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The Journal of Biological Chemistry Aug 1989The structural domains of human apolipoprotein B-100 in low density lipoproteins (LDL) and the conformational changes of B-100 that accompany the conversion of very low... (Comparative Study)
Comparative Study
The structural domains of human apolipoprotein B-100 in low density lipoproteins (LDL) and the conformational changes of B-100 that accompany the conversion of very low density lipoproteins (VLDL) to LDL were investigated by limited proteolysis with 12 endoproteases of various specificities, and their cleavage sites were determined. In B-100 of LDL, we identified two peptide regions that are highly susceptible to proteolytic cleavage. One region encompassed about 40 amino acids (residues 1280-1320, designated as the NH2-terminal region) and the other about 100 amino acids (residues 3180-3280, designated as the COOH-terminal region). IN LDL, the cleavage sites in both susceptible regions of B-100 were readily accessible to limited proteolysis; but in VLDL, only sites in the COOH-terminal region were readily accessible. Moreover, B-100 in VLDL appeared less degraded than B-100 in LDL by all enzymes used. Reduction of disulfide bonds of B-100 in both LDL and VLDL before digestion by Staphylococcus aureus V8 protease and clostripain exposed additional cleavage sites and increased the rate of B-100 degradation, suggesting that disulfide bonds probably exert conformational constraints. These results indicate the presence of three principal structural domains in B-100 of LDL that are relatively resistant to limited proteolysis. These three domains are connected by the two susceptible peptide regions. Our results also demonstrate differential accessibility of cleavage sites in B-100 of LDL and VLDL to limited proteolysis. This differential accessibility suggests that substantial changes in the conformation or environment of B-100 accompany the conversion of VLDL to LDL.
Topics: Amino Acid Sequence; Apolipoprotein B-100; Apolipoproteins B; Endopeptidases; Humans; Hydrolysis; Kinetics; Lipoproteins, LDL; Lipoproteins, VLDL; Metalloendopeptidases; Peptide Fragments; Protein Conformation
PubMed: 2668286
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1986The present study characterizes the substructural organization of apolipoprotein B-100 (B-100) of human plasma low density lipoproteins (LDL) and its relation to B-74...
The present study characterizes the substructural organization of apolipoprotein B-100 (B-100) of human plasma low density lipoproteins (LDL) and its relation to B-74 and B-26 of LDL and B-48 of chylomicrons and very low density lipoproteins. LDL were digested with human kallikrein and thrombin to yield two major fragments: K1 (Mr 410,000) and K2 (Mr 145,000) and T1 (Mr 385,000) and T2 (Mr 170,000), respectively. The antigenic sequences, Mr, and amino acid compositions of K1 and K2 were identical to those of plasma B-74 and B-26; B-26 and K2 had identical NH2-terminal sequences and correspond to the NH2-terminal region of B-100. K1 was further degraded by kallikrein to give K3 (Mr 235,000) and K4 (Mr 170,000); these peptides correspond immunochemically to two newly discovered plasma LDL peptides B-44 and B-30 and are assigned as complementary fragments of B-74. The thrombin cleavage fragments, T1 and T2, did not correspond to B-74 and B-26. Neither kallikrein nor thrombin generated a fragment from B-100 corresponding to B-48 in chylomicrons. However, B-48 showed antigenic homology with B-26 and to the of B-74 adjoining B-26, indicating that its structure is represented in the NH2-terminal half of B-100. The structural studies further clarify the relatedness among the B-100, B-74, B-26, and B-48 polypeptides and should now make possible the delineation of the functional domains mediating the interactions of apolipoprotein B in the circulation and arterial wall.
Topics: Amino Acid Sequence; Amino Acids; Antibodies; Antibodies, Monoclonal; Antigen-Antibody Complex; Apolipoprotein B-100; Apolipoprotein B-48; Apolipoproteins B; Chylomicrons; Humans; Kallikreins; Lipoproteins, VLDL; Molecular Weight; Peptide Fragments; Thrombin
PubMed: 3640763
DOI: No ID Found -
Gut Nov 1989Human apolipoprotein (apo)-B mRNA undergoes a novel tissue specific editing reaction which replaces a genomically templated cytidine with uridine. This substitution...
