Bovine Low Density Lipoprotein Receptor Research Articles

Localization of the N-terminal Domain of the Low Density Lipoprotein Receptor

The low density lipoprotein (LDL) receptor is a transmembrane glycoprotein performing "receptor-mediated endocytosis" of cholesterol-rich lipoproteins. At the N terminus, the LDL receptor has modular cysteine-rich repeats in both the ligand binding domain and the epidermal growth factor (EGF) precursor homology domain. Each repeat contains six disulfide-bonded cysteine residues, and this structural motif has also been found in many other proteins. The bovine LDL receptor has been purified and reconstituted into egg yolk phosphatidylcholine vesicle bilayers. Using gel electrophoresis and cryoelectron microscopy (cryoEM), the ability of the reconstituted LDL receptor to bind its ligand LDL has been demonstrated. After reduction of the disulfide-bonds in the N-terminal domain of the receptor, the reduced LDL receptor was visualized using cryoEM; reduced LDL receptors showed images with a diffuse density region at the distal end of the extracellular domain. Gold labeling of the reduced cysteine residues was achieved with monomaleimido-Nanogold, and the bound Nanogold was visualized in cryoEM images of the reduced, gold-labeled receptor. Multiple gold particles were observed in the diffuse density region at the distal end of the receptor. Thus, the location of the ligand binding domain of the LDL receptor has been determined, and a model is suggested for the arrangement of the seven cysteine-rich repeats of the ligand binding domain and two EGF-like cysteine-rich repeats of the EGF precursor homology domain.

Vesicle-reconstituted Low Density Lipoprotein Receptor: VISUALIZATION BY CRYOELECTRON MICROSCOPY

The low density lipoprotein (LDL) receptor is a key protein for maintaining cellular cholesterol homeostasis by binding cholesterol-rich lipoproteins through their apoB and apoE apoproteins. The LDL receptor is a transmembrane glycoprotein of M(r) approximately 115 kDa; based on its primary sequence, five distinct structural domains have been identified (Yamamoto, T., Davis, C. G., Brown, M. S., Schneider, W. J., Casey, M. L., Goldstein, J. L., and Russell, D. W. (1984) Cell 39, 27-38). As a first step toward providing a structural description of the intact LDL receptor, the receptor has been purified from bovine adrenal cortices, reconstituted into unilamellar egg yolk phosphatidylcholine vesicles, and imaged using cryoelectron microscopy (cryoEM). CryoEM has the advantage of providing images of the reconstituted LDL receptor in its frozen, fully hydrated state. LDL receptor molecules were visualized as elongated, stick-like projections from the vesicle surface with maximum dimensions approximately 120-A length by approximately 45-A width. In some of the images, a short arm (or arms) was visible at the distal end of the stick-like projections. The LDL receptor was labeled via accessible free cysteine residues, probably including that corresponding to Cys-431 of the known full-length sequence of the human LDL receptor. The accessible cysteine was demonstrated using a maleimide-biotin.streptavidin conjugate and confirmed by labeling with monomaleimido-Nanogold. Images obtained by cryoEM showed that the extracellular stick-like domain of the reconstituted LDL receptor was labeled by Nanogold. This combined cryoEM-Nanogold labeling study has provided the first low resolution structural images of the reconstituted, full-length bovine LDL receptor.

Structural studies of detergent-solubilized and vesicle-reconstituted low-density lipoprotein (LDL) receptor.

