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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.6.9.3268 ◽

1986 ◽

Vol 6 (9) ◽

pp. 3268-3277 ◽

Cited By ~ 23

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

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Unusual forms of low density lipoprotein receptors in hamster cell mutants with defects in the receptor structural gene.

The Journal of Cell Biology ◽

10.1083/jcb.102.5.1567 ◽

1986 ◽

Vol 102 (5) ◽

pp. 1567-1575 ◽

Cited By ~ 35

Author(s):

K F Kozarsky ◽

H A Brush ◽

M Krieger

Keyword(s):

Chinese Hamster Ovary ◽

Chinese Hamster Ovary Cells ◽

Low Density Lipoprotein ◽

Chinese Hamster ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Low Density ◽

Wild Type ◽

Ovary Cells ◽

Ldl Receptors

The structure and processing of low density lipoprotein (LDL) receptors in wild-type and LDL receptor-deficient mutant Chinese hamster ovary cells was examined using polyclonal anti-receptor antibodies. As previously reported for human LDL receptors, the LDL receptors in wild-type Chinese hamster ovary cells were synthesized as precursors which were extensively processed by glycosylation to a mature form. In the course of normal receptor turnover, an apparently unglycosylated portion of the cysteine-rich N-terminal LDL binding domain of the receptor is proteolytically removed. The LDL receptor-deficient mutants fall into four complementation groups, ldlA, ldlB, ldlC, and ldlD; results of the analysis of ldlB, ldlC, and ldlD mutants are described in the accompanying paper (Kingsley, D. M., K. F. Kozarsky, M. Segal, and M. Krieger, 1986, J. Cell. Biol, 102:1576-1585). Analysis of ldlA cells has identified three classes of mutant alleles at the ldlA locus: null alleles, alleles that code for normally processed receptors that cannot bind LDL, and alleles that code for abnormally processed receptors. The abnormally processed receptors were continually converted to novel unstable intracellular intermediates. We also identified a compound-heterozygous mutant and a heterozygous revertant which indicate that the ldlA locus is diploid. In conjunction with other genetic and biochemical data, the finding of multiple mutant forms of the LDL receptor in ldlA mutants, some of which appeared together in the same cell, confirm that the ldlA locus is the structural gene for the LDL receptor.

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Addition of a mannose-6-phosphate-containing oligosaccharide alters cellular processing of low density lipoprotein by parental and LDL-receptor-defective Chinese hamster ovary cells

Journal of Cell Science ◽

10.1242/jcs.68.1.183 ◽

1984 ◽

Vol 68 (1) ◽

pp. 183-194

Author(s):

A.M. Leichtner ◽

M. Krieger

Keyword(s):

Chinese Hamster Ovary ◽

Cho Cells ◽

Low Density Lipoprotein ◽

Chinese Hamster ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Receptor Activity ◽

Low Density ◽

Cellular Processing ◽

Mannose 6 Phosphate

Low density lipoprotein (LDL) was chemically modified by the addition of omega-(6-phospho)-tetra(alpha 1–3)mannosyl-(alpha 1–2)mannose (M56P), a phosphorylated oligosaccharide containing a terminal mannose 6-phosphate residue. Uptake and degradation of this modified LDL (M56P-LDL) by Chinese hamster ovary (CHO) cells occurred via the lysosomal enzyme (mannose 6-phosphate) receptor pathway. Cellular processing of M56P-LDL was saturable, specific for the mannose 6-phosphate marker, and occurred with approximately threefold higher affinity than that of native LDL by the LDL receptor pathway. Mannose 6-phosphate receptor activity, as measured by degradation of M56P-LDL, was ninefold lower than the LDL receptor activity. Degradation of M56P-LDL was more sensitive to inhibition by the lysosomotropic agent chloroquine than was degradation of LDL, suggesting differences in the intracellular processing of mannose 6-phosphate-bearing ligands and LDL. Previously isolated CHO cell lines defective in LDL receptor activity resembled parental CHO cells in their ability to process M56P-LDL. The potential use of M56P-LDL in the isolation of cells with pleiotropic mutations affecting receptor-mediated endocytosis is discussed.

