The use of monoclonal antibodies to probe human apolipoprotein B structure and function

1985 ◽  
Vol 63 (8) ◽  
pp. 906-912 ◽  
Author(s):  
Ross W. Milne ◽  
Yves L. Marcel
Keyword(s):  
Monoclonal Antibodies ◽  
Limited Proteolysis ◽  
Ldl Receptor ◽  
Plasma Lipid ◽  
Density Lipoprotein ◽  
Lipid Vesicles ◽  
Cholesterol Homeostasis ◽  
Antigenic Determinants ◽  
Lipoprotein Subfractions ◽  
Apo B

Apolipoprotein (apo) B plays an important role in plasma lipid transport and in the maintenance of cholesterol homeostasis. Attempts to determine the structure of apo B have been hampered by technical obstacles resulting from its chemical and physical properties. Recently monoclonal antibodies (Mabs) against human apo B have been used as probes to study apo B structure and heterogeneity. Certain Mabs are capable of blocking binding of low density lipoprotein (LDL) apo B to the cell surface LDL receptor, which presumably reflects the proximity of their antigenic determinants to the receptor recognition domain. The distribution of antigenic determinants recognized by Mabs has been studied on the hepatic (apo B-100) and intestinal (apo B-48) forms of apo B and on fragments generated by limited proteolysis of apo B. Some Mabs are specific for apo B-100, whereas others cross-react with apo B-48. Apo B-100 specific Mabs coupled to Sepharose have been used to isolate separately apo-B-containing lipoproteins of intestinal and hepatic origin and their respective lipid and apolipoprotein compositions have been determined. Using the separated fractions it has been shown that apo B-100, but not apo B-48, can react with the LDL receptor. Most Mabs failed to react with apo B which had been delipidated and resolubilized, but in some cases immunoreactivity could be recovered if the solubilized apo B were reincorporated into lipid vesicles. These experiments showed that different determinants had different lipid requirements for their expression. Within an individual there is immunochemical heterogeneity in apo-B-containing lipoproteins in the expression of apo B antigenic determinants which can be detected by Mabs. Intersubject differences in reactivity of lipoprotein subfractions with Mabs have also been observed and in some cases appear to represent genetic polymorphism of apo B.

scholarly journals Ligand selectivity of 105 kDa and 130 kDa lipoprotein-binding proteins in vascular-smooth-muscle-cell membranes is unique

1996 ◽  
Vol 317 (1) ◽  
pp. 297-304 ◽  
Author(s):  
Valery N. BOCHKOV ◽  
Vsevolod A. TKACHUK ◽  
Maria P. PHILIPPOVA ◽  
Dimitri V. STAMBOLSKY ◽  
Fritz R. BÜHLER ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Binding Proteins ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Dextran Sulphate ◽  
Apo B ◽  
Ligand Selectivity ◽  
Lipoprotein Binding

Using ligand blotting techniques, with low-density lipoprotein (LDL) as ligand, we have previously described the existence of atypical lipoprotein-binding proteins (105 kDa and 130 kDa) in membranes from human aortic medial tissue. The present study demonstrates that these proteins are also present in membranes from cultured human (aortic and mesenteric) and rat (aortic) vascular smooth-muscle cells (VSMCs). To assess the relationship of 105 and 130 kDa lipoprotein-binding proteins to known lipoprotein receptors, ligand binding specificity was studied. We tested effects of substances known to antagonize ligand binding to either the LDL [apolipoprotein B,E (apo B,E)] receptor (dextran sulphate, heparin, pentosan polysulphate, protamine, spermine, histone), the scavenger receptor (dextran sulphate, fucoidin), the very-low-density-lipoprotein (VLDL) receptor [receptor-associated protein (RAP)], or LDL receptor-related protein (RAP, α2-macroglobulin, lipoprotein lipase, exotoxin-A). None of these substances, with the exception of dextran sulphate, influenced binding of LDL to either 105 or 130 kDa proteins. Sodium oleate or oleic acid, known stimuli for the lipoprotein binding activity of the lipolysis-stimulated receptor, were also without effect. LDL binding to 105 and 130 kDa proteins was inhibited by anti-LDL (apo B) antibodies. LDL and VLDL bound to 105 and 130 kDa proteins with similar affinities (蝶50 μg/ml). The unique ligand selectivity of 105 and 130 kDa proteins supports the existence of a novel lipoprotein-binding protein that is distinct from all other currently identified LDL receptor family members. The similar ligand selectivity of 105 and 130 kDa proteins suggests that they may represent variant forms of an atypical lipoprotein-binding protein.

