Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor.

Cummings, Richard D.; Kornfeld, Stuart; Schneider, Wolfgang J.; Hobgood, Kim K.; Tolleshaug, Helge; Brown, Michael S.; Goldstein, Joseph L.

doi:10.1016/s0021-9258(17)43802-6

Cited by 347 publications

(68 citation statements)

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“…1, top). Previous experiments have established that the precursor is an endoglycosidase H sensitive ER protein, that is processed to an endoglycosidase H resistant, sialylated, mature protein during transport through the Golgi apparatus to the cell surface (Tolleshaug et al, 1982;Cummings et al, 1983;Kozarsky et al, 1986). The shift in electrophoretic mobility between the precursor and mature forms is due to maturation of the numerous O-linked and several N-linked oligosaccharides on the receptor.…”

Section: Human Ldlc Cdna Corrects the Abnormal Phenotypes Of Idic Cellsmentioning

confidence: 99%

LDLC encodes a brefeldin A-sensitive, peripheral Golgi protein required for normal Golgi function.

Podos

Reddy

Ashkenas

et al. 1994

The Journal of Cell Biology

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Abstract. Two genetically distinct classes of low density lipoprotein (LDL) receptor-deficient Chinese hamster ovary cell mutants, ldlB and ldlC, exhibit nearly identical pleiotropic defects in multiple medial and trans Golgi-associated processes (Kingsley, D., K. F. Kozarsky, M. Segal, and M. Krieger. 1986. J. Cell Biol. 102:1576-1585. In these mutants, the synthesis of virtually all N-and O-linked glycoproteins and of the major lipid-linked oligosaccharides is abnormal. The abnormal glycosylation of LDL receptors in ldlB and ldlC cells results in their dramatically reduced stability and thus very low LDL receptor activity. We have cloned and sequenced a human cDNA (LDLC) which corrects the mutant phenotypes of ldlC, but not ldlB, cells. Unlike wild-type CHO or ldlB cells, ldlC cells had virtually no detectable endogenous LDLC mRNA, indicating that LDLC is likely to be the normal human homologue of the defective gene in ldlC cells. The predicted sequence of the human LDLC protein (ldlCp, m83 kD) is not similar to that of any known proteins, and contains no major common structural motifs such as transmembrane domains or an ER translocation signal sequence. We have also determined the sequence of the Caenorhabditis elegans ldlCp by eDNA cloning and sequencing. Its similarity to that of human ldlCp suggests that ldlCp mediates a well-conserved cellular function. Immunofluorescence studies with anti-ldlCp antibodies in mammalian cells established that ldlCp is a peripheral Golgi protein whose association with the Golgi is brefeldin A sensitive. In ldlB cells, ldlCp was expressed at normal levels; however, it was not associated with the Golgi. Thus, a combination of somatic cell and molecular genetics has identified a previously unrecognized protein, ldlCp, which is required for multiple Golgi functions and whose peripheral association with the Golgi is both LDLB dependent and brefeldin A sensitive.

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Section: Human Ldlc Cdna Corrects the Abnormal Phenotypes Of Idic Cellsmentioning

confidence: 99%

LDLC encodes a brefeldin A-sensitive, peripheral Golgi protein required for normal Golgi function.

Podos

Reddy

Ashkenas

et al. 1994

The Journal of Cell Biology

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“…Thus the majority of the negative charge in the oligosaccharides is due to the presence of sialic acid residues. The existence of small fractions of oligosaccharides eluted with 140 mm-and 200 mM-NaCl could indicate the presence of additional negatively charged groups, such as sulphate [16] or phosphate [35]. However, we have detected no incorporation of radioactivity into apo B-48 when human jejunal explants in organ culture were incubated with [35S]sulphate, suggesting either the absence of or negligible oligosaccharide sulphation.…”

Section: Fractionation Of Oligosaccharides By Con A-sepharose Chromatographymentioning

confidence: 61%

“…Fractionation of oligosaccharides on the basis of their negative charge by QAE-Sephadex chromatography QAE-Sephadex can be used to separate oligosaccharides on the basis of their.negative charge. Neutral species are not retained by the column, and oligosaccharides with one, two, three and four negative charges are eluted with 20 mm-, 70 mm-, 140 mMand 200 mM-NaCl respectively [16,22]. The oligosaccharide standard asialo biantennary complex did not bind to the QAE-Sephadex column, whereas disialylated oligosaccharide standard did bind and was eluted with 70 mM-NaCl (results not shown).…”

Section: Fractionation Of Oligosaccharides By Con A-sepharose Chromatographymentioning

confidence: 89%

“…4). After acid treatment under conditions to effect oligosaccharide desialylation (10 mM-HCl for 1 h at 80°C) [16,22], 88% of the material was not retained by the QAE-Sephadex column, whereas 6 %, 5 % and 1 % were eluted with 20 mm-, 70 mm-and 140 mM-NaCl respectively (results not shown). Thus the majority of the negative charge in the oligosaccharides is due to the presence of sialic acid residues.…”

