Denaturation and proteolytic digestion of porcine low-density lipoprotein in aqueous guanidine hydrochloride solutions.

The denaturation of porcine low-density lipoprotein (LDL) in aqueous guanidine hydrochloride (GuHCl) was studied by flotation velocity experiments, optical rotatory dispersion and fluorescence spectroscopy. The denaturation of LDL occurred between 2 and 4M GuGCl, where small sigmoidal changes in iptical rotation and fluorescence intensity were noted. The hydrated density of the native LDL was 1.036g/cm-3 and this remained constant upon denaturation in 4M GuHCl. The slope of the flotation coefficient-solvent density curve was 35 per cent less for denatured LDL than for the native LDL. Since there is no indication of splitting of LDL in 4M GuHCl, it is natural to interpret the result in terms of an increase of the translational frictional coefficient by 50 per cent. The observed changes in optical rotation, fluorescence intensity and flotation coefficient in 4M GuHCl were readily reversed and native LDL was recovered after removal of GuHCl by dialysis. Proteolytic treatment of denatured LDL produced digested LDL which had a hydrated density of 1.021g/cm-3 corresponding to the loss of 30 per cent of apo-LDL. The digested LDL behaved like a compact, globular particle in aqueous NaCl solution and in 4M GuHCl. These results can best be interpreted by a model of the LDL particle in which approximately 30 per cent of apo-LDL is exposed to the solvent, such that it can be reversibly denatured by GuHCl and at the same time is easily avalable to proteolytic enzymes, whereas the rest of apo-LDL is tightly associated with lipids and possibly buried inside the lipid moiety. SDS-polyacrylamide gel electrophoresis of the digested LDL revealed four major peptide fragments with sizes ranging from 70,000 to 100,000 daltons. We believe that the method and results described in this paper will have meaningful applications in the study of membrane proteins.     

Studies on pig serum lipoproteins. I. Separation and properties of low-density lipoproteins.

The low-density lipoproteins in pig serum were separated into two subclasses (LDL1 and LDL2) by 2 to 7% pore size gradient gel electrophoresis. Preparative gel electrophoresis in 2 to 4% gradient gel made it possible to isolate these components as distinct entities. After delipidation by chromatography on Sepharose 4B in the presence of SDS, both apo-LDL1 and apo-LDL2 were found to have a molecular weight of 2.6X10(5). However, when these apoproteins were incubated in 10% sodium dodecyl sulfate, fragmentation occurred and the minimum fragment molecular weight was estimated to be 2.4X10(4). No essential difference was found in the amino acid compositions or fragmentation patterns of the apoproteins. However, the amounts of carbohydrates in the two apoproteins were different (7.09% in apo-LDL1 and 5.08% in apo-LDL2). The carbohydrate composition was 0.8% sialic acid, 2.38% N-acetyl-glucosamine, and 4.01% neutral sugars in apo-LDL1 and 0.5, 1.75, and 2.83% in apo-LDL2, respectively. In both apoproteins, mannose, galactose, and fucose were present in almost the same molar ratio of 4-5 : 2-3 : 1.     

Properties of human serum low density lipoproteins after modification by succinic anhydride

Human serum low density lipoprotein of d 1.019-1.063 (LDL(2)) treated with succinic anhydride at pH 7.5-8.0 showed the same chemical composition, hydrodynamic properties (flotation and sedimentation coefficients, intrinsic viscosity) and optical properties (circular dichroism) as untreated LDL(2). However, in contrast to LDL(2), the succinylated product (s-LDL(2)) failed to react with rabbit anti-LDL(2) antisera. Extraction with ethanol-ether 3:1 yielded the succinylated apoprotein (s-apo-LDL(2)), which was, unlike untreated apoprotein, soluble in aqueous buffers. Succinylated apoprotein, which was also immunologically unreactive, appeared to differ in structure from s-LDL(2), as assessed by the parameters of intrinsic viscosity and circular dichroism. The molecular weights of both LDL(2) and s-LDL(2) obtained by the technique of sedimentation equilibrium were 2.1-2.3 x 10(6). By the same method, s-apo-LDL(2) gave an uncorrected figure of 3.95-4.15 x 10(4) and, after correction for succinyl functions, of 3.60-3.80 x 10(4). Because of the assumptions made in the computations, the latter figure was considered approximate. The marked differences in molecular weight between s-apo-LDL(2) and whole apo-LDL(2) ( approximately 5 x 10(5)) were taken to support the subunit structure of apo-LDL(2), which is envisaged as an aggregate of about 12 subunits which dissociate upon succinylation. Further, the large percentage (about 90%) of the free amino groups of LDL(2) found to react with succinic anhydride suggests that these groups are at the surface of the molecule.     

