Role of calnexin, calreticulin, and endoplasmic reticulum mannosidase I in apolipoprotein(a) intracellular targeting

Apolipoprotein(a) [apo(a)] is a component of atherogenic lipoprotein(a) [Lp(a)]. Differences in the extent of endoplasmic reticulum (ER) associated degradation (ERAD) of apo(a) allelic variants contribute to the >1000-fold variation in plasma Lp(a) levels. Using human apo(a) transgenic mouse hepatocytes, we analyzed the role of the ER chaperones calnexin (CNX) and calreticulin (CRT), and ER mannosidase I in apo(a) intracellular targeting. Co-immunoprecipitation and pulse-chase analyses revealed similar kinetics of apo(a) interaction with CNX and CRT, peaking 15-30 min after apo(a) synthesis. Trapping of apo(a) N-linked glycans in their monoglucosylated form, by posttranslational inhibition of ER glucosidase activity with castanospermine (CST), enhanced apo(a)-CNX/CRT interaction and prevented both apo(a) secretion and ERAD. Delay of CST addition until 20 or 30 min after apo(a) synthesis [when no apo(a) had yet undergone degradation or Golgi-specific carbohydrate modification] allowed a portion of apo(a) to be secreted or degraded. These results are consistent with a transient apo(a)-CNX/CRT association and suggest that events downstream of CNX/CRT interaction determine apo(a) intracellular targeting. Inhibition of ER mannosidase I with deoxymannojirimycin or kifunensine had no effect on apo(a) secretion, but inhibited proteasome-mediated apo(a) ERAD even under conditions where apo(a)-CNX/CRT interaction was prevented. These results suggest a role for an additional, mannose-specific, ER lectin in targeting secretory proteins to the proteasome for destruction.     

Effect of tranexamic acid and delta-aminovaleric acid on lipoprotein(a) metabolism in transgenic mice

The assembly of lipoprotein(a) (Lp(a)) is a two-step process which involves the interaction of kringle-4 (K-IV) domains in apolipoprotein(a) (apo(a)) with Lys groups in apoB-100. Lys analogues such as tranexamic acid (TXA) or delta-aminovaleric acid (delta-AVA) proved to prevent the Lp(a) assembly in vitro. In order to study the in vivo effect of Lys analogues, transgenic apo(a) or Lp(a) mice were treated with TXA or delta-AVA and plasma levels of free and low density lipoprotein bound apo(a) were measured. In parallel experiments, McA-RH 7777 cells, stably transfected with apo(a), were also treated with these substances and apo(a) secretion was followed. Treatment of transgenic mice with Lys analogues caused a doubling of plasma Lp(a) levels, while the ratio of free:apoB-100 bound apo(a) remained unchanged. In transgenic apo(a) mice a 1. 5-fold increase in plasma apo(a) levels was noticed. TXA significantly increased Lp(a) half-life from 6 h to 8 h. Incubation of McA-RH 7777 cells with Lys analogues resulted in an up to 1. 4-fold increase in apo(a) in the medium. The amount of intracellular low molecular weight apo(a) precursor remained unchanged. We hypothesize that Lys analogues increase plasma Lp(a) levels by increasing the dissociation of cell bound apo(a) in combination with reducing Lp(a) catabolism.     

Sortilin enhances secretion of apolipoprotein(a) through effects on apolipoprotein B secretion and promotes uptake of lipoprotein(a)

