Apolipoprotein A-IV polymorphism and its effect on plasma lipid and apolipoprotein concentrations

Recently, we determined the apolipoprotein E (apoE) phenotype distribution in 2,000 randomly selected 35-year-old male individuals by slab gel isoelectric focusing of delipidated plasma samples, followed by immunoblotting using anti-apoE antiserum. These blots have been successfully re-used for immunovisualization of apoA-IV isoelectric focusing patterns. In a population sample of 1,393 individuals, four distinct apoA-IV isoforms were detected, encoded by the alleles A-IV*0, A-IV*1, A-IV*2, and A-IV*3 with gene frequencies of 0.002, 0.901, 0.079, and 0.018, respectively. The mean of plasma cholesterol, triglyceride, apoB and E levels did not differ significantly among the different apoA-IV phenotype groups. For these lipoprotein parameters, less than 0.1% of the total phenotypic variance could be accounted for by the APOA-IV gene locus. Our results did not show any effect of apoA-IV polymorphism on plasma apoA-I levels nor could we find any correlation between plasma levels of apoA-I and apoA-IV within the different apoA-IV phenotype groups. The plasma level of apoA-IV in subjects bearing the A-IV*3 allele is significantly lower than in subjects without the A-IV*3 allele (5 mg/dl versus 14 mg/dl). We therefore conclude that, in contrast to the apoE polymorphism, the polymorphism at the APOA-IV locus does not influence any of the levels of the lipoprotein parameters considered except apoA-IV.     

Plasma distribution of apoA-IV in patients with coronary artery disease and healthy controls

Recent studies showed lower apolipoprotein A-IV (apoA-IV) plasma concentrations in patients with coronary artery disease (CAD). The actual distribution of the antiatherogenic apoA-IV in human plasma, however, is discussed controversially and it was never investigated in CAD patients. We therefore developed a gentle technique to separate the various apoA-IV-containing plasma fractions. Using a combination of precipitation of all lipoproteins with 40% phosphotungstic acid and 4 M MgCl2, as well as immunoprecipitation of all apoA-I-containing particles with an anti-apoA-I antibody, we obtained three fractions of apoA-IV: lipid-free apoA-IV (about 4% of total apoA-IV), apoA-IV associated with apoA-I (LpA-I:A-IV, 12%), and apoA-I-unbound but lipoprotein-containing apoA-IV (LpA-IV, 84%). We compared these three apoA-IV fractions between 52 patients with a history of CAD and 52 age- and sex-matched healthy controls. Patients had significantly lower apoA-IV levels when compared to controls (10.28 +/- 3.67 mg/dl vs. 11.85 +/- 2.82 mg/dl, P = 0.029), but no major differences for the three plasma apoA-IV fractions. We conclude that our gentle separation method reveals a different distribution of apoA-IV than in many earlier studies. No major differences exist in the apoA-IV plasma distribution pattern between CAD patients and controls. Therefore, the antiatherogenic effect of apoA-IV has to be explained by other functional properties of apoA-IV (e.g., the antioxidative characteristics).     

A novel polymorphism of apolipoprotein A-IV is the result of an asparagine to serine substitution at residue 127

We have identified a hitherto genetic polymorphism of apolipoprotein A-IV (apo-IV). The molecular basis for this polymorphism is an A to G substitution at nucleotide 1687 resulting in an Asn to Ser change of amino acid 127. The frequencies of the two apoA-IV alleles (designated apoA-IV^127Asn and apoA-IV^127Ser), determined by Hinc II restriction analysis of PCR amplified exon three of the apoA-IV gene, were 0.788 and 0.212, respectively, in a Finnish population sample. Allele frequencies of another polymorphism due to a Thr to Ser substitution at amino acid 347 were determined using Hinf I restriction analysis. The allele frequencies were 0.823 for apoA-IV^347Thr and 0.177 for apoA-IV^247Ser. None of the apoA-IV polymorphisms (apoA-IV_{127:Asn→Ser}, apoA-IV_{347:Thr→Ser} and apoA-IV_{360:Gln→His}) had any effect of plasma lipid and lipoprotein concentrations in cohorts of dyslipidemic men and in a population sample of normolipidemic controls. There was also no association between the history of previous myocardial infarction and any of the apoA-IV alleles.     