Differences in the exposure of C- and N-terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies

1989 ◽  
Vol 92 (3) ◽  
pp. 519-528 ◽  
Author(s):  
P. Draber ◽  
E. Draberova ◽  
I. Linhartova ◽  
V. Viklicky
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Limited Proteolysis ◽  
Antigenic Determinants ◽  
Alpha Tubulin ◽  
Domain Specific ◽  
Double Label ◽  
Cytoplasmic Microtubules ◽  
C And N ◽  
Terminal Domains

A panel of 11 monoclonal antibodies specific to alpha- or beta-tubulin subunits was used to study the location of tubulin molecules in cytoplasmic microtubules. Specificity of antibodies was confirmed by immunoblotting and immunofluorescence experiments on fixed cells. The limited proteolysis of tubulin with trypsin and chymotrypsin followed by immunoblotting demonstrated that the antibodies discriminated between structural domains of both subunits. Epitope mapping of isolated alpha-tubulin revealed that a set of antibodies against the N-terminal domain of the alpha-subunit (TU-01, TU-02, TU-03, TU-09, 6–11B-1) recognized at least four different antigenic determinants. Immunofluorescence staining of unfixed detergent-extracted cells showed that antibodies to determinants on C-terminal domains labelled microtubules, but these were not decorated with antibodies to N-terminal domains. The same results were obtained after microinjection of antibodies into living cells. The unchanged distribution of microtubules in injected cells was confirmed by double-label immunofluorescence with polyclonal antibodies. The data indicate that while parts of C-terminal domains of both subunits are exposed on the exterior of the microtubules, considerable regions of the N-terminal domains are either not exposed on the surface of cytoplasmic microtubules, or are masked by interacting proteins.

Monoclonal antibodies can precipitate low-density lipoprotein. I. Characterization and use in determining apolipoprotein B.

1985 ◽  
Vol 31 (10) ◽  
pp. 1654-1658 ◽  
Author(s):  
S Marcovina ◽  
D France ◽  
R A Phillips ◽  
S J Mao
Keyword(s):  
Monoclonal Antibodies ◽  
Apolipoprotein B ◽  
Polyclonal Antibodies ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Radial Immunodiffusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Density ◽  
Apo B ◽  
Blotting Technique

Abstract We produced 20 mouse monoclonal antibodies against human plasma low-density lipoprotein (LDL). Individually they failed to precipitate LDL in agarose gel by the double-immunodiffusion technique; collectively they did, or as few as two combined monoclonal antibodies could do so. To mimic polyclonal antibodies in determination of apolipoprotein B (apo B) by radial immunodiffusion, a combination of four particular monoclonal antibodies (clones A, B, C, and D) was necessary. We characterized these four clones with respect to temperature dependency, affinity, total binding to 125I-labeled LDL, and specificity to the different species of apolipoprotein B. Two monoclonal antibodies (B and C) bound 100% of 125I-labeled LDL; clones A and D bound 80% and 87%, respectively. All four clones bound maximally to LDL at 4 degrees C. The affinity constants for clones A, B, C, and D were 0.6, 2.1, 3.8, and 2.3 X 10(9) L/mol, respectively. By the Western blotting technique, the four monoclonal antibodies all reacted with the species B-100 and B-74 of apolipoprotein B, and to various degrees with B-48 and B-26. Radial immunodiffusion (chi) and direct enzyme-linked immunosorbent assay (y) with a mixture of the four monoclonal antibodies gave almost identical results for 70 patients: y = 0.921 chi-2.58; r = 0.933.

scholarly journals Effect of reduced low-density lipoprotein receptor level on HepG2 cell cholesterol metabolism

1998 ◽  
Vol 329 (1) ◽  
pp. 81-89 ◽  
Author(s):  
Lahoucine IZEM ◽  
Eric RASSART ◽  
Lassana KAMATE ◽  
Louise FALSTRAULT ◽  
David RHAINDS ◽  
...  
Keyword(s):  
Hepg2 Cell ◽  
Hepg2 Cells ◽  
Cholesterol Metabolism ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Constitutive Expression ◽  
Density Lipoprotein ◽  
Low Density ◽  
Apo B ◽  
Acat Activity

