FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2072051243> ?p ?o ?g. }

• W2072051243 endingPage "22780" @default.
• W2072051243 startingPage "22771" @default.
• W2072051243 abstract "Scavenger receptor, class B, type I (SR-BI) mediates selective uptake of high density lipoprotein (HDL) cholesteryl ester. SR-BI recognizes HDL, low density lipoprotein (LDL), exchangeable apolipoproteins, and protein-free lipid vesicles containing negatively charged phospholipids. Lipopolysaccharides (LPS) are highly glycosylated anionic phospholipids contributing to septic shock. Despite significant structural similarities between anionic phospholipids and LPS, the role of SR-BI in LPS uptake is unknown. Cla-1, the human SR-BI orthologue, was determined to be a LPS-binding protein and endocytic receptor mediating the binding and internalization of lipoprotein-free, monomerized LPS. LPS strongly competed with HDL, lipidfree apoA-I and apoA-II for HDL binding to the mouse RAW cells. Stably transfected HeLa cells expressing Cla-1-bound LPS with a Kd of about 16 μg/ml, and had a 3–4-fold increase in binding capacity and LPS uptake. Bodipy-labeled LPS uptake was found to initially accumulate in the plasma membrane and subsequently in a perinuclear region identified predominantly as the Golgi complex. Bodipy-LPS and Alexa-apoA-I had staining that colocalized on the cell surface and intracellularly indicating similar transport mechanisms. When associated with HDL, LPS uptake was increased in Cla-1 overexpressing HeLa cells by 5–10-fold. Cla-1-associated 3H-LPS uptake exceeded 125I-apolipoprotein uptake by 5-fold indicating a selective LPS uptake. Upon interacting with Cla-1 overexpressing HeLa cells, the complex (Bodipy-LPS/Alexa 488 apolipoprotein-labeled HDL) bound and was internalized as a holoparticle. Intracellularly, LPS and apolipoproteins were sorted to different intracellular compartments. With LPS-associated HDL, intracellular LPS co-localized predominantly with transferrin, indicating delivery to an endocytic recycling compartment. Our study reveals a close similarity between Cla-1-mediated selective LPS uptake and the recently described selective lipid sorting by rodent SR-BI. In summary, Cla-1 was found to bind and internalize monomerized and HDL-associated LPS, indicating that Cla-1 may play important role in septic shock by affecting LPS cellular uptake and clearance. Scavenger receptor, class B, type I (SR-BI) mediates selective uptake of high density lipoprotein (HDL) cholesteryl ester. SR-BI recognizes HDL, low density lipoprotein (LDL), exchangeable apolipoproteins, and protein-free lipid vesicles containing negatively charged phospholipids. Lipopolysaccharides (LPS) are highly glycosylated anionic phospholipids contributing to septic shock. Despite significant structural similarities between anionic phospholipids and LPS, the role of SR-BI in LPS uptake is unknown. Cla-1, the human SR-BI orthologue, was determined to be a LPS-binding protein and endocytic receptor mediating the binding and internalization of lipoprotein-free, monomerized LPS. LPS strongly competed with HDL, lipidfree apoA-I and apoA-II for HDL binding to the mouse RAW cells. Stably transfected HeLa cells expressing Cla-1-bound LPS with a Kd of about 16 μg/ml, and had a 3–4-fold increase in binding capacity and LPS uptake. Bodipy-labeled LPS uptake was found to initially accumulate in the plasma membrane and subsequently in a perinuclear region identified predominantly as the Golgi complex. Bodipy-LPS and Alexa-apoA-I had staining that colocalized on the cell surface and intracellularly indicating similar transport mechanisms. When associated with HDL, LPS uptake was increased in Cla-1 overexpressing HeLa cells by 5–10-fold. Cla-1-associated 3H-LPS uptake exceeded 125I-apolipoprotein uptake by 5-fold indicating a selective LPS uptake. Upon interacting with Cla-1 overexpressing HeLa cells, the complex (Bodipy-LPS/Alexa 488 apolipoprotein-labeled HDL) bound and was internalized as a holoparticle. Intracellularly, LPS and apolipoproteins were sorted to different intracellular compartments. With LPS-associated HDL, intracellular LPS co-localized predominantly with transferrin, indicating delivery to an endocytic recycling compartment. Our study reveals a close similarity between Cla-1-mediated