Conformational analysis of serum apolipoprotein AII in lipoprotein complexes with bifunctional crosslinking reagents.

Apolipoprotein AII isolated from human serum high density lipoproteins was recombined with phosphatidylcholine to yield homogeneous particles of 80-120 A diameter. The radioactive bifunctional crosslinkers dimethyl [1,1'-14C]suberimidate and dimethyl 4,4'-dithiobis([1-14C]butyrimidate) were reacted with these particles. The kinetics of the reactions and the localisation of the crosslinked lysines of the polypeptide chains were determined. Thermolysin hydrolysis followed by two-dimensional separation of the peptides and isolation of the mono- and bifunctionally modified peptides allowed the assignment of the crosslinked peptides of the apoprotein AII seqeunce. The crosslinking pattern indicates a close-neighbour relationship (13-15 A) of the peptide chains between amino acid residues 3, 23, 46 and 55 of the symmetrical halves of the apo AII molecule. A reconstruction of the secondary structure of Apo AII in the lipoprotein complex on the basis of theoretical calculations is given and correlated with the chemical data.     

The human apolipoprotein AII gene: structural organization and sites of expression.

The complete nucleotide sequence of the human apolipoprotein All gene together with 911 bases of 5' flanking sequence and 687 bases of 3' flanking sequence have been determined. The mRNA coding region is interrupted by three introns of 169, 293 and 395bp. The Intro-exon structure of the apo All gene is similar to that of the apo AI, apo CIII and apo E genes: three introns separate 4 coding sequences specifying the 5' untranslated region, pre-peptide, a short N-terminal domain and a C-terminal domain composed of a variable number of lipid-binding amphipathic helices. Intron II carries a 33bp dG-dT repetitive element adjacent to the 3' splice junction which has the potential to adopt the Z-DNA conformation. The 5' and 3' terminuses of the mRNA have been identified by primer extension and S1 nuclease mapping. A number of short direct repeats are found in the 5' flanking region and an inverted repeat occurs between the CAAT and TATA boxes. Downstream of the the gene is an Alu family repeat containing a polymorphic MspI site, the deletion of which is associated with increased circulating levels of apoAII. ApoAII gene expression was demonstrated in adult human liver and HepG2 cells but not in human small intestine. Of ten Rhesus monkey tissues examined apo All mRNA was detected only in liver.     

Isolation and characterisation of a cDNA encoding the precursor for human apolipoprotein AII

cDNA clones encoding human apolipoprotein AII have been isolated from an adult liver cDNA library. Apo AII mRNA was shown to be approximately 600 bases in length by RNA blot hybridisation. The intracellular precursor of apo AII was inferred from the cDNA sequence to be a 100 amino acid polypeptide consisting of the 77 residue mature protein and an additional 23 amino terminal residues. The amino terminal extension, divisible into an 18 residue signal peptide and a 5 residue propeptide, is separated from the first amino acid of mature apo AII by dibasic residues. The 5' untranslated region of the message is 61 bases in length and the 3' untranslated region 113 bases. A polyadenylation signal is situated 14 bases 3' of the poly(A) tail.     

Synthesis and processing of human serum apolipoprotein AII in vitro and in Hep G2 cells

The synthesis and structure of the primary translation product of apo AII in a human liver poly(A+) mRNA primed cell-free system and its cotranslational modification was studied parallel to studies in vivo with Hep G2 cells, a human hepatoma cell line. The primary translation product is a preproprotein containing 100 amino acid residues, which is cleaved by the signal peptidase of endoplasmic reticulum to pro-apo AII with the loss of the N-terminal pre-sequence consisting of 18 amino acid residues. Hep G2 cells contain about equal amounts of the proform of apolipoprotein AII and of mature apo AII. Approximately in the same ratio pro- and mature apo AII are secreted into the medium. Determination of the partial amino-acid sequence by automated Edman degradation of the labelled prepro- and proforms of apo AII led to the segmentation of the N-terminus of the primary translation product, consisting of 23 amino acid residues, into the pre-sequence (18 residues) and the pro-sequence (5 residues) with terminal Arg-Arg-residues at the cleavage site to apo AII. We must therefore correct our previously postulated 17 and 6 residues containing segmentation. So far no information has been obtained in which compartment and at what stage of posttranslational events the dimerization occurs by formation of the single disulfide bond at position Cys6 in the mature apo AII structure, leading to the symmetrical molecule.     

