Sandip K. Basu

Receptor-mediated endocytosis of low-density lipoprotein in cultured cells.

Publisher Summary Studies of the cell surface binding, internalization, and metabolism of low-density lipoprotein (LDL) in cultured cells have provided useful information regarding the general aspects of receptor-mediated endocytosis. The study of the LDL receptor has been facilitated by analysis of mutant fibroblasts obtained from human subjects with disorders of cholesterol metabolism. The most informative cells, obtained from patients with familial hypercholesterolemia (FH), have defects in the gene encoding the LDL receptor. The existence of three classes of mutant alleles at the LDL receptor locus has been deduced on the basis of genetic and kinetic data. One of these alleles specifies a receptor that is unable to bind LDL. The second type of allele specifies a receptor that can bind small amounts of LDL; and the third type of allele specifies a receptor that can bind LDL but cannot be incorporated into coated pits and hence cannot carry the LDL into the cell. The first two alleles are common among FH patients, whereas the third allele is extremely rare.

Monensin Interrupts the Recycling of Low Density Lipoprotein Receptors in Human Fibroblasts

In cultured human fibroblasts, each LDL receptor mediates the internalization of approximately 100 particles of LDL every 20 hr. We provide evidence that this reutilization of LDL receptors involves the recycling of receptors into and out of the cell and that the carboxylic ionophore monensin blocks the return of the receptors to the surface. In the presence of monensin and LDL, 75% of the receptors disappeared from the cell surface within 15 min and more than 90% disappeared within 60 min. The receptors that left the surface were trapped intracellularly within perinuclear vacuoles, as visualized by indirect immunofluorescence with the use of LDL, monensin caused about 50% of the receptors to be trapped intracellularly within 15 min. The receptors that remained on the surface after monensin treatment could be trapped within the cell if LDL was added subsequently in the continued presence of monensin. Monensin did not decrease surface LDL receptors in fibroblasts from a patient (J.D.) with the internalization-defective form of familial hypercholesterolemia. In these mutant cells, LDL receptors are not localized to coated pits. The current data are interpreted to indicate that: in normal fibroblasts about 50% of surface LDL receptors absence of LDL; the remaining 50% of surface receptors can be induced to recycle by the presence of LDL; and monensin interrupts this recycling by preventing the receptor from returning to the surface, thereby causing the receptors to accumulate within the cell.

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.

Live Salmonella modulate expression of Rab proteins to persist in a specialized compartment and escape transport to lysosomes.

We investigated the intracellular route ofSalmonella in macrophages to determine a plausible mechanism for their survival in phagocytes. Western blot analysis of isolated phagosomes using specific antibodies revealed that by 5 min after internalization dead Salmonella-containing phagosomes acquire transferrin receptors (a marker for early endosomes), whereas by 30 min the dead bacteria are found in vesicles carrying the late endosomal markers cation-dependent mannose 6-phosphate receptors, Rab7 and Rab9. In contrast, liveSalmonella-containing phagosomes (LSP) retain a significant amount of Rab5 and transferrin receptor until 30 min, selectively deplete Rab7 and Rab9, and never acquire mannose 6-phosphate receptors even 90 min after internalization. Retention of Rab5 and Rab18 and selective depletion of Rab7 and Rab9 presumably enable the LSP to avoid transport to lysosomes through late endosomes. The presence of immature cathepsin D (48 kDa) and selective depletion of the vacuolar ATPase in LSP presumably contributes to the less acidic pH of LSP. In contrast, proteolytically processed cathepsin D (M r17,000) was detected by 30 min on the deadSalmonella-containing phagosomes. Morphological analysis also revealed that after uptake by macrophages, the deadSalmonella are transported to lysosomes, whereas the live bacteria persist in compartments that avoid fusion with lysosomes, indicating that live Salmonella bypass the normal endocytic route targeted to lysosomes and mature in a specialized compartment.