A previously reported polymorphic plasma protein of dogs and horses, identified as apolipoprotein A-IV

By using immunoblotting with antiserum specific to human plasma apolipoprotein A-IV (apoA-IV), a previously reported polymorphic plasma protein of dogs viz postalbumin-2 (Pa2) and one of horses viz serum protein 2 (SP2), were identified as apoA-IV of these species. This along with earlier published results implied that: (1) both dog and horse show a high degree of polymorphism at the APOA4 locus with three common alleles in each of the two species; and (2) apoA-IV phenotyping in these two species can be done by analysing plasma/serum samples by a simple method of two-dimensional electrophoresis, conducted under non-denaturing conditions, followed by general-protein staining of gels.     

A previously reported polymorphic plasma protein of dogs and horses, identified as apolipoprotein A-IV

Summary. By using immunoblotting with antiserum specific to human plasma apolipoprotein A-IV (apoA-IV), a previously reported polymorphic plasma protein of dogs viz postalbumin-2 (Pa2) and one of horses viz serum protein 2 (SP2), were identified as apoA-IV of these species. This along with earlier published results implied that: (1) both dog and horse show a high degree of polymorphism at the APOA4 locus with three common alleles in each of the two species; and (2) apoA-IV phenotyping in these two species can be done by analysing plasma/serum samples by a simple method of two-dimensional electrophoresis, conducted under non-denaturing conditions, followed by general-protein staining of gels.     

Radioimmunoassay of rat apolipoprotein A-IV

We have developed a specific and sensitive radioimmunoassay for rat apolipoprotein A-IV (apoA-IV). The protocol includes treatment of the samples for 1 h at 60 degrees C with 0.7% Tween 20. Under these conditions, linear logit-log plots have been obtained for apoA-IV in lymph and plasma lipoprotein fractions as well as for purified apoA-IV. The sensitivity of the assay is to 20 ng. Absolute mass values obtained with the assay were validated by comparison with values obtained with an independent method of colorimetric reading of apoA-IV separated by polyacrylamide gel electrophoresis from plasma high density lipoproteins. The concentration of apoA-IV in fasting plasma averaged 10.2 mg/dl and in the mesenteric duct lymph 15.8 and 12.6 mg/dl during the fasting and the fat absorption states, respectively.     

Genetic polymorphism of human plasma apolipoprotein A-IV is due to nucleotide substitutions in the apolipoprotein A-IV gene

Genetic polymorphism of human plasma apolipoprotein A-IV has been detected by isoelectric focusing techniques followed by immunoblotting. The molecular basis for this apoA-IV polymorphism has been elucidated. Analysis of the protein coding sequences of the apoA-IV alleles 1 and 2 revealed a single G to T substitution in the apoA-IV-2 allele. The point mutation, occurring in a region highly conserved among the mouse, rat, and human A-IV apolipoproteins, converts the glutamine at position 360 of the mature protein to a histidine. This amino acid substitution adds one positive charge unit to the apoA-IV-1 isoprotein (pI 4.97) thus creating the more basic apoA-IV-2 isoprotein (pI 5.02). Computer analysis of the apoA-IV-2 allele revealed that the single G to T substitution results in the loss of a BbvI and a Fnu4HI restriction enzyme site and in the formation of a new restriction site for the enzyme SfaNI. Protein primary and secondary structure predictions were largely unaffected by this amino acid exchange. These results on the structure of the apoA-IV-1 and apoA-IV-2 alleles suggest that the three other rare isoproteins (apoA-IV-0, apoA-IV-3, and apoA-IV-4) are also due to nucleotide and subsequent amino acid substitutions in the apoA-IV sequence.     

