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Immunoprecipitation of the low-density-lipoprotein (LDL) receptor and its precursor from human monocyte-derived
Abstract:
Synthesis of the low-density-lipoprotein (LDL) receptor protein by cultured human monocyte-derived macrophages was demonstrated by immunoprecipitation of [35S]methionine-labelled cell extracts with a monoclonal antibody to the bovine adrenal LDL receptor. Although the antibody does not bind to or inhibit binding of 125I-LDL to the LDL receptor on intact fibroblasts, it specifically binds to a protein in extracts of human skin fibroblasts, of Mr approx. 130,000 under non-reducing conditions, that is able to bind LDL. In monocyte-derived macrophages, as in fibroblasts, the receptor is synthesized as a low-Mr precursor that is converted into the mature protein. The half-life of the precursor in human macrophages is approx. 44 min. In cells from two homozygous familial-hypercholesterolaemic subjects, only the precursor form of the receptor is synthesized. Detection of abnormalities of LDL-receptor synthesis in human mononuclear cells may be a useful aid in diagnosis of familial hypercholesterolaemia that is simpler and quicker than methods requiring growth of cultured skin fibroblasts.
Insights
Researchers identified a simpler diagnostic method for familial hypercholesterolaemia by analyzing low-density-lipoprotein (LDL) receptor protein synthesis in human mononuclear cells, detecting precursor forms indicative of the condition.
Area of Science:
- Biochemistry
- Cell Biology
- Genetics
Background:
- Low-density-lipoprotein (LDL) receptors are crucial for cholesterol homeostasis.
- Familial hypercholesterolaemia (FH) is a genetic disorder characterized by high LDL levels.
- Current FH diagnosis can be complex and time-consuming.
Purpose of the Study:
- To investigate the synthesis of LDL receptors in human monocyte-derived macrophages.
- To explore the potential of analyzing LDL receptor synthesis for FH diagnosis.
Main Methods:
- Immunoprecipitation of [35S]methionine-labelled cell extracts using a monoclonal antibody to the LDL receptor.
- Analysis of LDL receptor precursor and mature protein forms in cultured human cells.
- Comparison of LDL receptor synthesis in healthy individuals versus FH patients.
Main Results:
- LDL receptor protein synthesis was confirmed in human monocyte-derived macrophages.
- A low-Mr precursor is synthesized and converted to a mature protein, similar to fibroblasts.
- Cells from homozygous FH subjects exclusively synthesized the precursor form.
- The half-life of the LDL receptor precursor in macrophages is approximately 44 minutes.
Conclusions:
- Abnormalities in LDL receptor synthesis are detectable in human mononuclear cells.
- This method offers a potentially simpler and quicker diagnostic aid for familial hypercholesterolaemia compared to fibroblast culture.
- Mononuclear cell analysis represents a promising approach for FH screening and diagnosis.