Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles

Human apolipoprotein A-I (apo A-I) and its engineered constructs form discoidal lipid bilayers upon interaction with lipids in vitro. We now report the cloning, expression, and purification of apo A-I derived from zebrafish (Danio rerio), which combines with phospholipids to form similar discoidal bilayers and may prove to be superior to human apo A-I constructs for rapid reconstitution of seven-transmembrane helix receptors into nanoscale apolipoprotein bound bilayers (NABBs). We characterized NABBs by gel-filtration chromatography, native polyacrylamide gradient gel electrophoresis, UV-visible photobleaching difference spectroscopy, and fluorescence spectroscopy. We used electron microscopy to determine the stoichiometry and orientation of rhodopsin (rho)-containing NABBs prepared under various conditions and correlated stability and signaling efficiency of rho in NABBs with either one or two receptors. We discovered that the specific activity of G protein coupling for single rhos sequestered in individual NABBs was nearly identical with that of two rhos per NABB under conditions where stoichiometry and orientation could be inferred by electron microscopy imaging. Thermal stability of rho in NABBs was superior to that of rho in various commonly used detergents. We conclude that the NABB system using engineered zebrafish apo A-I is a native-like membrane mimetic system for G-protein-coupled receptors and discuss strategies for rapid incorporation of expressed membrane proteins into NABBs.     

Intracellular supply of phospholipids for biliary secretion: evidence for a nonvesicular transport component

Phospholipids (PL) for biliary secretion could be supplied from the endoplasmic reticulum (ER) to the plasma membrane by cytosolic transfer proteins or transport vesicles. Therefore, we studied whether biliary secretions of PL and apolipoprotein A-I (apo A-I), as markers for the ER-to-Golgi vesicular transport pathway, are tightly coupled in isolated perfused rat livers with enhanced secretion (+60%) of PL after withdrawal of the cholesterol synthesis inhibitor pravastatin (0.1% of chow, fed for 7 days). Blocking agents dissociated the secretion of apo A-I and PL. Brefeldin A as well as cycloheximide inhibited biliary secretion of apo A-I (-52%; -68%), however, not of PL. Both bilirubin ditaurate and taurodehydrocholic acid reduced biliary secretion of PL (-27%; -79%), but not of apo A-I. The data support the concept that PL destined for biliary secretion bypass the vesicular transport pathway of apo A-I through the Golgi compartment, most likely via cytosolic transfer proteins.     

Hypobetalipoproteinemia with an apparently recessive inheritance due to a "de novo" mutation of apolipoprotein B

Familial hypobetalipoproteinemia (FHBL) is a co-dominant disorder either linked or not linked to apolipoprotein (apo) B gene. Abetalipoproteinemia (ABL) is a recessive disorder due to mutations of microsomal triglyceride transfer protein (MTP) gene. We investigated a patient with apparently recessive hypobetalipoproteinemia consistent with symptomatic heterozygous FHBL or a "mild" form of ABL. The proband had fatty liver associated with LDL-cholesterol (LDL-C) and apo B levels <5th percentile but no truncated apo B forms detectable in plasma. MTP gene sequence revealed that he was a carrier of the I128T polymorphism and an unreported amino acid substitution (V168I) unlikely to be the cause of hypobetalipoproteinemia. Apo B gene sequence showed that he was heterozygous for two single base substitutions in exon 9 and 22 resulting in a nonsense (Q294X) and a missense (R1101H) mutation, respectively. Neither of his parents carried the Q294X; his father and paternal grandmother carried the R1101H mutation. Analysis of polymorphic genetic markers excluded non-paternity. In conclusion, the proband has a "de novo" mutation of apo B gene resulting in a short truncated apo B form (apo B-6.46). Sporadic cases of FHBL with an apparently recessive transmission may be caused by "de novo" mutations of apo B gene.     

Inhibition of apolipoprotein B net synthesis and secretion from cultured rat hepatocytes by the calcium-channel blocker diltiazem.

1. The effect of the Ca2+-channel blocker diltiazem on hepatic apolipoprotein B (apo B) synthesis and secretion was studied in 12-18 h cultures of collagenase-dispersed rat hepatocytes. 2. The presence of diltiazem in the medium decreased apo B secretion by hepatocytes in a concentration-dependent manner. At 25 microM, diltiazem inhibited apo B secretion by approx. 36%, but there was no evidence of intracellular accumulation of apo B. 3. The inhibition of apo B secretion by hepatocytes was significantly correlated with cell-associated diltiazem (r = 0.72, P less than 0.01). 4. The rate of apo B secretion remained linear over 16 h even in the presence of 50 microM-diltiazem. 5. At diltiazem concentrations in the medium which were inhibitory for apo B secretion, [14C]acetate incorporation into cellular lipids and [35S]methionine incorporation into protein were enhanced. 6. Diltiazem inhibited the secretion of the apo B variants with a preferential inhibition of the higher-molecular-mass form of apo B (apo BH) over the lower-molecular-mass form (apo BL) at diltiazem concentrations in the medium greater than 25 microM. 7. Together, these results suggest that Ca2+ may play an important role in the synthesis and secretion of apo B-containing lipoproteins.     