Stimulation of cholesteryl ester synthesis in macrophages by extracts of atherosclerotic human aortas and complexes of albumin/cholesteryl esters.

Cholesteryl ester-rich particles extracted from human atherosclerotic plaques were shown to increase the rate of incorporation of [14C]oleate into cholesteryl [14C]oleate and to cause massive accumulation of cholesteryl esters in monolayers of mouse peritoneal macrophages. This stimulation showed saturation kinetics and susceptibility to competition by polyanions (polylnosinic acid, fucoidin, dextran sulfate), suggesting that cell surface binding was required. Cellular uptake and lysosomal hydrolysis of the cholesteryl esters were also required, as Indicated by the finding that stimulation of cholesteryl ester formation was prevented by the lysosomal inhibitor, chloroqulne. The cholesterol esterlflcation-stlmulatlng activity of the aortic extracts was excluded on a 2% agarose column and floated In the density range of 1.006 to 1.063 g/ml. Cholesterol-rich extracts from human adrenal glands and liver did not stimulate cholesteryl ester formation In macrophages. The aortic extracts did not stimulate cholesteryl ester synthesis In human fibroblasts. Complexes of 126l-labeled albumin and cholesteryl llnoleate formed In vitro were taken up and degraded in macrophages, but not in fibroblasts, by a process resembling the uptake of the aortic extracts. The current data suggest that macrophages express mechanisms for internalizing certain types of cholesteryl ester-rich lipid/protein complexes, Including those present In atherosclerotic plaques.

Hemoglobin Endocytosis in Leishmania Is Mediated through a 46-kDa Protein Located in the Flagellar Pocket

Four lines of evidence indicate that a specific high affinity binding site on the surface of Leishmania donovani promastigotes mediates rapid internalization and degradation of hemoglobin. 1) Binding and uptake of125I-hemoglobin by Leishmania followed saturation kinetics and were competed by unlabeled hemoglobin but not by globin or hemin or other heme- or iron-containing proteins. 2) Immunogold labeling studies revealed that, at 4 °C, hemoglobin binding was localized in the flagellar pocket of the promastigotes. Indirect immunofluorescence assays showed that, at 37 °C, the bound hemoglobin in such cells entered an endocytic compartment within 2 min and dispersed throughout the cell body by 15 min. 3) After incubation with hemoglobin-gold conjugates at 25 °C or 37 °C, the particles accumulated in discrete intracellular vesicles. 4) A single biotinylated protein of 46 kDa was revealed when solubilized membranes from surface biotinylated intact Leishmania adsorbed by hemoglobin-agarose beads were subjected to SDS-polyacrylamide gel electrophoresis and Western blotting with avidin-horseradish peroxidase. Considered together, these data indicate that this 46-kDa protein on the cell surface of L. donovani promastigotes mediates the binding of hemoglobin and its rapid internalization through a vesicular pathway characteristic of receptor-mediated endocytosis.

Rab5-mediated endosome–endosome fusion regulates hemoglobin endocytosis in Leishmania donovani

To understand the trafficking of endocytosed hemoglobin (Hb) in Leishmania, we investigated the characteristics of in vitro fusion between endosomes containing biotinylated Hb (BHb) and avidin–horseradish peroxidase (AHRP). We showed that early endosome fusion in Leishmania is temperature and cytosol dependent and is inhibited by ATP depletion, ATPγS, GTPγS and N‐ethylmaleimide treatment. The Rab5 homolog from Leishmania donovani, LdRab5, was cloned and expressed. Our results showed that homotypic fusion between the early endosomes in Leishmania is Rab5 dependent. Early endosomes containing BHb fused efficiently with late endosomes in a process regulated by Rab7, whereas no fusion between early and late endosomes was detected using fluid phase markers. Pre‐treatment of early endosomes containing BHb with monoclonal antibody specific for the C‐terminus of the Hb receptor (HbR) or the addition of the C‐terminal cytoplasmic fragment of the HbR specifically inhibited the fusion with late endosomes, suggesting that signal(s) mediated through the HbR cytoplasmic tail promotes the fusion of early endosomes containing Hb with late endosomes.

