Truncated apo B-70.5–containing lipoproteins bind to megalin but not the LDL receptor
Zhouji Chen, Jeffrey E. Saffitz, Mickey A. Latour, Gustav Schonfeld
Zhouji Chen, Jeffrey E. Saffitz, Mickey A. Latour, Gustav Schonfeld
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Truncated apo B-70.5–containing lipoproteins bind to megalin but not the LDL receptor

Abstract

Apo B-100 of LDL can bind to both the LDL receptor and megalin, but the molecular interactions of apo B-100 with these 2 receptors are not completely understood. Naturally occurring mutant forms of apo B may be a source of valuable information on these interactions. Apo B-70.5 is uniquely useful because it contains the NH2-terminal portion of apo B-100, that includes only one of the two putative LDL receptor–binding sites (site A). The lipoprotein containing apo B-70.5 (Lp B-70.5) was purified from apo B-100/apo B-70.5 heterozygotes by sequential ultracentrifugation combined with immunoaffinity chromatography. Cell culture experiments, ligand blot analysis, and in vivo studies all consistently showed that Lp B-70.5 is not recognized by the LDL receptor. The kidney was identified as a major organ in catabolism of Lp B-70.5 in New Zealand white rabbits. Autoradiographic analysis revealed that renal proximal tubular cells selectively removed Lp B-70.5. On ligand blotting of renal cortical membranes, Lp B-70.5 bound only to megalin. The ability of megalin to mediate cellular endocytosis of Lp B-70.5 was confirmed using retinoic acid/dibutyryl cAMP–treated F9 cells. This study suggests that the putative LDL receptor–binding site A on apo B-100 might not by itself be a functional binding domain and that the apo B–binding sites recognized by the LDL receptor and by megalin may be different. Moreover, megalin may play an important role in renal catabolism of apo B truncations, including apo B-70.5.

Authors

Zhouji Chen, Jeffrey E. Saffitz, Mickey A. Latour, Gustav Schonfeld

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Binding of 125I-megalin to immobilized thrombin-cleavage fragments of ap...
Binding of 125I-megalin to immobilized thrombin-cleavage fragments of apo B-100. Thrombin-treated LDL (tLDL) was prepared by incubating LDL with thrombin at 15°C for 16 hours. Untreated LDL (LDL) and tLDL were electrophoresed on 3–6% SDS-PAGE under reducing conditions. (a) Coomassie blue staining of LDL (5 μg) or tLDL (5 μg). (b) Binding of 125I-megalin (2 μg/mL in ligand blot binding buffer) to LDL (2 μg) or tLDL (2 μg) after electrophoresis and transfer onto Immobilon-P membranes. The binding assay was performed using the ligand blot procedure described in Methods.