Apolipoprotein B mRNA editing: a new tier for the control of gene expression.

Two forms of apolipoprotein (apo) B are found in mammals. The shorter form is translated from an edited mRNA in which a specific cytidine base is deaminated to a uridine, creating a new stop codon. Apo B mRNA editing is mediated by a site-specific cytidine deaminase that recognizes a downstream target sequence in the RNA. The enzyme has no energy or cofactor requirements and no RNA component, and thus bears no obvious relationship to RNA processing events such as splicing or polyadenylation. While apo B mRNA editing activity may have arrived late in evolution to target dietary lipid to the liver in mammals, the discovery of the editing activity in tissues and cells that do not express apo B suggests a more widespread role in the generation of RNA and protein diversity.     

Characterization of the apolipoprotein B mRNA editing enzyme: no similarity to the proposed mechanism of RNA editing in kinetoplastid protozoa.

Intestinal apolipoprotein B mRNA is edited at nucleotide 6666 by a C to U transition resulting in a translational stop codon. The enzymatic properties of the editing activity were characterised in vitro using rat enterocyte cytosolic extract. The editing activity has no nucleotide or ion cofactor requirement. It shows substrate saturation with an apparent Km for the RNA substrate of 2.2 nM. The editing enzyme requires no lag period prior to catalysis, and does not assemble into a higher order complex on the RNA substrate. In crude cytosolic extract editing activity is completely abolished by treatment with micrococcal nuclease or RNAse A. Partially purified editing enzyme is no longer sensitive to nucleases, but is inhibited in a dose dependent manner by nuclease inactivated crude extract. The buoyant density of partially purified editing enzyme is 1.3 g/ml, that of pure protein. Therefore, the apolipoprotein B mRNA editing activity consists of a well defined enzyme with no RNA component. The nuclease sensitivity in crude cytosolic extract is explained by the generation of inhibitors for the editing enzyme. The editing of apo B mRNA has little similarity to complex mRNA processing events such as splicing and unlike editing in kinetoplastid protozoa does not utilise guide RNAs.     

Apolipoprotein B gene variants are involved in the determination of blood glucose and lipid levels in patients with non-insulin dependent diabetes mellitus

We have examined the frequency of the EcoRI, XbaI and MspI RFLPs of the apolipoprotein B (apo B) gene in 110 type 2 diabetic patients and 91 healthy control subjects in order to ascertain whether variation in this gene may influence the development of non-insulin dependent diabetes mellitus (type 2 diabetes). Serum lipids including total-cholesterol (T-Chol), triacylglycerol (TAG), apolipoprotein E (apo E), apolipoprotein AI (apo AI), apolipoprotein B and lipoprotein (a) (Lp(a)) were analysed. Genomic DNA was extracted and the apo B polymorphic regions amplified by the polymerase chain reaction. Regions carrying EcoRI, XbaI, and MspI restriction sites present in the apo B gene were amplified and digested separately by the respective enzymes. No significant difference for genotypic frequencies was observed for the EcoRI, XbaI and MspI restriction sites in type 2 diabetic patients as compared to controls. Type 2 diabetic patients and controls with EcoRI +/+ and XbaI +/+ genotypes had higher apo E levels. The MspI +/+ genotype is more frequent in the patient and control groups with elevated T-Chol. Furthermore, the EcoRI -/-, XbaI -/-, and MspI +/+ genotypes were found to be significantly more frequent in type 2 diabetic patients with higher blood glucose levels. This study identifies the apo B gene polymorphisms in modulating plasma lipid/lipoprotein and glucose levels in patients with type 2 diabetes.     

