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.     

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.     

Apolipoprotein(a) stimulates vascular endothelial cell growth and migration and signals through integrin alphaVbeta3

Elevated plasma concentrations of Lp(a) [lipoprotein(a)] are an emerging risk factor for atherothrombotic disease. Apo(a) [apolipoprotein(a)], the unique glycoprotein component of Lp(a), contains tandem repeats of a plasminogen kringle (K) IV-like domain. In the light of recent studies suggesting that apo(a)/Lp(a) affects endothelial function, we evaluated the effects of apo(a)/Lp(a) on growth and migration of cultured HUVECs (human umbilical-vein endothelial cells). Two full-length r-apo(a) [recombinant apo(a)] variants (12K and 17K), as well as Lp(a), were able to stimulate HUVEC growth and migration to a comparable extent; 17K r-apo(a) also decreased the levels of total and active transforming growth factor-beta secreted by these cells. Using additional r-apo(a) variants corresponding to deletions and/or site-directed mutants of various kringle domains in the molecule, we were able to determine that the observed effects of full-length r-apo(a) on HUVECs were dependent on the presence of a functional lysine-binding site(s) in the apo(a) molecule. With respect to signalling events elicited by apo(a) in HUVECs, we found that 17K treatment of the cells increased the phosphorylation level of FAK (focal adhesion kinase) and MAPKs (mitogen-activated protein kinases), including ERK (extracellular-signal-regulated kinase), p38 and JNK (c-Jun N-terminal kinase). In addition, we showed that LM609, the function-blocking antibody to integrin alphaVbeta3, abrogated the effects of 17K r-apo(a) and Lp(a) on HUVECs. Taken together, the results of the present study suggest that the apo(a) component of Lp(a) signals through integrin alphaVbeta3 to activate endothelial cells.     

Antifibrinolytic effect of single apo(a) kringle domains: relationship to fibrinogen binding

Elevated plasma concentrations of lipoprotein(a) [Lp(a)] are associated with an increased risk for the development of atherosclerotic disease which may be attributable to the ability of Lp(a) to attenuate fibrinolysis. A generally accepted mechanism for this effect involves direct competition of Lp(a) with plasminogen for fibrin(ogen) binding sites thus reducing the efficiency of plasminogen activation. Efforts to determine the domains of apolipoprotein(a) [apo(a)] which mediate fibrin(ogen) interactions have yielded conflicting results. Thus, the purpose of the present study was to determine the ability of single KIV domains of apo(a) to bind plasmin-treated fibrinogen surfaces as well to determine their effect on fibrinolysis using an in vitro clot lysis assay. A bacterial expression system was utilized to express and purify apo(a) KIV (2), KIV (7), KIV (9) DeltaCys (which lacks the seventh unpaired cysteine) and KIV (10) which contains a strong lysine binding site. We also expressed and examined three mutant derivatives of KIV (10) to determine the effect of changing critical residues in the lysine binding site of this kringle on both fibrin(ogen) binding and fibrin clot lysis. Our results demonstrate that the strong lysine binding site in apo(a) KIV (10) is capable of mediating interactions with plasmin-modified fibrinogen in a lysine-dependent manner, and that this kringle can increase in vitro fibrin clot lysis time by approximately 43% at a concentration of 10 microM KIV (10). The ability of the KIV (10) mutant derivatives to bind plasmin-modified fibrinogen correlated with their lysine binding capacity. Mutation of Trp (70) to Arg abolished binding to both lysine-Sepharose and plasmin-modified fibrinogen, while the Trp (70) -->Phe and Arg (35) -->Lys substitutions each resulted in decreased binding to these substrates. None of the KIV (10) mutant derivatives appeared to affect fibrinolysis. Apo(a) KIV (7) contains a lysine- and proline-sensitive site capable of mediating binding to plasmin-modified fibrinogen, albeit with a lower apparent affinity than apo(a) KIV (10). However, apo(a) KIV (7) had no effect on fibrinolysis in vitro. Apo(a) KIV (2) and KIV (9) DeltaCys did not bind measurably to plasmin-modified fibrinogen surfaces and did not affect fibrinolysis in vitro.     

