Abstract
Very low density lipoproteins (VLDL) are heterogeneous, triglyceride-rich particles that are precursors of low density lipoproteins (LDL). Before conversion to LDL, the majority of VLDL are irreversibly cleared from plasma by uncertain mechanisms. To investigate one potential mechanism for VLDL clearance, we studied the ability of LDL receptors to mediate VLDL uptake in vitro. Small, intermediate, and large VLDL from normolipidemic humans were found to bind and undergo catabolism via LDL receptors on normal human fibroblasts. Binding to cell surfaces was up-regulated by lovastatin, an inducer of LDL receptors. Both LDL and a monoclonal antibody against the LDL receptor (IgG-C7) prevented binding of 125I-VLDL. Also, VLDL binding to mutant fibroblasts lacking LDL receptors was low. Thus, LDL receptors mediated VLDL interactions with cells. Binding affinity decreased near saturation, and the apparent number of high affinity sites decreased with increasing VLDL particle size. Because LDL receptors are small (M(r) 115,000) relative to VLDL (M(r) 9-24 x 10(6)) and are clustered in clathrin-coated pits, these findings suggest that steric hindrance becomes an important binding determinant near saturation and are consistent with a lattice model for LDL receptor-ligand interactions. The capacity for cellular catabolism of VLDL decreased with increasing particle size, consistent with a lattice model. The lattice model was also supported by differences between 125I-VLDL binding to cell surfaces and binding to partially purified LDL receptors in solid-phase assays in which steric constraints resulting from clustering in clathrin-coated pits are not present. In both cell-surface and solid-phase assays, VLDL bound via apoE, not apoB-100. Our studies establish that normal VLDL interact with LDL receptors and that steric hindrance due to crowding of particles on clustered LDL receptors is an important determinant of their binding and catabolism. These findings suggest that LDL receptors may participate in normal VLDL clearance in vivo.
Highlights
LDL receptors are stronglyimplicated as binding shown in Fig. 5 became apparent when binding was mediators ofVLDL binding and catabolism by experiments studied by Scatchard analysis (Fig. 7 and Table IV)
On aver- showing that thesephenomena are regulatedby lovastatin, are relatively low in fibroblasts lacking LDL receptors, share sites used by LDL or IgG-C7, and correlate with binding to LDL
Iits unknown whether normal fibroblasts express theVLDL receptor [20] or whether this receptor is regulated by lovastatin, it is unlikely that binding to VLDL receptors confounds our results
Summary
With medium containing one of the following: 50-fold excessunlabeled lipoproteins, 10 m g h l polyphosphate, 10 mg/ml heparin, 15 rn sura-. The hybridomas were used to make ascitic fluid in mice, from LDL receptors in fibroblasts were up-regulated by growth in medium which antibodies were isolated using protein A-Sepharose as previously containing 4 mg/ml lipoprotein-deficientserum for 48 h and 1 pg/ml described [37].Monoclonal antibodies 1D7 and 4G3, which lovastatin for 24h prior to use.Microtiter wells were coated with buffer block apoE- and apoB-100-mediatedbinding to LDL receptors [38,39], containing 30 pg/ml IgG-4A4T. Cell-surface and Solid-phase Binding Assays-Human fibroblasts assays was near equilibrium at 3 h as determined by time course exwere isolated from the foreskin of normal infants, cultured in 35-mm periments with association times varying from 1 t o 5 h (data not plastic wells, and incubated with lipoprotein-deficientserum and lovas- shown).For each assay,binding to BSA-coated wellswas measured as tatin t o up-regulate LDL receptors as previouslydescribed [40, 41]. B,, values for Sf 20-60 and 100400 lipoproteins significantly differ from that for LDL ( p < 0.05)
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