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.     

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.     

Effects of the apolipoprotein(a) size polymorphism on the lipoprotein(a) concentration in 7 ethnic groups

Summary Apolipoprotein(a) [apo(a)] exhibits a genetic size polymorphism explaining about 40% of the variability in lipoprotein(a) [Lp(a)] concentration in Tyroleans. Lp(a) concentrations and apo(a) phenotypes were determined in 7 ethnic groups (Tyrolean, Icelandic, Hungarian, Malay, Chinese, Indian, Black Sudanese) and the effects of the apo(a) size polymorphism on Lp(a) levels were estimated in each group. Average Lp(a) concentrations were highly significantly different among these populations, with the Chinese (7.0mg/dl) having the lowest and the Sudanese (46mg/dl) the highest levels. Apo(a) phenotype and derived apo(a) allele frequencies were also significantly different among the populations. Apo(a) isoform effects on Lp(a) levels were not significantly different among populations. Lp(a) levels were however roughly twice as high in the same phenotypes in the Indians, and several times as high in the Sudanese, compared with Caucasians. The size variation of apo(a) explains from 0.77 (Malays) to only 0.19 (Sudanese) of the total variability in Lp(a) levels. Together these data show (I) that there is considerable heterogeneity of the Lp(a) polymorphism among populations, (II) that differences in apo(a) allele frequencies alone do not explain the differences in Lp(a) levels among populations and (III) that in some populations, e.g. Sudanese Blacks, Lp(a) levels are mainly determined by factors that are different from the apo(a) size polymorphism.     

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.     

Effects of cultivation practice on floristic and flowering diversity of spontaneously growing plant species on arable fields

In the past, the floristic diversity of arable fields has been described in terms of species diversity (SD) and their degree of coverage (C), but never in combination with the recording of the actually flowered species (FS) and their flowering intensity (FI) to striking differences in the cultivation methods on arable land. In relation to SD and C, however, FS and FI may provide important additional information on the functional biodiversity of fields. The aim was therefore to investigate the effects of (a) conventional, (b) organic, and (c) smallholder (never application of herbicides) on the floristic diversity. Using a region in Germany, we investigated SD, C, FS, and FI synchronously in (a), (b), and (c), by 356 vegetation surveys (5 × 5 m plots) conducted in spring and summer in 2019 in winter cereals. Statistical tests were used to analyze the differences between (a), (b), and (c). The medians were used to compare the floristic diversity of (a), (b), and (c) and finally relationships of FS and FI to SD were analyzed in relation to the cultivation methods. Significant differences in SD, C, FS, and FI were found between the (a), (b), and (c) in spring and summer characterized by sharp declines from (c) to (b) to (a). A drastic reduction in floristic diversity from (c) 100 to (b) 52 to (a) 3 was determined. Plants in flower (FS, FI) were very poorly in (a), moderately well to well in (b), and well to very well represented in (c). (C) to (a) was characterized by a sharp decline and from (a) to (b) by sharp increase in floristic diversity. With current acreage proportions of (a) in mind, this would affect, about one third of land area in Germany, associated with a drastic reduction in functional biodiversity for insects.     

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.     

Lipoprotein(a) levels, apo(a) isoform size, and coronary heart disease risk in the Framingham Offspring Study

The aim of this study was to assess the independent contributions of plasma levels of lipoprotein(a) (Lp(a)), Lp(a) cholesterol, and of apo(a) isoform size to prospective coronary heart disease (CHD) risk. Plasma Lp(a) and Lp(a) cholesterol levels, and apo(a) isoform size were measured at examination cycle 5 in subjects participating in the Framingham Offspring Study who were free of CHD. After a mean follow-up of 12.3 years, 98 men and 47 women developed new CHD events. In multivariate analysis, the hazard ratio of CHD was approximately two-fold greater in men in the upper tertile of plasma Lp(a) levels, relative to those in the bottom tertile (P < 0.002). The apo(a) isoform size contributed only modestly to the association between Lp(a) and CHD and was not an independent predictor of CHD. In multivariate analysis, Lp(a) cholesterol was not significantly associated with CHD risk in men. In women, no association between Lp(a) and CHD risk was observed. Elevated plasma Lp(a) levels are a significant and independent predictor of CHD risk in men. The assessment of apo(a) isoform size in this cohort does not add significant information about CHD risk. In addition, the cholesterol content in Lp(a) is not a significant predictor of CHD risk.     