Characterization of the interaction of recombinant apolipoprotein(a) with modified fibrinogen surfaces and fibrin clots.

Elevated levels of lipoprotein(a) [Lp(a)] in plasma are a significant risk factor for the development of atherosclerotic disease, a property which may arise from the ability of this lipoprotein to inhibit fibrinolysis. In the present study we have quantitated the binding of recombinant forms of apolipoprotein(a) [17K and 12K r-apo(a); containing 8 and 3 copies, respectively, of the major repeat kringle sequence (kringle IV type 2)] to modified fibrinogen surfaces. Iodinated 17K and 12K r-apo(a) bound to immobilized thrombin-modified fibrinogen (i.e., fibrin) surfaces with similar affinities (Kd approximately 1.2-1.6 microM). The total concentration of binding sites (Bmax) present on the fibrin surface was approximately 4-fold greater for the 12K than for the 17K (Bmax values of 0.81 +/- 0.09 nM, and 0.20 +/- 0.01 nM respectively), suggesting that the total binding capacity on fibrin surfaces is reduced for larger apolipoprotein(a) (apo(a)) species. Interestingly, binding of apo(a) to intact fibrin was not detected as assessed by measurement of intrinsic fluorescence of free apo(a) present in the supernatants of sedimented fibrin clots. In other experiments, the total concentration apo(a) binding sites available on plasmin-modified fibrinogen surfaces was shown to be 13.5-fold higher than the number of sites available on unmodified fibrin surfaces (Bmax values of 2.7 +/- 0.3 nM and 0.20 +/- 0.01 nM respectively) while the affinity of apo(a) for these surfaces was similar. The increase in Bmax was correlated with plasmin-mediated exposure of C-terminal lysines since treatment of plasmin-modified fibrinogen surfaces with carboxypeptidase B produced a significant decrease in total binding signal as detected by ELISA (enzyme linked immunosorbent assay). Taken together, these data suggest that apo(a) binds to fibrin with poor affinity (low microM) and that the total concentration of apo(a) binding sites available on modified-fibrinogen surfaces is affected by both apo(a) isoform size and by the increased availability of C-terminal lysines on plasmin-degraded fibrinogen surfaces. However, the low affinity of apo(a) for fibrin indicates that Lp(a) may inhibit fibrinolysis through a mechanism distinct from binding to fibrin, such as binding to plasminogen.     

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.     

Baboon lipoprotein(a) binds very weakly to lysine-agarose and fibrin despite the presence of a strong lysine-binding site in apolipoprotein(a) kringle IV type 10

Human apolipoprotein(a) kringle IV type 10 [apo(a) KIV(10)] contains a strong lysine-binding site (LBS) that mediates the interaction of Lp(a) with biological substrates such as fibrin. Mutations in the KIV(10) LBS have been reported in both the rhesus monkey and chimpanzee, and have been proposed to explain the lack of ability of the corresponding Lp(a) species to bind to lysine and fibrin. To further the comparative analyses with other primate species, we sequenced a segment of baboon liver apo(a) cDNA spanning KIV(9) through the protease domain. Like rhesus monkey apo(a), baboon apo(a) lacks a kringle V (KV)-like domain. Interestingly, we found that the baboon apo(a) KIV(10) sequence contains all of the canonical LBS residues. We sequenced the apo(a) KIV(10) sequence from an additional 10 unrelated baboons; 17 of 20 alleles encoded Trp at position 70, whereas only two alleles encoded Arg at this position and thus a defective LBS. Despite the apparent presence of a functional KIV(10) LBS in most of the baboons, none of the Lp(a) in the plasma of the corresponding baboons bound specifically to lysine-Sepharose (agarose) even upon partial purification. Moreover, baboon Lp(a) bound very poorly to plasmin-modified fibrinogen. Expression of baboon and human KIV(10) in bacteria allowed us to verify that these domains bind comparably to lysine and lysine analogues. We conclude that presentation of KIV(10) in the context of apo(a) lacking KV may interfere with the ability of KIV(10) to bind to substrates such as fibrin; this paradigm may also be present in other non-human primates.     

