Release of low density lipoprotein from its cell surface receptor by sulfated glycosaminoglycans.

The sulfated glycosaminoglycan, heparin, was found to release 125I-labeled low density lipoprotein (125I-LDL) from its receptor site on the surface of normal human fibroblasts. Measurement of the amount of 125I-LDL released by heparin permitted the resolution of the total cellular uptake of 125I-LDL at 37 degrees C into two components: first, an initial rapid, high affinity binding of the lipoprotein to the surface receptor, from which the 125I-LDL could be released by heparin, and second, a slower process attributable to an endocytosis of the receptor-bound lipoprotein, which rendered it resistant to heparin release. At 4 degrees C the amount of heparin-releasable 125I-LDL was similar to that at 37 degrees C, but interiorization of the lipoprotein did not occur at the lower temperature. The physiologic importance of the cell surface LDL receptor was emphasized by the finding that mutant fibroblasts from a subject with homozygous Familial Hypercholesterolemia, which lack the ability to take up 125I-LDL at 37 degrees C, did not show cell surface binding of 125I-LDL, as measured by heparin release, at either 4 degrees C or 37 degrees C. Although heparin released 125I-LDL from its binding site, it did not release 3H-concanavalin A from its surface receptor, and conversely, alpha-methyl-D-mannopyranoside, which released 3H-concanavalin A, did not release surface-bound 125I-LDL. When added to the culture medium simultaneously with LDL, heparin prevented the binding of LDL to its receptor and hence prevented the LDL-mediated suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. The uptake of LDL by fibroblasts is proposed as a model of receptor-mediated adsorptive endocytosis of macromolecules in human cells.     

Relationship between heparin binding characteristics and ability of human spermatozoa to penetrate hamster ova

Heparin binding site affinity and density on human spermatozoa were compared between fertile and infertile men with normal or abnormal results in the zona-free hamster ova-sperm penetration assay (SPA). A portion of fresh semen from fertile donors and potentially infertile men was processed through the SPA while the remainder of the ejaculate was used to quantitate heparin binding on spermatozoa. Saturation binding assays with [3H]heparin (15-375 nM) were analysed for 3 groups of men: (1) infertile patients with abnormal SPA results, (2) infertile patients with normal SPA results and (3) fertile donors. The heparin binding site density was significantly higher in men who possessed normal SPA results (infertile men and fertile donors) than in infertile men with abnormal scores in the SPA. There was no difference in heparin binding affinity between the three groups. These findings suggest that the heparin binding-site density may be related to the ability of human spermatozoa to undergo successfully the acrosome reaction.     

The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.     

Determination of the LDL receptor binding capacity of human lymphocytes by immunocytofluorimetric assay

The determination of the LDL receptor binding capacity of human blood lymphocytes was assessed by indirect immunocytofluorimetric assay. To produce the maximal synthesis of the LDL receptor, the cholesterol efflux was enhanced by incubation of lymphocytes with HDL3 subfractions. The binding capacity of the LDL receptor was measured by incubation at 4 degrees C either with LDL and rabbit anti-LDL immunoglobulins or with peptide receptor antibody (ARP-Ig) raised against the NH2-terminal sequence of the LDL receptor. Thereafter complexes were incubated with fluorescein-labelled anti-rabbit immunoglobulin (FITC-Ig). Fluorescence flow cytometry was used to quantify the number of fluorescent lymphocytes and results were expressed as the percentage of lymphocytes with a fluorescent intensity above the threshold. Using preimmune rabbit immunoglobulin and then FITC-Ig, only 5-10% of cells were fluorescent. Neither LDL nor ARP-Ig could bind to homozygous familial hypercholesterolemia (FH) lymphocytes. Normal lymphocytes preincubated with HDL3 could bind LDL or ARP-Ig, the number of fluorescent cells being 59 and 39.2% respectively. Subjects with confirmed or suspected heterozygous FH demonstrated cell fluorescence at about half the normal level.     