The low-density lipoprotein (LDL) receptor plays a key role in maintaining circulating and cellular cholesterol homeostasis. The LDL receptor is a transmembrane glycoprotein whose biochemical and genetic properties have been extensively studied notably by Brown, Goldstein and colleagues [Brown, M. S., & Goldstein, J. L., (1986) Science 232, 34-47]. However, few if any structural studies of the LDL receptor have been reported, and details of its secondary and tertiary structure are lacking. In an attempt to determine the low-resolution structure of the LDL receptor, we have purified the receptor from bovine adrenal cortices using modifications of the method of Schneider et al. [Schneider, W. J., Goldstein, J. L., & Brown, M. S. (1985) Methods in Enzymol.109, 405-417]. Using circular dichroism, the secondary structure of the detergent-solubilized bovine LDL receptor at 25 degrees C was shown to be 19% alpha-helix, 42% beta-sheet, and 39% random coil. Interestingly, the detergent-solubilized receptor appeared to be quite resistant to changes in secondary structure over the temperature range 10-90 degrees C, with only minor but reversible changes being observed. In contrast, a more pronounced unfolding of the detergent-solubilized receptor was observed in the presence of guanidinium hydrochloride. Using the complete sequence of the human LDL receptor, sequence analysis by the Chou-Fasman prediction algorithm showed quite good agreement with the experimentally determined secondary structure of the bovine LDL receptor at 25 degrees C. Finally, the purified, bovine LDL receptor was reconstituted into large unilamellar vesicles of egg yolk phosphatidylcholine using a procedure exploiting preformed vesicles and detergent dialysis. We showed previously using negative stain electron microscopy that reconstituted vesicles bind LDL. Now, using cryoelectron microscopy of frozen hydrated reconstituted vesicles evidence of an extended, stick-like morphology (length approximately 120 A) for the extracellular domain of the LDL receptor has been obtained. Successful purification of the receptor, its incorporation into single bilayer vesicles, and its direct visualization by cryoelectron microscopy pave the way for more detailed structural studies of the LDL receptor and the receptor-LDL complex.

Uptake of endoneurial lipoprotein into Schwann cells and sensory neurons is mediated by low density lipoprotein receptors and stimulated after axonal injury.

During Wallerian degeneration of rat sciatic nerve, the expression of apolipoprotein E increases and apolipoprotein E-containing endoneurial lipoproteins accumulate in the distal nerve segment. In established primary cultures dissociated from dorsal root ganglia, Schwann cells and sensory neurons internalized rhodamine-labeled lipoproteins isolated from crushed rat sciatic nerve as well as low density lipoprotein (LDL) from human serum. The uptake of endoneurial lipoproteins could be inhibited by an excess of LDL or at low temperature (4 degrees C). After transection of nerve fibers in dorsal root ganglia explant cultures, the uptake of lipoproteins was markedly stimulated in Schwann cells that were in close proximity to degenerating neurites. A specific monoclonal antibody directed to the bovine LDL receptor (clone C7) was shown to cross-react with LDL receptor preparations of rat endoneurial cells. LDL receptor immunoreactivity was expressed by cell bodies and processes of cultured Schwann cells, sensory neurons, and fibroblasts from dorsal root ganglia. Incubation of Schwann cells and neurons with the LDL receptor antibody strongly inhibited the uptake of endoneurial lipoproteins. Our results provide direct evidence for the important role of the LDL receptor-mediated pathway to internalize endoneurial lipoproteins into Schwann cells and peripheral neurons required for reuse of cholesterol and other lipids in myelin and plasma membrane biogenesis during nerve repair.

Antipeptide antibody against the human low-density-lipoprotein receptor. Characterization and cross-reactivity with bovine lymphocytes.

A dodecapeptide corresponding to the external N-terminal sequence of the human low-density-lipoprotein (LDL) receptor was synthesized. Antibodies raised in rabbits against the peptide were purified and were shown to react specifically with the peptide and with human LDL receptor of fibroblasts, HeLa cells and lymphocytes using binding studies and immunoblotting. By indirect immunogold analysis, antibodies bound to the LDL receptor of human lymphocytes could be revealed as clusters. Anti-receptor peptide immunoglobulins specifically bound to the human HeLa cell's LDL receptor with a lower affinity than LDL (Kd x 3). The anti-receptor peptide immunoglobulins and 125I-labelled-LDL competed with each other for the LDL-receptor sites. Antibodies failed to react with lymphocytes of subjects with the homozygous form of familial hypercholesterolaemia. Cross-reactivity with the dodecapeptide of the bovine LDL receptor was limited, but this cross-reactivity was confirmed by the binding of anti-receptor peptide immunoglobulins to the LDL receptor from bovine lymphocytes.

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.

MRNA for low density lipoprotein receptor in brain and spinal cord of immature and mature rabbits.

