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Immunocytochemical localization of mutant low density lipoprotein receptors that fail to reach the Golgi complex
R K Pathak1, R K Merkle, R D Cummings
1Department of Cell Biology, University of Texas, Southwestern Medical Center, Dallas 75235.
Abstract:
In the low density lipoprotein (LDL) receptor system, blocks in intracellular movement of a cell surface receptor result from naturally occurring mutations. These mutations occur in patients with familial hypercholesterolemia. One class of mutant LDL receptor genes (class 2 mutations) produces a receptor that is synthesized and glycosylated in the endoplasmic reticulum (ER) but does not reach the cell surface. These receptors contain serine/threonine-linked (O-linked) carbohydrate chains with core N-acetylgalactosamine residues and asparagine-linked (N-linked) carbohydrate chains of the high mannose type that are only partially trimmed. To determine the site of blockage in transport, we used electron microscope immunohistochemistry to compare the intracellular location of LDL receptors in normal human fibroblasts with their location in class 2 mutant fibroblasts. In normal cells, LDL receptors were located in coated pits, coated vesicles, endosomes, multivesicular bodies, and portions of the Golgi complex. In contrast, the mutant receptors in class 2 cells were almost entirely confined to rough ER and irregular extensions of the rough ER. Metabolic labeling studies with [3H]glucosamine confirmed that these mutant receptors contain core O-linked sugars, suggesting that the enzymes that attach these residues are located in the rough ER or the transitional zone of the ER. These studies establish that naturally occurring mutations in cell surface receptors can cause the receptors to remain trapped in the ER, thereby preventing their normal function and producing a genetic disease.
Insights
Mutant low density lipoprotein (LDL) receptors get stuck in the endoplasmic reticulum (ER) due to familial hypercholesterolemia mutations. This ER retention prevents receptors from reaching the cell surface, causing genetic disease.
Area of Science:
- Cell Biology
- Molecular Genetics
- Biochemistry
Background:
- Familial hypercholesterolemia is a genetic disorder caused by mutations in the low density lipoprotein (LDL) receptor.
- Class 2 mutations result in LDL receptors synthesized in the endoplasmic reticulum (ER) but unable to reach the cell surface.
- These mutant receptors exhibit abnormal glycosylation patterns, including core O-linked and partially trimmed N-linked carbohydrate chains.
Purpose of the Study:
- To determine the intracellular site of transport blockage for class 2 mutant LDL receptors.
- To compare the localization of normal and mutant LDL receptors within human fibroblasts.
- To investigate the implications of ER retention on LDL receptor function and disease pathogenesis.
Main Methods:
- Electron microscope immunohistochemistry was employed to visualize and localize LDL receptors in normal and mutant fibroblasts.
- Metabolic labeling with [3H]glucosamine was used to analyze the glycosylation status of mutant receptors.
- Comparative analysis of intracellular receptor distribution between normal and class 2 mutant cells.
Main Results:
- Normal LDL receptors were found in various cellular compartments including coated pits, vesicles, endosomes, and Golgi.
- Mutant LDL receptors in class 2 cells were predominantly retained within the rough ER and its extensions.
- Metabolic labeling confirmed the presence of core O-linked sugars on mutant receptors, indicating early glycosylation within the ER.
Conclusions:
- Naturally occurring mutations can cause cell surface receptors to be trapped in the ER.
- ER retention of LDL receptors prevents their cell surface expression and normal function.
- This intracellular trafficking defect is a mechanism underlying familial hypercholesterolemia and related genetic diseases.