Folic acid-capped PEGylated magnetic nanoparticles enter cancer cells mostly via clathrin-dependent endocytosis

BackgroundThis work is focused on mechanisms of uptake in cancer cells of rationally designed, covalently assembled nanoparticles, made of superparamagnetic iron oxide nanoparticles (SPIONs), fluorophores (doxorubicin or Nile Blue), polyethylene glycol (PEG) and folic acid (FA), referred hereinafter as SFP-FA.MethodsSFP-FA were characterized by DLS, zetametry and fluorescence spectroscopy. The SFP-FA uptake in cancer cells was monitored using fluorescence-based methods like fluorescence-assisted cell sorting, CLSM with single-photon and two-photon excitation. The SFP-FA endocytosis was also analyzed with electron microscopy approaches: TEM, HAADF-STEM and EELS.ResultsThe SFP-FA have zeta potential below − 6 mW and stable hydrodynamic diameter close to 100 nm in aqueous suspensions of pH range from 5 to 8. They contain ca. 109 PEG-FA, 480 PEG-OCH₃ and 22–27 fluorophore molecules per SPION. The fluorophores protected under the PEG shell allows a reliable detection of intracellular NPs. SFP-FA readily enter into all the cancer cell lines studied and accumulate in lysosomes, mostly via clathrin-dependent endocytosis, whatever the FR status on the cells.ConclusionsThe present study highlights the advantages of rational design of nanosystems as well as the possible involvement of direct molecular interactions of PEG and FA with cellular membranes, not limited to FA-FR recognition, in the mechanisms of their endocytosis.General significanceComposition, magnetic and optical properties of the SFP-FA as well their ability to enter cancer cells are promising for their applications in cancer theranosis. Combination of complementary analytical approaches is relevant to understand the nanoparticles behavior in suspension and in contact with cells.     

Stepwise degradation of serum low denisty lipoprotein by sodium dodecyl sulfate.

The structure of human serum low density lipoprotein (LDL) was investigated by perturbing the LDL structure with sodium dodecyl sulfate (SDS). The change in LDL structure induced by the addition of SDS was monitored by sedimentation velocity measurements, ultraviolet difference spectroscopy, fluorescence spectroscopy and proteolytic digestion of apo-LDL with subtilisin BPN' [EC 3.4.21.14]. As the concentration of SDS was increased from 0.1 mg/ml to 3 mg/ml with LDL concentrations between 2.0 mg/ml and 4.4 mg/ml, the sedimentation coefficient of LDL changed in three distinct steps. It was found by chemical analyses that not more than 30% of the total lipid was lost from LDL in the second step, whereas the final step in the change of sedimentation coefficient corresponded to the complete removal of apo-LDL from the constituent lipids of LDL. The ultraviolet difference spectrum between the native and SDS-treated LDL and the quenching of LDL fluorescence underwent about 80% of the total change while the SDS concentration was only sufficient to cause the second of the three step changes in sedimentation coefficient. SDS-polyacrylamide gel electrophoresis of apo-LDL treated with subtilisin BPN' also showed that more than 70% of apo-LDL became susceptible to proteolysis under the same conditions. These results were interpreted as indicating that the solubilization of 20 to 30% of the lipids on the surface of LDL exposed nearly 80% or more of apo-LDL to the solvent. A small portion of apo-LDL was, however, still firmly anchored to the remaining lipid micelle as long as the concentration of SDS was less than that required to cause the final step of the change in sedimentation coefficient.     

