Abstract
Bovine milk lipoprotein lipase (LPL) induced binding, uptake, and degradation of 125I-labeled normal human triglyceride-rich lipoproteins by cultured mutant fibroblasts lacking LDL receptors. The induction was dose-dependent and occurred whether LPL and 125I-lipoproteins were added to incubation media simultaneously or LPL was allowed to bind to cell surfaces, and unbound LPL was removed by washing prior to the assay. Lipolytic modification of lipoproteins did not appear to be necessary for increased catabolism because the effect of LPL was not prevented by inhibitors of LPL's enzymatic activity, p-nitrophenyl N-dodecylcarbamate or phenylmethylsulfonyl fluoride. However, the effect was abolished by boiling LPL prior to the assay suggesting that major structural features of LPL were required. Also, LPL-induced binding to cells was blocked by an anti-LPL monoclonal antibody but not by antibodies that are known to block apolipoprotein E- or B-100-mediated binding to low density lipoprotein (LDL) receptors. This indicates that LPL itself mediated 125I-lipoprotein binding to cells. Cellular degradation of 125I-lipoproteins was partially or completely blocked by two previously described ligands for the LDL receptor-related protein/alpha 2-macroglobulin receptor (LRP): activated alpha 2-macroglobulin (alpha 2M*), and the 39-kDa receptor-associated protein. These data implicated LRP as mediating LPL-induced lipoprotein degradation and were confirmed by showing that LPL's effects were prevented by an immunoaffinity-isolated polyclonal antibody against LRP. Furthermore, LPL promoted binding of 125I-lipoproteins to highly purified LRP in a solid-phase assay. Heparin or heparinase treatment of cells markedly decreased LPL-induced binding, uptake, and degradation of lipoproteins, but had no effect on catabolism of alpha 2M*. Thus, cell-surface proteoglycans were obligatory participants in the effects of LPL but were not required for LRP-mediated catabolism of alpha 2M*. Taken together, these in vitro findings establish that through interaction with cell-surface proteoglycans, LPL induces catabolism of normal human triglyceride-rich lipoproteins via LRP.
Highlights
From the $Departmentof Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, the Ulepartmentof
A monoclonal antibody, MAB-7, cally interact with LDL receptor-related protein (LRP), we show that LRP mediates LPLinduced degradation of Sf 100-400 lipoproteins in a process facilitated by cell-surface proteoglycans
These data represent the first demonstration that normal plasma lipoproteins can be catabolized in uitro by an intracellular pathway mediated by LRP.Priortothecurrent studies, LRP-mediated lipoprote,in catabolism had been established in uitro only for apoE-enriched P-VLDL produced in cholesterol-fed rabbits ( 5, 6)
Summary
Vol 268, No 19, Issue of July 5,pp. 14168-14175, 1993 Printed in U.S.A. (Received for publication, December 17, 1992, and in revised form, March 15, 1993). LRP has been implicated in the uptakeof chylomicompletelyblockedby twopreviouslydescribedligands for theLDL receptor-related protein/a2-macroglobulin receptor(LRP):activatedaz-macroglobulin (azM*),and the 39-kDa receptor-associatedprotein These data implicated LRaPs mediating LPL-induced lipoprotein degradation and wereconfirmed by showing that LPL's effects were prevented by an immunoaffinity-isolated polyclonal antibody against LRP. The possibility that LPL might provide a recognition site for lipoprotein removal by a remnant receptor was originally suggested by Felts et al [8] This hypothesis received new attention when Beisiegel et al [9] cross-linked cell-surfacebound LPL to a protein that resembled LRP in size. We showed thatculturedfibroblastslackingLDL receptors degrade LPL via LRP, and LPLitself binds to LRP with high affinity in a solid-phase cell-free assay [10] This binding could be partially displaced by twopreviously described ligands for LRP (IO), activated as-macroglobulin tory participants in the effects of LPL but were not (az") andthe39-kDareceptor-associatedprotein(RAP). Proteins-Human S, 100-400 lipoproteins from fasted normolipidemic subjects were isolated from the d < 1.006 g/ml plasma fraction
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