Low-density lipoproteins (LDL) are taken up by both LDL receptor (LDLr)-dependent and -independent pathways. In order to determine the importance of these pathways in the activity of the various enzymes that are important in maintaining the cellular cholesterol level in hepatic cells, we created HepG2 cells expressing lower levels of LDLr. Thus HepG2 cells were transfected with a constitutive expression vector (pRc/CMV) containing a fragment of LDLr cDNA inserted in an antisense manner. Stable transformants were obtained that showed significant reductions of 42, 72 and 85% of LDLr protein levels compared with the control, as demonstrated by immunoblotting and confirmed by the LDL binding assay. The best inactivation was achieved with the construct containing the first 0.7 kb of LDLr cDNA. Incubating the different HepG2 cell subtypes with LDL showed similar association of apolipoprotein B (apo B) or cholesteryl esters from LDL with the cells, indicating that the LDLr deficiency did not significantly affect LDL uptake by the cell. However, apoB degradation was reduced significantly by 71-82% in the most LDLr-deficient HepG2 cells. We also found that 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA red) activity is significantly increased by 32-35% in HepG2 cells expressing very low levels of LDLr that also demonstrate a significant decrease of 20% in acyl-CoA:cholesterol acyltransferase (ACAT) activity. However, these effects are moderate compared with those observed when cells were incubated in lipoprotein-depleted medium, where a > 900% increase in HMGCoA red activity and a loss of 60% of ACAT activity was observed. Thus, in HepG2 cells, different levels of LDLr affect LDL-apoB degradation, but have very little effect on LDL association, HMGCoA red and ACAT activities, revealing that LDLr is more important in the clearance of LDL-apoB than in HepG2 cell cholesterol homoeostasis, a role that should be attributable to both LDLr-dependent and -independent pathways.

The LDL-Receptor and its Molecular Properties: From Theory to Novel Biochemical and Pharmacological Approaches in Reducing LDL-cholesterol

2020 ◽  
Vol 27 (2) ◽  
pp. 317-333 ◽  
Author(s):  
Dimitrios Petroglou ◽  
Ilias Kanellos ◽  
Christos Savopoulos ◽  
Georgia Kaiafa ◽  
Anastasios Chrysochoou ◽  
...  
Keyword(s):  
Transmembrane Protein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Therapeutic Agents ◽  
Molecular Properties ◽  
Cholesterol Homeostasis ◽  
Lipid Homeostasis ◽  
Pharmacological Interventions ◽  
Therapeutic Goals ◽  
Pubmed Database

Background: The Low-Density Lipoprotein (LDL) Receptor (LDL-R) is a transmembrane protein playing a crucial role in effective lipid homeostasis. Various therapeutic agents have been used in the management of dyslipidemias, however, the outcome of therapeutic target is debated. Objective: The aim of this review is to summarize and fully understand the current concept regarding LDL-R and its molecular properties, metabolic pathway, factors affecting LDL-R activity and all available pharmacological interventions. Additionally, non-lipid related properties of LDL-R are also referred. Methods: Literature from the PubMed database was extracted to identify papers between 1984 to 2017 regarding LDL-R and therapeutic agents on dyslipidemia management. Results: We analyzed basic data regarding agents associated with LDL-R (Sterol Regulating Element-Binding Proteins - SREBPs, Protein ARH, IDOL, Thyroid Hormones, Haematologic Disorders, Protein convertase subtilisin kexintype 9 - PCSK-9, ApoC-III) as well as non-lipid related properties of LDL-R, while all relevant (common and novel) pharmacological interventions (statins, fibrates, cholesterol absorption inhibitors, bile acid sequestrants and PCSK- 9) are also referred. Conclusion: LDL-R and its molecular properties are involved in lipid homeostasis, so potentially sets the therapeutic goals in cardiovascular patients, which is usually debated. Further research is needed in order to fully understand its properties, as well as to find the potential pharmacological interventions that could be beneficial in cholesterol homeostasis and various morbidities in order to reach the most appropriate therapeutic goal.

scholarly journals LRP1 integrates murine macrophage cholesterol homeostasis and inflammatory responses in atherosclerosis

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Xunde Xian ◽  
Yinyuan Ding ◽  
Marco Dieckmann ◽  
Li Zhou ◽  
Florian Plattner ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Apoptotic Cell ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Vascular Wall ◽  
Cholesterol Homeostasis ◽  
Cell Surface Receptor ◽  
Wide Range ◽  
Surface Receptor

Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor with diverse physiological roles, ranging from cellular uptake of lipoproteins and other cargo by endocytosis to sensor of the extracellular environment and integrator of a wide range of signaling mechanisms. As a chylomicron remnant receptor, LRP1 controls systemic lipid metabolism in concert with the LDL receptor in the liver, whereas in smooth muscle cells (SMC) LRP1 functions as a co-receptor for TGFβ and PDGFRβ in reverse cholesterol transport and the maintenance of vascular wall integrity. Here we used a knockin mouse model to uncover a novel atheroprotective role for LRP1 in macrophages where tyrosine phosphorylation of an NPxY motif in its intracellular domain initiates a signaling cascade along an LRP1/SHC1/PI3K/AKT/PPARγ/LXR axis to regulate and integrate cellular cholesterol homeostasis through the expression of the major cholesterol exporter ABCA1 with apoptotic cell removal and inflammatory responses.