selective LPS uptake and the recently described selective lipid sorting by rodent SR-BI. In summary, Cla-1 was found to bind and internalize monomerized and HDL-associated LPS, indicating that Cla-1 may play important role in septic shock by affecting LPS cellular uptake and clearance. Sepsis results from bacteria and their products entering the bloodstream and causing an overwhelming inflammatory response. Bacterial infections, as well as antibiotic therapy, cause the release of bacterial cell wall components including endotoxin (lipopolysaccharide (LPS), 1The abbreviations used are: LPS, lipopolysaccharide; IL, interleukin; HDL, high density lipoprotein; LDL, low density lipoprotein; SR-BI, scavenger receptor, class B, type I; Cla-1, CD36 and LIMPII analogous-1; DMEM, Dulbecco's modified Eagle's medium; CE, cholesterol ester; HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine. 1The abbreviations used are: LPS, lipopolysaccharide; IL, interleukin; HDL, high density lipoprotein; LDL, low density lipoprotein; SR-BI, scavenger receptor, class B, type I; Cla-1, CD36 and LIMPII analogous-1; DMEM, Dulbecco's modified Eagle's medium; CE, cholesterol ester; HBSS, Hanks' balanced salt solution; BSA, bovine serum albumin; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]glycine. lipoteichoic acid and peptidogycan (1Cohen J. McConnell J.S. Lancet. 1985; 2: 1069-1070Abstract PubMed Scopus (40) Google Scholar); see Refs. 2Periti P. Mazzei T. Int. J. Antimicrob. Agents. 1999; 12: 97-105Crossref PubMed Scopus (34) Google Scholar for review). Sepsis because of a Gram-negative bacterium is classically associated with endotoxemia, an acute phase reaction, and high mortality because of disseminated intravascular coagulation, multiple organ failure, and shock (3Burrell R. Circ. Shock. 1994; 43: 137-153PubMed Google Scholar). LPS induces a broad spectrum of biological effects associated with the activation of immune and inflammatory cells, such as macrophages, monocytes, and endothelial cells. Systemic LPS-related activation of macrophages leads to over-production of inflammatory mediators, such as leukocyte adhesion molecules, soluble cytokines and chemokines. LPS-activated phagocytes secrete tumor necrosis factor-α and IL-1β, which contributes to microcapillary damage, plasma leakage into tissue, hypotension and organ failure, the major manifestations of septic shock (for review see Refs. 4Ulevitch R.J. Immunol. Res. 2000; 21: 49-54Crossref PubMed Scopus (71) Google Scholar and 5Aderem A. Ulevitch R.J. Nature. 2000; 406: 782-787Crossref PubMed Scopus (2602) Google Scholar).The endotoxic activity of LPS, as well as its cellular uptake and metabolism, appear to be mediated by an interaction with specific cell surface receptor(s). Activation of LPS-competent cells is initiated by LPS-binding protein, which transfers LPS from the bacterial wall to membrane-associated CD14. LPS·CD14 complexes signal via Toll-like receptor 4 to activate NF-κB, as well as the c-Jun NH2-terminal kinase, and p38 mitogen-activated protein kinases (6Han J. Jiang Y. Li Z. Kravchenko V.V. Ulevitch R.J. Nature. 1997; 386: 296-299Crossref PubMed Scopus (678) Google Scholar, 7Ulevitch R.J. Tobias P.S. Annu. Rev. Immunol. 1995; 13: 437-457Crossref PubMed Scopus (1316) Google Scholar, 8Sweet M.J. Hume D.A. J. Leukocyte Biol. 1996; 60: 8-26Crossref PubMed Scopus (703) Google Scholar). The activation induces expression of genes encoding for tumor necrosis factor-α, IL-1β, IL-6, IL-8, leukocyte adhesion molecules (such as vascular cell adhesion molecular-1 and intracellular adhesion molecule-1), and chemotactic factors (such as monocyte chemoattractant protein-1), which are believed to be involved with the development and progression of septic shock.A large part of the host defense to septic shock involves the neutralization of LPS by its binding to high density lipoproteins (HDL), which ultimately results in the clearance of LPS by the liver (9Freudenberg M.A. Bog-Hansen T.C. Back U. Galanos C. Infect. Immun. 1980; 28: 373-380PubMed Google Scholar, 10Parker T.S. Levine D.M. Chang J.C. Laxer J. Coffin C.C. Rubin A.L. Infect. Immun. 1995; 63: 253-258Crossref PubMed Google Scholar). When incubated with human plasma, ∼90% of LPS is associated with lipoproteins, the majority (60%) of which are associated with HDL (11de Haas C.J. van Leeuwen H.J. Verhoef J. van Kessel K.P. van Strijp J.A. J. Immunol. Methods. 