Synthesis and secretion of apolipoprotein A1 by chick breast muscle

The present work shows that chick breast muscles synthesize and secrete a protein which is very similar to chicken serum apolipoprotein A1 (Apo-A1), the major protein constituent of serum "high density" lipoprotein particles. This conclusion is based on the following observations. 1) When chick breast muscle explants were incubated in the presence of radioactive amino acids, a labeled protein of the same size as serum Apo-A1 (Mr approximately equal to 27,000) accumulated in the incubation media; 2) the muscle-derived secretory protein and serum Apo-A1 generated the same size distribution of peptide fragments following digestion with Staphylococcus aureus V8 protease; and 3) antibodies raised against serum Apo-A1 specifically precipitated the radioactive muscle secretory protein. The newly secreted muscle-derived Apo-A1 was associated with lipid, as judged by its "flotation" behavior during centrifugation of the labeled incubation media in the presence of 0.2 g/ml of sodium bromide; this observation suggests that muscle explants secreted Apo-A1 molecules as part of lipoprotein particles or that these Apo-A1 molecules became associated with lipid shortly after their secretion. The present work, together with the very recent report by Blue et al. (Blue, M.L., Ostapchuk, P., Gordon, J.S., and Williams, D.L. (1982) J. Biol. Chem. 257, 11151-11159) demonstrate that avian tissues other than liver and intestinal mucosa synthesize and secrete Apo-A1. Results of short term amino acid incorporation experiments showed that chick breast muscles synthesize Apo-A1 at high rates only during the terminal stages of embryonic development and early stages of postembryonic maturation. Around the time of hatching, the relative rate of synthesis of Apo-A1 by chick breast muscle was found to be higher than in liver, a documented major site of synthesis of this apolipoprotein. Possible physiological implications of the present work will be considered.     

Expression and secretion of human apolipoprotein A-I in the heart

Nuclear envelope-peripheral heterochromatin fractions contain multiple histone kinase activities. In vitro assays and amino-terminal sequencing show that one of these activities co-isolates with heterochromatin protein 1 (HP1) and phosphorylates histone H3 at threonine 3. Antibodies recognizing this post-translational modification reveal that in vivo phosphorylation at threonine 3 commences at early prophase in the vicinity of the nuclear envelope, spreads to pericentromeric chromatin during prometaphase and is fully reversed by late anaphase. This spatio-temporal pattern is distinct from H3 phosphorylation at serine 10, which also occurs during cell division, suggesting segregation of differentially phosphorylated chromatin to different regions of mitotic chromosomes.     

Synthesis of recombinant high density lipoprotein with apolipoprotein A-I and apolipoprotein A-V

It has been shown that apolipoprotein A-V (apoA-V) over-expression significantly lowers plasma triglyceride levels and decreases atherosclerotic lesion development. To assess the feasibility of recombinant high density lipoprotein (rHDL) reconstituted with apoA-V and apolipoprotein A-I (apoA-I) as a therapeutic agent for hyperlipidemic disorder and atherosclerosis, a series of rHDL were synthesized in vitro with various mass ratios of recombinant apoA-I and apoA-V. It is interesting to find that apoA-V of rHDL had no effect on lipoprotein lipase (LPL) activation in vitro and very low density lipoprotein (VLDL) clearance in HepG2 cells and in vivo. By contrast, LPL activation and VLDL clearance were inhibited by the addition of apoA-V to rHDL. Furthermore, the apoA-V of rHDL could not redistribute from rHDL to VLDL after incubation at 37°C for 30 min. These findings suggest that an increase of apoA-V in rHDL could not play a role in VLDL clearance in vitro and in vivo, which could, at least in part, attribute to the lost redistribution of apoA-V from rHDL to VLDL and LPL binding ability of apoA-V in rHDL. The therapeutic application of rHDL reconstituted with apoA-V and apoA-I might need the construction of rHDL from which apoA-V could freely redistribute to VLDL.     