Human apolipoprotein (apo)-B mRNA undergoes a novel tissue specific editing reaction which replaces a genomically templated cytidine with uridine. This substitution converts codon 2153 from glutamine (CAA) in apo-B100 mRNA to a stop codon (UAA) in apo-B48 mRNA. This novel RNA editing process is responsible for the generation of hepatic apo-B100 and intestinal apo-B48. We have established the following concerning this process: (1) by transfection of a series of deletion mutants into the rat hepatoma cell line McArdle 7777, which makes both apo-B100 and apo-B48, we have defined a minimum sequence of 26 nucleotides that is required for apo-B mRNA editing. The sequence containing the modified nucleotide forms a 26 nucleotide highly conserved stem loop with the modified nucleotide occurring in an 8-base loop. (2) Conversion in vitro of apo-B mRNA has been established, using cell free S100 cytoplasmic extract and synthetic RNA templates. Activity was abolished by protease treatment. (3) Transgenic mice were created which expressed a human apo-B construct spanning the stop codon. Apo-B mRNA was found in all tissues examined and this was shown to undergo editing. (4) In the rat liver, which produces apo B-100 and apo-B48, modulation of the relative proportion of these proteins by thyroxine was demonstrated to be mediated at the level of the RNA editing mechanism. It is concluded that apo-B mRNA is edited by a generally expressed protein and editing is highly regulated.
Topics: Animals; Apolipoproteins B; Base Sequence; Biological Transport; Humans; Intestinal Mucosa; Lipid Metabolism; Liver; Mice; Mice, Transgenic; Molecular Sequence Data; RNA Processing, Post-Transcriptional; RNA, Messenger
PubMed: 2606364
DOI: 10.1136/gut.30.spec_no.35 -
Poultry Science Sep 2019Chronic heat stress can enhance fat synthesis in broilers, and excessive triglyceride (TG) synthesized by the liver needs to be transported to extrahepatic tissues by...
Chronic heat stress can enhance fat synthesis in broilers, and excessive triglyceride (TG) synthesized by the liver needs to be transported to extrahepatic tissues by very low density lipoprotein (VLDL) otherwise will accumulate in the liver, which may even result in hepatic steatosis. To investigate the molecular mechanisms by which chronic heat stress enhances fat synthesis and results in lipid accumulation in the liver of chickens, 144 broilers (Arbor Acres, 28-day-old) were randomly allocated to the normal control (NC, 22°C), heat stress (HS, consistent 32°C), or pair-fed (PF, 22°C) groups for a 14-D trial. The 7 D of heat exposure significantly increased the respiratory rate, relative weight of abdominal fat, the levels of glucose, TG, corticosterone, insulin, and VLDL in plasma, as well as the levels of TG, total cholesterol, acyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) in the liver, and mRNA expression levels of carbohydrate response element-binding protein (ChREBP), ACC, FAS, and microsomal triglyceride transfer protein (MTTP) in comparison with the other 2 groups. After 14 D of heat exposure, the relative weights of abdominal fat and liver and levels of TG and FAS in the liver were significantly higher in the HS group than in the other 2 groups, and there were no significant differences in the respiratory rate, plasma corticosterone concentration, apolipoprotein B (ApoB) level in the liver, and mRNA expression levels of key genes of fat synthesis among the 3 groups. In conclusion, chronic heat exposure activated LXRα pathway and enhanced fat synthesis in the liver after 7 D of heat exposure. After 14 D of heat exposure, heat-stressed broilers exhibited an adaptation to the high temperature in parameters of stress and fat synthesis gene expression levels. Moreover, chronic heat stress resulted in lipid accumulation in the liver of broilers, which is probably because the limited ApoB was not enough to transport the excessive TG synthesized by the liver in chronic heat-stressed broilers.
Topics: Animals; Apolipoproteins B; Avian Proteins; Chickens; Hot Temperature; Lipid Metabolism; Liver; Male; Random Allocation; Stress, Physiological
PubMed: 30809677
DOI: 10.3382/ps/pez056 -
Proceedings of the National Academy of... Aug 1986We report the characterization of intestine and liver cDNA clones for human apolipoprotein B (apoB) that map to the 5' end of the mRNA. The protein sequence encoded by...
