another Rho activator, farnysyl pyrophosphate, decreased EL expression only in the absence of atorvastatin. LXRalpha activation by T0901317 and 22(R)-hydroxycholesterol not only rescued but also significantly increased LPL expression in the presence and absence of atorvastatin, respectively, whereas LXRalpha inhibition by 22(S)-hydroxycholesterol decreased LPL expression. By contrast, EL expression was suppressed by LXRalpha activation in the presence or absence of atorvastatin. NF-kappaB inhibition by SN50 was associated with an approximately 30% reduction of EL expression. Furthermore, atorvastatin treatment significantly attenuated the lipid accumulation in macrophages treated with oxidized LDL. We conclude that atorvastatin reduces LPL and EL expression by reducing the activation of LXRalpha and NF-kappaB, respectively.