Expression of the human serum apolipoprotein AI and AII genes in Xenopus laevis oocytes. Lipid-associated secretion of gene products

The two major apolipoproteins of plasma high-density lipoproteins (HDL) are apolipoprotein AI (apo AI) and AII (apo AII). The apo AI and the correctly oriented apo CIII genes separated by 2.6 kb were obtained by fusion of two human lambda-genomic clones. The apo AII gene was isolated as a 3 kb clone. These apolipoprotein genes have been injected independently and together into Xenopus laevis oocytes and their expression studied. Both apolipoprotein genes were transcribed and translated into their preproforms and processed in Xenopus laevis oocytes to their proforms. They were secreted into the medium associated with newly synthesized phospholipids and neutral lipids as particles floating in the high-density lipoprotein range between 1.12 and 1.21 g/ml. Secreted apo AI is associated mainly with newly synthesized phosphatidylethanolamine and little triglyceride, apo AII with phosphatidylethanolamine, lysophosphatidylethanolamine and neutral lipids. Simultaneous injection of the apo AI and apo AII genes led to the secretion of both apoproteins which separated into two bands during CsCl-density gradient centrifugation. The heavier particles were associated with proapo AI and AII, phosphatidylethanolamine (greater than 90%) and traces of lysophosphatidylethanolamine as lipid components. Proapo AII was immunoprecipitated from the less dense fraction and found to be mainly associated with lysophosphatidylethanolamine. Radiolabelled newly synthesized apolipoproteins in secreted particles were characterized by immunoprecipitation after delipidation of the secreted lipoprotein particles. The oocyte-system proved very suitable for studies of the expression of serum apolipoprotein genes, the assembly of the apolipoproteins with specific lipids to lipoprotein particles and their secretion.     

DNA polymorphisms of the apolipoprotein AII and AI-CIII-AIV genes: a study in men selected for differences in high-density-lipoprotein cholesterol concentration.

We have investigated the frequencies of RFLPs of the apolipoprotein (apo) AII gene and of the apo AI-CIII-AIV gene cluster in 109 men, selected from a random sample of 1,910 men aged 45-59 years, to cover a wide range of plasma high-density-lipoprotein (HDL)-cholesterol concentration. There was no significant difference in apo AI or apo AII RFLP allele frequency between groups of individuals with high and low HDL-cholesterol concentration. However, the apo AI PstI RFLP showed an association with genetic variation determining the plasma concentration of apo AI in this sample. Genetic variation in the apo AI-CIII-AIV gene region, as defined by haplotypes, accounted for 16% of the phenotypic variance in the apo AI concentration and for 8% of the phenotypic variance in HDL-cholesterol concentration. There was no significant association between alleles of the apo AII MspI RFLP and genetic variation determining apo AII or HDL concentration. The data demonstrate that genetic variation in the apo AI-CIII-AIV gene cluster is involved in determining the serum concentration of apo AI in this sample of clinically well individuals.     