Ferritins can regulate the secretion of apolipoprotein B

Apolipoprotein B is secreted with atherogenic lipids as lipoprotein particles from hepatocytes. Regulation of the secretion of apolipoprotein B is largely post-translational and reflects the balance between processes that leads to particle assembly or to intracellular degradation. Previously, we conducted a proteomic screen to find proteins that bind apolipoprotein B in rat liver microsomes. We identified ferritin heavy and light chains in this screen among other proteins and showed that the two ferritins bind apolipoprotein B directly in vitro. In hepatocytes and other cells, ferritin heavy and light chains form cytosolic cages that store iron. We now show that ferritin heavy or light chains post-translationally inhibit the secretion of apolipoprotein B without altering the export of other hepatic proteins including albumin, factor XIII, and apolipoprotein A-I. This inhibition of apolipoprotein B secretion is not due to diminished lipid synthesis and can be partially overcome by stimulating triglyceride synthesis. The block in apolipoprotein B secretion by ferritins leads to an increase in endoplasmic reticulum-associated degradation of the apolipoprotein. Thus, despite being cytosolic proteins without known chaperone activity, ferritins can specifically regulate the secretion of apolipoprotein B post-translationally. The metabolic pathways for iron storage and intercellular cholesterol and triglyceride transport could intersect.     

Quantitation of endogenous liver apolipoprotein B mRNA editing

The mRNA for apolipoprotein B is translated into either a high molecular weight (apo BH) or low molecular weight (apo BL) form of the protein depending on a novel form of RNA processing known as RNA editing. Apo BH mRNA editing is both tissue-specific and hormonally regulated and involves transition of cytidine to uridine at codon 2153 thereby converting a glutamine codon (CAA) to a translational stop codon (UAA). Three methods for quantitating the endogenous levels of liver apo B mRNA editing were compared: (1) Southern blot hybridization with discriminative thermal washes, (2) competimer-hybridization with discriminative thermal washes and (3) competimer-polymerase chain reaction (competimer-PCR). The data suggest that hybridization and PCR can yield similar quantitation when competing oligonucleotides are used. Based on competimer-PCR it is proposed that 40% and 85% of normal rat liver and small intestine apo B mRNA (respectively) are edited.     

Variation at the apolipoprotein (apo) AI-CIII-AIV gene cluster and apo B gene loci is associated with lipoprotein and apolipoprotein levels in Italian children.

We have used RFLPs of the apolipoprotein (apo) B gene and apo AI-CIII-AIV gene cluster to estimate the genetic contribution of variation at these loci to the variability of plasmid lipid, lipoprotein, and apolipoprotein levels in 209 children from Sezze in central Italy. The sample was randomly divided into group I (107 children) and group II (102 children). Four site polymorphisms (PvuII, XbaI, MspI, and EcoRI) of the apo B gene and five site polymorphisms (XmnI, PstI, SstI, PvuII-CIII, and PvuII-AIV) of the apo AI-CIII-AIV gene cluster were examined in group I children. After adjustment for gender, age, and body-mass index, polymorphisms at both gene loci (PvuII-B, PvuII-CIII, and PvuII-AIV) were associated with significant effects on the levels of plasma apo AI, apo B, or high-density lipoprotein-cholesterol. RFLPs that showed significant effects in group I were genotyped in group II. All three polymorphisms were associated with similar effects on apolipoprotein levels, though for all RFLPs the magnitude of the effects was smaller in the group II children and only statistically significant for the effect of the PvuII-B genotype on apo AI levels. In the total sample of 209 children 7.4% of the sample variance in apo AI levels was explained by variation associated with the apo B PvuII-B RFLP. In addition, the PvuII-B RFLP was associated with significant effects on plasma apo B levels and explained 5.7% of the sample variance. The PvuII-CIII and PvuII-AIV polymorphisms were both associated with differences in apo AI levels, explaining 3.7%-5.7% of the sample variance. Taken together, the three PvuII polymorphisms explained 17.7% of the phenotypic variance in apo AI levels. There was significant evidence for an effect of nonlinearity of the PvuII-CIII genotypes on apo AI levels, with the individuals heterozygous for the polymorphism having the highest apo AI levels. No evidence of interaction between genotype and gender, age, and body-mass index was shown by covariance analysis. The molecular explanation of this effect is unclear. Our data show that variation at both the apo AI-CIII-AIV and apo B loci are associated with lipoprotein and apolipoprotein levels in this sample of Italian children.     

Separation and visualization of apolipoprotein B species by sodium dodecyl sulfate-agarose gel electrophoresis and immunoblotting

A method for separation and visualization of the different apolipoprotein B species using 0.2% sodium dodecyl sulfate-1.5% agarose gel electrophoresis and immunoblotting is described. The method is capable of demonstrating the different forms of apolipoprotein B (apo B) in plasma volumes of 10-50 microliters without prior ultracentrifugation. After ultracentrifugation of samples, estimation of the ratio between apo B 48 and apo B 100 is possible by scanning of Coomassie-stained gels or immunoblots.     

