Cholesterol uptake by human glioma cells via receptor-mediated endocytosis of low-density lipoprotein

1990 ◽  
Vol 73 (5) ◽  
pp. 760-767 ◽  
Author(s):  
Masaji Murakami ◽  
Yukitaka Ushio ◽  
Yosuke Mihara ◽  
Jun-Ichi Kuratsu ◽  
Seikoh Horiuchi ◽  
...  
Keyword(s):  
Malignant Glioma ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Glioma Cells ◽  
Low Density ◽  
Reductive Methylation ◽  
Content Type ◽  
Receptor Mediated Endocytosis ◽  
Fine Print

✓ Low-density lipoprotein (LDL) is a carrier of the cholesterol found in human plasma. Cells utilize cholesterol for membrane synthesis by taking up LDL via receptor-mediated endocytosis. In the present study, interactions of LDL with human malignant glioma cell lines (U-251MG and KMG-5) were investigated biochemically and morphologically. The LDL, labeled with the fluorescent dyes 1, l′-dioctadecy1–3,3,3′,3′-tetramethylindocarbocyanine (DiI) and fluorescein isothiocyanate (FITC), was internalized by both cell processes and cell bodies. Reductive methylation of DiI-labeled LDL, which abolishes the ability of the cell to bind to the LDL receptor, prevented the internalization of the cholesterol moiety of LDL. Cellular binding of 125I-LDL to U-251MG cells at 4°C revealed the presence of a specific saturable-associated receptor (dissociation constant (Kd) approximately 38 µg/ml). Endocytic uptake of 125I-LDL or 3H-cholesterol oleate-labeled LDL (3H-LDL) at 37°C demonstrated the cell-associated 125I-LDL and 3H-LDL increase. The intracellular degradation of protein moiety increased linearly with time. Reductive methylation of 3H-LDL led to a remarkable decrease in the cell-associated cholesterol moiety of LDL. The difference in uptake of the cholesterol moiety of LDL between U-251MG cells and KMG-5 cells showed that the U-251MG cells, which proliferate more actively than KMG-5 cells, take up more of the cholesterol moiety of LDL than do the KMG-5 cells. Thus, LDL cholesterol seems to be endocytosed predominantly via the LDL receptor present on the plasma membrane of malignant glioma cells. In addition, for growth, these cells may require large amounts of the cholesterol moiety of LDL.

scholarly journals Interaction of 4-hydroxynonenal-modified low-density lipoproteins with the fibroblast apolipoprotein B/E receptor

1986 ◽  
Vol 234 (1) ◽  
pp. 245-248 ◽  
Author(s):  
W Jessup ◽  
G Jurgens ◽  
J Lang ◽  
H Esterbauer ◽  
R T Dean
Keyword(s):  
Apolipoprotein B ◽  
Negative Charge ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Lipid Peroxidation Product ◽  
Modified Low Density Lipoproteins ◽  
Peroxidation Product

The incorporation of the lipid peroxidation product 4-hydroxynonenal into low-density lipoprotein (LDL) increases the negative charge of the particle, and decreases its affinity for the fibroblast LDL receptor. It is suggested that this modification may occur in vivo, and might promote atherogenesis.

scholarly journals Detection of the low-density-lipoprotein receptor with biotin-low-density lipoprotein. A rapid new method for ligand blotting

1985 ◽  
Vol 229 (3) ◽  
pp. 785-790 ◽  
Author(s):  
D P Wade ◽  
B L Knight ◽  
A K Soutar
Keyword(s):  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
High Sensitivity ◽  
Receptor Protein ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Cell Extracts ◽  
Density Lipoprotein Receptor ◽  
A New Technique

A new technique has been developed to identify low-density-lipoprotein (LDL) receptors on nitrocellulose membranes, after transfer from SDS/polyacrylamide gels, by ligand blotting with biotin-modified LDL. Modification with biotin hydrazide of periodate-oxidized lipoprotein sugar residues does not affect the ability of the lipoprotein to bind to the LDL receptor. Bound lipoprotein is detected with high sensitivity by a streptavidin-biotin-peroxidase complex, and thus this method eliminates the need for specific antibodies directed against the ligand. The density of the bands obtained is proportional to the amount of pure LDL receptor protein applied to the SDS/polyacrylamide gel, so that it is possible to quantify LDL receptor protein in cell extracts. Biotin can be attached to other lipoproteins, for example very-low-density lipoproteins with beta-mobility, and thus the method will be useful in the identification and isolation of other lipoprotein receptors.