Denaturation of apolipoprotein A-I and the monomer form of apolipoprotein A-I(Milano).

In this study the thermal and denaturant induced unfolding of apolipoprotein A-I (apo A-I) and the monomer form of apolipoprotein A-I(Milano) (apo A-I(M)) was followed. Dimer apo A-I(M) was reduced with dithiothreitol, which was present in the protein solutions in all experiments. Thermal denaturation is followed by differential scanning calorimetry (DSC) and far-UV and near-UV CD. Both apo A-I and monomer apo A-IM have a broad asymmetric DSC peak that could be deconvoluted into three non two-state transitions, apo A-I being more stable than the monomer apo A-IM. Estimation of melting of tertiary structure by near-UV CD is lower than that for secondary structure determined from far-UV. This together with the non two-state unfolding of the proteins observed with DSC is indicative of unfolding via a molten globular-like state. Apo A-I and monomer apo A-I(M) are equally susceptible to guanidinum chloride, half-unfolded at 1.2 M denaturant. The presence of 0.5 and 1.0 M denaturant, lower and equalize the denaturation temperatures of the proteins, respectively.     

Human apolipoprotein A-I. Post-translational modification by fatty acid acylation

The human apolipoproteins are secretory proteins some of which have been shown to undergo proteolytic processing and post-translational addition of carbohydrate. Apolipoprotein A-I (apo-A-I), the predominant protein associated with high density lipoproteins, undergoes co-translational proteolytic processing as well as post-translational conversion of proapo-A-I to mature apo-A-I following cellular secretion. Utilizing the human hepatoma cell line HEP-G2, we have established that, in addition to proteolytic processing, secreted nascent apo-A-I is acylated with palmitate. Uniformly labeled [14C]palmitate and [1-14C]palmitate were each incorporated into apo-A-I when analyzed by sodium dodecyl sulfate gel electrophoresis and autoradiography. The acylation of apo-A-I with palmitate was confirmed by immunoprecipitation and gas chromatography/mass spectrometry. Hydroxylamine treatment resulted in the deacylation of apo-A-I. Although three of the apo-A-I isoforms analyzed by two-dimensional gel electrophoresis were shown to contain radio-labeled palmitate, 80% of acylated apo-A-I was in the proapolipoprotein A-I isoform. [14C]Oleate was not incorporated in secreted apo-A-I, indicating the specificity of the acylation of apo-A-I. Incubation of [14C] palmitate-acylated apo-A-I in serum and plasma under conditions in which proapo-A-I is proteolytically cleaved to mature apo-A-I did not result in deacylation. These data establish that fatty acid acylation occurs in human secretory proteins in addition to the previously reported acylation of cellular membrane proteins. These results suggest that the covalent linkage of lipids to apolipoproteins may play a critical role in apolipoprotein and lipoprotein metabolism.     

A novel function of karyopherin beta3 associated with apolipoprotein A-I secretion

Human karyopherin beta3, highly homologous to a yeast protein secretion enhancer (PSE1), has often been reported to be associated with a mediator of a nucleocytoplasmic transport pathway. Previously, we showed that karyopherin beta3 complemented the PSE1 and KAP123 double mutant. Our research suggested that karyopherin beta has an evolutionary function similar to that of yeast PSE1 and/or KAP 123. In this study, we performed yeast two-hybrid screening to find a protein which would interact with karyopherin beta3 and identified apolipoprotein A-I (apo A-I), a secretion protein with a primary function in cholesterol transport. By using in vitro binding assay, co-immunoprecipitation, and colocalization studies, we defined an interaction between karyopherin beta3 and apo A-I. In addition, overexpression of karyopherin beta3 significantly increased apo A-I secretion. These results suggest that karyopherin beta3 plays a crucial role in apo A-I secretion. These findings may be relevant to the study of a novel function of karyopherin beta3 and coronary artery diseases associated with apo A-I.     

Radioimmunoassay of apolipoprotein A-I of rat serum.