2000; 242: 79-89Crossref PubMed Scopus (43) Google Scholar, 12Levels J.H. Abraham P.R. van den E.A. van Deventer S.J. Infect. Immun. 2001; 69: 2821-2828Crossref PubMed Scopus (167) Google Scholar). A smaller portion of LPS are associated with other plasma proteins including human serum albumin, LPS-binding protein, and soluble CD14 (11de Haas C.J. van Leeuwen H.J. Verhoef J. van Kessel K.P. van Strijp J.A. J. Immunol. Methods. 2000; 242: 79-89Crossref PubMed Scopus (43) Google Scholar). This non-lipoprotein-associated LPS is highly inflammatory, and it appears to represent the major form of LPS contributing to sepsis. Infusion of HDL has been shown to significantly reduce the endotoxin-induced release of tumor necrosis factor, IL-6, and IL-8 in a murine model of endotoxemia (13Levine D.M. Parker T.S. Donnelly T.M. Walsh A. Rubin A.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 12040-12044Crossref PubMed Scopus (424) Google Scholar, 14Pajkrt D. Doran J.E. Koster F. Lerch P.G. Arnet B. van der P.T. ten Cate J.W. van Deventer S.J. J. Exp. Med. 1996; 184: 1601-1608Crossref PubMed Scopus (341) Google Scholar) and has been proposed as a potential therapy.Bacterial LPS has been demonstrated to exist in high molecular weight (up to 1000 kDa) aggregates (cell wall debris) and in a monomerized state when it forms complexes with human serum albumin, CD14, LBP, low-density lipoproteins (LDL), or HDL. Aggregated LPS has been demonstrated to be rapidly taken up by the liver, lung, and spleen, organs with large reticuloendothelial cell populations, which abundantly express scavenger receptor class A (15van Oosten M. van de B.E. van Berkel T.J. Kuiper J. Infect. Immun. 1998; 66: 5107-5112Crossref PubMed Google Scholar, 16van Oosten M. van Amersfoort E.S. van Berkel T.J. Kuiper J. J. Endotoxin Res. 2001; 7: 381-384Crossref PubMed Google Scholar). Upon intravenous injection of iodinated LPS preparations that contain both partially monomerized LPS and aggregated LPS, the uptake of aggregated LPS by the reticuloendothelial system through scavenger receptor class A masks the participation of other receptors involved with the uptake of monomerized LPS in vivo. It has been reported that infusion of iodinated LPS monomerized by association with HDL results in an altered tissue uptake in mice (17Mathison J.C. Ulevitch R.J. Am. J. Pathol. 1985; 120: 79-86PubMed Google Scholar). Of significance, the association with steroid-producing tissues, such as adrenal gland and ovary was increased. These observations raise the possibility that LPS tissue targeting may also involve an HDL receptor, such as the scavenger receptor type B class I (SR-BI), which is highly expressed in steroid producing tissues and the liver (for review see Ref. 18Trigatti B.L. Rigotti A. Braun A. Biochim. Biophys. Acta. 2000; 1529: 276-286Crossref PubMed Scopus (55) Google Scholar).SR-BI is a well characterized HDL receptor that is highly expressed in the liver and steroidogenic tissues, including the adrenal, which is often affected during endotoxemia (19Munford R.S. Andersen J.M. Dietschy J.M. J. Clin. Invest. 1981; 68: 1503-1513Crossref PubMed Scopus (68) Google Scholar). Its human orthologue, CD36 and LIMPII analogous-1 (Cla-1), has also been shown as a human receptor for high density lipoprotein and apoptotic thymocytes (20Murao K. Terpstra V. Green S.R. Kondratenko N. Steinberg D. Quehenberger O. J. Biol. Chem. 1997; 272: 17551-17557Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Despite the fact that Cla-1 has not been studied as extensively as rodent SR-BI, the physiological role of Cla-1 is generally assumed to be similar to that of rodent SR-BI. The primary function of SR-BI has been previously demonstrated to be a selective uptake of HDL-free cholesterol and cholesteryl ester without the concomitant uptake of HDL apolipoproteins, which serve as ligands for SR-BI (21Xu S. Laccotripe M. Huang X. Rigotti A. Zannis V.I. Krieger M. J. Lipid Res. 1997; 38: 1289-1298Abstract Full Text PDF PubMed Google Scholar, 22Thuahnai S.T. Lund-Katz S. Williams D.L. Phillips M.C. J. Biol. Chem. 2001; 276: 43801-43808Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). The class A amphipathic α-helices of exchangeable apolipoproteins serve as the primary recognition motif for the interaction of HDL with SR-BI (23Williams D.L. Temel R.E. Connelly M.A. Endocr. Res. 2000; 26: 639-651Crossref PubMed Scopus (22) Google Scholar, 24Schulthess G. Compassi S. Werder M. Han C.H. Phillips M.C. Hauser H. Biochemistry. 