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.

Characterization of a family of gamma-ray-induced CHO mutants demonstrates that the ldlA locus is diploid and encodes the low-density lipoprotein receptor

1986 ◽  
Vol 6 (9) ◽  
pp. 3268-3277
Author(s):  
R D Sege ◽  
K F Kozarsky ◽  
M Krieger
Keyword(s):  
Gamma Irradiation ◽  
Cho Cells ◽  
Gamma Ray ◽  
Receptor Gene ◽  
Low Density Lipoprotein ◽  
Chinese Hamster ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Low Density ◽  
Ldl Receptors

The ldlA locus is one of four Chinese hamster ovary (CHO) cell loci which are known to be required for the synthesis of functional low-density lipoprotein (LDL) receptors. Previous studies have suggested that the ldlA locus is diploid and encodes the LDL receptor. To confirm this assignment, we have isolated a partial genomic clone of the Chinese hamster LDL receptor gene and used this and other nucleic acid and antibody probes to study a family of ldlA mutants isolated after gamma-irradiation. Our analysis suggests that there are two LDL receptor alleles in wild-type CHO cells. Each of the three mutants isolated after gamma-irradiation had detectable deletions affecting one of the two LDL receptor alleles. One of the mutants also had a disruption of the remaining allele, resulting in the synthesis of an abnormal receptor precursor which was not subject to Golgi-associated posttranslational glycoprotein processing. The correlation of changes in the expression, structure, and function of LDL receptors with deletions in the LDL receptor genes in these mutants directly demonstrated that the ldlA locus in CHO cells is diploid and encodes the LDL receptor. In addition, our analysis suggests that CHO cells in culture may contain a partial LDL receptor pseudogene.

Isolation and characterization of an extragenic suppressor of the low-density lipoprotein receptor-deficient phenotype of a Chinese hamster ovary cell mutant

1989 ◽  
Vol 9 (11) ◽  
pp. 4799-4806
Author(s):  
P Reddy ◽  
M Krieger
Keyword(s):  
Structural Gene ◽  
Chinese Hamster Ovary ◽  
Low Density Lipoprotein ◽  
Chinese Hamster ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Ovary Cell ◽  
Low Density ◽  
Extragenic Suppressor ◽  
Extragenic Suppression

ldlC cells are low-density lipoprotein (LDL) receptor-deficient Chinese hamster ovary cell mutants which express pleiotropic defects in Golgi-associated glycosylation reactions. The dramatically reduced stability of the abnormally glycosylated LDL receptors in ldlC cells was shown to be due, in part, to rapid proteolysis and release of a large extracellular fragment of the receptor into the medium. A set of spontaneously arising LDL receptor-positive revertants of ldlC cells has been isolated. One of these, RevC-13, exhibits the glycosylation defects characteristic of the original ldlC mutant, suggesting that restoration of receptor activity was due to extragenic suppression. This suppression was due to a dramatic increase in the rate of LDL receptor synthesis rather than to an increase in the stability of the abnormally glycosylated receptors. Increased receptor synthesis was not due to receptor gene amplification. The increased LDL receptor activity was subject to normal sterol regulation. Analysis of the RevC-13 extragenic suppressor activity in a series of hybrid cells showed that RevC-13 suppression was a codominant trait that acted in cis to the LDL receptor structural gene (ldlA). Thus, the extragenic suppression in RevC-13 cells has defined a genetic element which is either part of or linked to the LDL receptor structural gene and which can control LDL receptor expression.

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