Uptake of canine beta-very low density lipoproteins by mouse peritoneal macrophages is mediated by a low density lipoprotein receptor.

C Koo,M E Wernette-Hammond,T L Innerarity

doi:10.1016/s0021-9258(18)67367-3

Abstract

The receptor on mouse peritoneal macrophages that mediates the uptake of canine beta-very low density lipoproteins (beta-VLDL) has been identified in this study as an unusual apolipoprotein (apo-) B,E(LDL) receptor. Ligand blots of Triton X-100 extracts of mouse peritoneal macrophages using 125I-beta-VLDL identified a single protein. This protein cross-reacted with antibodies against bovine apo-B,E(LDL) receptors, but its apparent Mr was approximately 5,000 less than that of the human apo-B,E(LDL) receptor. Binding studies at 4 degrees C demonstrated specific and saturable binding of low density lipoproteins (LDL), beta-VLDL, and cholesterol-induced high density lipoproteins in plasma that contain apo-E as their only protein constituent (apo-E HDLc) to mouse macrophages. Apolipoprotein E-containing lipoproteins (beta-VLDL and apo-E HDLc) bound to mouse macrophages and human fibroblasts with the same high affinity. However, LDL bound to mouse macrophages with an 18-fold lower affinity than to human fibroblasts. Mouse fibroblasts also bound LDL with a similar low affinity. Compared with the apo-B,E(LDL) receptors on human fibroblasts, the apo-B,E(LDL) receptors on mouse macrophages were resistant to down-regulation by incubation of the cells with LDL or beta-VLDL. There are three lines of evidence that an unusual apo-B,E(LDL) receptor on mouse peritoneal macrophages mediates the binding and uptake of beta-VLDL: LDL with residual apo-E removed displaced completely the 125I-beta-VLDL binding to mouse macrophages, preincubation of the mouse macrophages with apo-B,E(LDL) receptor antibody inhibited both the binding of beta-VLDL and LDL to the cells and the formation of beta-VLDL- and LDL-induced cholesteryl esters, and binding of 125I-beta-VLDL to the cells after down-regulation correlated directly with the amount of mouse macrophage apo-B,E(LDL) receptor as determined on immunoblots. This unusual receptor binds LDL poorly, but binds apo-E-containing lipoproteins with normal very high affinity and is resistant to down-regulation by extracellular cholesterol.

Highlights

Toneal macrophages usin‘g261-j3-VLDLidentified a sin- Mouse peritoneal macrophages have been widely studied as gle protein
Receptor on cultured human fibroblasts is rapidly reduced by incubation with low concentrations of low density lipoproteins (LDL), whereas even high concentrations of LDL do not comparably reduce the binding and uptake of P-VLDL by mouse peritoneal macrophages [10]. These results suggested that a receptor other than the apo-B,E(LDLre)ceptor was responsible for the highaffinity uptake of P-VLDL by macrophages
Identification of Receptors for Canine p-VLDL by Western P Blotting-A receptor on mouse peritoneal macrophages that bound canine p-VLDL was identified by ligand blotting

Summary

RESULTS

Identification of Receptors for Canine p-VLDL by Western P Blotting-A receptor on mouse peritoneal macrophages that bound canine p-VLDL was identified by ligand blotting. Human low density lipoproteins (LDL), caninep-very low density lipoproteins (0-VLDL), or canine cholesterol-induced plasma high density lipoproteins containing apolipoprotein E as theonly protein constituent (apo-E HDL,) were incubated a t 4 "C with mouse peritoneal macrophages or human fibroblasts, and equilibrium dissociation constants ( K d ) for the binding were derived as described [29]. To test whether the lower binding affinity of mouse peritoneal macrophages for human LDL was due to cell type or species specificity, the binding of human LDL to mouse fibroblasts was examined. 100 pg/ml for macrophages versus 8 pg/ml for fibroblasts), Competitive Displacement of '251-V@-LDL Binding-To de- reflecting the lower binding affinity of LDL for mouse mactermine whether human LDL and canine@-VLDLbind to the rophage receptors (Table I and Fig. 3). Effect of Bovine Apo-B,E(LDL) Receptor Antibody on Lipoprotein-induced Cholesterol Esterification-The results described above showed that mouse macrophages contain the apo-B,E(LDL)receptor that may be responsible for the recep-

A Mouse LDL

DISCUSSION

Methods