2000; 39: 12623-12631Crossref PubMed Scopus (42) Google Scholar). However, lipid composition (especially the presence of negatively charged phospholipids) impacts HDL binding with SR-BI. Moreover, phospholipid vesicles containing no apolipoproteins, only negatively charged aminophospholipids, such as phosphatidylserines and, phospholipids containing a negative charge such as phosphatidylethanolamines, as well as the phospholipid probe DiI are also effective ligands for SR-BI (22Thuahnai S.T. Lund-Katz S. Williams D.L. Phillips M.C. J. Biol. Chem. 2001; 276: 43801-43808Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 25Urban S. Zieseniss S. Werder M. Hauser H. Budzinski R. Engelmann B. J. Biol. Chem. 2000; 275: 33409-33415Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Lipid A, the most conserved portion of endotoxin, is a phosphorylated glucosamine-based phospholipid, which resembles the physicochemical properties of phospholipids containing a negative charge, and may function as an independent ligand for SR-BI in adrenal epithelial cells, macrophages, and hepatocytes, the cells that highly express SR-BI. Additionally SR-BI can be involved with the selective uptake and excretion of HDL-associated LPS in the liver, an important mechanism of LPS clearance (26Read T.E. Harris H.W. Grunfeld C. Feingold K.R. Kane J.P. Rapp J.H. Eur. Heart J. 1993; 14: 125-129PubMed Google Scholar). In this study, we examined the potential role of Cla-1 in LPS metabolism and demonstrate that Cla-1 mediates the binding, endocytosis, and the cellular accumulation of both monomerized, lipoprotein-free LPS as well as LPS associated with HDL.MATERIALS AND METHODSLipopolysaccharides, Escherichia coli B4:0111, Salmonella minnesota Re 595, diphosphoryl lipid A, and monophosphoryl lipid A were purchased from Sigma. Lipopolysaccharides from E. coli K12 strain LCD25 (unlabeled and 3H-metabolically labeled) were purchased from List Biological Laboratories. Rabbit anti-SR-BI antibody cross-reacting with the human homologue Cla-1 was from Novus Biological.Raw Cells—Mouse monocyte-macrophages, RAW 264.7 (ATCC (American Type Culture Collection) TIB 71), were grown in 12-well plates in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml) in a humidified atmosphere containing 5% CO2 and 95% air at 37 °C.Cla-1 Overexpressing HeLa Cells—HeLa (Tet-off) cells (Clontech, Palo Alto, CA) were grown in DMEM (Invitrogen), supplemented with 10% fetal calf serum, 2 mm glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 100 μg/ml G418. Cells were transfected with Fu-GENE-6 (Roche Diagnostics), using the expression plasmid pTRE2 (Clontech), encoding a Cla-1 protein (pTRE2-CLA-1). Cells were co-transfected with pTRE2-CLA-1 and pTK-Hyg (Clontech), using a 1:20 ratio, and selected with 400 μg/ml hygromycin. Hygromycin-resistant cells were screened for the expression of the CLA-1 protein by utilizing rabbit anti-SR-BI (Novus Biological, Inc.) by Western blotting.HDL, Apolipoprotein Isolation, and Labeling—Human HDL2+3 (1.072 < d < 1.216) was isolated from the plasma of healthy donors by two repetitive centrifugations by the method of Ref. 27Redgrave T.G. Roberts D.C. West C.E. Anal. Biochem. 1975; 65: 42-49Crossref PubMed Scopus (865) Google Scholar. The HDL2+3 was passed through an agarose-heparin column (HiTrap, Amersham Biosciences), and an apoE-free HDL fraction was collected. Apolipoproteins were purified from human plasma (28Remaley A.T. Stonik J.A. Demosky S.J. Neufeld E.B. Bocharov A.V. Vishnyakova T.G. Eggerman T.L. Patterson A.P. Duverger N.J. Santamarina-Fojo S. Brewer Jr., H.B. Biochem. Biophys. Res. Commun. 2001; 280: 818-823Crossref PubMed Scopus (275) Google Scholar), and were over 99% pure, as determined by SDS-PAGE and amino-terminal sequence analysis. Labeling of HDL, apoA-I, and apoA-II with Na125I was performed by the N-bromosuccinimide method according to Sinn et al. (29Sinn H.J. Schrenk H.H. Friedrich E.A. Via D.P. Dresel H.A. Anal. Biochem. 