Alterations in high-density lipoprotein subfractions during postprandial lipidaemia induced by fat with and without ethanol

1988 ◽  
Vol 75 (2) ◽  
pp. 135-142 ◽  
Author(s):  
G. Franceschini ◽  
Y. Moreno ◽  
P. Apebe ◽  
L. Calabresi ◽  
E. Gatti ◽  
...  
Keyword(s):  
High Density Lipoprotein ◽  
Density Lipoprotein ◽  
Hdl Cholesterol ◽  
High Density ◽  
Acute Administration ◽  
Cholesteryl Esters ◽  
Lipoprotein Subfractions ◽  
Apo B ◽  
Postprandial Phase ◽  
High Density Lipoprotein Subfractions

1. Serum lipid and apolipoprotein levels, distribution and composition of high-density lipoprotein (HDL) sub-fractions and lecithin:cholesterol acryltransferase activity were analysed in nine normolipidaemic subjects, in whom a hypertriglyceridaemic state was induced by the acute administration of ethanol (40 g) plus fat (70 g) or of fat only. 2. Triglyceride (TG) levels increased by 180% 4–6 h after fat plus ethanol intake, the hypertriglyceridaemic response being inversely correlated with the basal HDL2 mass (r = −0.82). Serum apolipoprotein (apo) B levels rose by 8%, HDL–cholesterol decreased by 10% and HDL–TG increased by 57% at 6–8 h. 3. When ethanol was omitted, serum cholesterol and TG rose by 6% and 70%, respectively; both apo AI and apo B levels went up by 8%, whereas HDL-cholesterol rose progressively (15%) at 12 h. 4. The flotation rates of both HDL2 and HDL, increased, reaching a maximum 6–8 h after ethanol plus fat intake. These changes were due to an increase in TG and phospholipid contents, whereas cholesteryl esters and proteins decreased. 5. The alterations in HDL are attributable to the increase in TG-rich lipoproteins, to the stimulated cholesterol esterification (+ 15%) and to an enhanced transfer of newly formed cholesteryl esters to apo-B-containing lipoproteins in exchange for TG. 6. Changes in HDL properties were evident only when ethanol was given concomitantly with fat. 7. These findings suggest that in the postprandial phase lipoprotein changes may occur, which facilitate an improved removal of cholesterol from tissues.

scholarly journals Olive oil containing olive oil fatty acid esters of plant sterols and dietary diacylglycerol reduces low-density lipoprotein cholesterol and decreases the tendency for peroxidation in hypercholesterolaemic subjects

2007 ◽  
Vol 98 (3) ◽  
pp. 563-570 ◽  
Author(s):  
Yen-Ming Chan ◽  
Isabelle Demonty ◽  
Dori Pelled ◽  
Peter J. H. Jones
Keyword(s):  
Lipid Peroxidation ◽  
Fatty Acids ◽  
Lipid Profile ◽  
Olive Oil ◽  
Plasma Lipid ◽  
Density Lipoprotein ◽  
Plant Sterols ◽  
Fatty Acid Esters ◽  
Blood Lipid Profile ◽  
Apo B