Interfacial exclusion pressure determines the ability of apolipoprotein A-IV truncation mutants to activate cholesterol ester transfer protein

We used a panel of recombinant human apolipoprotein (apo) A-IV truncation mutants, in which pairs of 22-mer alpha-helices were sequentially deleted along the primary sequence, to examine the impact of protein structure and interfacial activity on the ability of apoA-IV to activate cholesterol ester transfer protein. Circular dichroism and fluorescence spectroscopy revealed that the secondary structure, conformation, and molecular stability of recombinant human apoA-IV were identical to the native protein. However, deletion of any of the alpha-helical domains in apoA-IV disrupted its tertiary structure and impaired its molecular stability. Surprisingly, determination of the water/phospholipid interfacial exclusion pressure of the apoA-IV truncation mutants revealed that, for most, deletion of amphipathic alpha-helical domains increased their affinity for phospholipid monolayers. All of the truncation mutants activated the transfer of fluorescent-labeled cholesterol esters between high and low density lipoproteins at a rate higher than native apoA-IV. There was a strong positive correlation (r = 0.790, p = 0.002) between the rate constant for cholesterol ester transfer and interfacial exclusion pressure. We conclude that molecular interfacial exclusion pressure, rather than specific helical domains, determines the degree to which apoA-IV, and likely other apolipoproteins, facilitate cholesterol ester transfer protein-mediated lipid exchange.     

Structure and function of the apoA-IV T347S and Q360H common variants

Human apolipoprotein A-IV (apoA-IV) is involved in chylomicron assembly and secretion, and in reverse cholesterol transport. Several apoA-IV isoforms exist, the most common in Caucasian populations being apoA-IV-1a (T347S) and apoA-IV-2 (Q360H). The objective of the present study was to investigate the impact of these common aminoacid substitutions on the ability of apoA-IV to bind lipids, to promote cell cholesterol efflux via ABCA1, and to maintain endothelial homeostasis. Recombinant forms of wild-type apoA-IV, apoA-IV Q360H, and apoA-IV T347S were produced in Escherichia coli. ApoA-IV Q360H and apoA-IV T347S showed a slightly higher α-helical content compared to wild-type apoA-IV, and associated with phospholipids faster than wild-type apoA-IV. The capacity to promote ABCA1-mediated cholesterol efflux was significantly greater for the apoA-IV T347S than the other apoA-IV isoforms. No differences were observed in the ability of apoA-IV isoforms to inhibit the production of VCAM-1 and IL-6 in TNFα-stimulated endothelial cells. In conclusion, the apoA-IV T347S common variant has increased lipid binding properties and cholesterol efflux capacity, while the apoA-IV Q360H variant has only slightly increased lipid binding properties. The two common aminoacid substitutions have no effect on the ability of apoA-IV to maintain endothelial homeostasis.     

Genotyping of apolipoprotein A-IV by digestion of amplified DNA with restriction endonuclease Fnu4HI: use of a tailored primer to abolish additional recognition sites during the gene amplification.

Apolipoprotein A-IV (apoA-IV) is involved in the metabolism of chylomicrons and high density lipoproteins. It displays genetic polymorphism due to two co-dominant alleles apoA-IV1 and apoA-IV2. The mutation that causes the polymorphism is a G to T substitution in the third base of codon 360 in the apoA-IV2 allele which results in a glutamine (Gln) to histidine (His) change of amino acid 360. This substitution leads to the abolition of a recognition site for the restriction enzyme Fnu4HI. Part of the third exon of the apoA-IV gene is amplified by the polymerase chain reaction (PCR) using a tailored primer, which abolishes the downstream recognition sites during the DNA amplification. The PCR products are digested with the restriction enzyme Fnu4HI and electrophoresed on a polyacrylamide gel. The apoA-IV genotypes are determined after staining with either ethidium bromide or silver. To validate the method, we determined the inheritance of the apoA-IV alleles in a three-generation kindred of 8 subjects and analyzed amplified DNA of 32 subjects of different apoA-IV phenotypes with this method. The results were compared to those obtained from isoelectric focusing and immunoblotting. In all cases studied, the two methods gave concordant results.     

The APOA4 T347S variant is associated with reduced plasma TAOS in subjects with diabetes mellitus and cardiovascular disease

