Chicken oocytes and fibroblasts express different apolipoproteins-B-specific receptors

We have previously characterized a 95-kDa plasma membrane receptor for low and very low density lipoproteins in chicken oocytes (George, R., Barber, D. L., and Schneider, W. J. (1987) J. Biol. Chem. 262, 16838-16847). We now report that somatic cells of chickens, such as fibroblasts, express a different receptor for these lipoproteins. This receptor has a Mr of 130,000 and is part of a regulatory system for cholesterol homeostasis analogous to the low density lipoprotein receptor pathway in mammalian cells. Oocytes produce only the 95-kDa receptor, while fibroblasts synthesize exclusively the 130-kDa receptor. In addition to their different Mr values, another distinctive feature of the two proteins was revealed by ligand blotting experiments: the oocyte receptor bound rabbit beta-VLDL (a class of apolipoprotein-B and -E containing lipoprotein particles), whereas the fibroblast receptor did not. Furthermore, polyclonal rabbit antibodies that recognize the oocyte 95-kDa receptor failed to cross-react with the 130-kDa protein on fibroblasts [corrected]. We suggest that different receptors have evolved in the chicken in order to facilitate the deposition of lipids into oocytes (i.e. yolk formation) with concomitant maintenance of cholesterol homeostasis in extraoocytic tissues.     

The laying hen expresses two different low density lipoprotein receptor-related proteins

We have identified, by a combination of ligand, 45Ca2+, and immunoblotting, two large membrane proteins akin to the mammalian so-called low density lipoprotein (LDL) receptor-related protein (LRP) in chicken tissues. LRP has thus far been demonstrated only in mammalian species where it is thought to act as a receptor for proteinase-alpha 2-macroglobulin complexes and/or chylomicron remnants, lipoproteins not produced in birds. One of the chicken LRPs was demonstrated in liver, and has the same apparent Mr and hallmark biochemical properties as rat liver LRP. The other chicken LRP is smaller (approximately 380 kDa) and is expressed in ovarian follicles, but is undetectable in liver. Immunological analysis demonstrated a lack of cross-reactivity between the two LRPs, as well as between them and the previously identified chicken oocyte-specific 95-kDa receptor for the yolk precursors, very low density lipoprotein, and vitellogenin (Stifani, S., Barber, D. L., Nimpf, J., and Schneider, W. J. (1989) Proc. Natl. Acad. Sci. U.S.A. 87, 1955-1959). As shown by ligand blotting, both chicken LRPs have the ability to interact with vitellogenin, a property they share not only with rat LRP, but also with mammalian LDL receptors. To obtain independent confirmation of the ligand blotting results, the smaller (follicular) LRP was purified and high-affinity binding of vitellogenin to it was demonstrated by a solid-phase filtration binding assay. Amino acid sequences of tryptic fragments of the smaller LRP were obtained, and its homology with human LRP demonstrated through unambiguous alignment of three fragments. Both chicken LRPs, the chicken oocyte 95-kDa receptor, as well as rat LRP, could be shown by ligand blotting to interact specifically with chicken serum alpha 2-macroglobulin. In addition, human apolipoprotein E, a ligand implicated in receptor-mediated metabolism of chylomicron remnants, also binds to the smaller chicken LRP, further emphasizing the similarities between LDL receptors and related proteins from a variety of species. In analogy to the known dichotomy of chicken LDL receptors, which is characterized by the production of the 95-kDa oocyte-specific receptor on one hand and a 130-kDa LDL receptor that is exclusively expressed in somatic cells (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139), it appears that the smaller and larger chicken LRPs also may be restricted to the oocyte and somatic cells, respectively.     

Characterization of the chicken oocyte receptor for low and very low density lipoproteins

The chicken oocyte receptor for low and very low density lipoproteins has been identified and characterized. Receptor activity present in octyl-beta-D-glucoside extracts of oocyte membranes was measured by a solid phase filtration assay, and the receptor was visualized by ligand blotting. The protein had an apparent Mr of 95,000 in sodium dodecyl sulfate-polyacrylamide gels under nonreducing conditions and exhibited high affinity for apolipoprotein B-containing lipoproteins, but not for high density lipoproteins or lipoproteins in which lysine residues had been reductively methylated. Binding of lipoproteins was sensitive to EDTA, suramin, and treatment with Pronase. In these aspects, the avian oocyte system was analogous to the mammalian low density lipoprotein receptor in somatic cells. Furthermore, a structural relationship between the mammalian and avian receptors was revealed by immunoblotting: polyclonal antibodies directed against the purified bovine low density lipoprotein receptor reacted selectively with the 95-kDa chicken receptor present in crude oocyte membrane extracts.     