Elevated plasma lipoprotein(a) (Lp(a)) is an independent, causal risk factor for atherosclerotic cardiovascular disease and calcific aortic valve stenosis. Lp(a) is formed in or on hepatocytes from successive noncovalent and covalent interactions between apo(a) and apoB, although the subcellular location of these interactions and the nature of the apoB-containing particle involved remain unclear. Sortilin, encoded by the SORT1 gene, modulates apoB secretion and LDL clearance. We used a HepG2 cell model to study the secretion kinetics of apo(a) and apoB. Overexpression of sortilin increased apo(a) secretion, while siRNA-mediated knockdown of sortilin expression correspondingly decreased apo(a) secretion. Sortilin binds LDL but not apo(a) or Lp(a), indicating that its effect on apo(a) secretion is likely indirect. Indeed, the effect was dependent on the ability of apo(a) to interact noncovalently with apoB. Overexpression of sortilin enhanced internalization of Lp(a), but not apo(a), by HepG2 cells, although neither sortilin knockdown in these cells or Sort1 deficiency in mice impacted Lp(a) uptake. We found several missense mutations in SORT1 in patients with extremely high Lp(a) levels; sortilin containing some of these mutations was more effective at promoting apo(a) secretion than WT sortilin, though no differences were found with respect to Lp(a) internalization. Our observations suggest that sortilin could play a role in determining plasma Lp(a) levels and corroborate in vivo human kinetic studies which imply that secretion of apo(a) and apoB are coupled, likely within the hepatocyte.     

Apolipoprotein[a] secretion from hepatoma cells is regulated in a size-dependent manner by alterations in disulfide bond formation

Apolipoprotein[a] (apo[a]) is a large disulfide linked glycoprotein synthesized by hepatocytes. We have examined the role of disulfide bond formation in the processing of apo[a] using human and rat hepatoma cells expressing apo[a] isoforms containing varying numbers of kringle 4 (K4) domains, following treatment with DTT. Hepatoma cells expressing 6- or 9-K4 isoforms revealed approximately 90% inhibition of apo[a] secretion following DTT treatment, although larger isoforms containing 13- or 17-K4 domains demonstrated continued secretion (up to 30% of control values), suggesting that a fraction of the larger isoforms is at least partially DTT resistant. Wash-out experiments demonstrated that these effects were completely reversible for all isoforms studied, with no enhanced degradation associated with prolonged intracellular retention. DTT treatment was associated with enhanced binding of apo[a] with the endoplasmic reticulum-associated chaperone proteins calnexin, calreticulin, and BiP, which was reversible upon DTT removal. The chemical chaperone 6-aminohexanoic acid, previously demonstrated by others to rescue defective apo[a] secretion associated with alterations in glycosylation, failed to alter the secretion of apo[a] following DTT treatment. The demonstration that DTT modulates apo[a] secretion in a manner influenced by both the type and number of K4 repeats extends understanding of the mechanisms that regulate its exit from the endoplasmic reticulum.     

Definition of the structural elements in plasminogen required for high-affinity binding to apolipoprotein(a): a study utilizing surface plasmon resonance

Lipoprotein(a) [Lp(a)] is suggested to link atherosclerosis and thrombosis owing to the similarity between the apolipoprotein(a) [apo(a)] moiety of Lp(a) and plasminogen. Lp(a) may interfere with tPA-mediated plasminogen activation in fibrinolysis, thereby generating a hypercoaguable state in vivo. The present study employed surface plasmon resonance (SPR) to examine the binding interaction between plasminogen and a physiologically relevant, 17-kringle recombinant apo(a) species [17K r-apo(a)] in real time. Native, intact Glu(1)-plasminogen bound to apo(a) with substantially higher affinity (K(D) approximately 0.3 microM) compared to a series of plasminogen fragments (K1-5, K1-3, K4, K5P, and tail domain) that interacted weakly with apo(a) (K(D) > 50 microM). Treatment of Glu(1)-plasminogen with citraconic anhydride (a lysine modification reagent) completely abolished binding to wild-type 17K r-apo(a), whereas citraconylated 17K r-apo(a) decreased binding to wild-type Glu(1)-plasminogen by approximately 50%; inhibition of binding was also observed using the lysine analogue epsilon-aminocaproic acid. Whereas native Glu(1)-plasminogen exhibited monophasic binding to 17K r-apo(a), truncated Lys(78)-plasminogen exhibited biphasic binding. Altering Glu(1)-plasminogen from its native, closed conformation (in chloride buffer) to an open conformation (in acetate buffer) also yielded biphasic isotherms. These SPR data are consistent with a two-state kinetic model in which a conformational change in the plasminogen-apo(a) complex may occur following the initial binding event. Differential binding kinetics between Glu(1)-/Lys(78)-plasminogen and apo(a) may explain why Lp(a) is a stronger inhibitor of tPA-mediated Glu(1)-plasminogen activation compared to Lys(78)-plasminogen activation.     