Apolipoprotein A-IV (apoA-IV) has been postulated to be antiatherogenic. Transgenic APOA4/Apoe-/- mice are protected against atherosclerosis, with plasma apoA-IV displaying antioxidant activity in vitro. In humans, there is an inverse relationship between apoA-IV levels and risk of coronary heart disease (CHD). Furthermore, the APOA4 T347S rare allele has been associated with increased risk of CHD and reduced apoA-IV levels. Reduced total antioxidant status (TAOS) due to increased oxidative stress is implicated in the process of atherogenesis. Thus, this study aimed to examine the association between the APOA4 T347S variant and TAOS in diabetic patients with (n = 196) or without (n = 509) cardiovascular disease (CVD). A higher percentage of CVD patients were present in the lowest quartile of TAOS, compared with the rest (P = 0.04). Overall, there was no association between genotype and TAOS. However, in patients with CVD, homozygotes for the S347 allele had significantly lower TAOS compared with TT and TS subjects (31.2 +/- 9.89% and 42.5 +/- 13.04% TAOS, respectively; P = 0.0024), an effect that was not seen in the patients without CVD. This study offers direct support for an antioxidant capacity of apoA-IV, thus providing some explanation for the antiatherogenic role of apoA-IV and the higher CVD risk in S347 homozygotes.     

Structure and interfacial properties of chicken apolipoprotein A-IV

To gain insight into the evolution and function of apolipoprotein A-IV (apoA-IV) we compared structural and interfacial properties of chicken apoA-IV, human apoA-IV, and a recombinant human apoA-IV truncation mutant lacking the carboxyl terminus. Circular dichroism thermal denaturation studies revealed that the thermodynamic stability of the alpha-helical structure in chicken apoA-IV (DeltaH = 71.0 kcal/mol) was greater than that of human apoA-IV (63.6 kcal/mol), but similar to that of human apoA-I (73.1 kcal/mol). Fluorescence chemical denaturation studies revealed a multiphasic red shift with a 65% increase in relative quantum yield that preceded loss of alpha-helical structure, a phenomenon previously noted for human apoA-IV. The elastic modulus of chicken apoA-IV at the air/water interface was 13.7 mN/m, versus 21.7 mN/m for human apoA-IV and 7.6 mN/m for apoA-I. The interfacial exclusion pressure of chicken apoA-IV for phospholipid monolayers was 31.1 mN/m, versus 33.0 mN/m for human A-I and 28.5 mN/m for apoA-IV.We conclude that the secondary structural features of chicken apoA-IV more closely resemble those of human apoA-I, which may reflect the evolution of apoA-IV by intraexonic duplication of the apoA-I gene. However, the interfacial properties of chicken apoA-IV are intermediate between those of human apoA-I and apoA-IV, which suggests that chicken apoA-IV may represent an ancestral prototype of mammalian apoA-IV, which subsequently underwent further structural change as an evolutionary response to the requisites of mammalian lipoprotein metabolism.     

Human apolipoprotein A-IV reduces gastric acid secretion and diminishes ulcer formation in transgenic mice

We have investigated the involvement of human apolipoprotein A-IV (apoA-IV) in gastric acid secretion and ulcer formation in recently generated apoA-IV transgenic mice. Compared to control littermates, transgenic animals showed a gastric acid secretion decreased by 43-77% whereas only slight variations were observed in the different cell population densities within the gastric mucosa. In addition, no variation in gastrin levels was observed. Transgenics were protected against indomethacin-induced ulcer formation, with lesions diminishing by 45 to 64% compared to controls. These results indicate that endogenous apoA-IV expression can regulate gastric acid secretion and ulcer development.     

Transport of lipid and apolipoproteins A-I and A-IV in intestinal lymph of the rat