Synthesis and biosensor performance of a near-IR thiol-reactive fluorophore based on benzothiazolium squaraine

Environmentally sensitive near-IR (NIR) dyes are useful fluorophores for various biosensor applications when tissue absorption, scattering, and autofluorescence are a leading concern. Biosensors operating in the NIR region (generally wavelengths >650 nm) would avoid interference from biological media and thereby facilitate relatively interference free sensing. Squaraine dyes are potential candidates to serve as reporter molecules due to their spectral properties in the NIR region, but none is commercially available for site-specific coupling to proteins through native or engineered thiols on cysteine. In this context, we have synthesized a thiol-reactive squaraine that displays fluorescence emission above 650 nm and have coupled the dye site-specifically to various mutants of glucose/galactose binding protein that contained an engineered cysteine for attachment. Mutant E149C/A213R/L238S ISQ GGBP gave a fluorescence change of +50% and a binding constant of 12 mM, which is in the human physiological range for glucose.     

Effects of cholesterol and lipoproteins on endocytosis by a monocyte-like cell line

The human monocyte/macrophage-like cell line U937 is a cholesterol auxotroph. Incubation of these cells in the growth medium in which delipidated fetal calf serum has been substituted for fetal calf serum depletes cellular cholesterol and inhibits growth. The cholesterol requirement of these cells for growth can be satisfied by human low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL), but not by high-density lipoprotein (HDL). U937 cells can bind and degrade LDL via a high-affinity site and this recognition is altered by acetylation of LDL. This indicates that these cells express relatively high LDL receptor activity and low levels of the acetyl-LDL receptor. The cells were used to study the role of cholesterol in lectin-mediated and fluid-phase endocytosis. Growth of the cells in the medium containing delipidated fetal calf serum results in impairment of both concanavalin A-mediated endocytosis of horseradish peroxidase and concanavalin A-independent endocytosis of Lucifer Yellow. Supplementation of the medium with cholesterol prevents cellular cholesterol depletion, supports growth and stimulates Lucifer Yellow endocytosis but fails to restore horseradish peroxidase endocytosis. However, if the cells are incubated in the presence of no less than 40 μg LDL protein/ml to maintain normal cell cholesterol levels, concanavalin A-mediated endocytosis of horseradish peroxidase is activated. The effect of LDL is specific since neither VLDL nor HDL₃ at the same protein concentration activates horseradish peroxidase uptake by the cells. Furthermore, the activation of endocytosis by LDL is not inhibited by the inclusion of heparin or acetylation of the LDL indicating that binding of LDL to the LDL receptor is not required for these effects. The mediation of activation of horseradish peroxidase endocytosis by the lectin is presumed to involve binding of LDL to concanavalin A associated with the cell surface which in turn stimulates horseradish peroxidase binding and uptake by adsorptive endocytosis. The rate of fluid endocytosis and endosome formation seems to depend on cellular cholesterol content presumably because cholesterol is involved in maintaining the appropriate plasma membrane structure and fluidity.     

Receptor-mediated endocytosis and intracellular trafficking of lipoproteins and transferrin in insect cells

While the intracellular pathways of ligands after receptor-mediated endocytosis have been studied extensively in mammalian cells, in insect cells these pathways are largely unknown. We transfected Drosophila Schneider line 2 (S2) cells with the human low-density lipoprotein (LDL) receptor (LDLR) and transferrin (Tf) receptor (TfR), and used endocytosis of LDL and Tf as markers. After endocytosis in mammalian cells, LDL is degraded in lysosomes, whereas Tf is recycled. Fluorescence microscopy analysis revealed that LDL and Tf are internalized by S2 cells transfected with LDLR or TfR, respectively. In transfectants simultaneously expressing LDLR and TfR, both ligands colocalize in endosomes immediately after endocytic uptake, and their location remained unchanged after a chase. Similar results were obtained with Spodoptera frugiperda Sf9 cells that were transfected with TfR, suggesting that Tf is retained intracellularly by both cell lines. The insect lipoprotein, lipophorin, is recycled upon lipophorin receptor (LpR)-mediated endocytosis by mammalian cells, however, not after endocytosis by LpR-expressing S2 transfectants, suggesting that this recycling mechanism is cell-type specific. LpR is endogenously expressed by fat body tissue of Locusta migratoria for a limited period after an ecdysis. A chase following endocytosis of labeled lipophorin by isolated fat body tissue at this developmental stage resulted in a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand.     