1988; 170: 186-192Crossref PubMed Scopus (78) Google Scholar). The specific radioactivities ranged from 1000 to 3000 cpm/ng of protein with more than 98% of the radioactivity being protein associated. Human HDL labeled with [3H]cholesteryl oleoyl ether (CE), a nonhydrolyzable cholesteryl ester analogue, was prepared by a modification of the procedure of Miyazaki et al. (30Miyazaki A. Sakai M. Suginohara Y. Hakamata H. Sakamoto Y. Morikawa W. Horiuchi S. J. Biol. Chem. 1994; 269: 5264-5269Abstract Full Text PDF PubMed Google Scholar). Fast protein liquid chromatography analysis demonstrated more than 95% of [3H]CE associated with the fraction corresponding to native HDL. The specific radioactivity for HDL-[3H]CE was 12–20 dpm/ng of HDL protein.Western Blot Analysis—Western blot analysis was performed, as previously described (31Bocharov A.V. Vishnyakova T.G. Baranova I.N. Patterson A.P. Eggerman T.L. Biochemistry. 2001; 40: 4407-4416Crossref PubMed Scopus (15) Google Scholar). Cell proteins were extracted with 2% Triton X-100 in Tris-buffered saline, pH 7.4. The extracts were precipitated by adding methanol to a final concentration of 90%. Precipitated proteins were dissolved in 2× SDS-PAGE sample buffer and applied on a 7.5% SDS-PAGE under reducing conditions. Anti-SR-BI antibody at a dilution of 1:1000 was used as the first antibody, and a sheep anti-rabbit IgG antibody conjugated with alkaline phosphatase (Sigma) was used as the second antibody. For protein normalization, mouse anti-human β-actin antibody at a dilution of 1:2500 was used as the first antibody, and a sheep anti-mouse IgG antibody conjugated with alkaline phosphatase (Sigma) was used as the second antibody.HDL Binding and Cholesteryl Oleoyl Uptake Assays—Saturation binding experiments were performed at 4 °C using 125I-HDL concentrations between 1.25 and 40 μg/ml. The cells were incubated with ice-cold Hanks' balanced salt solution (HBSS) containing 20 mg/ml BSA (HBSS/BSA) and labeled ligand in the presence or absence of a 20-fold excess unlabeled HDL. After a 2-h incubation on ice, specific binding was determined as previously reported (32Ulevitch R.J. Immunochemistry. 1978; 15: 157-164Crossref PubMed Scopus (63) Google Scholar).HDL-[3H]CE uptake experiments were performed in serum-free DMEM containing 0.2% BSA. Cell monolayers were incubated with various concentrations of HDL-[3H]CE in the presence (nonspecific uptake) or absence (total uptake) of a 25-fold excess of the unlabeled HDL for 20 h. Specific uptake was determined as the difference between total and nonspecific uptake.LPS Binding Assay—The lipopolysaccharide 0111:B4 (Sigma) was iodinated as reported earlier (32Ulevitch R.J. Immunochemistry. 1978; 15: 157-164Crossref PubMed Scopus (63) Google Scholar). Saturation binding experiments were performed at 4 °C using 125I-LPS concentrations between 1.25 and 40 μg/ml. All incubations were performed in HBSS containing 20 mg/ml BSA, which monomerized and formed complex with LPS. Nonspecific binding was determined in the presence of a 50-fold excess of unlabeled LPS. After a 2-h incubation on ice, the cells were rinsed with ice-cold HBSS and utilized for radioactivity measurements as reported earlier (32Ulevitch R.J. Immunochemistry. 1978; 15: 157-164Crossref PubMed Scopus (63) Google Scholar). Specific binding was determined as the difference between total and nonspecific binding, and normalized by protein content.Competition Binding Experiments—RAW cells were cultured for 24 h in serum-free DMEM before the experiment. After chilling on ice, cells were incubated in the presence of 5 μg/ml 125I-HDL, 1 μg/ml 125I-apoA-I, 1 μg/ml 125I-apoA-II, and increasing concentrations of cold ligands (HDL, apoA-I, apoA-II, and E. coli 0111:B4 LPS) for 1 h in HBSS/BSA. Cell radioactivity was measured as described under the “LPS-binding Assay.”LPS Uptake and Internalization Assays—For measurement of LPS uptake and internalization, cells were incubated in a CO2 incubator for different time periods in DMEM (20 mg/ml BSA) containing 1 μg/ml 125I-LPS in the presence or absence of 200× excess of unlabeled ligand. At specified time points, cells were chilled on ice and rinsed for three times with ice-cold PBS followed by a 20-min treatment with 0.05% trypsin, 5 mm EDTA, 150 mm NaCl solution on ice. Trypsin released radioactivity was determined as surface-bound