A double antibody radioimmunoassay technique was developed for quantification of apolipoprotein A-I, the major apoprotein of rat high density lipoprotein. Apo A-I was labeled with 125I by the chloramine-T method. 125I-labeled apo A-I had the same electrophoretic mobility as unlabeled apo A-I and more than 80% of the 125I was precipitated by rabbit anti apo A-I antibodies. The assay is sensitive at the level of 0.5-5 ng, and has intraassay and interassay coefficients of variation of 4.5 and 6.5% respectively. The specificity of the assay was established by competitive displacement of 125I-labeled apo A-I from its antibody by apo A-I and lipoproteins containing apo A-I, but not by rat albumin and other apoproteins. Immunoreactivity of high density lipoprotein and serum was only about 35% of that of their delipidated forms when Veronal buffer was used as a diluent. Inclusion of 5 mM sodium decyl sulfate in the incubation mixture brought out reactivity equivalent to that found after delipidation. Completeness of the reaction was verified by comparison with the amount of apo A-I in chromatographic fractions of the total apoprotein of high density lipoprotein. Content (weight %, mean values +/- S.D.) of immunoassayable apo A-I was: 62.3 +/- 5.9 in high density lipoprotein; 1.7 +/- 0.3 in low density lipoprotein; 0.09 +/- 0.03 in very low density lipoprotein and 25.0 +/- 5.0 in lymp chylomicrons. Concentration in whole serum was 51.4 +/- 8.9 mg/dl and 33.6 +/- 4.1 mg/dl for female and male rats, respectively (p less than 0.002), equivalent to the sex difference in concentration of high density lipoprotein. 95% of the apo A-I in serum was in high density lipoprotein, 5% in proteins of d greater than 1.21 g/ml and less than 1% in lipoproteins of d less than 1.063 g/ml.     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

Neutrophils activation can be diminished by apolipoprotein A-I

High density lipoprotein (HDL) has anti-inflammatory function. To investigate the effects of apolipoprotein A-I (ApoA-I), the major apolipoprotein of HDL, on activated neutrophils, we stimulated neutrophils in vitro with fMLP and PMA, as a receptor-binding and a nonreceptor-binding stimuli, respectively, and incubated ApoA-I with those neutrophils. Three conditions were utilized: 1) resting neutrophils + ApoA-I (0, 2.5,5, 10 microg/mL respectively), 2) fMLP(10(-7) mol/L)-activated neutrophils + ApoA-I (0, 2.5, 5, 10 microg/mL respectively), and 3) PMA(10(-7) mol/L)-activated neutrophils + ApoA-I (0, 2.5, 5, 10 microg/mL respectively). After incubation, we measured neutrophils adhesion to fibronectin, oxidative bust (O2- and H2O2 production), degranulation (release of MPO and elastase), and L929 cell mortality which were attacked by release-out of cytokines in activated neutrophils (using MTT). Our results showed that in vitro ApoA-I inhibits fMLP- and PMA- activated neutrophil adhesion, oxidative burst, degranulation and L929 cell mortality. These inhibition effects of ApoA-I on fMLP-activated neutrophils are more powerful than that on PMA-activated neutrophils. ApoA-I has no effect on resting neutrophils. We concluded that ApoA-I could diminish the function of activated neutrophils.     

Quantitative determination of apolipoprotein A-I in high-density lipoproteins and 'free' apolipoprotein A-I by two-dimensional agarose gel lipoprotein-'rocket' immunoelectrophoresis of human serum