A new human hereditary amyloidosis: the result of a stop-codon mutation in the apolipoprotein AII gene

Hereditary systemic amyloidosis may be caused by mutations in a number of plasma proteins including transthyretin, apolipoprotein AI, fibrinogen Aalpha-chain, lysozyme, and gelsolin. Each type of amyloidosis is inherited as an autosomal dominant disease and is associated with a structurally altered protein that aggregates to form amyloid fibrils. Here we report that the amyloid protein in a family with previously uncharacterized hereditary renal amyloidosis is apolipoprotein AII (apoAII) with a 21-residue peptide extension on the carboxyl terminus. Sequence analysis of the apoAII gene of affected individuals showed heterozygosity for a single base substitution in the apoAII stop codon. The mutation results in extension of translation to the next in-frame stop codon 60 nucleotides downstream and is predicted to give a 21-residue C-terminal extension of the apoAII protein identical to that found in the amyloid. This mutation produces a novel BstNI restriction site that can be used to identify individuals with this gene by restriction fragment length polymorphism analysis. This is the first report of apoAII amyloid in humans and the first mutation identified in apoAII protein. Amyloid fibril formation from apoAII suggests that this lipoprotein, which is predicted to have an amphipathic helical structure, must undergo a transition to a beta-pleated sheet by a mechanism shared by other lipoproteins that form amyloid.     

Rat ovarian apolipoprotein E: localization and gonadotropic control of messenger RNA.

Apolipoprotein E (apo E) is a 35-kDa protein found in association with various lipoproteins. It is synthesized by a wide variety of tissues, including the ovary. It can serve several functions, such as 1) transport of excess cholesterol from peripheral tissue to the liver; 2) directed movement of cholesterol from areas of high to low cholesterol concentration within tissue or organs; and 3) inhibition of the conversion of theca progesterone to androgen, thus acting as an autocrine or paracrine factor within the ovary. To better understand the physiological role of ovarian apo E, we employed the technique of in situ hybridization utilizing 35S-labeled apo E riboprobes to identify cells containing E mRNA. We studied ovaries of hypophysectomized immature rats administered various regimens of gonadotropins because of the uniform, predictable stimulation of follicular granulosa and theca development, ovulation, and corpus luteum formation. Apo E mRNA was localized predominantly in the theca, with an increase associated with theca hypertrophy. Apo E mRNA increased in granulosa cells with the development of preovulatory Graafian follicles, but decreased to baseline following ovulation and corpus luteum formation. These data are consistent with two roles for apo E in the ovary: 1) directing cholesterol to cells needing cholesterol as substrate for cell proliferation and steroidogenesis, and 2) acting as an autocrine regulatory factor to reduce theca androgen substrate for follicle estrogen production.     

Chromosomal localization of the human apoprotein CI gene and of a polymorphic apoprotein AII gene

Human apoprotein(apo) CI and apo AII cDNA probes have been used to analyze the segregation of the human genes in panels of human-mouse hybrids. The apo CI (APOCI) gene segregates with chromosome 19 and the apo AII (APOA2) gene with chromosome 1. Somatic cell hybrids containing chromosome translocations were used to map the apo AII gene to the 1p21-1qter region. Human APOA2 is polymorphic for the restriction endonuclease Msp I. Comparison of human and mouse chromosome 1 reveals a conserved group including apo AII, renin and peptidase genes and suggests that APOA2 will be found distal to this group on human chromosome 1. The mouse apo AII gene is closely linked with genes that regulate HDL structure. Similar HDL regulatory genes will probably be found near human APOA2.     

Angiotensin II (AII)-related idiotypic network. I. Common occurrence of AII internal image on rabbit anti-idiotypic antibodies

Internal images of foreign Ag have been demonstrated in a variety of systems as anticipated by the idiotypic network theory formulated by Jerne. However, they seem to be of rare occurrence. In order to estimate the actual frequency of antibodies bearing internal images (Ab2-beta) of angiotensin II (AII), a phylogenetically conserved peptide made up of eight amino acids, nine rabbits were immunized with affinity or protein A purified anti-AII antibodies (Ab1) from allotype-matched rabbits. Four of nine antiidiotypic antibodies (Ab2) exhibited internal image-like reactivity. They recognized all the polyclonal Ab1 tested, whatever the species (rabbit, mouse, guinea pig). In addition, they were strongly reactive with three mAb specific for a carboxy terminus epitope on AII (mAb 110, 199, and 211) and with a fourth monoclonal Ab1 (133) identifying a more central epitope. Advantage was taken of this reactivity with mAb1 to purify Ab2-beta by affinity chromatography of Ab2 on Sepharose 4B covalently linked to the three monoclonal Ab1 specific for the carboxy terminus epitope. The eluate displayed typical internal image properties: 1) it reacted with all the polyclonal Ab1 tested, 2) this reaction was completely abolished by AII, and 3) rabbits and mice immunized with the eluate all produced Ab1. The AII related idiotypic network is thus characterized by high frequency and immunogenicity of AII internal images. In addition, reactivity of the latter with monoclonal Ab1 indicates variable expression on Ab2-beta of the epitopes defined by the mAb on the nominal Ag.     