Cobalt(IV) corroles as catalysts for the electroreduction of O2: reactions of heterobimetallic dyads containing a face-to-face linked Fe(III) or Mn(III) porphyrin

A series of heterobinuclear cofacial porphyrin-corrole dyads containing a Co(IV) corrole linked by one of four different spacers in a face-to-face arrangement with an Fe(III) or Mn(III) porphyrin have been examined as catalysts for the electroreduction of O(2) to H(2)O and/or H(2)O(2) when adsorbed on the surface of a graphite electrode in air-saturated aqueous solutions containing 1M HClO(4). The examined compounds are represented as (PCY)M(III)ClCo(IV)Cl where P is a porphyrin dianion, C is a corrole trianion and Y is a biphenylene (B), 9,9-dimethylxanthene (X), dibenzofuran (O) or anthracene (A) spacer. The catalytic behavior of the seven investigated dyads in the two heterobimetallic (PCY)MClCoCl series of catalysts is compared on one hand to what was previously reported for related dyads with a single Co(III) corrole macrocycle linked to a free-base porphyrin with the same set of linking bridges, (PCY)H(2)Co, and on the other hand to dicobalt porphyrin-corrole dyads of the form (PCY)Co(2) which were shown to efficiently electrocatalyze the four electron reduction of O(2) at a graphite electrode in acid media. Comparisons between the four series of porphyrin-corrole dyads, (PCY)Co(2), (PCY)H(2)Co, (PCY)FeClCoCl and (PCY)MnClCoCl, show that in all cases the biscobalt dyads catalyze O(2) electroreduction at potentials more positive by an average 110mV as compared to the related series of compounds containing a Co(III) or Co(IV) corrole macrocycle linked to a free-base metalloporphyrin or a metalloporphyrin with an Fe(III) or Mn(III) central metal ion. The data indicates that the E(1/2) values where electrocatalysis is initiated is related to the initial site of electron transfer, which is the Co(III)/Co(II) porphyrin reduction process in the case of (PCY)Co(2) and the Co(IV)/Co(III) corrole reduction in the case of (PCY)MnClCoCl, (PCY)FeClCoCl and (PCY)H(2)Co. The overall data also suggests that the catalytically active form of the biscobalt dyad in (PCY)Co(2) contains a Co(II) porphyrin and a Co(IV) corrole.     

Distinctive and synergistic signaling of human adenosine A2a and dopamine D2L receptors in CHO cells

Adenosine A(2a) receptor (A(2a)R) colocalizes with dopamine D(2) receptor (D(2)R) in the basal ganglia and modulates D(2)R-mediated dopaminergic activities. A(2a)R and D(2)R couple to stimulatory and inhibitory G proteins, respectively. Their opposing roles in regulating neuronal activities, such as locomotion and alcohol consumption, are mediated by their opposite actions on adenylate cyclase, which often serves as "co-incidence detector" of various activators. On the other hand, the neural actions of A(2a)R and D(2)R are also, at least partially, independent of each other, as indicated by studies using D(2)R and A(2a)R knock-out mice. Here we co-expressed human A(2a)R and human D(2L)R in CHO cells and examined their signaling characteristics. Human A(2a)R desensitized rapidly upon agonist stimulation. A(2a)R activity (80%) was diminished after 2 hr of pretreatment with its agonist CGS21680. In contrast, human D(2L)R activity was sustained even after 2 hr and 18 hr pretreatment with its agonist quinpirole. Long-term (18 hr) stimulation of human D(2L)R also increased basal cAMP levels in CHO cells, whereas long-term (18 hr) activation of human A(2a)R did not affect basal cAMP levels. Furthermore, long-term (18 hr) activation of D(2L)R dramatically sensitized A(2a)R-induced stimulation of adenylate cyclase in a pertussis toxin-sensitive way. Forskolin-induced cAMP accumulation was significantly increased after short-term (2 hr) human D(2L)R stimulation and further elevated after long-term (18 hr) D(2L)R activation. However, neither short-term (2 hr) nor long-term (18 hr) stimulation of A(2a)R affected the inhibitory effects of D(2L)R on adenylate cyclase. Co-stimulation of A(2a)R and D(2L)R could not induce desensitization or sensitization of D(2L)R either. In summary, signaling through A(2a)R and D(2L)R is distinctive and synergistic, supporting their unique and yet integrative roles in regulating neuronal functions when both receptors are present.     