Mutation of lysine residues in apolipoprotein B-100 causes defective lipoprotein[a] formation

Lipoprotein[a] (Lp[a]) is assembled by a two-step process involving an initial lysine-dependent binding between apolipoprotein B-100 (apoB-100) and apolipoprotein[a] (apo[a]) that facilitates the formation of a disulphide bond between apoB-100Cys4,326 and apo[a]Cys4,057. Previous studies of transgenic mice expressing apoB-95 (4,330 amino acids) and apoB-97 (4,397 amino acids) have shown that apoB-100 amino acids 4,330-4,397 are important for the initial binding to apo[a]. Furthermore, a lysine-rich peptide spanning apoB-100 amino acids 4,372-4,392 has recently been shown to bind apo[a] and inhibit Lp[a] assembly in vitro. This suggests that a putative apo[a] binding site exists in the apoB-4,372-4,392 region. The aim of our study was to establish whether the apoB-4,372-4,392 sequence was important for Lp[a] assembly in the context of the full-length apoB-100. Transgenic mice were created that expressed a mutant human apoB-100, apoB-100K4-->S4, in which all four lysine residues in the 4,372-4,392 sequence were mutated to serines. The apoB-100K4-->S4 mutant showed a reduced capacity to form Lp[a] in vitro compared with wild-type human apoB-100. Double transgenic mice expressing both apoB-100K4-->S4 and apo[a] contained significant amounts of free apo[a] in the plasma, indicating a less-efficient assembly of Lp[a] in vivo. Taken together, these results clearly show that the apoB-4,372-4,392 sequence plays a role in Lp[a] assembly.     

Lipoprotein(a) accelerates atherosclerosis in uremic mice

Uremic patients have increased plasma lipoprotein(a) [Lp(a)] levels and elevated risk of cardiovascular disease. Lp(a) is a subfraction of LDL, where apolipoprotein(a) [apo(a)] is disulfide bound to apolipoprotein B-100 (apoB). Lp(a) binds oxidized phospholipids (OxPL), and uremia increases lipoprotein-associated OxPL. Thus, Lp(a) may be particularly atherogenic in a uremic setting. We therefore investigated whether transgenic (Tg) expression of human Lp(a) increases atherosclerosis in uremic mice. Moderate uremia was induced by 5/6 nephrectomy (NX) in Tg mice with expression of human apo(a) (n = 19), human apoB-100 (n = 20), or human apo(a) + human apoB [Lp(a)] (n = 15), and in wild-type (WT) controls (n = 21). The uremic mice received a high-fat diet, and aortic atherosclerosis was examined 35 weeks later. LDL-cholesterol was increased in apoB-Tg and Lp(a)-Tg mice, but it was normal in apo(a)-Tg and WT mice. Uremia did not result in increased plasma apo(a) or Lp(a). Mean atherosclerotic plaque area in the aortic root was increased 1.8-fold in apo(a)-Tg (P = 0.025) and 3.3-fold (P = 0.0001) in Lp(a)-Tg mice compared with WT mice. Plasma OxPL, as detected with the E06 antibody, was associated with both apo(a) and Lp(a). In conclusion, expression of apo(a) or Lp(a) increased uremia-induced atherosclerosis. Binding of OxPL on apo(a) and Lp(a) may contribute to the atherogenicity of Lp(a) in uremia.     