A method has been developed for quantitative analysis of 'free' apolipoprotein A-I and apolipoprotein A-I associated with high-density lipoprotein (HDL) in serum. The method utilizes the difference between the rate of electrophoretic migration of apolipoprotein A-I associated with HDL (alpha) and 'free' apolipoprotein A-I (pre-beta) in agarose gel. Apolipoprotein A-I is subsequently quantitated by electrophoresis in a second dimensional gel containing anti-apolipoprotein A-I antibodies. Using this method all apolipoprotein A-I of normal fasting serum was found associated with HDL (n = 16). By contrast, 'free' apolipoprotein A-I accounted for up to 12% of the total in the serum of patients with isolated hypertriglyceridemia (n = 8) or mixed hyperlipoproteinemia (n = 8). Between 30 and 35% of 'free' apolipoprotein A-I was found in one patient afflicted with the apolipoprotein C-II deficiency syndrome. Also, 'free' apolipoprotein A-I could be detected in normal postabsorptive serum. 30 and 90 min following heparin-enhanced lipolysis 'free' apolipoprotein A-I accounted for 23 and 20%, respectively, of the total apolipoprotein A-I of serum. Apolipoprotein A-I associated with HDL remained unaltered. It appears, therefore, that 'free' apolipoprotein A-I is liberated from triglyceride-rich lipoproteins during lipolysis.     

Proteolytic degradation and impaired secretion of an apolipoprotein A-I mutant associated with dominantly inherited hypoalphalipoproteinemia

We have devised a combined in vivo, ex vivo, and in vitro approach to elucidate the mechanism(s) responsible for the hypoalphalipoproteinemia in heterozygous carriers of a naturally occurring apolipoprotein A-I (apoA-I) variant (Leu(159) to Arg) known as apoA-I Finland (apoA-I(FIN)). Adenovirus-mediated expression of apoA-I(FIN) decreased apoA-I and high density lipoprotein cholesterol concentrations in both wild-type C57BL/6J mice and in apoA-I-deficient mice expressing native human apoA-I (hapoA-I). Interestingly, apoA-I(FIN) was degraded in the plasma, and the extent of proteolysis correlated with the most significant reductions in murine apoA-I concentrations. ApoA-I(FIN) had impaired activation of lecithin:cholesterol acyltransferase in vitro compared with hapoA-I, but in a mixed lipoprotein preparation consisting of both hapoA-I and apoA-I(FIN) there was only a moderate reduction in the activation of this enzyme. Importantly, secretion of apoA-I was also decreased from primary apoA-I-deficient hepatocytes when hapoA-I was co-expressed with apoA-I(FIN) following infection with recombinant adenoviruses, a condition that mimics secretion in heterozygotes. Thus, this is the first demonstration of an apoA-I point mutation that decreases LCAT activation, impairs hepatocyte secretion of apoA-I, and makes apoA-I susceptible to proteolysis leading to dominantly inherited hypoalphalipoproteinemia.     

Biosynthesis and compartmentalization of Po,apolipoprotein A-I,and lipids in the myelinating chick sciatic nerve

Myelin deposition in developing chick sciatic nerve is associated with rapid synthesis of lipids, the major myelin protein Po and apo A-I, a major constituent of plasma lipoproteins. In order to understand possible roles of apo A-I in myelin assembly the synthesis and appearance of Po, apo A-I and lipids was studied in an intracellular fraction, an intralamellar fraction thought to be related to, or derived from, myelin and compact myelin from rapidly myelinating sciatic nerve of 1 day chicks. Incorporation with methionine or pulse-chase experiments indicated that initial synthesis of Po occurs in the intracellular fraction followed by movement to the intralamellar fraction and myelin. Incorporation of labelled oleate into phospholipids suggested that initial synthesis occurs in the intracellular and intralamellar fractions with slow movement to myelin. Incorporation of labelled galactose into cerebrosides suggested that initial synthesis occurs partially in myelin with slow loss from this fraction to the intralamellar fraction. However, incorporation of methionine into apo A-I indicated that initial synthesis occurred in the intracellular fraction with some transfer to the intralamellar fraction and secretion of a major portion into the incubation medium. It is concluded that the subcellular distribution of nascent apo A-I is not well coordinated with the distribution of other nascent constitutents of the myelin membrane. The accumulation of nascent Po, phospholipids and cerebrosides in the intralamellar fraction compared to compact myelin suggests that this fraction may play a role as a precursor membrane or as a storage site for assembly of myelin constituents into compact myelin.Abbreviations PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - apo apolipoprotein - PC phosphatidylcholine - PE phosphatidylethanolamine     