Biphasic modulation of fatty acid synthase by hydrogen peroxide in Saccharomyces cerevisiae

Taking into account published contradictory results concerning the regulation of fatty acid synthase (Fas) by H(2)O(2), we carried out a systematic study where two methods of H(2)O(2) delivery (steady-state and bolus addition) and the effect of a wide range of H(2)O(2) concentrations were investigated. A decrease in Fas activity was observed for cells exposed to 100 and 150μM H(2)O(2) in a steady-state, while a bolus addition of the same H(2)O(2) concentrations did not alter Fas activity. Similar results were observed for the mRNA levels of FAS1, the gene that encodes Fas subunit β. However, the exposure to a steady-state 50μM H(2)O(2) dose lead to an increase in FAS1 mRNA levels, showing a biphasic modulation of Fas by H(2)O(2). The results obtained emphasize that cellular effects of H(2)O(2) can vary over a narrow range of concentrations. Therefore, a tight control of H(2)O(2) exposure, which can be achieved by exposing H(2)O(2) in a steady-state, is important for cellular studies of H(2)O(2)-dependent redox regulation.     

Acute impact of apheresis on oxidized phospholipids in patients with familial hypercholesterolemia

We measured oxidized phospholipids (OxPL), lipoprotein (a) [Lp(a)], and lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) pre- and postapheresis in 18 patients with familial hypercholesterolemia (FH) and with low(～10 mg/dl; range 10-11 mg/dl), intermediate (～50 mg/dl; range 30-61 mg/dl), or high (>100 mg/dl; range 78-128 mg/dl) Lp(a) levels. By using enzymatic and immunoassays, the content of OxPL and Lp-PLA(2) mass and activity were quantitated in lipoprotein density fractions plated in microtiter wells, as well as directly on apoB-100, Lp(a), and apoA-I immunocaptured within each fraction (i.e., OxPL/apoB and Lp-PLA(2)/apoB). In whole fractions, OxPL was primarily detected in the Lp(a)-containing fractions, whereas Lp-PLA(2) was primarily detected in the small, dense LDL and light Lp(a) range. In lipoprotein capture assays, OxPL/apoB and OxPL/apo(a) increased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apoB and Lp-PLA(2)/apoA-I levels were highest in the low Lp(a) group but decreased proportionally with increasing Lp(a) levels. Lp-PLA(2)/apo(a) was lowest in patients with low Lp(a) levels and increased proportionally with increasing Lp(a) levels. Apheresis significantly reduced levels of OxPL and Lp-PLA(2) on apoB and Lp(a) (50-75%), particularly in patients with intermediate and high Lp(a) levels. In contrast, apheresis increased Lp-PLA(2)-specific activity (activity/mass ratio) in buoyant LDL fractions. The impact of apheresis on Lp(a), OxPL, and Lp-PLA(2) provides insights into its therapeutic benefits beyond lowering apoB-containing lipoproteins.     

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.     

Tryptamine-gallic acid hybrid prevents non-steroidal anti-inflammatory drug-induced gastropathy: correction of mitochondrial dysfunction and inhibition of apoptosis in gastric mucosal cells

We have investigated the gastroprotective effect of SEGA (3a), a newly synthesized tryptamine-gallic acid hybrid molecule against non-steroidal anti-inflammatory drug (NSAID)-induced gastropathy with mechanistic details. SEGA (3a) prevents indomethacin (NSAID)-induced mitochondrial oxidative stress (MOS) and dysfunctions in gastric mucosal cells, which play a pathogenic role in inducing gastropathy. SEGA (3a) offers this mitoprotective effect by scavenging of mitochondrial superoxide anion (O(2)(·-)) and intramitochondrial free iron released as a result of MOS. SEGA (3a) in vivo blocks indomethacin-mediated MOS, as is evident from the inhibition of indomethacin-induced mitochondrial protein carbonyl formation, lipid peroxidation, and thiol depletion. SEGA (3a) corrects indomethacin-mediated mitochondrial dysfunction in vivo by restoring defective electron transport chain function, collapse of transmembrane potential, and loss of dehydrogenase activity. SEGA (3a) not only corrects mitochondrial dysfunction but also inhibits the activation of the mitochondrial pathway of apoptosis by indomethacin. SEGA (3a) inhibits indomethacin-induced down-regulation of bcl-2 and up-regulation of bax genes in gastric mucosa. SEGA (3a) also inhibits indometacin-induced activation of caspase-9 and caspase-3 in gastric mucosa. Besides the gastroprotective effect against NSAID, SEGA (3a) also expedites the healing of already damaged gastric mucosa. Radiolabeled ((99m)Tc-labeled SEGA (3a)) tracer studies confirm that SEGA (3a) enters into mitochondria of gastric mucosal cell in vivo, and it is quite stable in serum. Thus, SEGA (3a) bears an immense potential to be a novel gastroprotective agent against NSAID-induced gastropathy.     