ligand. Cell associated radioactivity counted after hydrolysis in 1 n NaOH was considered as internalized LPS. Specific binding and internalization were determined as the difference between total and nonspecific binding/internalization (the amount of radioactivity measured in the presence of ×200-fold excess of unlabeled ligand).Preparation of BODIPY-LPS and Alexa 568 HDL, Lipid-free ApoA-I and ApoA-II—HDL, apoA-I, and apoA-II were conjugated with Alexa-568/488, SE (Molecular Probes, protein labeling kit) following the kit instructions. The Alexa ligands were analyzed by 10–20% Tricine-SDS peptide gel electrophoresis. Gels were scanned using a Fluoroscan (model A, Hitachi). Alexa-labeled preparations of HDL and apolipoproteins were found in appropriate positions with molecular masses of 28 and 18 kDa for apoA-I and apoA-II, respectively (data not shown). Re-LPS was labeled using the BODIPY* FL, SE labeling kit from Molecular Probes, Inc. following the manufacturer's suggested procedure and modifications reported earlier (12Levels J.H. Abraham P.R. van den E.A. van Deventer S.J. Infect. Immun. 2001; 69: 2821-2828Crossref PubMed Scopus (167) Google Scholar).Uptake of BODIPY-LPS and Alexa 568-HDL; ApoA-I/LPS Co-localization Experiments—HeLa cells cultured on collagen-coated glass micro-slides were incubated with 5 μg/ml Alexa 568 HDL, 1–0.5 μg/ml Alexa 568 apoA-I, 1–0.5 μg/ml Alexa 568 apoA-II, or 0.5 μg/ml Bodipy-LPS for 1–2 h in a CO2 incubator in DMEM containing 20 mg/ml BSA. For quenching experiments, BODIPY-LPS (1 μg/ml) was incubated with cells for 30 min followed by being washed with ice-cold PBS and fixed with 4% paraformaldehyde. Imaging was performed in PBS containing 0.4 mg/ml trypan blue as the quenching agent. The effect of apoA-I on LPS uptake was studied by incubating HeLa cells with 0.5 μg/ml Bodipy-LPS in the presence of 100 μg/ml lipid-poor apoA-I for 1–2 h in a CO2 incubator. For co-localization, Bodipy-LPS and Alexa-568-apoA-I were used at the same concentration of 0.5 μg/ml. A Nikon video-imaging system, consisting of a phase-contrast inverted microscope equipped with a set of objectives and filters for immunofluorescence and connected to a digital camera and image processor, was used for recording Alexa 568-HDL and Bodipy-LPS uptake. For co-localization experiments, fluorescence was viewed with a Zeiss 510 laser scanning confocal microscope, using a krypton-argon-Omnichrome laser with excitation wavelengths of 488 and 568 nm for Bodipy-LPS and Alexa-568, respectively.Preparation of Bodipy-LPS/Alexa 488-Apolipoprotein-labeled HDL Complexes—Alexa 488-apolipoprotein-labeled HDL (5 mg) were mixed with Bodipy-LPS (5 μg) in a final volume of 1 ml followed by the addition of 2 ml of delipidated human plasma and incubated for 24 h at 37 °C. Bodipy-LPS/Alexa 488-apolipoprotein-labeled HDL complexes were re-isolated by a centrifugation in a NaBr gradient (1.072 < d < 1.216). After extensive dialysis against Ca2+,Mg2+-free PBS, the complexes were filtered (0.22 μm) and stored in a refrigerator up to 2 weeks. The purity of the complexes as determined by fluorescent scanning of native PAGE and agarose gel electrophoresis was close to 100%.Uptake of Bodipy-LPS/Alexa 488-Protein-labeled HDL Complexes— The surface binding of the LPS·HDL complex was studied by incubating 10 μg/ml doubly labeled HDL (Bodipy-LPS and Alexa 488-HDL) for 2 h with Cla-1 overexpressing or mock transfected HeLa cells at 4 °C and examined by confocal microscopy. Internalization of the complex was analyzed after three washings with ice-cold Ca2+,Mg2+-free PBS followed by incubation at 37 °C for a 4-h period in fresh serum-free culture medium. A separate sample of HDL (10 μg/ml) was incubated with HeLa cells at 37 °C for the 1- and 4-h periods.Preparation of 3H-LPS/HDL and LPS/125I-HDL Complexes—HDL (5 mg) were mixed with 3H-metabolically labeled LPS (150 μg, LCD25) in a final volume of 1 ml, followed by the addition of 2 ml of delipidated human plasma and incubation for 24 h at 37 °C. 30 μg of non-labeled LPS (LCD25) was incubated with 1 mg of 125I-HDL in a final volume of 200 μl, followed by the addition of 0.4 ml of delipidated human plasma and incubation for 24 h at 37 °C. Both 3H-LPS·HDL and LPS·125I-HDL complexes were re-isolated and analyzed as described above for Bodipy-LPS·Alexa HDL complexes. The specific radioactivity for HDL-3H-LPS was 12–14 dpm/ng of HDL protein.Selective LPS Uptake—The selective LPS uptake was examined by incubating 10 μg/ml LPS-labeled HDL (3H-metabolically labeled LPS) for 2 h with Cla-1 overexpressing or mock transfected HeLa cells at 37 °C in the presence or absence of a 100-fold excess of cold HDL. In a parallel experiment, the cells were incubated with 10 μg/ml LPS/125I-HDL in the presence of 100-fold excess of unlabeled HDL. Specific HDL uptake was determined as previously reported (32Ulevitch R.J. Immunochemistry. 