Oxidation of apolipoprotein(a) inhibits kringle-associated lysine binding: the loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site.

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein complex consisting of apolipoprotein(a) [apo(a)] disulfide-linked to apolipoprotein B-100. Lp(a) has been implicated in atherogenesis and thrombosis through the lysine binding site (LBS) affinity of its kringle domains. We have examined the oxidative effect of 2,2'-azobis-(amidinopropane) HCl (AAPH), a mild hydrophilic free radical initiator, upon the ability of Lp(a) and recombinant apo(a), r-apo(a), to bind through their LBS domains. AAPH treatment caused a time-dependent decrease in the number of functional Lp(a) or r-apo(a) molecules capable of binding to fibrin or lysine-Sepharose and in the intrinsic protein fluorescence of both Lp(a) and r-apo(a). The presence of a lysine analogue during the reaction prevented the loss of lysine binding and provided a partial protection from the loss of tryptophan fluorescence. The partial protection of fluorescence by lysine analogues was observed in other kringle-containing proteins, but not in proteins lacking kringles. No significant aggregation, fragmentation, or change in conformation of Lp(a) or r-apo(a) was observed as assessed by native or SDS-PAGE, light scattering, retention of antigenicity, and protein fluorescence emission spectra. Our results suggest that AAPH destroys amino acids in the kringles of apo(a) that are essential for lysine binding, including one or more tryptophan residues. The present study, therefore, raises the possibility that the biological roles of Lp(a) may be mediated by its state of oxidation, especially in light of our previous study showing that the reductive properties of sulfhydryl-containing compounds increase the LBS affinity of Lp(a) for fibrin.     

Nuclear magnetic resonance (NMR) solution structure, dynamics, and binding properties of the kringle IV type 8 module of apolipoprotein(a)

The plasma lipoprotein lipoprotein(a) [Lp(a)] comprises a low-density lipoprotein (LDL)-like particle covalently attached to the glycoprotein apolipoprotein(a) [apo(a)]. Apo(a) consists of multiple tandem repeating kringle modules, similar to plasminogen kringle IV (designated KIV1-KIV10), followed by modules homologous to the kringle V module and protease domain of plasminogen. The apo(a) KIV modules have been classified on the basis of their binding affinity for lysine and lysine analogues. The strong lysine-binding apo(a) KIV10 module mediates lysine-dependent interactions with fibrin and cell-surface receptors. Weak lysine-binding apo(a) KIV7 and KIV8 modules display a 2-3-fold difference in lysine affinity and play a direct role in the noncovalent step in Lp(a) assembly through binding to unique lysine-containing sequences in apolipoproteinB-100 (apoB-100). The present study describes the nuclear magnetic resonance solution structure of apo(a) KIV8 and its solution dynamics properties, the first for an apo(a) kringle module, and compares the effects of epsilon-aminocaproic acid (epsilon-ACA) binding on the backbone and side-chain conformation of KIV7 and KIV8 on a per residue basis. Apo(a) KIV8 adopts a well-ordered structure that shares the general tri-loop kringle topology with apo(a) KIV6, KIV7, and KIV10. Mapping of epsilon-ACA-induced chemical-shift changes on KIV7 and KIV8 indicate that the same residues are affected, despite a 2-3-fold difference in epsilon-ACA affinity. A unique loop conformation within KIV8, involving hydrophobic interactions with Tyr40, affects the positioning of Arg35 relative to the lysine-binding site (LBS). A difference in the orientation of the aromatic side chains comprising the hydrophobic center of the LBS in KIV8 decreases the size of the hydrophobic cleft compared to other apo(a) KIV modules. An exposed hydrophobic patch contiguous with the LBS in KIV8 and not conserved in other weak lysine-binding apo(a) kringle modules may modulate specificity for regions within apoB-100. An additional ligand recognition site comprises a structured arginine-glycine-aspartate motif at the N terminus of the KIV8 module, which may mediate Lp(a)/apo(a)-integrin interactions.     