Inhibition of apolipoprotein A-I gene expression by obesity-associated endocannabinoids

Obesity is associated with increased serum endocannabinoid (EC) levels and decreased high-density lipoprotein cholesterol (HDLc). Apolipoprotein A-I (apo A-I), the primary protein component of HDL is expressed primarily in the liver and small intestine. To determine whether ECs regulate apo A-I gene expression directly, the effect of the obesity-associated ECs anandamide and 2-arachidonylglycerol on apo A-I gene expression was examined in the hepatocyte cell line HepG2 and the intestinal cell line Caco-2. Apo A-I protein secretion was suppressed nearly 50% by anandamide and 2-arachidonoylglycerol in a dose-dependent manner in both cell lines. Anandamide treatment suppressed both apo A-I mRNA and apo A-I gene promoter activity in both cell lines. Studies using apo A-I promoter deletion constructs indicated that repression of apo A-I promoter activity by anandamide requires a previously identified nuclear receptor binding site designated as site A. Furthermore, anandamide-treatment inhibited protein-DNA complex formation with the site A probe. Exogenous over expression of cannabinoid receptor 1 (CBR1) in HepG2 cells suppressed apo A-I promoter activity, while in Caco-2 cells, exogenous expression of both CBR1 and CBR2 could repress apo A-I promoter activity. The suppressive effect of anandamide on apo A-I promoter activity in Hep G2 cells could be inhibited by CBR1 antagonist AM251 but not by AM630, a selective and potent CBR2 inhibitor. These results indicate that ECs directly suppress apo A-I gene expression in both hepatocytes and intestinal cells, contributing to the decrease in serum HDLc in obese individuals.     

Apolipoprotein A-I(Milano) and apolipoprotein A-I(Paris) exhibit an antioxidant activity distinct from that of wild-type apolipoprotein A-I

Apolipoprotein A-I(Milano) (apoA-I(Milano)) and apoA-I(Paris) are rare cysteine variants of apoA-I that produce a HDL deficiency in the absence of cardiovascular disease in humans. This paradox provides the basis for the hypothesis that the cysteine variants possess a beneficial activity not associated with wild-type apoA-I (apoA-I(WT)). In this study, a unique antioxidant activity of apoA-I(Milano) and apoA-I(Paris) is described. ApoA-I(Milano) was twice as effective as apoA-I(Paris) in preventing lipoxygenase-mediated oxidation of phospholipids, whereas apoA-I(WT) was poorly active. Antioxidant activity was observed using the monomeric form of the variants and was equally effective before and after initiation of oxidative events. ApoA-I(Milano) protected phospholipid from reactive oxygen species (ROS) generated via xanthine/xanthine oxidase (X/Xo) but failed to inhibit X/Xo-induced reduction of cytochrome c. These results indicate that apoA-I(Milano) was unable to directly quench ROS in the aqueous phase. There were no differences between lipid-free apoA-I(Milano,) apoA-I(Paris), and apoA-I(WT) in mediating the efflux of cholesterol from macrophages, indicating that the cysteine variants interacted normally with the ABCA1 efflux pathway. The results indicate that incorporation of a free thiol within an amphipathic alpha helix of apoA-I confers an antioxidant activity distinct from that of apoA-I(WT). These studies are the first to relate gain of function to rare cysteine mutations in the apoA-I primary sequence.     

Structure of the murine gene encoding apolipoprotein A-I.

A 1.6-kb DNA fragment containing the gene encoding apolipoprotein A-I from the mouse, Mus musculus, has been cloned and sequenced. It contains three exons separated by two introns and encodes a secreted polypeptide of 262 amino acids (aa), 238 of which constitute the mature protein. Comparisons with the rat and human proteins indicate moderate levels of shared identity (71 and 66%, respectively), although the overall aa compositions yield proteins with identical pIs (5.4). Kyte-Doolittle analyses of the three proteins indicate that there is no significant difference in the structure of these apolipoproteins.     