1978; 15: 157-164Crossref PubMed Scopus (63) Google Scholar). 125I-Apolipoprotein and 3H-LPS-lipoprotein uptake is expressed in terms of apparent particle uptake. Based on the specific activity of the labeled lipoprotein preparations, the amount of lipoprotein that would be necessary to deliver the observed amount of tracer was calculated.Degradation of HDL—Degradation of HDL was determined, using the following previously reported pulse-chase scheme (35Silver D.L. Wang N. Xiao X. Tall A.R. J. Biol. Chem. 2001; 276: 25287-25293Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Briefly, cultured Cla-1 overexpressing and mock transfected HeLa cells were pulsed for1hat37 °C with radiolabeled 125I-HDL·LPS complexes. Cells were then cooled on ice and washed 3 times with binding buffer. Following the washes, cells were returned to 37 °C and chased for 2 h in binding buffer in the absence of radiolabeled lipoproteins. At the completion of the chase period, cell media was collected and trichloroacetic acid-precipitable counts were determined as a measurement of degradation (35Silver D.L. Wang N. Xiao X. Tall A.R. J. Biol. Chem. 2001; 276: 25287-25293Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Cells were also lysed, and radioactivity and protein concentration was measured.Sites of LPS Delivery—For studying the sites of LPS delivery, cells were incubated with 1 μg/ml Bodipy-LPS at 37 °C for 2 h, then washed and chased at 37 °C for 30 min in the presence of Bodipy-transferrin or Bodipy-ceramide BSA complex. In separate experiments, instead of BSA-monomerized Bodipy-LPS, the cells were incubated with 10 μg/ml HDL-bound Bodipy-LPS to determine the sites of LPS transport when associated with HDL.RESULTSCompetition of LPS with SR-BI Ligands—The competition of LPS with HDL, which is known to bind to SR-BI, was analyzed in RAW cells, which have a high level of SR-BI expression (33Baranova I. Vishnyakova T. Bocharov A. Chen Z. Remaley A.T. Stonik J. Eggerman T.L. Patterson A.P. Infect. Immun. 2002; 70: 2995-3003Crossref PubMed Scopus (122) Google Scholar). As seen at Fig. 1, LPS (E. coli B4:0111) competed with iodinated HDL in a dose-dependent manner (Fig. 1A). Various forms of LPS were also evaluated, including 0111:B4, Re 595, Rc 595, and diphosphoryl lipid A, all of which similarly decreased 125I-HDL binding (Fig. 1B) in RAW cells. Because exchangeable HDL apolipoproteins are considered as the primary ligands for HDL binding, a competition of LPS with isolated lipid-poor apolipoproteins was studied. LPS efficiently competed with 125I-apoA-I and 125I-apoA-II (Fig. 2, top and bottom). Nearly similar competition curves were observed when unlabeled apolipoproteins were used as competitors. Because the experiments were conducted on ice, which prevents the formation of a complex between LPS and lipoproteins (34Netea M.G. Demacker P.N. Kullberg B.J. Jacobs L.E. Verver-Jansen T.J. Boerman O.C. Stalenhoef A.F. Van Der Meer J.W. Cytokine. 1998; 10: 766-772Crossref PubMed Scopus (37) Google Scholar), the HDL, apolipoproteins, and LPS interacted with HDL receptor as independent ligands.Fig. 2The LPS competition for apoA-I/apoA-II-binding sites in RAW cells. Top panel, RAW cells were incubated with 1 μg/ml 125I-apoA-I in t" @default.
• W2072051243 created "2016-06-24" @default.
• W2072051243 creator A5010241534 @default.
• W2072051243 creator A5011127352 @default.
• W2072051243 creator A5027032991 @default.
• W2072051243 creator A5051629012 @default.
• W2072051243 creator A5056787326 @default.
• W2072051243 creator A5064197056 @default.
• W2072051243 creator A5068951391 @default.
• W2072051243 creator A5081642385 @default.
• W2072051243 date "2003-06-01" @default.
• W2072051243 modified "2023-10-15" @default.