Apolipoprotein(a): expression and characterization of a recombinant form of the protein in mammalian cells

We have stably expressed a recombinant form of apo(a) in a human embryonic kidney cell line. The engineered protein (predicted mass of 250 kDa) contains 17 copies of the apo(a) domain, which resembles kringle 4 of plasminogen, followed by the plasminogen-like kringle 5 and protease-like domain of apo(a). The recombinant protein [r-apo(a)] was isolated from cell culture media by immunoaffinity chromatography, and its physical properties were studied. As is the case for apo(a) isolated from plasma-derived Lp(a), r-apo(a) is highly glycosylated (23% by weight), containing both N- and O-linked glycans, which results in an observed molecular mass of 500 kDa by SDS-PAGE. The high sialic acid content was reflected in a pI of 4.3 for the r-apo(a). Two subpopulations of r-apo(a) secreted by the permanent cell line were identified with respect to lysine-Sepharose binding; the majority of the r-apo(a) bound specifically to this matrix and was eluted with epsilon-aminocaproic acid (epsilon-ACA). When the r-apo(a) plasmid was used to transfect a human hepatoma cell line, lipoprotein particles were secreted containing the disulfide-linked complex of apoB-100 and the r-apo(a). The density of these particles was shown to be heterogeneous, with the majority of the r-Lp(a) floating in the density range of plasma-derived Lp(a).     

A role for apolipoprotein(a) in protection of the low-density lipoprotein component of lipoprotein(a) from copper-mediated oxidation

Low-density lipoprotein (LDL) oxidation is stimulated by copper. Addition of a recombinant form of apolipoprotein(a) (apo(a); the distinguishing protein component of lipoprotein(a)) containing 17 plasminogen kringle IV-like domains (17K r-apo(a)) protects LDL against oxidation by copper. Protection is specific to apo(a) and is not achieved by plasminogen or serum albumin. When Cu(2+) is added to 17K r-apo(a), its intrinsic fluorescence is quenched in a concentration-dependent and saturable manner. Quenching is unchanged whether performed aerobically or anaerobically and is reversible by ethylenediaminetetraacetate, suggesting that it is due to equilibrium binding of Cu(2+) and not to oxidative destruction of tryptophan residues. The fluorescence change exhibits a sigmoid dependence on copper concentration, and time courses of quenching are complex. At copper concentrations below 10 microM there is little quenching, whereas above 10 microM quenching proceeds immediately as a double-exponential decay. The affinity and kinetics of copper binding to 17K r-apo(a) are diminished in the presence of the lysine analogue epsilon -aminocaproic acid. We propose that copper binding to the kringle domains of 17K is mediated by a His-X-His sequence that is located about 5A from the closest tryptophan residue of the lysine binding pocket. Copper binding may account for the natural resistance to copper-mediated oxidation of lipoprotein(a) relative to LDL that has been previously reported and for the protection afforded by apo(a) from copper-mediated oxidation of LDL that we describe in the present study.     

Comparative analyses of the lysine binding site properties of apolipoprotein(a) kringle IV types 7 and 10.