The receptor for yolk lipoprotein deposition in the chicken oocyte.

The final rapid growth phase of the chicken oocyte is characterized by massive uptake of hepatically synthesized yolk precursor proteins from the plasma. The two major yolk-forming components, very low density lipoprotein (VLDL) and vitellogenin (VTG), have been shown to interact with a 95-kDa protein present in detergent extracts of ovarian membranes; this protein is absent in hens of a mutant nonlaying chicken strain (Nimpf, J., Radosavljevic, M., and Schneider, W. J. (1989) J. Biol. Chem. 264, 1393-1398). Here, we have purified the 95-kDa protein by ligand and immunoaffinity chromatography and demonstrated its role in receptor-mediated endocytosis by ultrastructural immunolocalization, structural, and functional studies. The receptor was visualized exclusively in the oocyte proper and was absent from somatic cells, in agreement with the previously reported expression of two different lipoprotein receptors in somatic cells and oocytes, respectively, of laying hens (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139). Amino acid sequences of tryptic fragments of the oocyte receptor were obtained, and its kinship to somatic low density lipoprotein receptors was confirmed through the demonstration of sequence conservation in three characteristic domains. In particular, the chicken receptor's internalization sequence, Phe-Asp-Asn-Pro-Val-Tyr, is identical with that in low density lipoprotein receptors from mammals as well as Xenopus laevis. The ligand-binding properties, specificity, and kinetic parameters of the oocyte receptor were characterized in filtration assays employing pure ligands and receptor. In conjunction with ligand-blotting experiments following limited protease digestion of the receptor, the binding assay data suggest that VTG recognizes a substructure of the VLDL-binding site. These studies establish that a cell-specific receptor mediates the endocytosis of VTG and VLDL into growing chicken oocytes and thus possibly plays a key role in control of oocyte growth.     

Apolipoprotein specificity of the chicken oocyte receptor for low and very low density lipoproteins: lack of recognition of apolipoprotein VLDL-II

At onset of egg-laying in the chicken, plasma levels of apolipoprotein VLDL-II (apoII) increase dramatically, suggesting a function of apoII in yolk deposition of triglyceride-rich lipoproteins. Thus, the possibility that this female-specific homodimeric protein (Mr of subunit, 9500) is recognized by the oocyte receptor for low and very low density lipoproteins was investigated. ApoII was purified from very low density lipoproteins by a novel, rapid procedure and reconstituted with egg phosphatidylcholine (PC) by detergent-dialysis. The resulting discoidal apoII/PC lipoprotein particles contained 3 mg of apoII per mg of PC and had a buoyant density of 1.062 g/ml. The ability of apoII/PC, as well as of physiological particles containing apoII but devoid of apolipoprotein B (apoB), namely high density lipoproteins (HDL) from laying hens, to interact with the oocyte receptor was tested. Both of these ligands failed to show saturable high affinity binding, in contrast to the apoB-containing ligands, low and very low density lipoproteins. Furthermore, neither laying-hen HDL which contain apoII and apoA-I nor apoII/PC were able to displace receptor-bound apoB-containing lipoproteins, as shown in competitive binding assays as well as by ligand blotting. Thus, we conclude that apoB, but not apoII, participates in binding and uptake of very low density lipoproteins via receptor-mediated endocytosis by growing chicken oocytes.     