Quantitative evaluation of the contribution of weak lysine-binding sites present within apolipoprotein(a) kringle IV types 6-8 to lipoprotein(a) assembly

During lipoprotein(a) (Lp(a)) assembly, non-covalent interactions between apolipoprotein(a) (apo(a)) and low density lipoprotein precede specific disulfide bond formation. Studies have shown that the non-covalent step involves an interaction between the weak lysine-binding sites (WLBS) present within each of apo(a) kringle IV types 6, 7, and 8 (KIV(6-8)), and two lysine residues (Lys(680) and Lys(690)) within the NH(2) terminus of the apolipoprotein B-100 (apoB) component of low density lipoprotein. In the present study, we introduced single point mutations (E56G) into each of the WLBS present in apo(a) KIV(6-8) and expressed these mutations in the context of a 17-kringle (17K) recombinant apo(a) variant. Single mutations that disrupt the WLBS in KIV(6), KIV(7), and KIV(8), as well as mutants that disrupt the WLBS in both KIV(6) and KIV(7), or both KIV(7) and KIV(8), were assessed for their ability to form non-covalent and covalent Lp(a) complexes. Our results demonstrate that both apo(a) KIV(7) and KIV(8), but not KIV(6), are required for maximally efficient non-covalent and covalent Lp(a) assembly. Single mutations in the WLBS of KIV(7) or KIV(8) resulted in a 3-fold decrease in the affinity of 17K recombinant apo(a) for apoB, and a 20% reduction in the rate of covalent Lp(a) formation. Tandem mutations in the WLBS in both KIV(7) and KIV(8) resulted in a 13-fold reduction in the binding affinity between apo(a) and apoB, and a 75% reduction in the rate of the covalent step of Lp(a) formation. We also showed that KIV(7) and KIV(8) specifically bind with high affinity to apoB-derived peptides containing Lys(690) or Lys(680), respectively. Taken together, our data demonstrate that specific interactions between apo(a) KIV(7) and KIV(8) and Lys(680) and Lys(690) in apoB mediate a high affinity non-covalent interaction between apo(a) and low density lipoprotein, which dictates the efficiency of covalent Lp(a) formation.     

Interaction of apolipoprotein(a) with apolipoprotein B-containing lipoproteins

Recombinant DNA-derived apolipoprotein(a) was used to demonstrate that the apo(a) moiety of lipoprotein(a) (Lp(a)) is responsible for the binding of Lp(a) to other apolipoprotein B-containing lipoproteins (apoB-Lp) including LDL2, a subclass of low density lipoproteins (d = 1.030-1.063 g/ml). The r-apo(a).LDL2 complexes exhibited the same binding constant as Lp(a).LDL2 (10(-8) M). Treatment of either recombinant apo(a) or Lp(a) with a reducing agent destroyed binding activity. A synthetic polypeptide corresponding to a portion of apo(a)'s kringle-4 inhibited the binding (K1 = 1.9 x 10(-4) M) of LDL2 to Lp(a). Therefore, we concluded that binding to apoB-Lp was mediated by the kringle-4-like domains on apo(a). Using ligand chromatography which can detect complexes having a KD as low as 10(-2) M, we demonstrated the binding of plasminogen to apoB-Lp. Like Lp(a), binding of plasminogen to apoB-Lp was mediated by the kringle domain(s). The differences in binding affinity may be due to amino acid substitutions in the kringle-4-like domain. In most of the kringle-4-like domains of apo(a), the aspartic residue critical for binding to lysine was substituted by valine. Consistent with this substitution, we found that L-proline and hydroxyproline, but not L-lysine, inhibited the binding of LDL2 to apo(a). Inhibition by L-proline could be reversed in the binding studies by increasing the amount of apo(a); and L-proline-Sepharose bound plasma Lp(a), suggesting that L-proline acted as a ligand for the kringle-4-like domain(s) of apo(a) involved in the binding of apoB-Lp. The binding of apo(a) to proline and hydroxyproline could be responsible for the binding of apo(a) to the subendothelial extracellular matrix, i.e. domains of proteins rich in proline or hydroxyproline (e.g. collagen and elastin).     