Surface localization of 5'-nucleotidase on the mouse lymphocyte.

The optimal conditions for the cytochemical localization of 5'-nucleotidase (AMPase) in the mouse lymphocyte have been established. Quantitative monitoring of the effects of fixation and the components of the cytochemical medium showed that the cytochemistry can be performed under conditions that do not lead to loss of AMPase activity, and also under conditions where penetration of the substrate into the cell has occurred. The cytochemical reaction product was seen only on the surface of a proportion of splenic lymphocytes, regardless of the fixative used. Biochemical data confirmed that AMPase is an ectoenzyme and is the only protein in splenic lymphocytes capable of catalysing the hydrolysis of AMP. The activity of 5'-nucleotidase was studied also by harvesting cells either from thymus or spleen of A/ST or Cd-1 mouse strains. The enzymatic activity in splenic lymphocytes was more than six time higher than the activity of intact thymus cells. Cytochemically it was evident that within splenic lymphocytes there was a distinct population of lymphocytes with readily demonstrable AMPase activity, and another with no cytochemically demonstrable AMPase activity. It was concluded that murine lymphocytes vary in their activity of AMPase, and that the enzyme is exclusively confined to the cell surface.     

On the molecular basis of the recognition of angiotensin II (AII). NMR structure of AII in solution compared with the X-ray structure of AII bound to the mAb Fab131.

The high-resolution 3D structure of the octapeptide hormone angiotensin II (AII) in aqueous solution has been obtained by simulated annealing calculations, using high-resolution NMR-derived restraints. After final refinement in explicit water, a family of 13 structures was obtained with a backbone RMSD of 0.73 +/- 0.23 A. AII adopts a fairly compact folded structure, with its C-terminus and N-terminus approaching to within approximately 7.2 A of each other. The side chains of Arg2, Tyr4, Ile5 and His6 are oriented on one side of a plane defined by the peptide backbone, and the Val3 and Pro7 are pointing in opposite directions. The stabilization of the folded conformation can be explained by the stacking of the Val3 side chain with the Pro7 ring and by a hydrophobic cluster formed by the Tyr4, Ile5 and His6 side chains. Comparison between the NMR-derived structure of AII in aqueous solution and the refined crystal structure of the complex of AII with a high-affinity mAb (Fab131) [Garcia, K.C., Ronco, P.M., Verroust, P.J., Brunger, A.T., Amzel, L.M. (1992) Science257, 502-507] provides important quantitative information on two common structural features: (a) a U-shaped structure of the Tyr4-Ile5-His6-Pro7 sequence, which is the most immunogenic epitope of the peptide, with the Asp1 side chain oriented towards the interior of the turn approaching the C-terminus; (b) an Asx-turn-like motif with the side chain aspartate carboxyl group hydrogen-bonded to the main chain NH group of Arg2. It can be concluded that small rearrangements of the epitope 4-7 in the solution structure of AII are required by a mean value of 0.76 +/- 0.03 A for structure alignment and approximately 1.27 +/- 0.02 A for sequence alignment with the X-ray structure of AII bound to the mAb Fab131. These data are interpreted in terms of a biological "nucleus" conformation of the hormone in solution, which requires a limited number of structural rearrangements for receptor-antigen recognition and binding.     