Plant sterols (PS) and MUFA are well-documented cholesterol lowering agents. We aimed to determine the effect of PS esterified to olive oil fatty acids (PS-OO) on blood lipid profile and lipid peroxidation in hypercholesterolaemic subjects. Twenty-one moderately overweight, hypercholesterolaemic subjects consumed three consecutive treatment diets, each lasting 28 d and separated by 4-week washout periods, using a randomized crossover design. Diets contained 30 % energy as fat, 70 % of which was provided by olive oil (OO), and differed only in the treatment oils: OO, PS esterified to sunflower oil fatty acids (PS-SO), and PS-OO. Both PS-SO and PS-OO treatments provided 1·7 g PS /d. PS-OO and PS-SO consumption resulted in a decrease (P = 0·0483) in LDL-cholesterol (LDL-C) concentrations compared with the OO diet. Although total cholesterol and apo B-100 levels were not significantly affected, PS-SO and, to some extent, PS-OO reduced the total:HDL-cholesterol (HDL-C) ratio (P = 0·0142) and the apo B-100:apo A-I ratio (P = 0·0168) compared with the OO diet. There were no differences across diets in lipoprotein(a) (Lp(a)) and lipid peroxidation levels. However, following consumption of OO and PS-SO, Lp(a) concentrations increased (P = 0·0050 and 0·0421, respectively), while PS-OO treatment did not affect Lp(a) levels. Furthermore, there was a decrease (P = 0·0097) in lipid peroxidation levels with PS-OO treatment during the supplementation phase. Our results suggest that supplementing an OO-rich diet with PS-OO favourably alters the plasma lipid profile and may decrease the susceptibility of LDL-C to lipid peroxidation in hypercholesterolaemic subjects.

scholarly journals The relation between ApoB/ApoA-1 ratio and the severity of coronary artery disease in patients with acute coronary syndrome

2021 ◽  
Vol 73 (1) ◽  
Author(s):  
Rehab Ibrahim Yaseen ◽  
Mohamed Hesham El-Leboudy ◽  
Hend Mohammed El-Deeb
Keyword(s):  
Acute Coronary Syndrome ◽  
Apolipoprotein B ◽  
Density Lipoprotein ◽  
Serum Levels ◽  
Cholesterol Homeostasis ◽  
Gensini Score ◽  
Structural Protein ◽  
Apo B ◽  
Coronary Syndrome ◽  
Apolipoprotein A

Abstract Background Apolipoprotein B is considered the primary protein constituent of low-density lipoprotein. LDL contains variable quantities of cholesterol, but each lipoprotein contains a single ApoB protein. Thus, ApoB is a better index for the LDL circulation if compared to LDL cholesterol. On the contrary, apolipoprotein A-1 is a main structural protein of high-density lipoprotein. It has a major role in reversing cholesterol flow and cellular cholesterol homeostasis once detected. The aim of the study is to measure apo B/apo A-1 ratio in patients with acute coronary syndrome and assess its relationship with the severity of CAD. A total of 90 patients were enrolled in the study and subdivided into 3 groups: 30 patients of STEMI, 30 patients of NSTEMI, and 30 patients presented with unstable angina. Serum levels of apolipoprotein A-1 and apolipoprotein B were properly measured upon admission, and apo B/apo A-1 ratio was calculated. Results Both of Apo B and Apo B/Apo A1 ratio correlated significantly with Gensini scores (P value <0.001). High Gensini score patients had significantly high Apo B/Apo A1 ratio with the best cutoff value of 0.8 with sensitivity of 90% and specificity of 70%. Conclusion Apo B is an independent risk predictor for the severity of CAD in patients with acute coronary syndromes. Moreover, the Apo B/Apo A1 ratio remains highly significant in patients with high Gensini score.

scholarly journals BLOS1 mediates kinesin switch during endosomal recycling of LDL receptor

2020 ◽  
Author(s):  
Chang Zhang ◽  
Chanjuan Hao ◽  
Guanghou Shui ◽  
Wei Li
Keyword(s):  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Cholesterol Homeostasis ◽  
Whole Body ◽  
Cellular Mechanisms ◽  
Anterograde Transport ◽  
Recycling Endosomes ◽  
Endosomal Recycling ◽  
Density Lipoprotein Receptor

AbstractLow-density lipoprotein receptor (LDLR) in hepatocytes plays a key role in normal clearance of circulating LDL and in whole body cholesterol homeostasis. The trafficking of LDLR is highly regulated in clathrin-dependent endocytosis, endosomal recycling and lysosomal degradation. Current studies have been focusing on its endocytosis and degradation. However, the detailed molecular and cellular mechanisms underlying its endosomal recycling are largely unknown. We found that BLOS1, a shared subunit of BLOC-1 and BORC, is involved in LDLR endosomal recycling. Loss of BLOS1 leads to less membrane LDLR and impairs LDL clearance from plasma in hepatocyte-specific BLOS1 knockout mice. BLOS1 interacts with kinesin-3, and that BLOS1 acts as a new adaptor for kinesin-2 to coordinate kinesin-3 and kinesin-2 during the long-range transport of recycling endosomes (REs) to plasma membrane along microtubule tracks to overcome hurdles at microtubule intersections. These findings provide new insights into RE’s anterograde transport and the pathogenesis of dyslipidemia.

Sign in / Sign up

Export Citation Format

Share Document