Oocytes from the mutant restricted ovulator hen lack receptor for very low density lipoprotein

Hens of the "Restricted Ovulator" (R/O) chicken strain are characterized by the absence of egg-laying and concomitant severe hyperlipidemia due to a single gene defect (Ho, K. J., Lawrence, W. D., Lewis, L. A., Liu, L. B., and Taylor, C. B. (1974) Arch. Pathol. 98, 161-172). However, the underlying biochemical defect has not been identified. Previous studies on receptor-mediated growth of chicken oocytes have led to the characterization of a 95-kDa oocyte plasma membrane receptor that binds very low density lipoproteins (VLDL) (George, R., Barber, D. L., and Schneider, W. J. (1987) J. Biol. Chem. 262, 16838-16847). The current experiments demonstrate the absence of this receptor from R/O oocytes. Ligand binding experiments showed that ovarian membranes from mutant hens failed to display high affinity, saturable, and specific binding of 125I-VLDL. Ligand blotting with 125I-VLDL and Western blotting with polyclonal anti-receptor antibodies visualized the 95-kDa receptor in normal oocytes, but R/O ovarian membranes were devoid of any cross-reactive protein. Finally, plasma clearance of intravenously injected 125I-VLDL was dramatically impaired in R/O in comparison to normal hens, with a concomitant decrease in the radioactivity accumulating in R/O oocytes. These data strongly suggest that the absence of the 95-kDa receptor for VLDL from oocytes is responsible for the R/O phenotype, and that the receptor not only binds VLDL, but also mediates its uptake. This animal model provides a powerful tool for investigations of receptor-mediated growth of chicken oocytes and for the elucidation of regulatory mechanisms in lipid and lipoprotein metabolism of laying hens.     

Evolution of lipoprotein receptors. The chicken oocyte receptor for very low density lipoprotein and vitellogenin binds the mammalian ligand apolipoprotein E

The laying hen expresses two different lipoprotein transport receptors in cell-specific fashion. On the one hand, a 95-kDa oocyte membrane protein mediates the uptake of the major yolk precursors, very low density lipoprotein, and vitellogenin; on the other hand, somatic cells synthesize a 130-kDa receptor that is involved in the regulation of cellular cholesterol homeostasis (Hayashi, K., Nimpf, J., and Schneider, W. J. (1989) J. Biol. Chem. 264, 3131-3139). Here we show that the oocyte-specific receptor binds, in addition to the yolk precursor proteins, an apolipoprotein of mammalian origin, apolipoprotein E. Ligand blotting, a solid-phase binding assay, and antireceptor antibodies were employed to demonstrate that binding of vitellogenin, very low density lipoprotein (via apolipoprotein B), and apolipoprotein E occurs to closely related, if not identical, sites on the 95-kDa oocyte receptor. The binding properties of lipovitellin, which harbors the receptor recognition site of vitellogenin, are analogous to those of apolipoprotein E: both require association with lipid for expression of functional receptor binding. The ligand specificity of the avian oocyte lipoprotein receptor supports the hypothesis that vitellogenin, which has evolved in oviparous species, represents a counterpart to mammalian apolipoprotein E.     

Extracellular matrices of the avian ovarian follicle. Molecular characterization of chicken perlecan

In egg-laying species, such as the chicken, the mode of transport of lipoprotein particles from the capillary plasma to endocytic receptors on the oocyte surface is largely unknown. Here we show by molecular characterization that the large prominent heparan sulfate proteoglycan of extracellular matrices, termed perlecan or HSPG2 (the product of the hspg2 gene), is a component of ovarian follicles that may participate in this process. However, although normally a major HSPG of basement membranes or basal laminae, in chicken follicles, perlecan is absent from the membranous structure between the theca interna and granulosa cell layers, which to date has been considered a bona fide basement membrane. Rather, the protein is localized in the extracellular matrix of theca externa cells, which produce this HSPG. Furthermore, in chicken testes, perlecan is localized in the peritubular spaces but in less organized fashion than the classical basement membrane components, agrin and laminin. All five domains and structural hallmarks of chicken perlecan (4071 residues) have been conserved in its mammalian counterparts. We have produced the recombinant domain II (containing low density lipoprotein (LDL) receptor-like binding repeats) of chicken perlecan and demonstrate its capacity to bind LDL and very low density lipoprotein (VLDL), apolipoprotein B-containing lipoproteins ultimately destined for uptake into oocytes via members of the low density lipoprotein receptor family. Binding to perlecan heparan sulfate side chains may facilitate the interaction of lipoproteins with domain II. Based on the current results and on domain-domain interactions revealed by recent ultrastructural investigations of the LDL receptor, nidogen, and laminin (Rudenko, G., Henry, L., Henderson, K., Ichtchenko, K., Brown, M. S., Goldstein, J. L., and Deisenhofer, J. (2002) Science 298, 2353-2358 and Takagi, J., Yang, Y., Liu, J. H., Wang, J. H., and Springer, T. A. (2003) Nature 424, 969-974), we propose a novel role of perlecan in mediating plasma-to-oocyte surface transport of VLDL particles.     

