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 l ipoproteins, and LDL receptor deficiency may be the underlying cause of sev eral primary and secondary dyslipidaemic conditions. Intervention strategie s are often targeted to increase hepatic LDL receptor expression. It is dif ficult to quantitate hepatic LDL receptor activity and to monitor changes p ost-therapy. In order to avoid liver biopsy, human skin fibroblasts or circ ulating mononuclear cells have often been used as surrogate markers for the hepatic receptor. Fibroblasts, and particularly mononuclear cells, are rel atively easy to isolate and can be stored for extensive lengths of time wit hout significant loss of LDL receptor expression. Leucocytes or fibroblasts are normally probed with isotopically or gold-labelled LDL. However, the s pecific activity of the LDL conjugate is usually too low to enable accurate quantitation of differences, or changes, in LDL receptor expression. In th is study, we describe an enhanced colloidal gold-labelling procedure for th e detection of LDL receptor binding activity. The binding of colloidal gold -labelled chylomicron remnants to human hepatocytes (HepG2 cells) was compa red 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 antise rum specific to the LDL receptor. The binding of gold-labelled remnants was substantially greater than that of gold-labelled LDL. Compared with gold-l abelled LDL, we found a much clearer demarcation of remnant binding with he patocytes incubated in the presence or absence of sterols. Our observations suggest that, because of the greater affinity of the LDL receptor for lipo proteins containing apolipoprotein E, changes in LDL receptor expression mi ght be more readily identified using gold-labelled remnants. We conclude th at gold-conjugated chylomicron remnants might provide a useful means of det ecting subtle changes in LDL receptor expression.