Macropinocytosis is the endocytic pathway that mediates macrophage foam cell formation with native low density lipoprotein

Previously, we reported that fluid-phase endocytosis of native LDL by PMA-activated human monocytederived macrophages converted these macrophages into cholesterol-enriched foam cells (Kruth, H. S., Huang, W., Ishii, I., and Zhang, W. Y. (2002) J. Biol. Chem. 277, 34573-34580). Uptake of fluid by cells can occur either by micropinocytosis within vesicles (<0.1 microm diameter) or by macropinocytosis within vacuoles ( approximately 0.5-5.0 microm) named macropinosomes. The current investigation has identified macropinocytosis as the pathway for fluid-phase LDL endocytosis and determined signaling and cytoskeletal components involved in this LDL endocytosis. The phosphatidylinositol 3-kinase inhibitor, LY294002, which inhibits macropinocytosis but does not inhibit micropinocytosis, completely blocked PMA-activated macrophage uptake of fluid and LDL. Also, nystatin and filipin, inhibitors of micropinocytosis from lipid-raft plasma membrane domains, both failed to inhibit PMA-stimulated macrophage cholesterol accumulation. Time-lapse video phase-contrast microscopy and time-lapse digital confocal-fluorescence microscopy with fluorescent DiI-LDL showed that PMA-activated macrophages took up LDL in the fluid phase by macropinocytosis. Macropinocytosis of LDL depended on Rho GTPase signaling, actin, and microtubules. Bafilomycin A1, the vacuolar H+-ATPase inhibitor, inhibited degradation of LDL and caused accumulation of undegraded LDL within macropinosomes and multivesicular body endosomes. LDL in multivesicular body endosomes was concentrated >40-fold over its concentration in the culture medium consistent with macropinosome shrinkage by maturation into multivesicular body endosomes. Macropinocytosis of LDL taken up in the fluid phase without receptor-mediated binding of LDL is a novel endocytic pathway that generates macrophage foam cells. Macropinocytosis in macrophages and possibly other vascular cells is a new pathway to target for modulating foam cell formation in atherosclerosis.     

The Cbl-interacting protein TULA inhibits dynamin-dependent endocytosis

The Cbl- and ubiquitin-interacting protein T-cell ubiquitin ligand (TULA) has been demonstrated to inhibit endocytosis and downregulation of ligand-activated EGF receptor (EGFR) by impairing Cbl-induced ubiquitination. We presently report that TULA additionally inhibited clathrin-dependent endocytosis in general, as both uptake of transferrin (Tf) and low-density lipoprotein (LDL) was inhibited. Additionally, endocytosis of the raft proteins CD59 and major histocompatibility complex class I (MHC-I), which we demonstrate were mainly endocytosed clathrin-independently, but dynamin-dependently, was blocked in cells overexpressing TULA. By contrast, the uptake of ricin, which is mainly endocytosed clathrin- and dynamin-independently, was not affected by overexpressed TULA. Consistently, TULA and dynamin co-immunoprecipitated and colocalized intracellularly, and upon overexpression of dynamin the TULA-mediated inhibitory effect on endocytosis of Tf, LDL, CD59 and MHC-I was counteracted. Overexpressed dynamin did not restore ubiquitination of the EGFR, and consistently dynamin did not rescue endocytosis of the EGFR in cells overexpressing TULA. We conclude that TULA inhibits both clathrin-dependent and clathrin-independent endocytic pathways by functionally sequestering dynamin via the SH3 domain of TULA binding proline-rich sequences in dynamin.     

The ins and outs of pertussis toxin

Pertussis toxin, produced and secreted by the whooping cough agent Bordetella pertussis, is one of the most complex soluble bacterial proteins. It is actively secreted through the B. pertussis cell envelope by the Ptl secretion system, a member of the widespread type IV secretion systems. The toxin is composed of five subunits (named S1 to S5 according to their decreasing molecular weights) arranged in an A-B structure. The A protomer is composed of the enzymatically active S1 subunit, which catalyzes ADP-ribosylation of the α subunit of trimeric G proteins, thereby disturbing the metabolic functions of the target cells, leading to a variety of biological activities. The B oligomer is composed of 1S2:1S3:2S4:1S5 and is responsible for binding of the toxin to the target cell receptors and for intracellular trafficking via receptor-mediated endocytosis and retrograde transport. The toxin is one of the most important virulence factors of B. pertussis and is a component of all current vaccines against whooping cough.     