Characterization of lipoprotein receptors on rat Fu5AH hepatoma cells

The rat hepatoma cell line Fu5AH has the unusual property of accumulating massive amounts of cholesteryl ester upon incubation with hypercholesterolemic serum, and especially when incubated with beta-very low density lipoproteins (beta-VLDL) from cholesterol-fed dogs. The present study was designed to identify and characterize the lipoprotein receptors that mediate the cholesteryl ester accumulation. The beta-VLDL and cholesterol-induced apolipoprotein (apo) E-containing high density lipoproteins (apoE HDLc) bound to Fu5AH cells with very high affinity (Kd approximately equal to 10(-10) M), whereas low density lipoproteins (LDL) bound with unusually low affinity (Kd approximately equal to 10(-8) M). Receptor binding activity of 125I-labeled beta-VLDL, 125I-labeled apoE HDLc, and 125I-labeled LDL was abolished by incubation in the presence of an excess of unlabeled LDL or of a polyclonal antibody to the bovine adrenal apoB,E(LDL) receptor. The receptors were completely down-regulated by preincubating Fu5AH cells with beta-VLDL, but much higher levels of beta-VLDL were required than for down-regulation of fibroblast apoB,E(LDL) receptors. Receptor binding was abolished by reductive methylation of the lysyl residues of the apolipoprotein of the beta-VLDL and by an apoE monoclonal antibody (1D7) that blocks receptor binding. The Fu5AH receptor was further characterized by using the bovine adrenal apoB,E(LDL) receptor antibody. A single protein (Mr approximately equal to 130,000) was identified in Triton extracts of whole cells, and two proteins (Mr approximately equal to 130,000 and 115,000) were found in Fu5AH cell membranes disrupted by homogenization. The Mr approximately equal to 115,000 protein was released from the membranes and did not react with an antibody to the carboxyl-terminal (cytoplasmic) domain of the apoB,E(LDL) receptors. These studies indicate that Fu5AH cells express apoB,E(LDL) receptors that have unusually low affinity for apoB-continuing lipoproteins, require large amounts of cholesterol to induce down-regulation, and are susceptible to specific proteolysis in cell homogenates. These apoB,E(LDL) receptors are responsible for the receptor-mediated uptake of beta-VLDL and chylomicron remnants by Fu5AH cells.     

Instability of crab vitellogenin and its immunological relatedness with mammalian atherogenic lipoproteins

Vitellogenesis is the process of accumulation of vitellogenin (Vg) in rapidly growing oocytes of oviparous animals and its' subsequent transformation into lipovitellin (Lv). Lipovitellin, which forms the major yolk protein, serves as a principal nutrient reserve for the developing embryo. In the present study, Vg and Lv were purified from the hemolymph and ovary, respectively of the crab Scylla serrata by gel filtration followed by preparative gel electrophoresis. It was observed that purified Vg, but not Lv, possessed an intrinsic protease activity with which it underwent autoproteolysis giving rise to several smaller proteins. Furthermore, urea-mediated unfolding studies by UV-spectral analysis revealed clearly that Vg was easily disrupted by urea whereas Lv was resistant. Taken together, these results suggest that although Lv had a stable conformation, its precursor Vg was labile and highly sensitive to degradation. Another aspect that was investigated in the present study was the immunological kinship of crab Vg and Lv to mammalian atherogenic lipoproteins, the low density lipoprotein (LDL), very low density lipoprotein (VLDL), and apolipoprotein B (apoB). By Western blot analysis, it was demonstrated that crab Vg and Lv were immunoreactive to antibodies to human LDL, VLDL, and apoB. These observations suggest the existence of common epitope recognition sites in crab Vg and mammalian lipid transferring proteins. This corroborates well with our earlier study on the recognition of crab Vg receptor by mammalian lipoproteins.     