• W2072051243 title "Binding and Internalization of Lipopolysaccharide by Cla-1, a Human Orthologue of Rodent Scavenger Receptor B1" @default.
• W2072051243 cites W1521655014 @default.
• W2072051243 cites W1822061653 @default.
• W2072051243 cites W1827361146 @default.
• W2072051243 cites W1833727215 @default.
• W2072051243 cites W1863056418 @default.
• W2072051243 cites W1930598094 @default.
• W2072051243 cites W1956848161 @default.
• W2072051243 cites W1978075735 @default.
• W2072051243 cites W1979300491 @default.
• W2072051243 cites W1983125054 @default.
• W2072051243 cites W1986439388 @default.
• W2072051243 cites W1988397920 @default.
• W2072051243 cites W1997678048 @default.
• W2072051243 cites W2000531499 @default.
• W2072051243 cites W2017120858 @default.
• W2072051243 cites W2020361370 @default.
• W2072051243 cites W2028653252 @default.
• W2072051243 cites W2030198124 @default.
• W2072051243 cites W2035634737 @default.
• W2072051243 cites W2043225342 @default.
• W2072051243 cites W2044343704 @default.
• W2072051243 cites W2057392671 @default.
• W2072051243 cites W2062201648 @default.
• W2072051243 cites W2071465162 @default.
• W2072051243 cites W2072506326 @default.
• W2072051243 cites W2075128127 @default.
• W2072051243 cites W2076490603 @default.
• W2072051243 cites W2079460307 @default.
• W2072051243 cites W2082943173 @default.
• W2072051243 cites W2084676774 @default.
• W2072051243 cites W2086525709 @default.
• W2072051243 cites W2092974710 @default.
• W2072051243 cites W2093099491 @default.
• W2072051243 cites W2094162221 @default.
• W2072051243 cites W2094858554 @default.
• W2072051243 cites W2096137537 @default.
• W2072051243 cites W2113828544 @default.
• W2072051243 cites W2121080720 @default.
• W2072051243 cites W2126359905 @default.
• W2072051243 cites W2128046304 @default.
• W2072051243 cites W2136707054 @default.
• W2072051243 cites W2151817114 @default.
• W2072051243 cites W2154167892 @default.
• W2072051243 cites W2158092179 @default.
• W2072051243 cites W2164724187 @default.
• W2072051243 cites W2166648967 @default.
• W2072051243 cites W2306393560 @default.
• W2072051243 cites W2327114865 @default.
• W2072051243 doi "https://doi.org/10.1074/jbc.m211032200" @default.
• W2072051243 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/12651854" @default.
• W2072051243 hasPublicationYear "2003" @default.
• W2072051243 type Work @default.
• W2072051243 sameAs 2072051243 @default.
• W2072051243 citedByCount "134" @default.
• W2072051243 countsByYear W20720512432012 @default.
• W2072051243 countsByYear W20720512432013 @default.
• W2072051243 countsByYear W20720512432014 @default.
• W2072051243 countsByYear W20720512432015 @default.
• W2072051243 countsByYear W20720512432016 @default.
• W2072051243 countsByYear W20720512432017 @default.
• W2072051243 countsByYear W20720512432018 @default.
• W2072051243 countsByYear W20720512432019 @default.
• W2072051243 countsByYear W20720512432020 @default.
• W2072051243 countsByYear W20720512432021 @default.
• W2072051243 countsByYear W20720512432022 @default.
• W2072051243 countsByYear W20720512432023 @default.
• W2072051243 crossrefType "journal-article" @default.
• W2072051243 hasAuthorship W2072051243A5010241534 @default.
• W2072051243 hasAuthorship W2072051243A5011127352 @default.
• W2072051243 hasAuthorship W2072051243A5027032991 @default.
• W2072051243 hasAuthorship W2072051243A5051629012 @default.
• W2072051243 hasAuthorship W2072051243A5056787326 @default.
• W2072051243 hasAuthorship W2072051243A5064197056 @default.
• W2072051243 hasAuthorship W2072051243A5068951391 @default.
• W2072051243 hasAuthorship W2072051243A5081642385 @default.
• W2072051243 hasBestOaLocation W20720512431 @default.
• W2072051243 hasConcept C139066938 @default.
• W2072051243 hasConcept C139770010 @default.
• W2072051243 hasConcept C170493617 @default.
• W2072051243 hasConcept C185592680 @default.
• W2072051243 hasConcept C18903297 @default.
• W2072051243 hasConcept C203014093 @default.
• W2072051243 hasConcept C2778163477 @default.
• W2072051243 hasConcept C2778754761 @default.
• W2072051243 hasConcept C2778914748 @default.

• next