Visualization of the binding, endocytosis, and transcytosis of low- density lipoprotein in the arterial endothelium in situ

We investigated the interaction and transport of low-density lipoprotein (LDL) through the arterial endothelium in rat aorta and coronary artery, by perfusing in situ native, untagged human, and rat LDL. The latter was rendered electron-opaque after it interacted with the endothelial cell and was subsequently fixed within tissue. We achieved LDL electron-opacity by an improved fixation procedure using 3,3'-diaminobenzidine, and mordanting with tannic acid. The unequivocal identification of LDL was implemented by reacting immunocytochemically the perfused LDL with anti LDL-horseradish peroxidase conjugate. Results indicate that LDL is taken up and internalized through two parallel compartmented routes. (a) A relatively small amount of LDL is taken up by endocytosis via: (i) a receptor-mediated process (adsorptive endocytosis) that involved coated pits/vesicles, and endosomes, and, probably, (ii) a receptor-independent process (fluid endocytosis) carried out by a fraction of plasmalemmal vesicles. Both mechanisms bringing LDL to lysosomes supply cholesterol to the endothelial cell itself. (b) Most circulating LDL is transported across the endothelial cell by transcytosis via plasmalemmal vesicles which deliver LDL to the other cells of the vessel wall. Endocytosis is not enhanced by increasing LDL concentration, but the receptor-mediated internalization decreases at low temperature. Transcytosis is less modified by low temperature but is remarkably augmented at high concentration of LDL. While the endocytosis of homologous (rat) LDL is markedly more pronounced than that of heterologous (human) LDL, both types of LDL are similarly transported by transcytosis. These results indicate that the arterial endothelium possesses a dual mechanism for handling circulating LDL: by a high affinity process, endocytosis secures the endothelial cells' need for cholesterol; by a low-affinity nonsaturable uptake process, transcytosis supplies cholesterol to the other cells of the vascular wall, and can monitor an excessive accumulation of plasma LDL. Since in most of our experiments we used LDL concentrations above those found in normal rats, we presume that at low LDL concentrations saturable high-affinity uptake would be enhanced in relation to nonsaturable pathways.     

The apolipoprotein E-dependent low density lipoprotein cholesteryl ester selective uptake pathway in murine adrenocortical cells involves chondroitin sulfate proteoglycans and an alpha 2-macroglobulin receptor

Cells acquire lipoprotein cholesterol by receptor-mediated endocytosis and selective uptake pathways. In the latter case, lipoprotein cholesteryl ester (CE) is transferred to the plasma membrane without endocytosis and degradation of the lipoprotein particle. Previous studies with Y1/E/tet/2/3 murine adrenocortical cells that were engineered to express apolipoprotein (apo) E demonstrated that apoE expression enhances low density lipoprotein (LDL) CE uptake by both selective and endocytic pathways. The present experiments test the hypothesis that apoE-dependent LDL CE selective uptake is mediated by scavenger receptor, class B, type I (SR-BI). Surprisingly, SR-BI expression was not detected in the Y1/E/tet/2/3 clone of Y1 adrenocortical cells, indicating the presence of a distinct apoE-dependent pathway for LDL CE selective uptake. ApoE-dependent LDL CE selective uptake in Y1/E/tet/2/3 cells was inhibited by receptor-associated protein and by activated alpha(2)-macroglobulin (alpha(2)M), suggesting the participation of the LDL receptor-related protein/alpha(2)M receptor. Reagents that inhibited proteoglycan synthesis or removed cell surface chondroitin sulfate proteoglycan completely blocked apoE-dependent LDL CE selective uptake. None of these reagents inhibited SR-BI-mediated LDL CE selective uptake in the Y1-BS1 clone of Y1 cells in which LDL CE selective uptake is mediated by SR-BI. We conclude that LDL CE selective uptake in adrenocortical cells occurs via SR-BI-independent and SR-BI-dependent pathways. The SR-BI-independent pathway is an apoE-dependent process that involves both chondroitin sulfate proteoglycans and an alpha(2)M receptor.     