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Tri-iodothyronine prevents the amiodarone-induced decrease in the expression of the liver low-density lipoprotein receptor gene

Journal of Endocrinology ◽

10.1677/joe.0.1520413 ◽

1997 ◽

Vol 152 (3) ◽

pp. 413-421 ◽

Cited By ~ 17

Author(s):

F Hudig ◽

O Bakker ◽

W M Wiersinga

Keyword(s):

Ldl Cholesterol ◽

Plasma Cholesterol ◽

Receptor Gene ◽

Low Density Lipoprotein ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Low Density ◽

Ldl Receptors ◽

Receptor Mrna ◽

Receptor Gene Expression

Treatment with amiodarone, a potent antiarrhythmic drug, is associated with a dose-dependent increase in plasma cholesterol resulting from a decreased number of liver low-density lipoprotein (LDL) receptors. Similar changes occur in hypothyroidism, and it has been suggested that amiodarone acts via induction of a local 'hypothyroid-like' state in extrathyroidal tissues. The present study was designed to evaluate whether exogenous tri-iodothyronine (T3) could prevent the effects of amiodarone on LDL cholesterol. Rats were treated for 14 days with water, amiodarone 10 mg/100 g body weight (BW), or amiodarone and 2·5, 5 or 10 μg T3/100 g BW respectively. Relative to controls, amiodarone increased plasma LDL cholesterol by 31% and decreased liver LDL receptor mRNA by 56% and protein by 45%; liver T3 content was reduced by 21%. Addition of T3 to the treatment with amiodarone dose-dependently reversed all these changes, with a return to control values of plasma cholesterol and the number of liver LDL receptors, although LDL receptor mRNA remained slightly lower. Treatment of rats for 14 days with T3 alone (5 μg/100 g BW) decreased plasma LDL cholesterol by 19% and increased liver LDL receptor mRNA by 41%. In conclusion, T3 prevents the amiodarone-induced changes in plasma LDL cholesterol and liver LDL receptor gene expression. These findings suggest that the inhibitory effect of amiodarone on LDL receptor gene expression is mediated by T3-dependent pathways. Journal of Endocrinology (1997) 152, 413–421

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.11.4799-4806.1989 ◽

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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Flow cytometry with a monoclonal antibody to the low density lipoprotein receptor compared with gene mutation detection in diagnosis of heterozygous familial hypercholesterolemia

Clinical Chemistry ◽

10.1093/clinchem/44.5.966 ◽

1998 ◽

Vol 44 (5) ◽

pp. 966-972 ◽

Cited By ~ 6

Author(s):

Bent Raungaard ◽

Finn Heath ◽

Jens Uffe Brorholt-Petersen ◽

Henrik Kjærulf Jensen ◽

Ole Faergeman

Keyword(s):

Flow Cytometry ◽

T Lymphocytes ◽

Receptor Gene ◽

Low Density Lipoprotein ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Receptor Expression ◽

Low Density ◽

Flow Cytometry Assay ◽

Stop Mutation

Abstract We used a fluorescence flow cytometry assay with a monoclonal low density lipoprotein (LDL) receptor-specific antibody to detect LDL receptor expression on blood T lymphocytes and monocytes. We prepared peripheral blood mononuclear cells from patients with genetically verified LDL receptor-defective (Trp66-Gly mutation, n = 17) or receptor-negative (Trp23-stop mutation, n = 17) heterozygous familial hypercholesterolemia (FH) and from healthy individuals (n = 24). The cells were stimulated to express the maximum amount of LDL receptor by preincubation in lipoprotein-deficient medium. A dual-labeling technique allowed flow cytometric analysis of LDL receptor expression on cells identified by fluorescently conjugated surface marker antibodies. Knowing the LDL receptor gene mutation of the FH patients allowed us to compare the diagnostic capability of this functional assay with the DNA diagnosis and to validate the assay with molecular genetics instead of clinical indices of heterozygous FH. T lymphocytes expressed more LDL receptors and gave better diagnostic results than monocytes, and cells from patients with either the Trp66-Gly or the Trp23-stop mutation had variable but significantly reduced LDL receptor expression. The data indicate that this fluorescence flow cytometry assay is unsuitable for diagnosis of individual cases of heterozygous FH but that it may be useful for functionally characterizing mutations in the LDL receptor gene.