Investigation of the mechanism of binding between internalin B and heparin using surface plasmon resonance

Listeria monocytogenes, a food-borne pathogen that infects immunocompromised patients, enters and proliferates within mammalian cells by taking advantage of host cell machinery. While entry into macrophages and other phagocytic cells occurs constitutively, intracellular invasion of nonphagocytic cells, such as epithelial and endothelial cells, occurs through induced phagocytosis. Invasion of these nonphagocytic cell types is under the control of the secreted L. monocytogenes protein internalin B (InlB), which directly associates with and activates the receptor tyrosine kinase Met. Activation of Met by InlB has previously been shown to be potentiated by binding of glycosaminoglycans to the GW domains of this protein. We studied the interaction between heparin and full-length InlB as well as a truncated, functional form of InlB to understand the mode of interaction between these two molecules. InlB preferred long-chain (>or=dp14) heparin oligosaccharides, and the interaction with heparin fit a complicated binding model with a dissociation constant in the nanomolar range. While there are various explanations for this complicated binding model, one supported by our data involves binding and rebinding of InlB to multiple binding sites on heparin in a positive and weakly cooperative manner. This mode is consistent with enhancement of interaction of InlB with glycosaminoglycans for activation of Met.     

Stimulation of the LDL receptor activity in the human hepatoma cell line Hep G2 by high-density serum fractions

The regulation of the LDL receptor activity in the human hepatoma cell line Hep G2 was studied. In Hep G2 cells, in contrast with fibroblasts, the LDL receptor activity was increased 2.5-fold upon increasing the concentration of normal whole serum in the culture medium from 20 to 100% by volume. Incubation of the Hep G2 cells with physiological concentrations of LDL (up to 700 micrograms/ml) instead of incubation under serum-free conditions resulted in a maximum 2-fold decrease in LDL receptor activity (10-fold decrease in fibroblasts). Incubation with physiological concentrations of HDL with a density of between 1.16 and 1.20 g/ml (heavy HDL) resulted in an approximately 7-fold increase in LDL receptor activity (1.5-fold increase in fibroblasts). This increased LDL receptor activity is due to an increase in the number of LDL receptors. Furthermore, simultaneous incubation of Hep G2 cells with LDL and heavy HDL (both 200 micrograms/ml) resulted in a 3-fold stimulation of the LDL receptor activity as compared with incubation in serum-free medium. 3-Hydroxy-3-methylglutaryl-CoA reductase activity was also stimulated after incubation of Hep G2 with heavy HDL (up to 3-fold). The increased LDL receptor activity in Hep G2 cells after incubation with heavy HDL was independent of the action of lecithin:cholesterol acyltransferase during that incubation. However, previous modification of heavy HDL by lecithin:cholesterol acyltransferase resulted in an enhanced ability of heavy HDL to stimulate the LDL receptor activity. Our results indicate that in Hep G2 cells the heavy HDL-mediated stimulation of the LDL receptor activity overrules the LDL-mediated down-regulation and raises the suggestion that in man the presence of heavy HDL and the action of lecithin:cholesterol acyltransferase in plasma may be of importance in receptor-mediated catabolism of LDL by the liver.     

The kinetics of binding of serum lipoproteins by immobilized heparin.

A model arterial system of heparin immobilized on an agarose gel was used to study the amount and kinetics of binding of porcine serum lipoproteins to heparin. Binding occurred to lipoproteins in the density range 1.006 less than d less than 1.062, but there was no binding with high density lipoprotein. A theoretical model of the kinetic experiments was formulated and used to demonstrate that the rate of the binding reaction could be considered instantaneous relative to the rate of transport of lipoproteins. Extrapolation of these results to arterial levels of glycosaminoglycans and lipoprotein indicate that complexes of lipoprotein and the glycosaminoglycans could account for much of the cholesterol entrapment in atherosclerotic lesions.     