Apolipoprotein(a) [apo(a)] shares extensive sequence similarity with plasminogen and consists of multiple tandem repeats of domains similar to plasminogen kringle IV (KIV), followed by domains homologous to the plasminogen KV and protease domains. The apo(a) KIV domains can be classified into 10 types on the basis of amino acid sequence (KIV(1)-KIV(10)) of which KIV(10) contains a canonical lysine binding site (LBS); KIV(10) mediates the lysine-dependent interaction of Lp(a) with certain biological substrates. Molecular modeling studies indicated the presence of weak LBS in each of KIV(5)-KIV(8), and subsequent biochemical studies have revealed contributions of these kringles to lysine-mediated interactions involving apo(a). The present study describes the direct demonstration of a weak LBS within KIV(7), as well as the first characterization of the ligand specificity of an LBS outside that of KIV(10). We have expressed both KIV(7) and KIV(10) from bacterial cells and purified them to homogeneity from cell lysates. Equilibrium binding analyses of the KIV(7) LBS using intrinsic fluorescence revealed an affinity for L-lysine and its analogues approximately 10-fold weaker (K(D) = 230 +/- 42 microM for epsilon-aminocaproic acid) than that of KIV(10) (K(D) = 33 +/- 4 microM for epsilon-aminocaproic acid). Moreover, we demonstrated differences in specificity of the LBS of KIV(7) in comparison with KIV(10) in that KIV(7) preferentially bound L-proline. Both kringles bind 4-aminobutyric acid with similar affinities albeit with apparently different mechanisms. Key Phe(62) --> Tyr and Asp(56) --> Glu substitutions in the KIV(7) LBS result in alterations in the size of the LBS and in the spatial relationship between the cationic and anionic centers in the LBS and thus account for the differences in the binding properties of KIV(7) and KIV(10).     

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).     

Apolipoprotein(a), a link between atherosclerosis and tumor angiogenesis

Lipoprotein (a) [Lp(a)] is a LDL-like particle with one apolipoprotein(a) [apo(a)] covalently bound to apolipoprotein B, the structural protein of Low Density Lipoprotein (LDL). Lewis Lung Carcinoma (LL/2) cells exhibited delayed growth and reduced angiogenesis in apo(a) transgenic mice, expressing a recombinant apo(a) [r-apo(a)] with 18 kringle 4 repeats. The mean microvessel density of subcutaneous LL/2 tumors from apo(a) transgenic mice was significantly lower than that of tumors from control wild type mice. CHO cells secreting a truncated apo(a) protein with only six kringle 4 repeats did not exhibit delayed tumor growth nor did it impair angiogenesis. These data point to an unappreciated role of human apo(a) in angiogenesis and cancer biology. As angiogenesis is necessary for reendothelialization following vascular injury, suppression of angiogenesis by apo(a) may also contribute to the atherogenicity of apo(a). The differences between the truncated apo(a) and r-apo(a) are consistent with the higher atherogenicity of higher molecular weight isoforms.     

The linkage with apolipoprotein (a) in lipoprotein (a) modifies the immunochemical and functional properties of apolipoprotein B

Lipoprotein (a) [Lp(a)] was isolated from several donors and its apolipoprotein (a) [apo(a)] dissociated by a reductive treatment, generating the apo(a)-free form of Lp(a) [Lp(a--)] that contains apolipoprotein B (apo B) as its sole protein. Using anti-apo B monoclonal antibodies, the properties of apo B in Lp(a), Lp(a--), and autologous low-density lipoprotein (LDL) were compared. Marked differences in apo B immunoreactivity were found between these lipoproteins, due to the presence of apo(a) in Lp(a). Apo(a) enhanced the expression of two epitopes in the amino-terminal part of apo B while it diminished the immunoreactivity of three other epitopes in the LDL receptor binding domain. Accordingly, the binding of the lipoproteins to the LDL receptor was also decreased in the presence of apo(a). In a different experimental system, the incubation of antibodies that react with 27 distinct epitopes distributed along the whole length of apo B sequence with plastic-bound Lp(a) and Lp(a--) failed to reveal any epitope of apo B that is sterically hindered by the presence of apo(a). Our results demonstrate that the presence of apo(a) modified the organization and function of apo B in Lp(a) particles. The data presented indicate that most likely the modification is not due to a steric hindrance but that some more profound conformational changes are involved. We suggest that the formation of the disulfide bridge between apo B and apo(a) in Lp(a) alters the system of disulfide bonds present in apo B and thereby modifies apo B structure.     