Low density lipoprotein turnover in swine.

The catabolism of intravenously injected 125I-labelled low density lipoproteins (LDL) was followed in normal miniature swine for 2 weeks. When compared with the two-exponential model, the decay curve of the plasma radioactivity associated with the LDL fraction was best described by a three-exponential model. In this system, the half-lives were 4.5 +/- 3.7, 19.7 +/- 6.6, and 127 +/- 70 h (mean of four studies). Assuming a kinetic model with metabolism of LDL in the rapidly equilibrating compartment and two slower equilibrating compartments (a model requiring three exponentials), the mean fractional catabolic rate for apo-LDL was calculated to be 0.015 h-1. Therefore, if at steady state, the synthetic rate for apo-LDL in the same pigs would be 5.6 +/- 4.1 mg/h. Different kinetic models using two or three exponentials would provide different values for the synthetic rate of apo-LDL. However, in view of the known existence of at least three major equilibrating pools for LDL in plasma, liver, and lymph, and in view of the present results, the kinetic model for LDL metabolism should be better represented by a three-exponential system.     

Fluorescence studies of macrophage recognition and endocytosis of native and acetylated low-density lipoprotein

Macrophage recognition and endocytosis of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labeled low-density lipoprotein (LDL) and acetyl LDL (Ac-LDL) was studied using fluorescence flow cytometry and fluorescence video intensification microscopy. RAW264 macrophages and U937 monocytes were grown in the tissue culture media in the presence and absence of LDL and Ac-LDL. Several lines of evidence indicate that receptor-mediated endocytosis of diI-labeled LDL or Ac-LDL was taking place. Binding can be distinguished from binding plus endocytosis by incubation at 4 and 37 degrees C, respectively. Binding was saturable at 4 degrees C and uptake at 37 degrees C was time- and ligand dose-dependent. Also, unlabeled LDL and Ac-LDL compete for their receptors. Macrophages grown in the presence or absence of LDL demonstrated distinct labeling patterns. LDL receptors were significantly increased by culture in defined medium without serum lipoproteins, while Ac-LDL receptors remained unaffected. Flow cytometry can provide an important tool to examine receptor levels, modulation of these levels and receptor-mediated endocytosis. Video intensification microscopy of similarly labeled cells has been performed. Receptors appear as punctate fluorescence, usually distributed randomly across the cell surface.     

Hyaluronic acid-polyethyleneimine conjugate for target specific intracellular delivery of siRNA

A novel target specific small interfering RNA (siRNA) delivery system was successfully developed using polyethyleneimine (PEI)-hyaluronic acid (HA) conjugate. Anti-PGL3-Luc siRNA was used as a model system suppressing the PGL3-Luc gene expression. The siRNA/PEI-HA complex with an average size of ca. 21 nm appeared to be formed by electrostatic interaction between the negatively charged siRNA and the positively charged PEI of PEI-HA conjugate. The cytotoxicity of siRNA/PEI-HA complex to B16F1 cells was lower than that of siRNA/PEI complex according to the MTT assay. When B16F1 and HEK-293 cells were treated with fluorescein isothiocyanate (FITC) labeled siRNA/PEI-HA complex, B16F1 cells, with a lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), showed higher green fluorescent intensity than HEK-293 cells because of the HA receptor mediated endocytosis of the complex. Accordingly, the PGL3-Luc gene silencing of anti-PGL3-Luc siRNA/PEI-HA complex was more efficient in B16F1 cells than in HEK-293 cells. In addition, the inhibited PGL3-Luc gene silencing effect in the presence of free HA in the transfection medium revealed that siRNA/HA-PEI complex was selectively taken up to B16F1 cells via HA receptor mediated endocytosis. All these results demonstrated that the intracellular delivery of anti-PGL3-Luc siRNA/PEI-HA complex could be facilitated by the HA receptor mediated endocytosis.     