We report the characterization of intestine and liver cDNA clones for human apolipoprotein B (apoB) that map to the 5' end of the mRNA. The protein sequence encoded by the 5011 nucleotides derived from sequence analysis of these clones includes 1643 amino acid residues of the mature protein of Mr 184,000. The amino acid sequence at the amino terminus of B-74 peptide was determined and mapped to residue 1298. The size (Mr 145,700) and amino acid composition of the B-26 region encoded by these clones (including amino acid residues 1-1297) closely match the values obtained from the B-26 peptide. The amino acid sequence of peptide B-100 at the junction of peptides B-26 and B-74 (Phe-Lys decreases- Ser) shows structural homology to the site on human kininogen (Phe-Arg decreases- Ser) that is cleaved by the protease plasma kallikrein. The encoded protein contains five potential N-glycosylation sites and several regions in which the hydroxyamino acids, serine and threonine, are present in high abundance. The protein sequence presented in this report represents approximately 30% of the total B-100 protein and will aid in the characterization of additional cDNA clones.
Topics: Amino Acid Sequence; Apolipoproteins B; Base Sequence; Cloning, Molecular; DNA; Humans; Intestines; Liver; Protein Conformation; Receptors, LDL
PubMed: 3461454
DOI: 10.1073/pnas.83.15.5678 -
Journal of Veterinary Internal Medicine Jul 2016Cholesterol deficiency (CD), a newly identified autosomal recessive genetic defect in Holstein cattle, is associated with clinical signs of diarrhea, failure to thrive,...
BACKGROUND
Cholesterol deficiency (CD), a newly identified autosomal recessive genetic defect in Holstein cattle, is associated with clinical signs of diarrhea, failure to thrive, and hypocholesterolemia.
HYPOTHESIS/OBJECTIVES
The objective is to describe the clinicopathological phenotype of affected Holstein cattle homozygous for the causative apolipoprotein B gene (APOB) mutation.
ANIMALS
Six Holstein cattle, 5 calves with a clinical history of chronic diarrhea, and 1 heifer with erosions in the buccal cavity and neurologic symptoms were admitted to the Clinic for Ruminants.
METHODS
This case review included a full clinical examination, a complete blood count, blood chemistry, and measurements of cholesterol and triglycerides. The animals were euthanized and necropsied. A PCR-based direct gene test was applied to determine the APOB genotype.
RESULTS
All 6 animals were inbred, could be traced back to the sire Maughlin Storm, and were confirmed homozygous for the APOB mutation. The clinical phenotype included poor development, underweight, and intermittent diarrhea in the calves, and neurologic signs in the heifer included hypermetria and pacing. Hypocholesterolemia and low triglycerides concentrations were present in all animals. The pathological phenotype of all animals was steatorrhea with enterocytes of the small intestine containing intracytoplasmic lipid vacuoles. The peripheral nervous system of the heifer displayed degenerative changes.
CONCLUSIONS AND CLINICAL IMPORTANCE
Suspicion of CD in Holstein cattle is based on the presence of chronic diarrhea with no evidence of primary infections. Confirmation of the associated APOB gene mutation is needed. Additionally, the heifer demonstrated primarily signs of neurologic disease providing an unexpected phenotype of CD.
Topics: Animals; Apolipoproteins B; Cattle; Cattle Diseases; Cholesterol; Diarrhea; Female; Genetic Predisposition to Disease; Homozygote; Inbreeding; Male; Mutation
PubMed: 27279263
DOI: 10.1111/jvim.13976 -
The Journal of Clinical Investigation Nov 1986The presence of apolipoprotein (apo) B in liver and intestine from a patient with abetalipoproteinemia was evaluated by immunohistochemistry with a polyclonal and six...