Angiotensin II (AII)-related idiotypic network. II. Heterogeneity and fine specificity of AII internal images analyzed with monoclonal antibodies

Although the structural basis of internal images borne by beta type monoclonal anti-idiotypic antibody (Ab2) begins to be elucidated, there is little information on the repertoire of epitopes which make up the internal images expressed by polyclonal Ab2. We addressed this question by using a two-way approach in the angiotensin II (AII)-related idiotypic network, a system characterized by common occurrence of internal images on rabbit Ab2. First, two sets of internal images were purified in parallel by affinity chromatography on Sepharose 4B covalently linked to either mAb 110 (S4B-110), a mAb specific for a phenylalanine requiring carboxy terminus epitope (Phe8) on AII, or mAb 133 (S4B-133), reactive with a more central epitope also expressed on Phe8 substituted peptide analogs. The respective eluates, EL1 110 and E11 133, exhibited only partially overlapping reactivity, as demonstrated by 1) a different pattern of inhibition by various AII peptide analogues of EL1 110 and E11 133 binding to the same anti-AII antibody (Ab1) (either the homologous polyclonal Ab1 102 or mAb 133), 2) and a distinct profile of EL1 110 and EL1 133 binding to 12 biotinylated monoclonal Ab1 identifying a variety of epitopes on AII. To analyze further the respective distribution of mAb 110 and mAb 133 defined epitopes on Ab2-beta molecules, Ab2 were submitted to sequential affinity chromatography on S4B-110 followed by S4B-133, and the fractionated internal images were characterized by the pattern of binding to the various monoclonal Ab1. It was thus possible to purify two Ab2-beta subpopulations that exclusively imaged the determinant identified by mAb 110 (ii 110) or that identified by mAb 133 (ii 133). A third subpopulation which was successively retained on S4B-110 and S4B-133 expressed both internal images (ii 110 + 133), and was additionally reactive with all the other monoclonal Ab1 tested. In any case, monoclonal Ab1 binding to the different sets of internal images was totally inhibited by an excess of AII. These results indicate that the repertoire of internal epitopes is similar to that of the nominal Ag, but is scattered over distinct subpopulations of Ab2-beta molecules that can be fractionated by affinity chromatography. Some of the latter seem to bear several epitopes and resemble the whole nominal Ag, whereas others appear to image only one determinant. Second, we raised 7 anti-anti-idiotypic mAb (monoclonal Ab3) against affinity-purified Ab2-beta and analyzed their fine specificity for AII.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ectopic localization of putative AII amacrine cells in the outer plexiform layer of the developing FVB/N mouse retina

The FVB/N mouse is a model of retinitis pigmentosa which shows a rapid loss of photoreceptors during early postnatal (P) life. We investigated the cellular localization of glycine transporter 1 (GlyT-1) in the developing FVB/N mouse retina. In control retinas, the developmental pattern of GlyT-1-immunoreactive amacrine cells was well in accordance with a previous report. However, in the FVB/N mouse retina, some GlyT-1-labeled amacrine cells sent their processes into the outer plexiform layer (OPL) from P14 onward. From P21 onward, GlyT-1-labeled cells were visible in the OPL. These cells were further characterized by double-label immunofluorescence experiments with an antiserum against disabled 1 (Dab-1), and showed Dab-1 immunoreactivity, indicating that these cells are putative AII amacrine cells. These results clearly demonstrate that AII amacrine cells have the potential capacity to respond to photoreceptor degeneration by migrating or sprouting their processes into the OPL in the developing FVB/N mouse retina.This study was supported by a Korea Research Foundation Grant (2001, PF0005) from the Ministry of Education     

Analysis of apolipoprotein E nuclear localization using green fluorescent protein and biotinylation approaches