Effect of tranexamic acid and δ-aminovaleric acid on lipoprotein(a) metabolism in transgenic mice

The assembly of lipoprotein(a) (Lp(a)) is a two-step process which involves the interaction of kringle-4 (K-IV) domains in apolipoprotein(a) (apo(a)) with Lys groups in apoB-100. Lys analogues such as tranexamic acid (TXA) or δ-aminovaleric acid (δ-AVA) proved to prevent the Lp(a) assembly in vitro. In order to study the in vivo effect of Lys analogues, transgenic apo(a) or Lp(a) mice were treated with TXA or δ-AVA and plasma levels of free and low density lipoprotein bound apo(a) were measured. In parallel experiments, McA-RH 7777 cells, stably transfected with apo(a), were also treated with these substances and apo(a) secretion was followed. Treatment of transgenic mice with Lys analogues caused a doubling of plasma Lp(a) levels, while the ratio of free:apoB-100 bound apo(a) remained unchanged. In transgenic apo(a) mice a 1.5-fold increase in plasma apo(a) levels was noticed. TXA significantly increased Lp(a) half-life from 6 h to 8 h. Incubation of McA-RH 7777 cells with Lys analogues resulted in an up to 1.4-fold increase in apo(a) in the medium. The amount of intracellular low molecular weight apo(a) precursor remained unchanged. We hypothesize that Lys analogues increase plasma Lp(a) levels by increasing the dissociation of cell bound apo(a) in combination with reducing Lp(a) catabolism.     

Mixed-disulfide folding intermediates between thyroglobulin and endoplasmic reticulum resident oxidoreductases ERp57 and protein disulfide isomerase

We present the first identification of transient folding intermediates of endogenous thyroglobulin (Tg; a large homodimeric secretory glycoprotein of thyrocytes), which include mixed disulfides with endogenous oxidoreductases servicing Tg folding needs. Formation of disulfide-linked Tg adducts with endoplasmic reticulum (ER) oxidoreductases begins cotranslationally. Inhibition of ER glucosidase activity blocked formation of a subgroup of Tg adducts containing ERp57 while causing increased Tg adduct formation with protein disulfide isomerase (PDI), delayed adduct resolution, perturbed oxidative folding of Tg monomers, impaired Tg dimerization, increased Tg association with BiP/GRP78 and GRP94, activation of the unfolded protein response, increased ER-associated degradation of a subpopulation of Tg, partial Tg escape from ER quality control with increased secretion of free monomers, and decreased overall Tg secretion. These data point towards mixed disulfides with the ERp57 oxidoreductase in conjunction with calreticulin/calnexin chaperones acting as normal early Tg folding intermediates that can be "substituted" by PDI adducts only at the expense of lower folding efficiency with resultant ER stress.     

The apolipoprotein(a) component of lipoprotein(a) mediates binding to laminin: contribution to selective retention of lipoprotein(a) in atherosclerotic lesions