The presence of apolipoprotein (apo) B in liver and intestine from a patient with abetalipoproteinemia was evaluated by immunohistochemistry with a polyclonal and six monoclonal antibodies to different apo B-48 and B-100 epitopes. In normal liver, apo B was present inside and outside hepatocytes. The patients liver exhibited staining in the cytoplasm with the polyclonal and three monoclonal antibodies. By immunoelectron-microscopy, apo B was found to be present in the smooth endoplasmatic reticulum and the Golgi complex. Normal intestinal epithelium was labeled with polyclonal and all monoclonal antibodies, including those specific for apo B-100. The patients epithelium stained with polyclonal and six monoclonals, and apo B was present in the Golgi complex. Thus, normal intestinal mucosa expressed apo B-48 and B-100 epitopes, which indicates apo B-100 synthesis in the gut. The synthesis of the apo B molecule in the patient seems to be retained in both liver and gut, which suggests a posttranslational defect.
Topics: Abetalipoproteinemia; Adult; Antibodies, Monoclonal; Apolipoprotein B-100; Apolipoprotein B-48; Apolipoproteins B; Duodenum; Epitopes; Fatty Acids, Nonesterified; Female; Genes, Recessive; Humans; Jejunum; Liver; Microscopy, Electron
PubMed: 2429992
DOI: 10.1172/JCI112727 -
The American Journal of Clinical... Jul 2006HIV lipodystrophy syndrome (HLS) is characterized by accelerated lipolysis, inadequate fat oxidation, increased hepatic reesterification, and a high frequency of growth...
BACKGROUND
HIV lipodystrophy syndrome (HLS) is characterized by accelerated lipolysis, inadequate fat oxidation, increased hepatic reesterification, and a high frequency of growth hormone deficiency (GHD). The effect of growth hormone (GH) replacement on these lipid kinetic abnormalities is unknown.
OBJECTIVE
We aimed to measure the effects of physiologic GH replacement on lipid kinetics in men with HLS and GHD.
DESIGN
Seven men with HLS and GHD were studied with the use of infusions of [13C1]palmitate, [2H5]glycerol, and [2H3]leucine to quantify total and net lipolysis, palmitate and free fatty acid (FFA) oxidation, and VLDL apolipoprotein B-100 synthesis before and after 6 mo of GH replacement (maximum: 5 microg x kg(-1) x d(-1)).
RESULTS
GH replacement decreased the rates of total lipolysis [FFA(total) rate of appearance (x +/- SE): from 4.80 +/- 1.24 to 3.32 +/- 0.76 mmol FFA x kg fat(-1) x h(-1); P < 0.05] and net lipolysis (FFA(net) rate of appearance: from 1.87 +/- 0.34 to 1.20 +/- 0.25 mmol FFA x kg fat(-1) x h(-1); P < 0.05). Fat oxidation decreased (from 0.28 +/- 0.02 to 0.20 +/- 0.02 mmol FFA x kg lean body mass(-1) x h(-1); P < 0.002), as did the rate of appearance of FFAs available for intrahepatic reesterification (from 0.50 +/- 0.13 to 0.29 +/- 0.09 mmol FFA x kg fat(-1) x h(-1); P < 0.03). Fractional and absolute synthetic rates of VLDL apolipoprotein B-100 were unaltered. These kinetic changes were associated with a decrease in the waist-to-hip ratio but no significant change in fasting plasma lipid concentrations. Fasting plasma glucose concentrations increased after treatment (from 5.2 +/- 0.2 to 5.8 +/- 0.3 mmol/L; P < 0.01).
CONCLUSIONS
Physiologic GH replacement has salutary effects on abnormal lipid kinetics in HLS. The effects are mediated by diminished lipolysis and hepatic reesterification rather than by increased fat oxidation.
Topics: Adipocytes; Apolipoprotein B-100; Apolipoproteins B; Body Composition; Carbon Isotopes; Deuterium; Esterification; Fatty Acids, Nonesterified; HIV-Associated Lipodystrophy Syndrome; Human Growth Hormone; Humans; Lipid Metabolism; Lipolysis; Liver; Male; Middle Aged; Oxidation-Reduction; Palmitates
PubMed: 16825697
DOI: 10.1093/ajcn/84.1.204 -
Proceedings of the National Academy of... Jul 1986The synthesis of apolipoprotein B (apoB) was examined in human fetal and adult intestine and liver. Intestine and liver were minced and then incubated with [3H]leucine,...