Intestinal lipid absorption is associated with marked increases in the synthesis and secretion of apolipoprotein A-IV (apoA-IV) by the small intestine. Whether the increased intestinal apoA-IV synthesis and secretion results from increased fat uptake, increased cellular triglyceride (TG) content, or increased secretion of TG-rich lipoproteins by the enterocytes is unknown. Previous work from this laboratory has shown that a hydrophobic surfactant, Pluronic L-81 (L-81), is a potent inhibitor of intestinal formation of chylomicrons (CM), without reducing fat uptake or re-synthesis to TG. Furthermore, this inhibition can be reversed quickly by the cessation of L-81 infusion. Thus L-81 offers a unique opportunity to study the relationship between lymphatic TG, apoA-I and A-IV secretion. In this study, we studied the lymphatic transport of TG, apoA-I, and apoA-IV during both the inhibitory phase (L-81 infused together with lipid) and the subsequent unblocking phase (saline infusion). Two groups of lymph fistula rats were used, the control and the experimental rats. In the experimental rats, a phosphate-buffered taurocholate-stabilized emulsion containing 40 mumol [3H]triolein, 7.8 mumol of phosphatidylcholine, and 1 mg L-81 per 3 ml was infused at 3 ml/h for 8 h. This was then replaced by glucose-saline infusion for an additional 12 h. The control rats received the same lipid emulsion as the experimental rats, but without L-81 added, for 8 h. Lymph lipid was determined both by radioactivity and by glyceride-glycerol determination, and the apoA-I and apoA-IV concentrations were determined by rocket electroimmunophoresis assay. L-81 inhibited the rise in lymphatic lipid and apoA-IV output in the experimental rats after the beginning of lipid + L-81 infusion. Upon cessation of L-81 infusion, the mucosal lipid accumulated as a result of L-81 treatment was rapidly cleared into lymph as CM. This was associated with a marked increase in apoA-IV output; the maximal output was about 3 times that of the fasting level. There was a time lag of 4-5 h between the peak lymph lipid output and the peak lymph apoA-IV output during the unblocking phase in the experimental rats. There was also a comparable time lag between the maximal lipid and apoA-IV outputs in the control animals. Incorporation studies using [3H]leucine showed that apoA-IV synthesis was not stimulated during lipid + L-81 infusion, perhaps explaining the lack of increase in lymphatic A-IV secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased plasma apoA-IV level is a marker of abnormal postprandial lipemia: a study in normoponderal and obese subjects.

Plasma apolipoprotein A-IV (apoA-IV) levels are found elevated in hypertriglyceridemic patients. However, the relationship between plasma apoA-IV level and postprandial lipemia is not well known and remains to be elucidated. Thus, our objective was to study the relationship between plasma apoA-IV and postprandial TG after an oral fat load test (OFLT). Plasma apoA-IV was measured at fast and during an OFLT in 16 normotriglyceridemic, normoglucose-tolerant android obese subjects (BMI = 34.6 +/- 2.9 kg/m(2)) and 30 normal weight controls (BMI = 22.2 +/- 2.3 kg/m(2)). In spite of not statistically different fasting plasma TG levels in controls and obese patients, the former group showed an altered TG response after OFLT, featuring increased nonchylomicron TG area under the curve (AUC) compared with controls (516 +/- 138 vs. 426 +/- 119 mmol/l x min, P < 0.05). As compared to controls, obese patients showed increased apoA-IV levels both at fast (138.5 +/- 22.4 vs. 124.0 +/- 22.8 mg/l, P < 0.05) and during the OFLT (apoA-IV AUC: 79,833 +/- 14,281 vs. 68,176 +/- 17,463 mg/l x min, P < 0.05). Among the whole population studied, as among the control and obese subgroups, fasting plasma apoA-IV correlated significantly with AUC of plasma TG (r = 0.60, P < 0.001), AUC of chymomicron TG (r = 0.45, P < 0.01), and AUC of nonchylomicron TG (r = 0.62, P < 0.001). In the multivariate analysis, fasting apoA-IV level constituted an independent and highly significant determinant of AUC of plasma TG, AUC of chymomicron TG, AUC of nonchylomicron TG, and incremental AUC of plasma TG. In conclusion, we show a strong link between fasting apoA-IV and postprandial TG metabolism. Plasma fasting apoA-IV is shown to be a good marker of TG response after an OFLT, providing additional information on post-load TG response in conjunction with other known factors such as fasting TGs.     

Human apolipoprotein A-IV: displacement from the surface of triglyceride-rich particles by HDL2-associated C-apoproteins