Lipoprotein(a) [Lp(a)] entrapment by vascular extracellular matrix may be important in atherogenesis. We sought to determine whether laminin, a major component of the basal membrane, may contribute to Lp(a) retention in the arterial wall. First, immunohistochemistry experiments were performed to examine the relative distribution of Lp(a) and laminin in human carotid artery specimens. There was a high degree of co-localization of Lp(a) and laminin in atherosclerotic specimens, but not in non-atherosclerotic sections. We then studied the binding interaction between Lp(a) and laminin in vitro. ELISA experiments showed that native Lp(a) particles and 17K and 12K recombinant apolipoprotein(a) [r-apo(a)] variants interacted strongly with laminin whereas LDL, apoB-100, and the truncated KIV(6-P), KIV(8-P), and KIV(9-P) r-apo(a) variants did not. Overall, the ELISA data demonstrated that Lp(a) binding to laminin is mediated by apo(a) and a combination of the lysine analogue epsilon-aminocaproic acid and salt effectively decreases apo(a) binding to laminin. Secondary binding analyses with 125I-labeled r-apo(a) revealed equilibrium dissociation constants (K(d)) of 180 and 360 nM for the 17K and 12K variants binding to laminin, respectively. Such similar K(d) values between these two r-apo(a) variants suggest that isoform size does not appear to influence apo(a) binding to laminin. In summary, our data suggest that laminin may bind to apo(a) in the atherosclerotic intima, thus contributing to the selective retention of Lp(a) in this milieu.     

Catalysis of covalent Lp(a) assembly: evidence for an extracellular enzyme activity that enhances disulfide bond formation

The assembly of lipoprotein(a) (Lp(a)) particles occurs via a two-step mechanism in which noncovalent interactions between apolipoprotein(a) (apo(a)) and the apolipoproteinB-100 component of low density lipoprotein precede the formation of a single disulfide bond. Although we have previously demonstrated that the rate constant for the covalent step of Lp(a) assembly can be enhanced by altering the conformational status of apo(a), the resultant rates of covalent Lp(a) particle formation measured in vitro are relatively slow. The large excess of Lp(a) (over apo(a)) observed in vivo can be accounted for by a preferential clearance of apo(a) over Lp(a) and/or a sufficiently high rate of covalent Lp(a) assembly. In the present study, we report that cultured human hepatoma cells secrete an oxidase activity that dramatically enhances the rate of covalent Lp(a) assembly. This activity is likely possessed by a protein because it is heat-sensitive and is retained in the concentrate following ultrafiltration through a 5 kDa cutoff filter. However, a small molecule cofactor for the activity is suggested by the observation that the activity is lost upon dialysis. Plots of Lp(a) assembly rate versus input apo(a) concentration gave rectangular hyperbolae; the reaction displayed an unusual dependence on the concentration of apoB-100, with increasing concentrations of apoB-100 resulting in slower rates of Lp(a) assembly at low concentrations of apo(a), an effect that was alleviated by higher apo(a) concentrations. Interestingly, V(max(app))/K(m(app)) ratios were insensitive to apoB-100 concentration, which is diagnostic of a ping-pong reaction mechanism. In this way, the putative Lp(a) oxidase may be functionally analogous to protein disulfide isomerase, which exhibits a similar mechanism during the catalysis of disulfide bond formation during protein folding, although we have ruled out a role for this enzyme in Lp(a) assembly.     

FGF21 inhibits apolipoprotein(a) expression in HepG2 cells via the FGFR1-ERK1/2-Elk-1 pathway

Lipoprotein(a) [Lp(a)] is a highly atherogenic lipoprotein, whose metabolism is poorly understood. Efficient and secure drugs that can lower elevated plasma Lp(a) concentrations are currently lacking. Fibroblast growth factor-21 (FGF-21), a member of the FGFS super family, regulates glucose and lipid metabolism in hepatocytes and adipocytes via FGFR-ERK1/2 signaling. In this study, we investigated the molecular mechanisms that influence apolipoprotein(a) [apo(a)] biosynthesis. We also determined the effects of FGF21 on HepG2 cell apo(a) expression and secretion, as well as the mechanism of FGF21 in these effects. Results showed that FGF21 inhibited apo(a) expression at both mRNA and protein levels in a dose- and time--dependent manner and then suppressed the secretion of apo(a). These effects were attenuated by PD98059 (ERK1/2 inhibitor) and Elk-1 siRNA. PD166866 (FGFR1 inhibitor) also attenuated the FGF21-mediated inhibition of apo(a) expression and inhibited ERK1/2 and Elk-1 activation. These results demonstrate that FGF21 suppresses apo(a) expression via the FGFR1-ERK1/2-Elk-1 pathway.     