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Measurement of the absolute number of functioning low-density lipoprotein receptors in vivo using a monoclonal antibody

Biochemical Journal ◽

10.1042/bj3050897 ◽

1995 ◽

Vol 305 (3) ◽

pp. 897-904 ◽

Cited By ~ 4

Author(s):

C Fitzsimmons ◽

R Bush ◽

D Hele ◽

C Godliman ◽

E Gherardi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Body Weight ◽

Low Density Lipoprotein ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Absolute Number ◽

Low Density ◽

Ldl Receptors ◽

The Absolute

MAC188 S/S is a monoclonal antibody which can be used in vivo to measure the absolute number of functioning low-density lipoprotein (LDL) receptors in a rabbit. The antibody binds to the extra-cellular domain of the LDL receptor and binding is not blocked by the presence of LDL. When the antibody-receptor complex is internalized, receptor recycling is inhibited for several hours. Thus when saturating doses of MAC188 S/S are administered intravenously, the amount of antibody removed from the blood (minus non-specific removal) is determined solely by the total number of LDL receptors in an animal. In this study MAC188 S/S was used to measure the number of LDL receptors in control rabbits and in animals treated with 17 alpha-ethinyl oestradiol. After treatment (which caused a 47% decrease in plasma cholesterol), receptor-mediated removal of MAC188 S/S from the blood was saturated in both groups following injection of 3.0 mg of antibody per kg body weight. Based on the amount of antibody removed via the LDL receptor at this dose, the total number of accessible LDL receptors was calculated as (2.0 +/- 0.3) x 10(15) receptors per kg body weight in control rabbits and (4.0 +/- 0.4) x 10(15) receptors per kg body weight in oestrogen-treated animals. The number of receptors in various organs was also determined. The monoclonal antibody approach therefore, allows accurate determination of LDL receptor numbers in animals with markedly different concentrations of circulating LDL, conditions in which the use of endogenous ligand would be subject to significant errors.

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Control of low density lipoprotein receptor gene expression in steroidogenic cells

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y89-153 ◽

1989 ◽

Vol 67 (8) ◽

pp. 968-973 ◽

Cited By ~ 9

Author(s):

Koichiro Takagi ◽

Jerome F. Strauss III

Keyword(s):

Gene Expression ◽

Cyclic Amp ◽

Granulosa Cells ◽

Receptor Gene ◽

Low Density Lipoprotein ◽

Ldl Receptor ◽

Density Lipoprotein ◽

Low Density ◽

Steroidogenic Cells ◽

Receptor Gene Expression

Low density lipoprotein (LDL)-carried cholesterol is a primary substrate for steroid hormone synthesis by luteinized human granulosa cells. Chorionic gonadotropin and 8-bromo-cAMP both increase LDL receptor levels in granulosa cells by stimulating accumulation of the receptor mRNA. LDL and 25-hydroxycholesterol reduce LDL receptor expression, but this suppressive effect is partially overcome by 8-bromo-cAMP. Using fusion gene constructs containing the LDL receptor gene promoter transfected into JEG-3 cells, a cyclic AMP responsive enhancer could not be identified in the LDL receptor gene upstream promoter in transfection studies. We suggest that the LDL receptor gene in human steroidogenic cells is under negative control by a sterol effector, but that a cyclic AMP triggered process overcomes, to some extent, the sterol-mediated suppression. The detailed mechanisms by which sterol and cyclic AMP modulate LDL receptor gene expression remain to be elucidated.Key words: low density lipoproteins, low density lipoprotein receptors, cholesterol, steroidogenesis, gonadotropins.

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