Structural features of apolipoprotein B synthetic peptides that inhibit lipoprotein(a) assembly

Lipoprotein(a) [Lp(a)] is assembled via an initial noncovalent interaction between apolipoprotein B100 (apoB) and apolipoprotein(a) [apo(a)] that facilitates the formation of a disulfide bond between the two proteins. We previously reported that a lysine-rich, alpha-helical peptide spanning human apoB amino acids 4372-4392 was an effective inhibitor of Lp(a) assembly in vitro. To identify the important structural features required for inhibitory action, new variants of the apoB4372-4392 peptide were investigated. Introduction of a central leucine to proline substitution abolished the alpha-helical structure of the peptide and disrupted apo(a) binding and inhibition of Lp(a) formation. Substitution of hydrophobic residues in the apoB4372-4392 peptide disrupted apo(a) binding and inhibition of Lp(a) assembly without disrupting the alpha-helical structure. Substitution of all four lysine residues in the peptide with arginine decreased the IC50 from 40 microM to 5 microM . Complexing of the arginine-substituted peptide to dimyristoylphosphatidylcholine improved its activity further, yielding an IC50 of 1 microM. We conclude that the alpha-helical structure of apoB4372-4392, in combination with hydrophobic residues at the lipid/water interface, is crucial for its interaction with apo(a). Furthermore, the interaction of apoB4372-4392 with apo(a) is not lysine specific, because substitutions with arginine result in a more effective inhibitor.     

Apolipoprotein(a) and plasminogen interactions with fibrin: a study with recombinant apolipoprotein(a) and isolated plasminogen fragments.

Lipoprotein(a) [Lp(a)], but not low-density lipoprotein (LDL), was previously shown to impair the generation of fibrin-bound plasmin [Rouy et al. (1991) Arterioscler. Thromb. 11, 629-638] by a mechanism involving binding of Lp(a) to fibrin. It was therefore suggested that the binding was mediated by apolipoprotein(a) [apo(a)], a glycoprotein absent from LDL which has a high degree of homology with plasminogen, the precursor of the fibrinolytic enzyme plasmin. Here we have evaluated this hypothesis by performing comparative fibrin binding studies using a recombinant form of apo(a) containing 17 copies of the apo(a) domain resembling kringle 4 of plasminogen, native Lp(a), and Glu-plasminogen (Glu1-Asn791). Attempts were also made to identify the kringle domains involved in such interactions using isolated elastase-derived plasminogen fragments. The binding experiments were performed using a well-characterized model of an intact and of a plasmin-digested fibrin surface as described by Fleury and Anglés-Cano [(1991) Biochemistry 30, 7630-7638]. Binding of r-apo(a) to the fibrin surfaces was of high affinity (Kd = 26 +/- 8.4 nM for intact fibrin and 7.7 +/- 4.6 nM for plasmin-degraded fibrin) and obeyed the Langmuir equation for adsorption at interfaces. The binding to both surfaces was inhibited by the lysine analogue AMCHA and was completely abolished upon treatment of the degraded surface with carboxypeptidase B, indicating that r-apo(a) binds to both the intrachain lysines of intact fibrin and the carboxy-terminal lysines of degraded fibrin. As expected from these results, both r-apo(a) and native Lp(a) inhibited the binding of Glu-plasminogen to the fibrin surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue-type plasminogen activator binds to and is inhibited by surface-bound lipoprotein(a) and low-density lipoprotein