Previous results indicate that apoE (apolipoprotein E) may be associated with the nucleus in specific cell types, particularly under stress conditions such as serum starvation. In addition, nuclear apoE localization in ovarian cancer was recently shown to be correlated with patient survival. In order to better understand the factors associated with apoE nuclear localization, we examined intracellular apoE trafficking using live-cell imaging of CHO (Chinese-hamster ovary) cells that constitutively expressed apoE-GFP (green fluorescent protein). In addition, we used biotinylated apoE (in a lipid-free state and as a lipidated discoidal complex) to track the uptake and potential nuclear targeting of exogenous apoE. Our results indicate that a small proportion of apoE-GFP is detected in the nucleus of living apoE-GFP-expressing CHO cells and that the level of apoE-GFP in the nucleus is increased with serum starvation. Exposure of control CHO cells to exogenous apoE-GFP did not result in nuclear apoE-GFP localization in the recipient cells. Similarly, biotinylated apoE did not reach the nucleus of control CHO cells or SK-N-SH neurons. In contrast, when biotinylated apoE was delivered to recipient cells as a lipidated apoE disc, apoE was detected in the nucleus, suggesting that the lipoprotein complex alters the intracellular degradation or trafficking of apoE. Biotinylated apoE discs containing each of the three common human apoE isoforms (E2, E3 and E4) were also tested for nuclear trafficking. All three apoE isoforms were equally detected in the nucleus. These studies provide new evidence that apoE may be targeted to the nucleus and shed light on factors that regulate this process.     

Surface and intracellular localization of concanavalin A in human lymphocytes

Normal human lymphocytes were treated at +4 °C with concanavalin A and subsequently washed. The cells were then incubated for various periods at +37 °C in a culture medium and the fate of the agglutinin was followed by an electron microscopic stain specific for concanavalin A. At zero incubation time, a positive reaction was noted on the entire cell membrane and on numerous pinocytic vesicles; 80% of the lymphocytes were stained. Within 15 min of incubation, the cell surface appeared discontinuously labelled while all the intracytoplasmic vesicles were strongly positive. After 2 h of incubation, the cell surface was almost free of label and positive vesicles were found to be concentrated at one pole of the cell. After 3 h of incubation, no label was found on the cell membrane; instead, large vesicles communicating with the cell membrane contained labelled material.     

Intracellular modification of human apolipoprotein AII (apoAII) and sites of apoAII mRNA synthesis: comparison of apoAII with apoCII and apoCIII isoproteins

We have studied the intracellular modifications of human apoAII by pulse-chase labeling of HepG2 cell cultures with [35S]methionine or [3H]arginine followed by two-dimensional analysis and autoradiography of the radiolabeled apoAII isoproteins. A short (5.0-min) pulse showed the presence of a precursor form of apoAII (pI = 5.75) designated proapoAII or apoAII3. A 5-10-min chase resulted in a decrease in the relative concentration of the proapoAII coupled with an increase in the relative concentration of a new form (pI = 5.3) designated modified proapoAII or apoAII1. Longer chase resulted in the appearance of the plasma apoAII form and at least five other acidic apoAII isoproteins in the cell lysate and the culture medium. Labeling with [3H]arginine showed that apoAII isoproteins designated 3, 1, -1, and -3 contained the prosegment whereas isoproteins designated 1a, 0, -1a, -2a, -3a, and -4a did not. Comparison of nascent apoAII, apoCII, and apoCIII isoproteins revealed that they were distinctly different on the two-dimensional gels. Neuraminidase treatment converted the acidic apoAII isoproteins to isoproteins 1a and 0 (modified and plasma apoAII forms). The combined data are consistent with the following intra- and/or extracellular modifications of apoAII: (a) modification of the apoAII which results in the net loss of two positive charges; (b) glycosylation of the modified proapoAII with carbohydrate chains containing sialic acid; (c) proteolytic removal of the prosegment and cyclization of the N-terminal glutamine. Analysis of apoAII mRNA distribution in 13 fetal human tissues as well as in cell lines of human origin showed abundance of apoAII mRNA in liver and HepG2 cells and only traces in the intestine.