Studies on the surface structure of the intracytoplasmic membrane in the photosynthetic purple bacterium Chromatium vinosum by means of chemical modification

The chemical and physical properties of bullfrog serum low density lipoprotein (LDL) were investigated. On a weight percentage basis, LDL contained cholesterol ester, 30.3%; cholesterol, 5.6%; triglyceride, 12.5%; phospholipids, 23.3%; and protein, 22.4%. The fatty acid compositions of triglycerides and major phospholipids from the bullfrog serum LDL were quite similar to those of human serum LDL. However, the fatty acid composition of the chlesterol ester from the bullfrog serum LDL was quite different from that of the human serum LDL. The average particle weight, determined by gel filtration, was 2 X 10(6) daltons. This value is very close to that of human LDL. In the fluorescence emission spectrum of bullfrog serum LDL, the emission maximum was 324 nm. The amino acid composition of the apo-LDL resembled that of human apo-LDL.     

Inducible macropinocytosis of hyaluronan in B16-F10 melanoma cells

Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Endocytosis is thought to play an essential role in the catabolism of HA due to the intracellular compartmentalization of the HA degrading hyaluronidase enzymes. Previous investigations have shown that keratinocytes, chondrocytes and breast tumor cell lines endocytose HA via the cell surface glycoprotein, CD44. However, other cell types endocytose HA using a CD44-independent mechanism that remains to be defined. The purpose of this study was to investigate HA endocytosis in B16-F10 melanoma cells. We found that B16-F10 melanoma cells expressed CD44 on their surfaces. Unexpectedly, CD44 did not play a role in the endocytosis of HA. Electron microscopy studies revealed that B16-F10 melanoma cells exhibited membrane ruffling, a characteristic feature of macropinocytosis, only after incubating the cells with the HA co-polymer. Moreover, B16-F10 melanoma cells endocytosed HA via macropinocytosis as assessed by drug inhibition studies and the co-localization of fluorescently labeled HA with fluorescent tracers under confocal microscopy. Based on these results, we conclude that induced macropinocytosis may provide a previously unrecognized avenue for HA endocytosis in some cell types.     

The adaptor protein beta-arrestin2 enhances endocytosis of the low density lipoprotein receptor

Endocytosis of the low density lipoprotein (LDL) receptor (LDLR) in coated pits employs the clathrin adaptor protein ARH. Similarly, agonist-dependent endocytosis of heptahelical receptors in coated pits employs the clathrin adaptor beta-arrestin proteins. In mice fed a high fat diet, we found that homozygous deficiency of beta-arrestin2 increased total and LDL plus intermediate-density lipoprotein cholesterol levels by 23 and 53%, respectively (p < 0.05), but had no effect on high density lipoprotein cholesterol levels. We therefore tested whether beta-arrestins could affect the constitutive endocytosis of the LDLR. When overexpressed in cells, beta-arrestin1 and beta-arrestin2 each associated with the LDLR, as judged by co-immunoprecipitation, and augmented LDLR endocytosis by approximately 70%, as judged by uptake of fluorescent LDL. However, physiologic expression levels of only beta-arrestin2, and not beta-arrestin1, enhanced endogenous LDLR endocytosis (by 65%) in stably transfected beta-arrestin1/beta-arrestin2 double-knockout mouse embryonic fibroblasts (MEFs). Concordantly, when RNA interference was used to suppress expression of beta-arrestin2, but not beta-arrestin1, LDLR endocytosis was reduced. Moreover, beta-arrestin2-/- MEFs demonstrated LDLR endocytosis that was 50% less than cognate wild type MEFs. In fusion protein pull-down assays, beta-arrestin2 bound to the LDLR cytoplasmic tail stoichiometrically, and binding was abolished by mutation of LDLR Tyr807 to Ala. Mutation of LDLR cytoplasmic tail Ser833 to Asp enhanced both the affinity of LDLR fusion protein binding to beta-arrestin2, and the efficiency of LDLR endocytosis in cells expressing beta-arrestin2 physiologically. We conclude that beta-arrestin2 can bind to and enhance endocytosis of the LDLR, both in vitro and in vivo, and may thereby influence lipoprotein metabolism.     

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.

The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis.