Studies on the structure and function of the apolipoprotein(a) gene

Lp(a) is an LDL-like lipoprotein that is a major inherited risk factor for atherosclerosis. It is distinguished from Lp(a) by the addition of apolipoprotein(a). The gene structure of apolipoprotein(a) is homologous to plasminogen, and competition with plasminogen activity may account for some of the pathophysiology associated with Lp(a). Six highly related genes have now been identified, and at least four are found in close proximity in overlapping genomic clones. Studies have begun on the regulation of apolipoprotein (a) gene expression, and the human apolipoprotein(a) gene has been inserted into transgenic mice, where it leads to the development of arterial lesions.     

Inherited quantitative DNA variation in the LPA ("apolipoprotein (a)") gene

The Lp(a) antigen resides in a polypeptide chain that is attached to apolipoprotein B (apoB) by a disulfide bridge. Recently, cDNA for this polypeptide chain (frequently referred to as the Lp(a) polypeptide chain, Lp(a) apolipoprotein or apolipoprotein (a] was cloned and extensive homology to plasminogen was uncovered. This homology creates significant difficulties in studying DNA variation in the gene (the LPA gene) for this polypeptide and the plasminogen gene. We have studied a variant 2 kilobase (kb) DNA fragment detectable after digestion with the restriction enzyme MspI, which appears to originate from the LPA gene since it is detected by LPA probes but not with plasminogen probes. It is related to the "kringle IV" region of the LPA gene since it is detected with an LPA probe that only contains "kringle IV" repeats. A proportion of people appears to lack (or have an undetectable level of) the 2 kb fragment and there are significant quantitative differences between samples from people who have the fragment. Presence and amount of this fragment appear to segregate in families as a Mendelian trait. This quantitative DNA variation is likely to reflect differences between individuals in number of "kringle IV" repeats at the LPA locus.     

How often has Lp(a) evolved?

The lipoprotein Lp(a) is associated with increased risk of atherosclerosis and myocardial infarction in humans. Lp(a) is mostly confined to primate species, due to the limited phylogenetic distribution of its distinguishing protein component, apolipoprotein(a) which is a close homolog of plasminogen. The known properties of Lp(a) are reviewed here. Many of these derive from the ability of Lp(a) to bind to the same substrates as plasminogen. A possible new animal model of Lp(a) is the hedgehog, which contains an Lp(a)-like particle that is the apparent product of independent evolution of a multi-kringle, apolipoprotein(a)-like protein by duplication and modification of portions of the hedgehog plasminogen gene.     

Lp(a) lipoprotein is a major risk factor for cardiovascular disease: pathogenic mechanisms and clinical significance

The results of two previous and two recent studies of middle-aged males and females are presented to exemplify the clinical importance of lipoprotein (a) (Lp(a)) as a risk factor for atherosclerosis and coronary heart disease. In these studies various conventional and recently suggested risk factors were included and different methods for Lp(a) quantification were used. Lp(a) was a significant risk factor in all four studies. In the recent prospective case-control study, Lp(a) and cholesterol were found to act synergistically and predict primary acute myocardial infarction in Swedish males. A cholesterol level above 6.5 mmol/1 increased the risk of acute myocardial infarction if the Lp(a) level was above 200 mg/1. The plasma apo A-I level was a protective factor. In the other recent case-control study, an Lp(a) level above 500 mg/1 was a highly significant risk factor in Black and White US women with myocardial infarction or advanced coronary artery disease in addition to low density lipoprotein cholesterol levels above 130 mg/dl. A high apo A-I level was a protective factor. In these studies no other factors tested reached significance in multivariate logistic regression analysis. A hypothetical association between high Lp(a) levels and intracellular infection with Chlamydia pneumoniae is discussed. The results suggest that the Lp(a) level is useful in identifying high-risk individuals. Lowering low density lipoprotein cholesterol below 100 mg/dl (7lt;2.6 mmol/1) seems to be most important in both males and females with high-risk Lp(a) levels.     

APO(a) variants and lipoprotein(a) in men with or without myocardial infarction

The lipoprotein Lp(a) with high plasma concentration is an independent genetic determinant for cardiovascular diseases. It was investigated as a quantitative factor of risk for myocardial infarction. A total of 345 Italian subjects, 127 Cases and 218 Controls, were studied. Lipids and lipoproteins were compared. Cases had atherogenic traits, such as lower HDL cholesterol and higher triglycerides than Controls. In particular, they had Lp(a) concentrations over the risk threshold, (median, 27 mg/dl in Cases vs 17 mg/dl in Controls; P = 0.0075, Mann-Whitney test) which confirmed the association of this parameter with the disease. Two main functional variants of the apo(a) gene, KringleIV and penta-nucleotide repeat, (PNR) were analyzed. Allele and genotype frequency distributions differed between Cases and Controls. Lp(a) concentrations differed according to PNR genotypes in Controls: subjects having alleles >8 showed lower Lp(a). This was not found in Cases. They had a higher prevalence of the smaller KringleIV alleles, the high Lp(a)-expressing ones. In Cases, genotypes consisting of two small KringleIV alleles were prevalently associated to PNR 8/9 and 8/10, thus preventing Lp(a) lowering. The putative apo(a) enhancer within LINE1 in the apo(a)-plasminogen intergenic region was investigated for functional polymorphisms. No variants that could be associated to the Lp(a) variability were found.     

The lysine-binding function of Lp(a)

The atherogenicity of Lp(a) is attributable to the binding of its apolipoprotein(a) component to fibrin and other plasminogen substrates. It can attenuate the activation of plasminogen, diminishing plasmin-dependent fibrinolysis and transforming growth factor-β activation. Apolipoprotein(a) contains a major lysine-binding site in one of its kringle domains. Destroying this site by site-directed mutagenesis greatly reduces the binding of apolipoprotein(a) to lysine and fibrin. Transgenic mice expressing wild-type apolipoprotein(a) have a 5-fold increase in the development of lipid lesions, as well as a large increase in the focal deposition of apolipoprotein(a) in the aorta, compared to the lysine-binding site mutant strain and to non-transgenic litter mates. Although the adaptive function of apolipoprotein(a) remains obscure, a gene with similar structure has evolved by independent remodeling of the plasminogen twice during the course of mammalian evolution.     

Lp(a) lipoprotein enters cultured fibroblasts independently of the plasma membrane low density lipoprotein receptor

Lp(a) lipoprotein shares the apoB antigen with low density lipoprotein (LDL). The Lp(a) antigen is unique for Lp(a) lipoprotein. Fibroblast association (i.e. plasma membrane binding plus intracellular accumulation), plasma membrane binding, intracellular accumulation and degradation of 125I-Lp(a) lipoprotein were studied in strains from subjects with or without autosomal dominant hypercholesterolemia (HC). Subjects without HC (non-HCs) have cell surface receptors for low density lipoprotein (LDL receptors). On the average, HC heterozygotes have half-normal LDL receptor activity and "receptor-negative" HC homozygous cell strains lack functional receptors. Fibroblast processing of 125I-Lp(a) lipoprotein was compared to fibroblast processing of 125I-LDL. LDL receptor-dependent processing of 125I-LDL was saturated at about 50 microgram apo 125I-LDL.ml-1 in non-HC fibroblasts. 125I-Lp(a) lipoprotein was, however, largely processed independently of receptor mechanisms by non-HC cells (highest concentration examined 150 microgram apo 125I-Lp(a) lipoprotein . ml-1). Lp(a) lipoprotein did not interfere with 125I-LDL for fibroblast association, but inhibited 125I-LDL degradation. The interference with 125I-LDL degradation was time dependent. Only slightly higher 125I-Lp(a) lipoprotein processing values were found in non-HC and HC heterozygous strains than in "receptor-negative" HC homozygous strains. However, non-HC cells had more than tenfold higher 125I-LDL processing values than "receptor-negative" HC homozygous cells.     

Cardiovascular risk factors in people with different genotypes in the insertion/deletion (I/D) polymorphism at the locus for angiotensin I-converting enzyme (ACE)

The deletion (D) allele of an insertion/deletion (I/D) polymorphism at the locus for angiotensin I-converting enzyme (ACE) has been reported to be an independent risk factor for myocardial infarction (MI), particularly in people lacking traditional risk factors. Furthermore, a borderline association between Lp(a) lipoprotein level and the I/D polymorphism at the ACE locus was reported in one study. We have searched for possible "level gene" or "variability gene" effects of ACE genes on Lp(a) lipoprotein, total cholesterol (TC), high density lipoprotein (HDL) cholesterol (HDLC), low density lipoprotein (LDL) cholesterol (LDLC), triglycerides (TG), apolipoprotein B (apoB), apolipoprotein A-I (apoA-I), and body mass index (BMI). None of these variables differed significantly between genotypes in the I/D polymorphism in any of three population samples. A single population sample created by combining the three series, exhibited an insignificant trend towards individuals carrying the D-allele having a higher level of Lp(a) lipoprotein than those lacking it, and DD homozygotes had a significantly higher Lp(a) lipoprotein level than the combined group of ID/II individuals (p = 0.03). These results may indicate that the D-allele of the I/D polymorphism at the ACE locus could influence the level of Lp(a) lipoprotein.     

In vitro studies of the interaction of calcium ions and other divalent cations with the Lp(a) lipoprotein and other isolated serum lipoproteins

The interaction of isolated Lp(a) lipoprotein with different divalent cations was studied and compared to that of other isolated lipoprotein classes.
Purified Lp(a) lipoprotein was found to be most sensitive to the metal ions tested, and the Lp(a) lipoprotein was the only lipoprotein which was precipitated by calcium ions alone. The precipitation apparently depends on the ionic radii of the cations used as well as on the lipoprotein class tested. The precipitation reaction between calcium ions and the Lp(a) lipoprotein, and the interaction between calcium ions and LDL (without precipitation) seem to follow the known rules for small ion - macromolecule interaction reasonably well. The calcium ion - Lp(a) lipoprotein interaction results in a small aggregate. The binding is of ionic type and the precipitation reaction is initially reversible. It was estimated that LDL particles have a mean of 290 equivalent and non-interacting binding sites for calcium ions.
The above observations concerning the Lp(a) lipoprotein may be of interest in view of the significantly higher frequency of early coronary heart disease in Lp(a+) than in Lp(a-) individuals, and in view of the previously reported biochemical differences between individuals of different Lp phenotype.     

The inheritance of sinking-pre-beta lipo-protein and its relation to the Lp(a) antigen*

We have investigated the genetics of plasma sinking-pre-beta lipoprotein (spβ) as determined by the method of Breckenridge and Maguire, using several approaches: (i) a population study, (ii) a twin study and (iii) the use of family data. In addition, by the use of split samples, the spβ level as determined by us was correlated with Lp(a) typing carried out in Oslo by Dr. Kåre Berg. Although the spβ level is a continuous character, the results clearly showed it to be to a considerable extent under the control of the major autosomal gene pair constituted by the alleles Lp^a and Lp, which mainly control the production of the Lp(a) antigen, Lp^a being dominant. In our data the boundary between the LpLp and Lp^a Lp genotypes appeared to fall between the spβ 2 and 3 mg% levels, while that between Lp^a Lp and Lp^a Lp^a was in the 15 mg% region. These boundaries, which were inferred from both typing and population statistics, received good confirmation from the family data. It appears that some 88% of the variation in spp level is directly ascribed to segregation of Lp^a and Lp. On the basis of the twin study and other data, we conclude that the residual observed variation in spβ is almost entirely ascribed to analytical error of determination and polygenic effects, the influence of environment being negligible. The heritability is close to 100%.     

Effects of apoE gene polymorphism on Lp(a) concentrations depend on the size of apo(a): a study of 466 white men

 Polymorphisms in the genes for the low-density lipoprotein (LDL) receptor ligands, apolipoprotein E (apoE), and apolipoprotein B (apoB) are associated with variation in plasma levels of LDL cholesterol. Lp(a) lipoprotein(a) [Lp(a)] is LDL in which apoB is attached to a glycoprotein called apolipoprotein(a) [apo(a)]. Apo(a) has several genetically determined isoforms differing in molecular weight, which are inversely correlated with Lp(a) concentrations in blood. The interaction of apo(a) with triglyceride-rich lipoproteins differs with the size of apo(a), and therefore the effects of apoE gene polymorphism on Lp(a) levels could also depend on apo(a) size. We have investigated the possible effect of genetic variation in the apoE and apoB genes on plasma Lp(a) concentrations in 466 white men with different apo(a) phenotypes. Overall there was no significant association between the common apoE polymorphism and Lp(a), but in the subgroup with apo(a)-S4, concentrations of Lp(a) differed significantly among the apoE genotypes (P=0.05). Lp(a) was highest in the apoE genotypes ɛ₂ɛ₃ and ɛ₃ɛ₃ and lowest in genotype ɛ₃ɛ₄, and the apoE polymorphism was estimated to account for about 2.4% of the variation in Lp(a). In contrast, in the subgroup with apo(a)-S2 Lp(a) was significantly lower (P=0.04) in apoE genotype ɛ₂ɛ₃ than in genotype ɛ₃ɛ₃. Lp(a) concentrations did not differ among the XbaI (P=0.65) or SP 24/27 (P=0.26) polymorphisms of the apoB gene. The expected effects of both apoE and apoB polymorphism on LDL levels were significant in the whole population sample and in subjects with large-sized apo(a) isoforms (P<0.01), whereas no effect was seen in those with low molecular weight apo(a) isoforms. We conclude that the influence of apoE genotypes on Lp(a) concentrations depends on the size of the apo(a) molecule in Lp(a), possibly because both apo(a)-S4 and apoE4 have high affinity for triglyceride-rich lipoproteins and may be taken up and degraded rapidly by remnant receptors. Received: 12 January 1995 / Accepted: 12 July 1996     

Lack of association of apolipoprotein E polymorphism with plasma Lp(a) levels in the Chinese

Apolipoprotein E (apoE) polymorphism and its influence on plasma lipids, lipoproteins, lipoprotein (a) [Lp(a)] and apolipoproteins was studied in 536 (270 males and 266 females) healthy Chinese in Singapore. From analysis of variance with age and BMI as covariates, apoE genotype was found to exert a significant influence on plasma total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and apoB in females. Its effect in males was marginally significant only on LDL-C. In both sexes, plasma TC, LDL-C and apoB were lower in those who were E2-3 than in those who were E3-3. There was no significant difference in log-transformed Lp(a) level between the apoE genotypes after adjusting for the confounding effect of LDL-C in addition to age and BMI. The percentage variance (R2times100) of the lipid traits explained by apoE polymorphism in the females was 4.94% for plasma TC, 5.85% for LDL-C and 4.25% for apoB. We conclude that: 1) ε2 allele had a lowering effect on plasma TC, LDL-C and apoB; 2) apoE polymorphism did not have any significant influence on Lp(a) concentration; and 3) the effect of apoE polymorphism on plasma TC, LDL-C and apoB was gender-specific, with a stronger influence in females than in males.     

In vitro studies of the interaction of isolated Lp(a) lipoprotein and other serum lipoproteins with glycosaminoglycans

The interaction of isolated Lp(a) lipoprotein or other lipoprotein classes with different glycosaminoglycans (GAG) bound to activated Sepharose was studied. In contrast to LDL, the Lp(a) lipoprotein did not bind to the GAG tested if sodium was used as a buffer cation. In the presence of Ca++, however, even the Lp(a) lipoprotein was bound to GAG. This type of binding, probably mediated by divalent cation bridges, is apparently not a simple function of the GAG used. Addition of GAG in solution revealed that this binding may be the only one existing under physiological conditions, and it appears possible that the Lp(a) lipoprotein is bound more firmly to GAG than is LDL under such conditions.     

Apolipoprotein(a): structural and functional consequences of mutations in kringle type 10 (or kringle 4–37)

The size polymorphism of Lp(a) is well recognized. It is now apparent that there is an additional polymorphism resulting from mutations occurring at the kringle level. One of these mutations involves a trp72 to arg substitution in apo(a) kringle type 10 and is attended by a defective binding of Lp(a) to immobilized lysine/fibrin. Other mutations affecting the other amino acids of the "lysine-binding pocket" may have similar functional consequences and may be important at the clinical level in terms of thrombogenesis.     

Changes in Lp(a) lipoprotein and other plasma proteins during acute myocardial infarction

The sequential changes of Lp(a) lipoprotein concentrations in patients (n=59) suffering acute myocardial infarction (AMI) were examined and compared with other plasma proteins. The temporal and quantitative characteristics of the responses in concentration of acute phase reactants (CRP, haptoglobin, α₁-antitrypsin, α-acid glycoprotein), lipids (total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol) and apolipoproteins AI and B were similar to previous reports. Lp(a) lipoprotein showed transient changes with an initial decrease of 10–25% compared to the 3-month control value, followed by rebound on day 7–11 above admission level, before again declining. We were able to demonstrate a quantitative relationship between infarct size and alterations in plasma levels of acute phase reactants. However, in addition to rather unusual significant fluctuations during AMI, Lp(a) lipoprotein changes seemed unrelated to infarct size. These findings do not support the view that Lp(a) lipoprotein acts as an acute phase reactant.     

Confirmation of an influence of the inherited Lp(a) variation on serum insulin and glucose levels

Previously reported analyses on three different series of people suggested that fasting serum insulin levels are lower in males with (high levels of) serum Lp(a) lipoprotein (Lp(a+)) than in males without detectable Lp(a) lipoprotein (Lp(a-)). The same was observed during an oral glucose tolerance test. Also, blood glucose concentrations tended to be lower in males with high levels of Lp(a) lipoprotein than in those in whose serum no Lp(a) lipoprotein could be detected. In this paper, we present data which appear to confirm the previously reported results. A significant correlation was found between the fasting triglyceride level and the sum of insulin values determined during the oral glucose tolerance test in healthy Lp(a-) but not in Lp(a+) individuals. The present data, together with those previously reported on an effect of the Lp(a) locus on serum lipid levels and on propensity to contract coronary heart disease, indicate that the genetically determined Lp(a) lipoprotein may be of considerable clinical importance.     

─株特异抗人血浆Lp（a）单克隆抗体（A5g3）的制备及特征分析

用序列超速离心和柱层析分离纯化的人血浆脂蛋白（ａ）（Ｌｐ（ａ））免疫Ｂａｌｂ／ｃ小鼠，得到具有对Ｌｐ（ａ）高抗体活性的小鼠脾细胞，与小鼠ＮＳ１骨髓瘤细胞经聚乙二醇（ＰＥＧ）杂文融合，建立分泌特异抗人血浆Ｌｐ（ａ）的单克隆杂交瘤细胞系，对其中的─株单克隆细胞株──Ａ５ｇ３进行了初步分析。Ａ５ｇ３分泌的单克隆抗体类型为ＩｇＧ（２ａ），经间接ＥＬＩＳＡ法分析与Ｌｐ（ａ）有高度反应性，与低密度脂蛋白（ＬＤＬ）及血浆纤溶酶原（Ｐｇ）无反应，免疫印迹结果也表明，Ａ５ｇ３可与纯化的或血浆中的Ｌｐ（ａ）结合，与血浆中的其它成分和纯化的ＬＤＬ及Ｐｇ均不结合，因此Ａ５ｇ３是特异抗人血浆Ｌｐ（ａ）的单克隆抗体。Ａ５ｇ３与还原后的Ｌｐ（ａ）也不结合，说明其与Ｌｐ（ａ）的结合依赖于抗原的完整结构，具有此种特性的Ｌｐ（ａ）特异抗体国内少见报道。     

LDL-unbound apolipoprotein(a) and carotid atherosclerosis in hemodialysis patients

High lipoprotein{a) [Lp(a)] plasma concentrations, which are genetically determined by apo(a) size polymorphism, are directly associated with an increased risk for atherosclerosis. Patients with end-stage renal disease (ESRD), who show an enormous prevalence of cardiovascular disease, have elevated plasma concentrations of Lp(a). In recent studies we were able to show that apo(a) size polymorphism is a better predictor for carotid atherosclerosis and coronary artery disease in hemodialysis patients than concentrations of Lp(a) and other lipoproteins. Less than 5% of apo(a) in plasma exists in a low-density lipoprotein (LDL)-unbound form. This “free” apo(a) consists mainly of disintegrated apo(a) molecules of different molecular weight, ranging from about 125 to 360 kDa. LDL-unbound apo(a) molecules are elevated in patients with ESRD. The aim of this study was therefore to investigate whether the LDL-unbound form of apo(a) contributes to the prediction of carotid atherosclerosis in a group of 153 hemodialysis patients. The absolute amount of LDL-unbound apo(a) showed a trend to increasing values with the degree of carotid atherosclerosis, but the correlation of Lp(a) plasma concentrations with atherosclerosis was more pronounced. In multivariate analysis the two variables were related to neither the presence nor the degree of atherosclerosis. Instead, the apo(a) phenotype took the place of Lp(a) and LDL-unbound apo(a). After adjustment for other variables, the odds ratio for carotid atherosclerosis in patients with a low molecular weight apo(a) phenotype was about 5 (p < 0.01). This indicates a strong association between the apo(a) phenotype and the prevalence of carotid atherosclerosis. Finally, multivariate regression analysis revealed age, angina pectoris and the apo(a) phenotype as the only significant predictors of the degree of atherosclerosis in these patients. In summary, it seems that LDL-unbound apo(a) levels do not contribute to the prediction of carotid atherosclerosis in hemodialysis patients. However, this does not mean that “free”, mainly disintegrated, apo(a) has no atherogenic potential.     

The effect of percutaneous oestrogen replacement therapy on Lp(a) and other lipoproteins

Objectives: To determine the effect of percutaneous oestrogen replacement therapy on lipoprotein (a) and other plasma lipoproteins. Methods: Open longitudinal prospective study conducted at the hormone replacement clinic of the Prince of Wales Hospital, New Territories, Hong Kong. Thirty women who had undergone a total abdominal hysterectomy and bilateral salpingo-oophorectomy for benign gynaecological conditions were treated with 1.5 mg of percutaneous 17β-oestradiol gel applied daily for a period of 12 consecutive months. Measurements of plasma lipoproteins were made before the commencement of treatment and repeated at 6- and 12-month intervals. Results: There was a significant reduction in the concentrations of Lp(a) during the first 6 months of treatment, with median values falling from 7.87 mg dL⁻¹ to 6.16 mg dL⁻¹ (P = 0.004, 0–6 months). During the second 6 months, the median concentration increased to 9.38 mg dL⁻¹, (P = 0.072, 66-12 months), which did not significantly differ from the baseline level (P = 0.545, 0–12 months). Significant reductions in the concentrations of apoprotein A-I (apo A-I), apoprotein B (apo B), high density lipoprotein cholesterol (HDL-C), and HDL₃-C were also present after 6 months (P = 0.043, 0.049, 0.028, 0.013, respectively), but there were no differences between the baseline values of these lipoproteins and those at the completion of the study (P = 0.948, 0.244, 0.839, 0.117 respectively). Drug compliance was maintained throughout the study, with similar mean oestradiol concentrations at 6 and 12 months. Conclusions: The percutaneous administration of 17β-oestradiol has variable short term effects on plasma lipoproteins which are not maintained over a longer duration of treatment. By avoiding the ‘first pass’ effect on the liver, this method of delivery does not appear to produce the sustained changes in lipoproteins seen with oral treatment.