The synthesis of apolipoprotein B (apoB) was examined in human fetal and adult intestine and liver. Intestine and liver were minced and then incubated with [3H]leucine, homogenized, and subjected to immunoprecipitation with antiserum that recognized both apoB-100 and apoB-48 (forms of apoB found in low density lipoproteins and in chylomicrons, respectively). Immunoprecipitates of fetal and adult liver contained radioactivity in a single apoB-100 peak when examined by NaDodSO4/polyacrylamide gel electrophoresis. Intestine from fetuses at 11 weeks of gestation incorporated radioactivity mainly into apoB-100, with little incorporation into apoB-48. Sixteen-week fetal intestine showed both apoB-100 and apoB-48, whereas adult intestine incorporated radioactivity only into apoB-48. Pulse-chase experiments with 11- and 16-week fetal intestine showed no evidence for the conversion of apoB-100 to apoB-48. Incubation of intestinal homogenates with fetal liver apoB-100 did not result in the conversion of apoB-100 to smaller forms of apoB. A cDNA probe to hepatic apoB-100 identified a single, 18-kilobase transcript in poly(A)+ RNA from fetal and adult liver and fetal intestine of all ages. These studies define the developmental pattern of apoB synthesis in human fetal and adult liver and intestine. No evidence could be found for the conversion of apoB-100 to apoB-48. The finding of a single mRNA transcript despite the form of apoB synthesized in each tissue is discussed.
Topics: Adult; Apolipoprotein B-100; Apolipoprotein B-48; Apolipoproteins B; DNA; Duodenum; Fetus; Genetic Markers; Humans; Intestinal Mucosa; Intestines; Liver; Organ Specificity; Protein Processing, Post-Translational; RNA Processing, Post-Transcriptional; Transcription, Genetic
PubMed: 3460091
DOI: 10.1073/pnas.83.14.5296 -
Journal of Lipid Research Aug 1993Very low density lipoproteins (VLDL) are a heterogenous population of particles differing in size and composition. Heparin-Sepharose chromatography yields three VLDL...
Very low density lipoproteins (VLDL) are a heterogenous population of particles differing in size and composition. Heparin-Sepharose chromatography yields three VLDL subfractions. Two subfractions, VLDLNR-1 and VLDLNR-2, which are not retained by heparin, contain little or no detectable apolipoprotein (apo)E. According to negative stain electron microscopy, VLDLNR-1 is slightly larger than VLDLNR-2. The third fraction, VLDLR, is composed of smaller particles that are retained by the heparin-Sepharose and contain apoE. The C apolipoproteins of the respective VLDL subfractions transfer to 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) single bilayer vesicles giving three subfractions designated VLDLNR-1-C, VLDLNR-2-C, and VLDLR-C. The protein, phospholipid, and cholesterol (free + esterified) contents decrease in the order VLDLR > VLDLNR-2 > VLDLNR-1. Triglyceride content decreases in the opposite order. POPC treatment of each VLDL subfraction increases the phospholipid and decreases the protein, triglyceride, and cholesteryl ester contents, while free cholesterol remains unchanged. According to immunological analysis of each subfraction with well-characterized monoclonal antibodies, the accessibility of some epitopes of apoB-100 on VLDL is changed by POPC treatment. Electron-microscopic analysis of POPC-treated VLDL subfraction reveals vacancies on the surfaces of each particle. VLDLNR-1, VLDLNR-2, and VLDLR are resistant to thrombin cleavage, whereas the lipoproteins lacking C apolipoproteins are not. Thrombin cleavage (8 h) of apoB-100 of VLDLNR-2-C and VLDLR-C gives two fragments, T1 and T2, that are converted to smaller fragments only after prolonged treatment. In contrast, apoB-100 of VLDLNR-1-C is converted into small fragments after 8 h thrombin treatment. These results suggest that removal of apoCs affects the accessibility and conformation of apoB-100 in the individual VLDL subfractions in the region near residue 3249, which is the primary thrombin cleavage site and the epitope of monoclonal antibody 4C11.
Topics: Apolipoprotein B-100; Apolipoproteins B; Apolipoproteins C; Chromatography; Enzyme-Linked Immunosorbent Assay; Humans; Lipoproteins, VLDL; Microscopy, Electron; Peptide Fragments; Thrombin
PubMed: 8409765
DOI: No ID Found