Hybridization studies with [32P]cDNA probes revealed detectable amounts of mRNA for the low density lipoprotein (LDL) receptor in the central nervous system (CNS) of rabbits. mRNA levels were highest in the medulla/pons and spinal cord, which were the most heavily myelinated regions that were studied. Lower, but detectable levels were present in cerebral cortex, hypothalamus, thalamus, midbrain, and cerebellum. In the medulla/pons and spinal cord, the levels of receptor mRNA were in a range comparable to that detected in the liver. The levels of receptor mRNA in whole brain were constant from 3 days of age to adulthood and, thus, did not vary in proportion to the rate of myelin synthesis. LDL receptor mRNA in the CNS was produced by the same gene that produced the liver and adrenal mRNA as revealed by the demonstration of a deletion in the neural mRNA of Watanabe-heritable hyperlipidemic (WHHL) rabbits identical to the deletion in the LDL receptor gene of these mutant animals. Using antibodies directed against the bovine LDL receptor, we showed that LDL receptor protein is present in the medulla/pons of adult cows. The cell types that express LDL receptors in the CNS and the functions of these receptors are unknown.

Binding of low-density lipoprotein and chylomicron remnants to the hepatic low-density lipoprotein receptor of dogs, rats and rabbits demonstrated by ligand blotting. Failure to detect a distinct chylomicron-remnant-binding protein by ligand blotting.

In this paper, human low-density lipoprotein (LDL), rat chylomicron remnants and very-low-density lipoproteins of beta-mobility from cholesterol-fed rabbits (beta VLDL) have been shown to bind strongly to a protein present in solubilised liver membranes of rats, rabbits and dogs by ligand blotting with biotin-modified lipoproteins. This binding protein was identified as the LDL-receptor on several criteria. First, binding of the lipoproteins to the receptor was saturable and Ca2+-dependent; secondly, the apparent relative molecular mass of the binding protein (ranging from 128,000 in the rabbit, 145,000 in the rat to 147,000 in the dog) was similar to that of the purified bovine LDL receptor. Finally, binding activity was greatly increased in the livers of rats treated with oestrogen in pharmacological doses and absent from the liver of Watanabe heritable hyperlipidaemic (WHHL) rabbits that have a genetic defect in the LDL receptor. Some binding was also observed to a high-molecular-mass protein present in solubilised liver membranes of rats and rabbits, which, in rabbits at least, shared antigenic determinants with rabbit apoB and was not likely to be related to the LDL receptor as it was present in equal amounts in normal and WHHL rabbits. No evidence was obtained for a specific chylomicron remnant binding protein, distinct from the LDL receptor, whose activity could be detected in solubilised liver membranes by ligand blotting although a variety of solubilisation and fractionation conditions were employed.

The low density lipoprotein receptor on human peripheral blood monocytes and lymphocytes: visualization by ligand blotting and immunoblotting techniques.

Western blotting and immunoprecipitation techniques were used to study low density lipoprotein (LDL) receptors from cultured human monocytes, lymphocytes, and fibroblasts. After incubation in lipoprotein-deficient media to allow induction, receptors were solubilized, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose paper, and detected by incubation with apolipoprotein B-containing lipoproteins followed by radiolabeled antiapolipoprotein B antibody. LDL receptors of identical apparent mol wt were demonstrated in monocyte and lymphocyte cell extracts; the receptors bound LDL as well as human post-prandial lipoprotein (density, less than 1.0063) on Western blots, but did not bind acetyl-LDL. LDL receptors in mononuclear cells could also be detected by immunoblotting using immunoglobulin G-C7, a monoclonal antibody raised against the bovine LDL receptor. When cell extracts were blotted, the mononuclear cell receptors had a lower mol wt than the fibroblast receptor in unreduced sodium dodecyl sulfate-polyacrylamide gels, with an apparent mol wt difference of 5,000 [134,000 +/- 1,400 (+/- SE) for mononuclear cells vs. 139,000 +/- 1600 for fibroblasts]. Immunoprecipitation and electrophoresis of L-[35S]methionine-labeled cell extracts revealed more rapid conversion of the receptor precursor to the mature receptor in monocytes than in fibroblasts. Western blotting of mononuclear cells from a patient with an abnormally high mol wt receptor characterized in fibroblasts demonstrated that the same mutation was expressed in monocytes and lymphocytes. This report represents the first visualization of human mononuclear cell LDL receptors by Western blotting and immunoprecipitation. These techniques may find application in population screening for LDL receptor variability.