The low density lipoprotein receptor prevents secretion of dense apoB100-containing lipoproteins from the liver

The assembly and secretion of very low density lipoproteins (VLDL) require microsomal triglyceride transfer protein (MTP). Recent evidence also suggests a role for the low density lipoprotein (LDL) receptor in this process. However, the relative importance of MTP in the two steps of VLDL assembly and the specific role of the LDL receptor still remain unclear. To further investigate the role of MTP and the LDL receptor in VLDL assembly, we bred mice harboring "floxed" Mttp alleles (Mttpflox/flox) and a Cre transgene on a low-density lipoprotein receptor-deficient background to generate mice with double deficiency in the liver (Ldlr-/- MttpDelta/Delta). In contrast to the plasma of Ldlr+/+ MttpDelta/Delta mice, the plasma of Ldlr-/- MttpDelta/Delta mice contained apoB100. Accordingly, Ldlr-/- MttpDelta/Delta but not Ldlr+/+ MttpDelta/Delta hepatocytes secreted apoB100-containing lipoprotein particles. The secreted lipoproteins were of LDL and HDL sizes but no VLDL-sized lipoproteins could be detected. These findings indicate that hepatic LDL receptors function as "gatekeepers" targeting dense apoB100-containing lipoproteins for degradation. In addition, these results suggest that very low levels of MTP are insufficient to mediate the second step but sufficient for the first step of VLDL assembly.     

Purification of catalytic subunit of low density lipoprotein receptor kinase and identification of heat-stable activator protein

Bovine adrenal cortex contains a high molecular weight casein kinase II-like enzyme (Mr 500,000) that phosphorylates a specific serine residue in the cytoplasmic domain of the low density lipoprotein (LDL) receptor (Kishimoto, A., Brown, M. S., Slaughter, C. A., and Goldstein, J. L. (1987) J Biol. Chem. 262, 1344-1351). In the current paper, we provide evidence to suggest that this 500-kDa kinase can be dissociated into two subunits, a catalytic subunit and an activator subunit, by treatment with 1 M NaCl. The catalytic subunit was purified to homogeneity (greater than 100,000-fold) using affinity chromatography on GTP-agarose plus several other chromatography steps. It had an Mr of 50,000 by gel filtration and 35,000 by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The catalytic subunit phosphorylated casein actively, but it phosphorylated the LDL receptor with only low affinity. The affinity for the LDL receptor was increased 10-fold (saturation at 10 nM LDL receptor) by addition of a second protein that was released from a high molecular weight 500-kDa complex by 1 M NaCl. This activator protein (Mr 120,000 by gel filtration) was extremely heat stable but was destroyed by trypsin. It appeared to be required in stoichiometric amounts with relation to the LDL receptor. It did not increase the ability of the 50-kDa subunit to phosphorylate casein nor did it activate phosphorylation of the LDL receptor or casein by classic casein kinase II. The current data raise the possibility that the specificity of the 500-kDa LDL receptor kinase is attributable to a heat-stable activator subunit that binds to the LDL receptor and thereby renders it a better substrate for the catalytic subunit of the kinase.     

Receptor binding activity of lipid recombinants of apolipoprotein B-100 thrombolytic fragments

Apolipoprotein (apo) B-100, the protein constituent of low density lipoproteins (LDL), is the determinant responsible for LDL binding to the apoB,E(LDL) receptor on cells. The current study was designed to identify the region(s) of apoB-100 that interact with the apoB,E(LDL) receptor. Apolipoprotein B-100 was fragmented by thrombin digestion, and the isolated fragments (T2, T3, T4) were recombined with cholesterol-induced canine high density lipoproteins (HDLc). Before the recombination, the receptor binding activity of apoE of the HDLc was abolished by reductive methylation and extensive trypsin treatment. This treatment permitted almost complete replacement of the small residual apoE fragments by the large apoB fragments. Recombinant apoB particles were isolated by ultracentrifugation and tested for binding to receptors on cultured human fibroblasts. The recombinant particles had chemical and physical properties similar to those of native HDLc. Recombinants of both the whole thrombolytic digest and of isolated fragments displayed specific binding to the apoB,E (LDL) receptor. Anti-apoB,E(LDL) receptor antibodies abolished 90% of the binding, and there was almost no specific binding to receptor-negative fibroblasts or to cells in which the receptors had been down-regulated. The binding of apoB-100 recombinants to the receptor also demonstrated calcium dependency; in addition, the surface binding of the recombinants was released by polyanionic compounds. All these recombinants had binding affinities comparable to one another but less than that of native LDL. Although T2, T3 and T4 recombinants can all bind specifically to the apoB,E(LDL) receptor, it remains to be established whether their activity represents physiologically relevant binding. Nevertheless, the present findings illustrate the potential of the recombinant method using HDLc lipids to reconstitute biological activity.     