Section: Fractionation Of Oligosaccharides By Con A-sepharose Chromatographymentioning

confidence: 97%

“…Chylomicrons prepared from pooled human lymph were delipidated and apo B-48 was isolated by preparative SDS/ PAGE. After strong acid hydrolysis (2 M-HCI at 100 'C, for 6 h), excess acid was removed and the monosaccharide mixture was re-N-acetylated [16], followed by reduction with NaB3H4 [17]. Radiolabelled monosaccharide alditols were fractionated by h.p.l.c.…”

Section: Monosaccharide Compositionmentioning

confidence: 99%

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Human small-intestinal apolipoprotein B-48 oligosaccharide chains

Sasak

Lown

Colburn

1991

Biochemical Journal

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Hepatic apolipoprotein (apo) B-100 isolated from human plasma is known to contain N-linked oligosaccharides of high-mannose-type and complex-type structures. Sequencing data have revealed that apo B-48 of small-intestinal origin, which represents about 48% of apo B-100 polypeptide from the N-terminus, possesses six potential sites for N-linked oligosaccharides, of which five are likely to be glycosylated. The characterization of the carbohydrate moiety of apo B-48 is the focus of this study. Apo B-48 was labelled with L-[35S]methionine and D-[3H]glucosamine in organ culture of human small-intestinal explants. N-Glycanase treatment resulted in loss of radioactivity from D-[3H]glucosamine-labelled but not L-[35S]methionine-labelled apo B-48 secreted into the medium, and caused no distinct change in mobility of apo B-48 upon electrophoresis on 5% polyacrylamide gel. Analysis of monosaccharide content revealed the presence of 16.8, 17.8, 13.4, 3.4, 2.4 and 2.3 residues of N-acetylglucosamine, mannose, galactose, fucose, xylose and N-acetylgalactosamine respectively. Small-intestinal apo B-48 from human lymph chylomicrons bound to [14C]concanavalin A, and the binding could be inhibited with methyl alpha-D-mannoside. In addition, wheat-germ, peanut, Limulus, soya-bean and Ulex lectins bound apo B-48 specifically. To characterize the carbohydrate moiety further, N-linked oligosaccharides were released by N-Glycanase treatment and reduced with NaB3H4. Labelled oligosaccharides were separated on a concanavalin A-Sepharose column. The majority (78%) were biantennary complex-type structures, 16% were high-mannose type and 6% (not retained by the column) most probably represented higher-branched oligosaccharides. These results suggest the presence of one high-mannose-type and four biantennary complex-type oligosaccharides, as well as probable O-linked sugars in apo B-48. By the use of h.p.l.c., exoglycosidase treatments and ion-exchange chromatography, a mixture of high-mannose-type species with predominant Man8GlcNAc2 as well as monosialylated, desialylated and fucosylated forms of complex-type oligosaccharides were detected.

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LDL Receptor and Its Role in Inherited Disease

Soutar

2010

Encyclopedia of Life Sciences

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The low‐density lipoprotein (LDL) receptor mediates the specific uptake of LDL from the circulation and its intracellular degradation by a process known as the LDL receptor pathway. LDL receptor gene expression is regulated by intracellular sterol content, and is limited mainly to the liver in vivo . Mutations in LDLR , the gene encoding the LDL receptor, cause familial hypercholesterolaemia (FH), a dominantly inherited disease where accumulation of LDL in the circulation increases the risk of coronary heart disease. Defects in other genes, including APOB , encoding the ligand for the LDL receptor, ARH , encoding a protein required for its internalisation and proprotein convertase subtilisin/kexin type 9 ( PCSK9 ), encoding a protein that reduces LDL receptor protein levels, cause a similar disorder because the encoded proteins are involved in the LDL receptor pathway. FH can readily be treated with cholesterol‐lowering drugs to reduce cardiovascular risk effectively, and identification of the causal genetic defect allows unequivocal and early diagnosis. Key Concepts: The LDL receptor mediates specific uptake and intracellular degradation of serum LDL. Inherited defects in the LDL receptor pathway cause familial hypercholesterolaemia (FH), characterised by increased serum LDL and increased risk of coronary heart disease. Most FH patients have mutations in LDLR , but defects in other genes cause a similar disorder. A recessive form of FH is caused by null mutations in ARH, a clathrin adaptor protein required for LDL receptor internalisation. A dominant form of FH is caused by gain‐of‐function mutations in PCSK9 , which encodes a protein that promotes degradation of LDL receptor protein. Dominant loss‐of‐function mutations in PCSK9 reduce serum cholesterol and risk of coronary heart disease. One relatively common dominant mutation in APOB , encoding the ligand for the LDL receptor, causes familial defective apoB (FDB). LDLR transcription is regulated by sterol response element‐binding proteins (SREBP) in a complex mechanism involving several other proteins. FH can readily be treated with cholesterol‐lowering drug therapy to abolish the increased risk of coronary disease, but is currently under‐diagnosed. Identifying causal mutations in known patients and screening their relatives should identify more affected individuals.

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Biosynthesis of N- and O-linked oligosaccharides of the low density lipoprotein receptor.

Cited by 347 publications

References 55 publications

LDLC encodes a brefeldin A-sensitive, peripheral Golgi protein required for normal Golgi function.

LDLC encodes a brefeldin A-sensitive, peripheral Golgi protein required for normal Golgi function.

Human small-intestinal apolipoprotein B-48 oligosaccharide chains

LDL Receptor and Its Role in Inherited Disease

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