Plasma apolipoprotein A-I levels and bile lipid secretion during thoracic duct drainage in the rat

A large part of the circulating apolipoprotein A-I (apoA-I) is produced by the intestine. Yet the plasma levels of apoA-I are retained or even increased in rats with thoracic duct drainage (Johansson, B. and Nilsson, A, (1981) FEBS Lett. 130, 305-308 and Franzén, J. et al. (1987) Biochim. Biophys. Acta 918, 11-15). In this study we examined the effects of biliary drainage and of combined biliary and lymphatic drainage on the plasma apoA-I levels, and also the effects of lymphatic drainage on the output of biliary lipids in the rat. 63 h of biliary drainage caused a 40% decrease of the serum apoA-I concentration. In contrast the concentration in rats with combined thoracic duct and biliary drainage was 153% of that in control rats. The biliary secretion of bile acids, phosphatidylcholine and cholesterol declined to a lower level in rats with combined thoracic duct and biliary drainage, but increased at the later time intervals to the same levels as in rats with bile fistulas only. Intravenous chyle infusion 3-36 h after commencing the biliary drainage did not prevent the decrease in biliary lipid output. The study thus provided no evidence that the reduced hepatic inflow of apoB-containing lipoproteins during biliary drainage is of importance for the reduced biliary lipid output. The loss of all the chyle lipoproteins leads, however, to an even more pronounced decrease in the biliary lipid secretion. The drainage of all the chyle constituents also leads to an increased apoA-I synthesis that more than compensates for the apoA-I loss in chyle, whereas biliary drainage only lowers the plasma apoA-I levels.     

Structural analysis of human apolipoprotein A-I variants. Amino acid substitutions are nonrandomly distributed throughout the apolipoprotein A-I primary structure

In the course of an electrophoretic mutation screening program of 32,000 dried blood samples from newborns, 17 genetic variants of apolipoprotein A-I (apoA-I) were found and structurally analyzed. The following defects were identified by the combined use of high performance liquid chromatography, time-of-flight secondary ion mass spectrometry, and sequence analysis: Pro3----Arg (1 x), Pro4----Arg (1 x), Asp89----Glu (1 x), Lys107----0 (4 x), Lys107----Met (2 x), Glu139----Gly (2 x), Glu147----Val (1 x), Pro165----Arg (4 x), and Glu198----Lys (1 x). The distribution of point mutations in the apoA-I gene leading to these 9 and 11 other variants of apoA-I reported previously was statistically analyzed. Substitutions are overrepresented in the 10 amino-terminal amino acids (p less than 0.001, chi 2-test) and in residues 103-177 (p less than 0.025, chi 2-test) or residues 103-198 (p less than 0.05, chi 2-test), respectively. We further noted the following. (i) Prolines were substituted by arginine or histidine residues at a frequency much higher than expected on the basis of random nucleotide substitutions (5 out of 18 "electrically non-neutral" amino acid substitutions, p less than 0.001, chi 2-test). These substitutions are the result of transversions of cytosines contained within stretches of at least 5 consecutive cytosines in the apoA-I gene. The observed hypervariability of the apoA-I amino terminus, therefore, might be caused by a hot spot for mutation formed by the 7 subsequent cytosines in codons 3, 4, and 5. (ii) CpG dinucleotides were overrepresentatively affected by C----T transitions (5 out of 18 electrically nonneutral amino acid substitution, p less than 0.001, chi 2-test). The hypervariability of the apoA-I alpha-helical domain might therefore be caused by CpG dinucleotides predominantly occurring in codons 120-208 of apoA-I (82 out of 125). (iii) Comparison of mutation sites in the human apoA-I gene with sites of nonsynonymous substitutions revealed that amino acid substitutions found in human apoA-I were predominantly localized in areas that were little conserved during mammalian evolution. These regions may therefore represent areas of less structural constraint for the function of apoA-I.