Elevated levels of lipoprotein(a) [Lp(a)] are associated with an increased risk of atherothrombotic disease, but the mechanism(s) by which Lp(a) potentiates atherogenesis is unknown. The extensive homology of apolipoprotein(a) [apo(a)] to plasminogen has led us and others to postulate that Lp(a) may impair fibrinolysis. We have previously shown that Lp(a) inhibits fibrin stimulation of plasminogen activation by tissue-type plasminogen activator (t-PA); however, we and other investigators have been unable to demonstrate direct inhibition of t-PA by Lp(a) in solution. We now report that t-PA binds reversibly and saturably to surface-bound Lp(a) and to low-density lipoprotein (LDL) and that as a result of this binding activation of plasminogen by t-PA is inhibited. The catalytic efficiency (kcat/Km) of t-PA when bound to polystyrene surface-bound fibrinogen increased 2.9-fold compared to t-PA bound to control wells. When bound to surface-bound Lp(a), however, the catalytic efficiency of t-PA was reduced 9.5-fold compared to t-PA bound to control wells; likewise, by binding to surface-bound LDL, the catalytic efficiency of t-PA was reduced 16-fold compared to the control. Studies with defined monoclonal antibodies suggest that major determinants of t-PA binding are its active site, the LDL receptor binding domain of apolipoprotein B-100 (apoB-100), and apo(a). These data suggest a unique mechanism by which Lp(a) and LDL incorporated in an atheroma can inhibit endogenous fibrinolysis and thereby contribute to the genesis of atherothrombotic disease.     

Identification of a critical lysine residue in apolipoprotein B-100 that mediates noncovalent interaction with apolipoprotein(a)

We have previously shown that lipoprotein(a) (Lp(a)) assembly involves an initial noncovalent interaction between sequences within apolipoprotein(a) (apo(a)) kringle IV types 5-8 and the amino terminus of apolipoprotein B-100 (sequences between amino acids 680 and 781 in apoB-100), followed by formation of a disulfide bond. In the present study, citraconylation of lysine residues in apoB-100 abolished the ability of the modified low density lipoprotein to associate with apo(a), thereby demonstrating a direct role for lysine residues in apoB in the first step of Lp(a) assembly. To identify specific lysine residues in the amino terminus of apoB that are required for the noncovalent interaction, we initially used an affinity chromatography method in which recombinant forms of apo(a) (r-apo(a)) were immobilized on Sepharose beads. Assessment of the ability of carboxyl-terminal truncations of apoB-18 to bind to r-apo(a)-Sepharose revealed that a 25-amino acid sequence in apoB (amino acids 680-704) bound specifically to apo(a) in a lysine-dependent manner; citraconylation of the lysine residues in the apoB derivative encoding this sequence abolished the binding interaction. Using fluorescence spectrometry, we found that a synthetic peptide corresponding to this sequence bound directly to apo(a); the peptide also reduced covalent Lp(a) formation. Lysine residues present in this sequence (Lys(680) and Lys(690)) were mutated to alanine in the context of apoB-18. We found that the apoB-18 species containing the Lys(680) mutation was incapable of binding to r-apo(a)-Sepharose columns, whereas the apoB-18 species containing the Lys(690) mutation exhibited slightly reduced binding to these columns. Taken together, our data indicate that Lys(680) is critical for the noncovalent interaction of apo(a) and apoB-100 that precedes covalent Lp(a) formation.     

Assembly of lipoprotein (a) in transgenic rabbits expressing human apolipoprotein (a)

The study of human lipoprotein (a) [Lp(a)] has been hampered due to the lack of appropriate animal models since apolipoprotein (a) [apo(a)] is found only in primates and humans. In addition, human apo(a) in transgenic mice can not bind to murine apoB to form Lp(a) particles. In this study, we generated three independent transgenic rabbits expressing human apo(a) in their plasma at 1.8-4.5 mg/dl. In the plasma of transgenic rabbits, unlike the plasma of transgenic mice, about 80% of the apo(a) was covalently associated with rabbit apo-B and was contained in the fractions with density 1.02-1.10 g/ml, indicating the formation of Lp(a). These results suggest that transgenic rabbits expressing human apo(a) exhibit efficient assembly of Lp(a) and can be used as an animal model for the study of human Lp(a).