Angiotensin II increases adipose angiotensinogen expression

In addition to the well-defined contribution of the liver, adipose tissue has been recognized as an important source of angiotensinogen (AGT). The purpose of this study was to define the angiotensin II (ANG II) receptors involved in regulation of adipose AGT and the relationship of this control to systemic AGT and/or angiotensin peptide concentrations. In LDL receptor-deficient (LDLR(-/-)) male mice, adipose mRNA abundance of AGT was 68% of that in liver, and adipose mRNA abundance of the angiotensin type 1a (AT(1a)) receptor (AT(1a)R) was 38% of that in liver, whereas mRNA abundance of the angiotensin type 2 (AT(2)) receptor (AT(2)R) was 57% greater in adipose tissue than in liver. AGT and angiotensin peptide concentrations were decreased in plasma of AT(1a)R-deficient (AT(1a)R(-/-)) mice and were paralleled by reductions in AGT expression in liver. In contrast, adipose AGT mRNA abundance was unaltered in AT(1a)R(-/-) mice. AT(2)R(-/-) mice exhibited elevated plasma angiotensin peptide concentrations and marked elevations in adipose AGT and AT(1a)R mRNA abundance. Increases in adipose AGT mRNA abundance in AT(2)R(-/-) mice were abolished by losartan. In contrast, liver AGT and AT(1a)R mRNA abundance were unaltered in AT(2)R(-/-) mice. Infusion of ANG II for 28 days into LDLR(-/-) mice markedly increased adipose AGT and AT(1a)R mRNA but did not alter liver AGT and AT(1a)R mRNA. These results demonstrate that differential mRNA abundance of AT(1a)/AT(2) receptors in adipose tissue vs. liver contributes to tissue-specific ANG II-mediated regulation of AGT. Chronic infusion of ANG II robustly stimulated AT(1a)R and AGT mRNA abundance in adipose tissue, suggesting that adipose tissue serves as a primary contributor to the activated systemic renin-angiotensin system.     

Conformational effects of C(alpha,alpha)-dipropargylglycine as a constrained residue

A useful synthon to approach artificial phenylalanyl peptides in a [2 + 2 + 2] cycloaddition reaction, C(alpha,alpha)-dipropargylglycine (Dprg) is examined for its conformational preferences as a constrained residue. Crystal structure analysis and preliminary NMR results establish possible preference of the residue for folded (alpha) rather than extended (beta) region of the straight phi,psi conformational space. Boc-Dprg-L-Leu-OMe (1) displays two molecular conformations within the same crystallographic asymmetric unit, with Dprg in the alpha(R) or alpha(L) conformation, participating in a type I beta-turn or an alpha(L)-alpha(R)-type fold, in which Leu(2) assumes the alpha(R) conformation stereochemically favored for an L-chiral residue. Boc-Dprg-D-Val-L-Leu-OMe (2) displays a type I' beta-turn conformation in crystal, with both Dprg(1) and D-Val(2) assuming the alpha(L) conformation stereochemically favored for a D-chiral residue, with 4 --> 1 type hydrogen bond linking L-Leu(3) NH with Boc CO. NMR analysis using temperature variation, solvent titration, and a spin probe study suggests a fully solvent-exposed nature of Dprg NH, ruling out a fully extended C(5)-type conformation for this residue, and solvent sequestered nature of L-Leu(3) NH, suggesting possibility of a beta-turn due to Dprg assuming a folded conformation.     

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.     

Reconstitution of lipoprotein(a) by infusion of human low density lipoprotein into transgenic mice expressing human apolipoprotein(a).