Human apolipoprotein A-IV rapidly dissociates from the surface of lymph chylomicrons following their entry into circulation by an unknown mechanism. We have therefore investigated the binding of human apoA-IV to triglyceride-rich particles and the interaction of these apoA-IV/lipid complexes with human HDL2. Human apoA-IV was purified from lipoprotein depleted serum (J. Lipid Res. 1983. 24:52-59). Triglyceride-rich particles of well-defined properties were isolated from Intralipid, a commercially available phospholipid-triglyceride emulsion. Various concentrations of radiolabeled human apoA-IV were incubated at 24 degrees C with a fixed quantity of lipid particles; the particles were reisolated by centrifugation, and bound and free apoA-IV were quantitated. In 50 mM Tris, pH 7.4, apoA-IV bound to the triglyceride-rich particles in a non-cooperative manner, with a Kd of 2.0 microM. The calculated maximal binding was 4.96 X 10(-4) mol of apoA-IV bound per mol of phospholipid. The addition of increasing amounts of human HDL2 to the incubations caused the progressive dissociation of apoA-IV from the triglyceride-rich particles. Analysis of the reisolated particles by isoelectric focusing demonstrated the presence of C-apoproteins, suggesting their transfer from HDL2. Addition of purified apoC-III-1 to the incubations at concentrations equivalent to those present in HDL2 caused a similar dissociation of apoA-IV. HDL2 was modified to selectively remove C-apoproteins, without alteration of other physical characteristics. This modified HDL2 was four times less effective in causing apoA-IV dissociation. These results demonstrate that the lipid binding properties of human apoA-IV may be quantitatively examined using triglyceride-rich particles as model chylomicrons. This approach reproduces in vitro the dissociation of apoA-IV that occurs in vivo when mesenteric lymph chylomicrons enter the circulation, and suggests that the primary mechanism for this phenomenon is the transfer of C-apoproteins from high density lipoproteins to the triglyceride-rich particle surface. We hypothesize that this mechanism may play an important role in the modulation of chylomicron apoA-IV content in man.     

Distribution of apolipoprotein A-IV among lipoprotein subclasses in rat serum

The distribution of apolipoproteins (apo) A-I, A-IV, and E in sera of fed and fasted rats was studied using various methods for the isolation of lipoproteins. Serum concentrations of apoA-I and apoA-IV decreased significantly during fasting (16 and 31%, respectively), while apoE concentrations remained essentially the same. Chromatography of sera on 6% agarose columns showed that apoA-IV is present on HDL and as so-called "free" apoA-IV. The concentration of "free" apoA-IV decreased six- to seven-fold during fasting, explaining the decrease in total serum apoA-IV. Serum apoA-I and apoE are almost exclusively associated with HDL-sized particles. When sera are centrifuged at a density of 1.21 g/ml, marked quantities of apoA-I (8-9%) and apoE (11-22%) are recovered in the "lipoprotein-deficient" infranatant, suggesting that ultracentrifugation affects the integrity of serum HDL. The nature of the chromatographically separated carriers of serum apoA-IV was investigated by quantitative immunoprecipitation. From these studies, it is concluded that apoA-IV in rat serum is present in at least three fractions: 1) particles with the size and composition of HDL, containing both apoA-I and apoA-IV and possibly minor quantities of apoE; 2) HDL-sized particles containing apoA-IV, but no apoA-I or apoE; 3) "free" apoA-IV probably containing small amounts of bound cholesterol and phospholipid.     

Apolipoprotein E and A-IV polymorphisms in ethnic Russians living in Estonia

137 Russians living in Estonia was screened by isoelectric focusing and immunoblotting procedures to determine the distribution of genetic variations in apolipoprotein E (apoE) and apolipoprotein A-IV (apoA-IV) genes. The apoA-IV-2 allele and epsilon4 allele frequency of the Russians tended to be lower than in most other European populations.     

Apolipoprotein A-IV is a novel substrate for matrix metalloproteinases

Screening of matrix metalloproteinase (MMP)-14 substrates in human plasma using a proteomics approach previously identified apolipoprotein A-IV (apoA-IV) as a novel substrate for MMP-14. Here, we show that among the tested MMPs, purified apoA-IV is most susceptible to cleavage by MMP-7, and that apoA-IV in plasma can be cleaved more efficiently by MMP-7 than MMP-14. Purified recombinant apoA-IV (44-kDa) was cleaved by MMP-7 into several fragments of 41, 32, 29, 27, 24, 22 and 19 kDa. N-terminal sequencing of the fragments identified two internal cleavage sites for MMP-7 in the apoA-IV sequence, between Glu(185) and Leu(186), and between Glu(262) and Leu(263). The cleavage of lipid-bound apoA-IV by MMP-7 was less efficient than that of lipid-free apoA-IV. Further, MMP-7-mediated cleavage of apoA-IV resulted in a rapid loss of its intrinsic anti-oxidant activity. Based on the fact that apoA-IV plays important roles in lipid metabolism and possesses anti-oxidant activity, we suggest that cleavage of lipid-free apoA-IV by MMP-7 has pathological implications in the development of hyperlipidemia and atherosclerosis.