A protein partially expressed on the surface of HepG2 cells that binds lipoproteins specifically is nucleolin

Nucleolin, a major nucleolar protein of rapidly growing eukaryotic cells, has been thought to be predominantly if not exclusively located in the nucleolus. Recent data however [Borer, R.A., Lehner, C.F., Eppenberger, H.M., & Nigg, N.A. (1989) Cell 56, 379-390] suggest that the protein shuttles constantly between the nucleus and cytoplasm. Ligand blotting studies of whole cell extracts of HepG2 cells identified, in addition to the LDL receptor, another LDL binding protein of Mr 109,000. The 109-kDa protein was partially purified by HPLC and, like the LDL receptor, bound apoB- and apoE-containing lipoproteins but not HDL. However, unlike the LDL receptor, the 109-kDa protein bound lipoproteins in the presence of EDTA and reducing agents, had a lower affinity for lipoproteins than the LDL receptor, and did not react with two antibodies raised against the LDL receptor. The protein sequences of three separate peptides derived from the partially purified 109-kDa species were determined and were identical except for one residue to three separate regions of the published sequence of nucleolin. On immunoblot analysis the 109-kDa protein reacted with a nucleolin-specific antibody, and purified nucleolin reacted both with anti-109-kDa antibody and with LDL. When intact HepG2 cells were treated with Pronase before harvest, there was a 46% decrease in 109-kDa protein while recovery of actin, an intracellular protein, was unaffected. When intact HepG2 cells were surface iodinated and the proteins subjected to HPLC fractionation, the 109-kDa protein was found to be iodinated.(ABSTRACT TRUNCATED AT 250 WORDS)     

The peroxisome proliferator-activated receptor alpha (PPARalpha) agonist ciprofibrate inhibits apolipoprotein B mRNA editing in low density lipoprotein receptor-deficient mice: effects on plasma lipoproteins and the development of atherosclerotic lesions

Low density lipoprotein receptor (LDLR)-deficient mice fed a chow diet have a mild hypercholesterolemia caused by the abnormal accumulation in the plasma of apolipoprotein B (apoB)-100- and apoB-48-carrying intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Treatment of LDLR-deficient mice with ciprofibrate caused a marked decrease in plasma apoB-48-carrying IDL and LDL but at the same time caused a large accumulation of triglyceride-depleted apoB-100-carrying IDL and LDL, resulting in a significant increase in plasma cholesterol levels. These plasma lipoprotein changes were associated with an increase in the hepatic secretion of apoB-100-carrying very low density lipoproteins (VLDL) and a decrease in the secretion of apoB-48-carrying VLDL, accompanied by a significant decrease in hepatic apoB mRNA editing. Hepatic apobec-1 complementation factor mRNA and protein abundance were significantly decreased, whereas apobec-1 mRNA and protein abundance remained unchanged. No changes in apoB mRNA editing occurred in the intestine of the treated animals. After 150 days of treatment with ciprofibrate, consistent with the increased plasma accumulation of apoB-100-carrying IDL and LDL, the LDLR-deficient mice displayed severe atherosclerotic lesions in the aorta. These findings demonstrate that ciprofibrate treatment decreases hepatic apoB mRNA editing and alters the pattern of hepatic lipoprotein secretion toward apoB-100-associated VLDL, changes that in turn lead to increased atherosclerosis.     

The expressed human hepatic receptor for low-density lipoproteins differs from the fibroblast low-density lipoprotein receptor