Lipoprotein(a) (Lp(a)) is an atherosclerosis-causing lipoprotein that circulates in human plasma as a complex of low density lipoprotein (LDL) and apolipoprotein(a) (apo(a)). It is not known whether apo(a) attaches to LDL within hepatocytes prior to secretion or in plasma subsequent to secretion. Here we describe the development of a line of mice expressing the human apo(a) transgene under the control of the murine transferrin promoter. The apo(a) was secreted into the plasma, but circulated free of lipoproteins. When human (h)-LDL was injected intravenously, the circulating apo(a) rapidly associated with the lipoproteins, as determined by nondenaturing gel electrophoresis. Human HDL and mouse LDL had no such effect. When h-VLDL was injected, there was a delayed association of apo(a) with the lipoprotein fraction which suggests that apo(a) preferentially associated with a metabolic product of VLDL. The complex of apo(a) with LDL formed both in vivo and in vitro was resistant to boiling in the presence of detergents and denaturants, but was resolved upon disulfide reduction. These studies suggest that apo(a) fails to associate with mouse lipoproteins due to structural differences between human and mouse LDL, and that Lp(a) formation can occur in plasma through the association of apo(a) with circulating LDL.     

6-Aminohexanoic acid as a chemical chaperone for apolipoprotein(a)

Apolipoprotein (a) (apo(a)) is a component of the atherogenic lipoprotein, Lp(a). The efficiency with which apo(a) escapes the endoplasmic reticulum (ER) and is secreted by the liver is a major determinant of plasma Lp(a) levels. Apo(a) contains a series of domains homologous to plasminogen kringle (K) 4, each of which possesses a potential lysine-binding site. By using primary mouse hepatocytes expressing a 17K4 human apo(a) protein, we found that high concentrations (25-200 mM) of the lysine analog, 6-aminohexanoic acid (6AHA), increased apo(a) secretion 8-14-fold. This was accompanied by a decrease in apo(a) presecretory degradation. 6AHA inhibited accumulation of apo(a) in the ER induced by the proteasome inhibitor, lactacystin. Thus, 6AHA appeared to inhibit degradation by increasing apo(a) export from the ER. Significantly, 6AHA overcame the block in apo(a) secretion induced by the ER glucosidase inhibitor, castanospermine. 6AHA may therefore circumvent the requirement for calnexin and calreticulin interaction in apo(a) secretion. Sucrose gradients and a gel-based folding assay were unable to detect any influence of 6AHA on apo(a) folding. However, non-covalent or small, disulfide-dependent changes in apo(a) conformation would not be detected in these assays. Proline also increased the efficiency of apo(a) secretion. We propose that 6AHA and proline can act as chemical chaperones for apo(a).     

Mutations of nonconserved residues within the calcium channel alpha1-interaction domain inhibit beta-subunit potentiation

Voltage-dependent calcium channels consist of a pore-forming subunit (Ca(V)alpha(1)) that includes all the molecular determinants of a voltage-gated channel, and several accessory subunits. The ancillary beta-subunit (Ca(V)beta) is a potent activator of voltage-dependent calcium channels, but the mechanisms and structural bases of this regulation remain elusive. Ca(V)beta binds reversibly to a conserved consensus sequence in Ca(V)alpha(1), the alpha(1)-interaction domain (AID), which forms an alpha-helix when complexed with Ca(V)beta. Conserved aromatic residues face to one side of the helix and strongly interact with a hydrophobic pocket on Ca(V)beta. Here, we studied the effect of mutating residues located opposite to the AID-Ca(V)beta contact surface in Ca(V)1.2. Substitution of AID-exposed residues by the corresponding amino acids present in other Ca(V)alpha(1) subunits (E462R, K465N, D469S, and Q473K) hinders Ca(V)beta's ability to increase ionic-current to charge-movement ratio (I/Q) without changing the apparent affinity for Ca(V)beta. At the single channel level, these Ca(V)1.2 mutants coexpressed with Ca(V)beta(2a) visit high open probability mode less frequently than wild-type channels. On the other hand, Ca(V)1.2 carrying either a mutation in the conserved tryptophan residue (W470S, which impairs Ca(V)beta binding), or a deletion of the whole AID sequence, does not exhibit Ca(V)beta-induced increase in I/Q. In addition, we observed a shift in the voltage dependence of activation by +12 mV in the AID-deleted channel in the absence of Ca(V)beta, suggesting a direct participation of these residues in the modulation of channel activation. Our results show that Ca(V)beta-dependent potentiation arises primarily from changes in the modal gating behavior. We envision that Ca(V)beta spatially reorients AID residues that influence the channel gate. These findings provide a new framework for understanding modulation of VDCC gating by Ca(V)beta.