Selective delivery of drugs to macrophages through a highly specific receptor. An efficient chemotherapeutic approach against leishmaniasis.

Methotrexate (Mtx) conjugated with maleylated bovine serum albumin (Mtx-MBSA) was taken up and degraded by cultured hamster peritoneal macrophages through the polyanion binding site for acetylated low density lipoprotein. Mtx-MBSA also eliminated intracellular amastigotes of Leishmania donovani in cultured hamster peritoneal macrophages about three times more efficiently than free Mtx. The antileishmanial effect of Mtx-MBSA on parasitized macrophages was blocked by MBSA, lysosomal inhibitors (chloroquine and monensin), and metabolic antagonists of Mtx (folic and folinic acids). The primary sites of accumulation of radioactivity of injected 125I-labeled Mtx-MBSA were the macrophage rich tissues, viz. liver and spleen. These results suggest that Mtx-MBSA would ensure rapid and effective killing of intracellular parasites harbored by macrophages. Furthermore, these results also indicate the feasibility of a general approach for rapid intracellular delivery of desired agents to macrophages for various purposes.

IL-6 and IL-12 specifically regulate the expression of Rab5 and Rab7 via distinct signaling pathways.

Recent studies have shown that phagosome maturation depends on the balance between pro‐inflammatory and anti‐inflammatory cytokines, indicating that cytokine modulates phagosome maturation. However, the mechanism of cytokine‐mediated modulation of intracellular trafficking remains to be elucidated. Here, we have shown that treatment of macrophages with IL‐6 specifically induce the expression of Rab5 through the activation of extracellular signal‐regulated kinase, whereas IL‐12 exclusively upregulate the expression of Rab7 through the activation of p38 MAPK. We have cloned the 5′‐flanking regions of the rab5c or rab7 into the promoterless reporter vector. Our results have shown that cells transfected with rab5c chimera are transactivated by IL‐6, and IL‐12 specifically transactivates cells containing rab7 chimera. Moreover, our results also show that IL‐12 induces lysosomal transport, whereas IL‐6 stimulates the fusion between early compartments in macrophages and accordingly modulates Salmonella trafficking and survival in macrophages. This is the first demonstration showing that cytokine differentially regulates endocytic trafficking by controlling the expression of appropriate Rab GTPase, and provides insight into the mechanism of cytokine‐mediated regulation of intracellular trafficking.

Leishmania requires Rab7-mediated degradation of endocytosed hemoglobin for their growth

Leishmania is unable to synthesize heme and must acquire it from exogenous source, the mechanism of which is not known. We have shown that Leishmania endocytoses hemoglobin (Hb) and subsequently degrade it probably to generate heme. To understand how internalized Hb is degraded, we have cloned and expressed Rab7 homolog from Leishmania donovani. Interestingly, Rab7 in Leishmania is found to be localized both on early and late endocytic compartment and regulates both uptake and degradation of endocytosed Hb demonstrating that Rab7 in Leishmania play a very unique role connecting both early and late events of Hb endocytosis. Our data also indicate that overexpression of Rab7:WT in Leishmania induces transport of Hb to lysosomes and rapidly degrade internalized Hb. Whereas Hb transport to lysosomes and its degradation is significantly inhibited in cells overexpressing Rab7:T21N, a GDP locked mutant of Rab7. Moreover, cells overexpressing Rab7:T21N grow at a slower rate (<50%) compared with control Leishmania. Addition of exogenous hemin recovers the growth of Rab7:T21N mutant cells almost to the control level, suggesting that intracellular heme generated by Rab7-mediated Hb degradation is required for optimal growth of the parasites. Thus, our results identify a potential target which might be exploited to suppress the growth of Leishmania.