Detection of LDL receptor by ligand blotting with chylomicron remnants labelled with colloidal gold.
The LDL receptor plays a pivotal role in the clearance of pro-atherogenic lipoproteins, and LDL receptor deficiency may be the underlying cause of several primary and secondary dyslipidaemic conditions. Intervention strategies are often targeted to increase hepatic LDL receptor expression. It is difficult to quantitate hepatic LDL receptor activity and to monitor changes post-therapy. In order to avoid liver biopsy, human skin fibroblasts or circulating mononuclear cells have often been used as surrogate markers for the hepatic receptor. Fibroblasts, and particularly mononuclear cells, are relatively easy to isolate and can be stored for extensive lengths of time without significant loss of LDL receptor expression. Leucocytes or fibroblasts are normally probed with isotopically or gold-labelled LDL. However, the specific activity of the LDL conjugate is usually too low to enable accurate quantitation of differences, or changes, in LDL receptor expression. In this study, we describe an enhanced colloidal gold-labelling procedure for the detection of LDL receptor binding activity. The binding of colloidal gold-labelled chylomicron remnants to human hepatocytes (HepG2 cells) was compared with that of gold-conjugated LDL. Labelled remnants bound specifically to a cell surface protein with a molecular weight of approximately 130 kDa. Binding was blocked in the presence of unlabelled remnants, LDL, or antiserum specific to the LDL receptor. The binding of gold-labelled remnants was substantially greater than that of gold-labelled LDL. Compared with gold-labelled LDL, we found a much clearer demarcation of remnant binding with hepatocytes incubated in the presence or absence of sterols. Our observations suggest that, because of the greater affinity of the LDL receptor for lipoproteins containing apolipoprotein E, changes in LDL receptor expression might be more readily identified using gold-labelled remnants. We conclude that gold-conjugated chylomicron remnants might provide a useful means of detecting subtle changes in LDL receptor expression.
Pal
S
S
Department of Nutrition, Dietetics and Food Sciences, Curtin University of Technology, Perth, Australia.
Elsegood
C L
CL
Proctor
S D
SD
Mamo
J C
JC
D001057:Apolipoproteins E / Q000378:metabolism; D015415:Biomarkers; D002624:Chemistry, Clinical / Q000379:methods*; D053298:Chylomicron Remnants; D002914:Chylomicrons / Q000378:metabolism*; D003101:Collodion / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000378:metabolism; D017979:Gold Colloid / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D008074:Lipoproteins / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D011485:Protein Binding; D011973:Receptors, LDL / Q000032:analysis* / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D001057:Apolipoproteins E; D015415:Biomarkers; D053298:Chylomicron Remnants; D002914:Chylomicrons; D017979:Gold Colloid; D008024:Ligands; D008074:Lipoproteins; D011973:Receptors, LDL; D003101:Collodion