The role of the cellular receptor for the low-density lipoproteins (LDL) in cholesterol transport was initially defined through the study of nonhepatic cells in vitro. Since the liver is central in plasma lipoprotein metabolism, an investigation of hepatic lipoprotein receptors is important for understanding normal lipoprotein transport. Utilizing human hepatic and fibroblast membranes, the characteristics of receptors for LDL from hepatic and nonhepatic tissues were directly compared. Human hepatic membranes reversibly bound LDL within 5 min. Although both fibroblast and hepatic membranes saturably bound LDL at 37 degrees C, the fibroblast LDL receptor affinity (Kd = 2.5 X 10(-8) M) and number (5.5 X 10(12) sites/mg membrane protein) were greater than the hepatic receptor affinity (Kd = 10.8 X 10(-8) M) and number (0.5 X 10(12) sites/mg membrane protein). In contrast to the fibroblast LDL receptor which was unable to bind LDL in the presence of EDTA, the hepatic LDL receptor binding of LDL was only partially blocked by EDTA. The binding of LDL to its hepatic receptor is highly temperature-dependent, and studies utilizing both radiolabeled LDL and colloidal gold-labeled LDL indicate that little, if any, binding of LDL hepatic membranes occur at 0-4 degrees C. The hepatic membrane receptor(s) (Mr approximately equal to 270 000 and 330 000) differ from that of the fibroblast LDL receptor (Mr approximately equal to 130 000) and these proteins are present in hepatic membranes from a patient lacking the fibroblast LDL receptor. These data indicate that an expressed hepatic LDL receptor has unique properties different from those of the fibroblast LDL receptor and that the expressed protein(s) is genetically distinct from the fibroblast receptor.     

Interactions of low density lipoprotein2 and other apolipoprotein B-containing lipoproteins with lipoprotein(a)

Studies were undertaken to investigate potential interactions among plasma lipoproteins. Techniques used were low density lipoprotein2 (LDL2)-ligand blotting of plasma lipoproteins separated by nondenaturing 2.5-15% gradient gel electrophoresis, ligand binding of plasma lipoproteins by affinity chromatography with either LDL2 or lipoprotein(a) (Lp(a)) as ligands, and agarose lipoprotein electrophoresis. Ligand blotting showed that LDL2 can bind to Lp(a). When apolipoprotein(a) was removed from Lp(a) by reduction and ultracentrifugation, no interaction between LDL2 and reduced Lp(a) was detected by ligand blotting. Ligand binding showed that LDL2-Sepharose 4B columns bound plasma lipoproteins containing apolipoproteins(a), B, and other apolipoproteins. The Lp(a)-Sepharose column bound lipoproteins containing apolipoprotein B and other apolipoproteins. Furthermore, the Lp(a) ligand column bound more lipoprotein lipid than the LDL2 ligand column, with the Lp(a) ligand column having a greater affinity for triglyceride-rich lipoproteins. Lipoprotein electrophoresis of a mixture of LDL2 and Lp(a) demonstrated a single band with a mobility intermediate between that of LDL2 and Lp(a). Chemical modification of the lysine residues of apolipoprotein B (apoB) by either acetylation or acetoacetylation prevented or diminished the interaction of LDL2 with Lp(a), as shown by both agarose electrophoresis and ligand blotting using modified LDL2. Moreover, removal of the acetoacetyl group from the lysine residues of apoB by hydroxylamine reestablished the interaction of LDL2 with Lp(a). On the other hand, blocking of--SH groups of apoB by iodoacetamide failed to show any effect on the interaction between LDL2 and Lp(a). Based on these observations, it was concluded that Lp(a) interacts with LDL2 and other apoB-containing lipoproteins which are enriched in triglyceride; this interaction is due to the presence of apolipoprotein(a) and involves lysine residues of apoB interacting with the plasminogen-like domains (kringle 4) of apolipoprotein(a). Such results suggest that Lp(a) may be involved in triglyceride-rich lipoprotein metabolism, could form transient associations with apoB-containing lipoproteins in the vascular compartment, and alter the intake by the high affinity apoB, E receptor pathway.     

Apolipoprotein E Structure and Substrate and Receptor-Binding Activities of Triglyceride-Rich Human Plasma Lipoproteins in Normo- and Hypertriglyceridemia

Cysteine-arginine interchanges along the primary sequence of human plasma apolipoprotein E (apoE) play an important role in determining its biological functions due to a high mutation frequency of cytosine in CGX triplet that codes 33 of 34 apolipoprotein arginine residues. The contribution of apoE secondary structure to apolipoprotein-lipid interaction is described. The significance of apolipoprotein in triglyceride synthesis, lipoprotein lipolysis, and receptor-mediated clearance of lipolytic remnants of triglyceride-rich lipoproteins is discussed as well. The metabolic flow of lipoproteins in normo- and hypertriglyceridemia can be described by separate compartments that contribute to lipoprotein interaction with at least six different receptors: 1) low density lipoprotein (LDL) receptor; 2) LDL receptor-related protein (LRP); 3) apoB(48) macrophage receptor for hypertriglyceridemic very low density lipoproteins (VLDL); 4) scavenger receptors; 5) VLDL receptor; 6) lipolysis-stimulated receptor. The contribution of the exposure of apoE molecules on the surface of triglyceride-rich particles sensitive both to lipolysis and plasma triglyceride content to the interaction with LDL receptor and LRP is emphasized.     