TRAM1 is involved in disposal of ER membrane degradation substrates

ER quality control consists of monitoring protein folding and targeting misfolded proteins for proteasomal degradation. ER stress results in an unfolded protein response (UPR) that selectively upregulates proteins involved in protein degradation, ER expansion, and protein folding. Given the efficiency in which misfolded proteins are degraded, there likely exist cellular factors that enhance the export of proteins across the ER membrane. We have reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, participates in HCMV US2- and US11-mediated dislocation of MHC class I heavy chains (Oresic, K., Ng, C.L., and Tortorella, D. 2009). Consistent with the hypothesis that TRAM1 is involved in the disposal of misfolded ER proteins, cells lacking TRAM1 experienced a heightened UPR upon acute ER stress, as evidenced by increased activation of unfolded protein response elements (UPRE) and elevated levels of NF-κB activity. We have also extended the involvement of TRAM1 in the selective degradation of misfolded ER membrane proteins Cln6^M241T and US2, but not the soluble degradation substrate α₁-antitrypsin null_HK. These degradation model systems support the paradigm that TRAM1 is a selective factor that can enhance the dislocation of ER membrane proteins.     

Determinants of binding of oxidized phospholipids on apolipoprotein (a) and lipoprotein (a)

Oxidized phospholipids (OxPLs) are present on apolipoprotein (a) [apo(a)] and lipoprotein (a) [Lp(a)] but the determinants influencing their binding are not known. The presence of OxPLs on apo(a)/Lp(a) was evaluated in plasma from healthy humans, apes, monkeys, apo(a)/Lp(a) transgenic mice, lysine binding site (LBS) mutant apo(a)/Lp(a) mice with Asp^55/57→Ala^55/57 substitution of kringle (K)IV₁₀)], and a variety of recombinant apo(a) [r-apo(a)] constructs. Using antibody E06, which binds the phosphocholine (PC) headgroup of OxPLs, Western and ELISA formats revealed that OxPLs were only present in apo(a) with an intact KIV₁₀ LBS. Lipid extracts of purified human Lp(a) contained both E06- and nonE06-detectable OxPLs by tandem liquid chromatography-mass spectrometry (LC-MS/MS). Trypsin digestion of 17K r-apo(a) showed PC-containing OxPLs covalently bound to apo(a) fragments by LC-MS/MS that could be saponified by ammonium hydroxide. Interestingly, PC-containing OxPLs were also present in 17K r-apo(a) with Asp⁵⁷→Ala⁵⁷ substitution in KIV₁₀ that lacked E06 immunoreactivity. In conclusion, E06- and nonE06-detectable OxPLs are present in the lipid phase of Lp(a) and covalently bound to apo(a). E06 immunoreactivity, reflecting pro-inflammatory OxPLs accessible to the immune system, is strongly influenced by KIV₁₀ LBS and is unique to human apo(a), which may explain Lp(a)’s pro-atherogenic potential.     

Degradation of newly synthesized apolipoprotein B-100 in a pre-Golgi compartment

The synthesis and secretion of apolipoprotein (apo) B-100 have been studied in a human hepatoblastoma cell line, the Hep G2 cells. Pulse-chase analysis showed that apoB-100 was not quantitatively recovered in the culture medium. To reveal the intracellular degradation of apoB-100 prior to secretion, cells were incubated with 1 microgram/ml Brefeldin A (BFA) which impeded protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus and the fate of apoB-100 retained in the cells was traced at 37 degrees C. A significant amount of intracellular apoB-100 (40-60%/h) was degraded during the chase period, whereas apoA-1 remained intact. ApoB-100 degradation was temperature dependent, no degradation was observed below 20 degrees C. This degradation process was not inhibited by chloroquine, leupeptin, pepstatin, and chymostatin, suggesting that lysosomal proteases were not involved and that apoB-100 was degraded in a pre-Golgi compartment which is either part of, or closely related to, the ER. Preincubation of cells with low density lipoproteins (LDL) induced a 22-32% increase in the degradation of apoB-100. This result raised the possibility that secretion of apoB-100 might be regulated through the intracellular degradation of apoB-100. These results suggest the existence of the degradation pathway for apoB-100 in a pre-Golgi compartment and an unique regulatory mechanism for apoB-100 secretion.     