CDNA cloning of the bovine low density lipoprotein receptor: feedback regulation of a receptor mRNA.

The low density lipoprotein (LDL) receptor belongs to a class of migrant cell surface proteins that mediate endocytosis of macromolecular ligands. No cDNAs for this class of proteins have been isolated to date. In the current paper, we report the isolation of a cDNA clone for the LDL receptor from a bovine adrenal cDNA library. The library was constructed by the Okayama-Berg method from poly(A)+ RNA that had been enriched in receptor mRNA by immunopurification of polysomes. Mixtures of synthetic oligonucleotides encoding the amino acid sequence of two neighboring regions of a single cyanogen bromide fragment were used as hybridization probes to identify a recombinant plasmid containing the LDL receptor cDNA. This plasmid, designated pLDLR-1, contains a 2.8-kilobase (kb) insert that includes a sequence which corresponds to the known amino acid sequence of a 36-residue cyanogen bromide fragment of the receptor. pLDLR-1 hybridized to a mRNA of approximately equal to 5.5 kb in the bovine adrenal gland. This mRNA, like the receptor protein, was 9-fold more abundant in bovine adrenal than in bovine liver. pLDLR-1 cross-hybridized to a mRNA of approximately equal to 5.5 kb in cultured human epidermoid carcinoma A-431 cells. This mRNA was markedly reduced in amount when sterols were added to the culture medium, an observation that explains the previously observed feedback regulation of LDL receptor protein. Southern blot analysis of bovine genomic DNA with 32P-labeled pLDLR-1 revealed a simple pattern of hybridization, consistent with a single-copy gene containing introns.

Use of antipeptide antibodies to demonstrate external orientation of the NH2-terminus of the low density lipoprotein receptor in the plasma membrane of fibroblasts.

The low density lipoprotein (LDL) receptor is a member of a class of receptors that bind macromolecules at the cell surface and facilitate their cellular uptake by receptor-mediated endocytosis. The orientation of the LDL receptor in the plasma membrane is unknown. In the current studies the sequence of amino acids at the NH2-terminus of the bovine adrenal LDL receptor was determined, and a synthetic peptide corresponding to amino acids 1-16 was prepared. Antibodies against this peptide were raised in rabbits and were shown by immunoblotting analysis to react specifically with the bovine LDL receptor. The anti-receptor peptide antibodies also bound to the LDL receptor on the outer surface of the plasma membrane of intact human fibroblasts, as visualized by indirect immunofluorescence. Specificity of this binding reaction was confirmed by the observation that the anti-receptor peptide antibodies did not bind to mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack LDL receptors. These data demonstrate that the LDL receptor is oriented in the plasma membrane with its NH2-terminus facing the extracellular surface.

Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia.

Monoclonal antibodies directed against the low density lipoprotein (LDL) receptor have been prepared by immunization of mice with a partially purified receptor from bovine adrenal cortex. Spleen cells from the mice were fused with the Sp2/0-Ag14 line of mouse myeloma cells. The most extensively studied monoclonal antibody, designated immunoglobulin-C7, reacts with the human and bovine LDL receptor, but not with receptors from the mouse, rat, Chinese hamster, rabbit, or dog. 125I-labeled monoclonal antibody binds to human fibroblasts in amounts that are equimolar to 125I-LDL. In fibroblasts from 6 of 8 patients with the receptor-negative form of homozygous familial hypercholesterolemia, which have less than 5% of normal LdL binding, the amount of monoclonal antibody binding was also less than 5% of normal. Fibroblasts from the other two receptor-negative homozygotes bound an amount of monoclonal antibody that was much greater than expected on the basis of LDL binding, suggesting that these two patients produce a structurally altered receptor that binds the antibody, but not LDL. In normal fibroblasts, the receptor-bound monoclonal antibody was taken up and degraded at 37 degrees C at rapid rate similar to that for LDL. Fibroblasts from a patient with the internalization defective form of familial hypercholesterolemia bound the monoclonal antibody, but did not internalize or degrade it. The current data demonstrate the usefulness of monoclonal antibodies as probes for the study of the cellular and genetic factors involved in receptor-mediated endocytosis.