Recycling of apolipoprotein E in mouse liver

Following the internalization of low density lipoprotein (LDL) by the LDL receptor within cells, both the lipid and the protein components of LDL are completely degraded within the lysosomes. Remnant lipoproteins are also internalized by cells via the LDL receptor as well as other receptors, but the events following the internalization of these complexes, which use apolipoprotein E (apoE) as their ligand for receptor capture, have not been defined. There is evidence that apoE-containing beta-very low density lipoproteins follow differential intracellular routing depending on their size and apoE content and that apoE internalized with lipoproteins can be resecreted by cultured hepatocytes and fibroblasts. In the present studies, we addressed the question of apoE sparing or recycling as a physiologic phenomenon. Remnant lipoproteins (d < 1.019 g/ml) from normal mouse plasma were iodinated and injected into normal C57BL/6 mice. Livers were collected at 10, 30, 60, and 120 min after injection, and hepatic Golgi fractions were prepared for gel electrophoresis analysis. Golgi preparations were analyzed for galactosyltransferase enrichment (>40-fold above cell homogenate) and by appearance of the Golgi stacks and vesicles on electron microscopy. Iodinated apoE was consistently found in the Golgi fractions peaking at 10 min and disappearing by 2 h after injection. Although traces of apoB48 were present in the Golgi fractions, the apoE/apoB ratio in the Golgi was 50-fold higher compared with serum. Quantitatively similar results were obtained when the very low density lipoprotein remnants were injected into mice deficient in either apoE or the LDL receptor, indicating that the phenomenon of apoE recycling is not influenced by the production of endogenous apoE and is not dependent on the presence of LDL receptors. In addition, radioactive apoE in the Golgi fractions was part of d = 1.019-1.21 g/ml complexes, indicating an association of recycled apoE with either newly formed lipoproteins or the internalized complexes. These studies show that apoE recycling is a physiologic phenomenon in vivo and establish the presence of a unique pathway of intracellular processing of apoE-containing remnant lipoproteins.     

Identification of lipoprotein-binding proteins in rat liver Golgi apparatus membranes

The low density lipoprotein (LDL) receptor has been shown to be a plasma membrane glycoprotein responsible for the cellular binding and endocytosis of plasma lipoproteins. Inasmuch as the Golgi apparatus has been shown to participate in glycoprotein processing and in the assembly of plasma lipoproteins by hepatic and intestinal epithelial cells, the present studies were designed to test the hypothesis that lipoprotein receptors are present within Golgi membranes. Utilizing ligand blotting with a variety of iodinated lipoproteins, several lipoprotein-binding proteins were identified in rat liver Golgi membranes at apparent molecular weights (Mr) 200,000, 160,000, 130,000, 120,000, 100,000, 80,000, and 70,000. The 130,000 protein was the most prominent and was identified as the mature LDL receptor by its binding characteristics and an Mr characteristic of the plasma membrane receptor. Enzymatic deglycosylation studies suggested that the 120,000 and 100,000 proteins were LDL receptor precursors lacking sialic acid. Antibody to the LDL receptor recognized all the bands on immunoblots except the 70,000 protein, with the 130,000 protein being the most prominent. Isolation of the Golgi fractions in the presence of protease inhibitors did not eliminate any of the proteins recognized by the antibody but did result in sharper bands on the blots. Additionally, we investigated the hypothesis that conditions that regulate plasma membrane LDL receptors also cause detectable changes in receptors in Golgi membranes. All the binding proteins were increased in Golgi membranes from rats treated with 17-alpha-ethynylestradiol. Colchicine caused an accumulation of 120,000 Mr protein, suggesting blockage of final sialylation in the trans Golgi. When protein synthesis was inhibited by cycloheximide, there was no reduction of mature LDL receptors in Golgi membranes, consistent with recycling of receptors through this organelle.