IL-6 and lipoprotein(a) [LP(a)] concentrations are related only in patients with high APO(a) isoforms in monoclonal gammopathy

We have investigated the influence of apo(a) genetics on the relationship between interleukin (IL)-6, and lipoprotein (a) [Lp(a)] levels in 154 patients with monoclonal gammopathy and 189 healthy subjects. No significant differences in Lp(a) levels and distribution of subjects with different sizes of apo(a) isoforms were found between patients and healthy controls. Relationship between IL-6 and Lp(a) levels was strongly dependent on the size of apo(a) isoforms. In patients with high-size apo(a) isoforms Lp(a) levels positively correlated (r=0.475, P=0.0007) to IL-6 concentrations, whereas no correlation was found in patients with low apo(a) isoforms. Our present finding may provide a plausible explanation for the contradictory findings about the acute phase protein nature of Lp(a).     

Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding

Lipoprotein (a) [Lp(a)] belongs to the class of highly thrombo-atherogenic lipoproteins. The assembly of Lp(a) from LDL and the specific apo(a) glycoprotein takes place extracellularly in a two-step process. First, an unstable complex is formed between LDL and apo(a) due to the interaction of the unique kringle (K) IV-type 6 (T6) in apo(a) with amino groups on LDL, and in the second step this complex is stabilized by a disulfide bond between apo(a) KIV-T9 and apoB(100). In order to understand this process better, we overexpressed and purified apo(a) KIV-T6 in Escherichia coli. Recombinant KIV-T6 was expressed as a His-tag fusion protein under control of the T7 promoter in BL21 (DE3) strain. After one-step purification by affinity chromatography the yield was 7 mg/l of bacterial suspension. Expressed fusion apo(a) KIV-T6 was insoluble in physiological buffers and it also lacked the characteristic kringle structure. After refolding using a specific procedure, high-resolution (1)H-NMR spectroscopy revealed kringle structure-specific signals. Refolded KIV-T6 bound to Lys-Sepharose with a significantly lower affinity than recombinant apo(a) (EC(50) with epsilon-ACA 0.47 mM versus 2-11 mM). In competition experiments a 1000-fold molar excess of KIV-T6 was needed to reach 60% inhibition of Lp(a) assembly.     

Intracellular supply of phospholipids for biliary secretion: evidence for a nonvesicular transport component

Phospholipids (PL) for biliary secretion could be supplied from the endoplasmic reticulum (ER) to the plasma membrane by cytosolic transfer proteins or transport vesicles. Therefore, we studied whether biliary secretions of PL and apolipoprotein A-I (apo A-I), as markers for the ER-to-Golgi vesicular transport pathway, are tightly coupled in isolated perfused rat livers with enhanced secretion (+60%) of PL after withdrawal of the cholesterol synthesis inhibitor pravastatin (0.1% of chow, fed for 7 days). Blocking agents dissociated the secretion of apo A-I and PL. Brefeldin A as well as cycloheximide inhibited biliary secretion of apo A-I (-52%; -68%), however, not of PL. Both bilirubin ditaurate and taurodehydrocholic acid reduced biliary secretion of PL (-27%; -79%), but not of apo A-I. The data support the concept that PL destined for biliary secretion bypass the vesicular transport pathway of apo A-I through the Golgi compartment, most likely via cytosolic transfer proteins.