Murine lymphocyte and embryonal carcinoma cell surface antigens recognised by rabbit anti-murine embryonal carcinoma serum

Some recent studies have suggested that murine embryonal carcinoma (EC) cells which lack classical H-2 molecules must express some novel class-I-MHC (major histocompatibility complex) type antigens. We have investigated whether rabbit anti-EC sera may recognize such components. Molecules of 40 and 48 kd were immunoprecipitated from EC cells and lymphocytes. The possibility that these molecules may be coded for by genes in the Qa-Tla locus which contains many MHC class-I genes or pseudogenes was investigated. The 40 and 48 kd molecules were not associated with beta 2-microglobulin on EC cells, nor with each other or other molecules through S-S bonds, although they appeared to have intradisulphide structures. Peptide map analysis suggested that the lymphocyte 40 and 48 kd molecules were related to the EC cell 40 and 48 kd molecules. However, these molecules were different from H-2, Ia or Qa-2 molecules from the same mouse strain.     

Relationship between fertilizing ability of frozen human spermatozoa and capacity for heparin binding and nuclear decondensation.

Nuclear decondensation of spermatozoa induced by heparin, reduced glutathione (GSH) or a mixture of heparin and GSH was studied using frozen-thawed human spermatozoa. The percentages of decondensed spermatozoa in controls and after treatment for 60 min with 30 mumol heparin l-1, 5 mmol GSH l-1, or heparin-GSH mixture were 1.5, 22.1, 4.3 and 37.6%, respectively. Most of the decondensed spermatozoa were eliminated by Percoll gradient centrifugation of samples previously treated with heparin or heparin-GSH mixture. However, comparable numbers of motile spermatozoa were recovered in the control and in each treated sample, demonstrating that a major proportion of motile spermatozoa was resistant to heparin (or heparin-GSH) effects on nuclear decondensation of spermatozoa. Fertilization of hamster oocytes was attempted using spermatozoa recovered in the 90% Percoll fraction and resistant to heparin-GSH decondensing mixture. Although insemination used a constant number of motile spermatozoa, fertilization rates were higher after treatments with heparin and GSH alone than in control or heparin-GSH-treated samples. In addition the number of spermatozoa that attached to the oocyte plasma membrane was a sixth or a half for sperm pretreated with heparin-GSH or heparin alone, respectively compared with untreated values. However, there was no evidence for induced acrosomal reaction by heparin and GSH, at least at the concentrations used. Qualitative analyses of heparin-binding sites were performed on untreated spermatozoa recovered in the 90% Percoll fraction by incubating spermatozoa in the presence of heparin covalently linked to albumin and coupled to colloidal gold (5 nm). Among this population of spermatozoa, 40.5% bound heparin-gold and labelling was mainly observed on the sperm head surface (88% of labelled spermatozoa) with (59.5%) or without (28.5%) tail labelling. Only a small proportion (23%) of spermatozoa that attached to the oocyte plasma membrane bound heparin-gold conjugate and only weak labelling was observed on the sperm head. Moreover, the proportion of spermatozoa that bound heparin-gold conjugate decreased (r = -0.77, P less than 0.0001) in relation to increasing concentrations of motile spermatozoa in the sample.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inhibition of binding of lactoferrin to the human promonocyte cell line THP-1 by heparin: the role of cell surface sulphated molecules

Lactoferrin, an iron-binding protein of the transferrin family, is a highly basic protein which interacts with many acidic molecules, including heparin proteoglycans. Such interactions may modify some of the biological properties of lactoferrin. In the present work we found that heparin caused a dose-dependent inhibition of specific binding of both human and bovine lactoferrin to human monocytic THP-1 cells. Low-affinity binding sites (Kd 500 nM) were more susceptible to inhibition by heparin than the high-affinity sites (Kd 100 nM). The effect was mediated by interaction between lactoferrin and heparin rather than by competition between heparin and lactoferrin for common binding sites on the cells. Pretreatment of cells with NaClO3 to prevent sulphation of surface glycosaminoglycans reduced lactoferrin binding, and de-N-sulphated heparin did not inhibit binding of lactoferrin to THP-1 cells. These results suggest that heparin binding and monocyte/macrophage binding by lactoferrin both involve interactions between basic regions in the N1 domain of lactoferrin and sulphate groups. The N-terminal Arg2-Arg5 sequence of human lactoferrin may be involved, but it does not seem to be the key element in these interactions.     

Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor

LPL mediates the uptake of lipoproteins into different cell types independent of its catalytic activity. The mechanism of this process and its physiological relevance are not clear. Taking into account the importance of the endothelial barrier for lipoprotein uptake, in vitro studies with primary aortic endothelial cells from wild-type and low density lipoprotein receptor (LDLR)-deficient (LDLR(-/-)) mice were performed. Addition of LPL almost doubled the uptake of LDL into wild-type cells. However, there was virtually no LPL-mediated change of LDL uptake into LDLR(-/-) cells. Upregulation of LDLR by lipoprotein-deficient serum/lovastatin in wild-type cells resulted in a 7-fold increase of LPL-mediated LDL uptake. Uptake of chylomicron remnants was not affected by LDLR expression. In proteoglycan-deficient cells, LPL did not increase the uptake of lipoproteins. The physiological relevance of this pathway was studied in mice that were both LDLR(-/-) and transgenic for catalytically inactive LPL in muscle. In the presence of LDLR, inactive LPL reduced LDL cholesterol significantly (13-24%). In the absence of LDLR, LDL cholesterol was not affected by transgenic LPL. Metabolic studies showed that in the presence of LDLR, LPL increased the muscular uptake of LDL by 77%. In the absence of LDLR, transgenic LPL did not augment LDL uptake. Chylomicron uptake was not affected by the LDLR genotype. We conclude that LPL-mediated cellular uptake of LDL, but not of chylomicrons, is dependent on the presence of both LDLR and proteoglycans.     

LDL receptors in bovine tissues assayed as the heparin-sensitive binding of 125I-labeled LDL in homogenates: relation between liver LDL receptors and serum cholesterol in the fetus and post term

The heparin-sensitive binding of 125I-labeled LDL in homogenates of bovine tissues was determined using a membrane filter assay. The binding fulfilled several criteria which have been established for the binding of LDL to its receptor, namely: saturability, dependence on Ca2+, sensitivity to proteolytic destruction and heat sensitivity. The adrenal cortex and the active corpus luteum exhibited the highest binding activity of the 22 different tissues assayed. Tissues from the central nervous system had low binding activity. Livers from fetal animals had higher binding than livers from young and adult animals and the binding of 125I-LDL to fetal liver homogenates showed an inverse correlation to the serum cholesterol levels, indicating that the LDL receptors in fetal liver may play a role in the regulation of the serum cholesterol level in the fetus during gestation. After birth, the binding of 125I-LDL to calf liver homogenates decreased to levels found in adult animals and this was paralleled by an increase of total serum cholesterol, suggesting that the rapid rise in serum cholesterol in mammals observed soon after birth may be caused by a decrease of the receptor-mediated catabolism of LDL in the liver.     

Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)‐expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293‐EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (K_D, 8.49 ± 0.11 × 10^?7 M) > erlotinib (K_D, 1.07 ± 0.02 × 10^?6 M) > canertinib (K_D, 1.41 ± 0.07 × 10^?6 M) > afatinib (K_D, 1.80 ± 0.12 × 10^?6 M) > vandetanib (K_D, 1.99 ± 0.03 × 10^?6 M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.     

Cooperation between fixed and low pH-inducible interfaces controls lipoprotein release by the LDL receptor

Low-density lipoprotein (LDL) receptors bind lipoprotein particles at the cell surface and release them in the low pH environment of the endosome. The published structure of the receptor determined at endosomal pH reveals an interdomain interface between its beta propeller and its fourth and fifth ligand binding (LA) repeats, suggesting that the receptor adopts a closed conformation at low pH to release LDL. Here, we combine lipoprotein binding and release assays with NMR spectroscopy to examine structural features of the receptor promoting release of LDL at low pH. These studies lead to a model in which the receptor uses a pH-invariant scaffold as an anchor to restrict conformational search space, combining it with flexible linkers between ligand binding repeats to interconvert between open and closed conformations. This finely tuned balance between interdomain rigidity and flexibility is likely to represent a shared structural feature in proteins of the LDL receptor family.