scholarly journals 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

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.

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.

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

1986 ◽  
Vol 102 (5) ◽  
pp. 1567-1575 ◽  
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.

scholarly journals 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.

scholarly journals 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.

scholarly journals A study of the chylomicron metabolism in WHHL rabbits after fat loading. Discrepancy between results based on measurement of apoprotein B-48 or retinyl palmitate

1992 ◽  
Vol 285 (2) ◽  
pp. 641-646 ◽  
Author(s):  
P N M Demacker ◽  
P J van Heijst ◽  
A F H Stalenhoef
Keyword(s):  
New Zealand ◽  
Strong Correlation ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Retinyl Palmitate ◽  
Receptor Activity ◽  
Low Density ◽  
Apoprotein B ◽  
Chylomicron Metabolism

We studied the metabolism of chylomicrons in homozygous Watanabe heritable hyperlipidaemic (WHHL) rabbits and in cholesterol-fed or normally fed New Zealand White (NZW) rabbits by measuring the concentrations of apoprotein B-48 and of retinyl palmitate in their serum after feeding fat plus this vitamin according to two different protocols. Compared with NZW controls, retinyl palmitate accumulated in both hyperlipidaemic groups under study, not only in the d less than 1.019 fraction but also in the low-density lipoprotein (LDL) fraction. A strong correlation was found between the retinyl palmitate concentration in either the d less than 1.019 fraction or the LDL fraction of the WHHL rabbits and the concentrations of cholesterol and triacylglycerols in these fractions. This suggests that retinyl palmitate is exchanged rapidly between exogenous and endogenous lipoproteins. This is supported by the lack of a correlation between the retinyl palmitate concentrations and the intensity of the apoprotein B-48 band in the respective d less than 1.019 fractions or LDL fractions; in most fractions, in which large amounts of retinyl palmitate were present, the intensity of the apoprotein B-48 band was not increased compared with the fasting concentrations. Assuming that retinyl palmitate is a marker for the transfer of exogenous lipids, the results of our experiments indicate that the removal of exogenous lipids is delayed by complexing to endogenously synthesized lipoproteins. However, the clearance of apoprotein B-48 is normal and thus independent of the LDL-receptor activity.

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