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

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

Isolation and characterization of two sub-species of Lp(a), one containing apo E and one free of apo E.

Lipoprotein Lp(a) was isolated by immunoaffinity chromatography using anti apolipoprotein B and anti apolipoprotein (a) immunosorbents. Besides apolipoproteins (a) and B, this fraction was shown to contain apolipoproteins C and E. Therefore, it was decided to further purify this crude Lp(a) into particles containing apolipoprotein E and particles free of apo E, using chromatography with an anti apolipoprotein E immunosorbent. Lp(a), free of apolipoprotein E was cholesterol ester rich and triacylglycerol poor and was found mainly in the LDL size range. In contrast, Lp(a) containing apolipoprotein E was triacylglycerol rich and was distributed mainly in the VLDL and IDL size range. Binding of these two fractions, one containing apo E and one free of it, to the apo B/E receptor of HeLa cells was studied. Both fractions bound to the receptor but the one containing apo E had a better affinity than the one free of apo E. Further studies are needed to identify the clinical importance of these two different entities.     

A targeted apolipoprotein B-38.9-producing mutation causes fatty livers in mice due to the reduced ability of apolipoprotein B-38.9 to transport triglycerides

Nonphysiological truncations of apolipoprotein (apo) B-100 cause familial hypobetalipoproteinemia (FHBL) in humans and mice. An elucidation of the mechanisms underlying the FHBL phenotypes may provide valuable information on the metabolism of apo B-containing lipoproteins and the structure-function relationship of apo B. To generate a faithful mouse model of human FHBL, a subtle mutation was introduced into the mouse apo B gene by targeting embryonic stem cells using homologous recombination followed by removal of the selection marker gene by Cre-loxP-mediated site-specific recombination. The engineered mice bear a premature stop codon at residue 1767 and a 42-base pair loxP inserted into intron 24 of the apo B gene, thus closely resembling the apo B-38.9-producing mutation in humans. Apo B-38.9 was the sole apo B protein in homozygote (apob(38.9/38.9)) plasma. In heterozygotes (apob(+/)(38. 9)), apo B-100 and apo B-48 were reduced by 75 and 40%, respectively, and apo B-38.9 represented 20% of total circulating apo B. Hepatic apo B-38.9 mRNA levels were reduced by 40%. In cultured apob(+/)(38. 9) hepatocytes, apo B-100 was produced in trace quantities, and the synthesis rate of apo B-38.9 relative to apo B-48 was reduced by 40%. However, almost equimolar amounts of apo B-38.9 and apo B-48 were secreted into the media. Pulse-chase studies revealed that apo B-38. 9 was secreted at a faster rate and more efficiently than apoB-48. Nevertheless, both apob(+/)(38.9) and apob(38.9/38.9) mice had reduced hepatic triglyceride secretion rates and fatty livers. Thus, low mRNA levels or defective secretion of apo B-38.9 may not be responsible for the FHBL phenotypes caused by the apo B-38.9 mutation. Rather, a reduced capacity of apo B-38.9 for triglyceride transport may account for the fatty livers in these mice.     

Pause transfer: a topogenic sequence in apolipoprotein B mediates stopping and restarting of translocation.

Previously, we described the stepwise translocation of a large amino-terminal fragment of apolipoprotein B (apo B15) in which the nascent secretory chain translocates through a series of distinct, nonintegrated transmembrane intermediates with large domains exposed to the cytoplasm. Thus, apo B15 appears to stop and restart translocation at several points. We have identified a sequence of amino acids in apo B15 that confers this behavior on a heterologous chimeric protein. In addition, we dissect pausing into two distinct steps, stopping and restarting, thereby trapping otherwise transient intermediates. Finally, we demonstrate the function of a second "pause transfer" sequence over 200 amino acids downstream in apo B15 that restarts translocation posttranslationally, suggesting that multiple pause transfer sequences are involved in the biogenesis of apolipoprotein B.     

Hepatitis B virus surface antigen binds to apolipoprotein H.

We have previously demonstrated that a plasma membrane-enriched fraction isolated from human liver is capable of binding recombinant hepatitis B surface antigen (rHBsAg) (P. Pontisso, M. A. Petit, M. Bankowski, and M. E. Peeples, J. Virol. 63:1981-1988, 1989). In this study we have separated the plasma membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used a ligand-blotting technique to identify a 46-kDa rHBsAg-binding protein. This protein could be removed from the membranes with a weakly acidic buffer, implying that it is peripherally bound. Examination of human serum revealed that the 46-kDa binding protein is a serum protein. Isolation of plasma lipoproteins revealed that the binding protein is in part associated with chylomicrons and high-density lipoproteins, both of which are targeted to the hepatocyte during the normal course of lipid metabolism. The binding protein was identified as apolipoprotein H (apo H), also known as beta 2-glycoprotein I, on the basis of copurification of the rHBsAg-binding activity with the apo H protein and the ability of cDNA-expressed apo H to bind rHBsAg. Serum-derived HBsAg also binds to apo H, indicating that binding is not unique to rHBsAg. Binding is saturable, requires only the small S protein of rHBsAg, and is inhibited by excess rHBsAg, antibodies to HBsAg, and antibodies to apo H. The binding activity of apo H is destroyed upon reduction, indicating that 1 or more of its 22 disulfide bonds are required for interaction with rHBsAg. The possibility that an interaction between hepatitis B virus particles and lipoprotein particles may facilitate entry of the virus into hepatocytes is discussed.     

Apolipoprotein synthesis and secretion in Hep G2 cells: effects of monensin and cycloheximide.

Hep G2 cells were used to study the relationship between apolipoprotein synthesis and secretion, as revealed by their interaction with agents modulating these processes. Cycloheximide inhibited the secretion of both apolipoproteins (apo) AI and B, but the reduction in apo AI secretion was evident at earlier times. Monensin also inhibited secretion of apo AI and apo B, but only apo AI accumulated intracellularly. Pulse-chase studies showed that, at concentrations of monensin that had no effect on total protein synthesis, apo B synthesis was specifically inhibited. Triacylglycerol synthesis was inhibited to the same extent as apo B synthesis, but this preceded the latter inhibition and unlike apo B there was an accumulation of intracellular triglyceride. These results suggest that distinctive mechanisms modulate the synthesis and secretion of apo AI and apo B, and that apo B synthesis can be specifically inhibited by mechanisms that initially block triglyceride production.     

Biogenesis of very-low-density lipoproteins in rat liver. Intracellular distribution of apolipoprotein B

The hepatic subcellular distribution of apolipoprotein B (apo B) was studied quantitatively by using an enzyme immunoassay developed for apo B and by immunoadsorption-precipitation of [3H]leucine-labelled apo B. Over 50% (of 0.59 microgram/mg protein) of the apo B was located in the microsomal fraction. Further subfractionation of the microsomes revealed that 47% of the microsomal apo B was in the Golgi apparatus, while another 43% was associated with the rough endoplasmic reticulum. The smooth endoplasmic reticulum accounted for only 4% of the total. When rat livers were labelled with [3H]leucine for 10 min, the rough endoplasmic reticulum accounted for 80% of the total immunoadsorbed precipitable apo B radioactivity while the smooth accounted for 20%, with no contribution from the Golgi. However, only 8.7% of the total radioactive immunoadsorbed precipitable apo B was lipoprotein-associated, the remainder being membrane-bound. Lipoprotein-associated apo B radioactivity in the smooth endoplasmic reticulum accounted for 40%, with the rough contribution attributed at 50% and the Golgi at 9%. We concluded that (a) there are two major pools of apo B in rat liver microsomes; (b) although the apo B mass may be negligible in the smooth endoplasmic reticulum, the latter does play a role in lipoprotein biogenesis. The possible function of apo B associated with membranes of the microsomes is also discussed.     

Apolipoproteins and their electrophoretic pattern throughout the reproductive cycle in the green frog Rana esculenta

Lipoprotein fractions in Rana esculenta were separated using the same salt intervals currently applied for human lipoproteins. Very low density lipoproteins (VLDL), low density lipoproteins (LDL) and high density lipoproteins (HDL) were analyzed with reference to the electrophoretic pattern. The lipoprotein electrophoretic pattern in males and females throughout the reproductive cycle showed minor differences. In general, each fraction was characterized by a specific apolipoprotein content. VLDL and LDL fractions were dominated by a high molecular weight (MW) band, most likely the counterpart of human Apolipoprotein B (apo B). The apo B in R. esculenta cross reacted, although weakly, with antibodies raised against chicken apo B. The HDL fraction showed a band with an apparent MW of 29 kDa. The electrophoretic mobility of the protein moiety of HDL was similar to human apolipoprotein A-I (apo A-I). However, HDL apolipoprotein of R. esculenta did not cross react with antibodies against chicken apo A-I under either denaturing or native conditions. The HDL apolipoprotein of R. esculenta was purified by DEAE-Sephacel chromatography followed by HPLC. Its amino acid composition showed a moderate correlation with trout, salmon, chicken and human apo A-I.