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• W2029360079 abstract "African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and phospholipids to trypanosomes. We show evidence for the existence of a trypanosome lipoprotein scavenger receptor, which facilitates the endocytosis of both native and modified lipoproteins, including HDL and LDL. This lipoprotein scavenger receptor also exhibits selective lipid uptake, whereby the uptake of the lipid components of the lipoprotein exceeds that of the protein components. Trypanosome lytic factor (TLF1), an unusual HDL found in human serum that protects from infection by lysing Trypanosoma brucei brucei, is also bound and endocytosed by this lipoprotein scavenger receptor. HDL and LDL compete for the binding and uptake of TLF1 and thereby attenuate the trypanosome lysis mediated by TLF1. We also show that a mammalian scavenger receptor facilitates lipid uptake from TLF1 in a manner similar to the trypanosome scavenger receptor. Based on these results we propose that HDL, LDL, and TLF1 are all bound and taken up by a lipoprotein scavenger receptor, which may constitute the parasite's major pathway mediating the uptake of essential lipids. African trypanosomes are lipid auxotrophs that live in the bloodstream of their human and animal hosts. Trypanosomes require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions. Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol, and phospholipids to trypanosomes. We show evidence for the existence of a trypanosome lipoprotein scavenger receptor, which facilitates the endocytosis of both native and modified lipoproteins, including HDL and LDL. This lipoprotein scavenger receptor also exhibits selective lipid uptake, whereby the uptake of the lipid components of the lipoprotein exceeds that of the protein components. Trypanosome lytic factor (TLF1), an unusual HDL found in human serum that protects from infection by lysing Trypanosoma brucei brucei, is also bound and endocytosed by this lipoprotein scavenger receptor. HDL and LDL compete for the binding and uptake of TLF1 and thereby attenuate the trypanosome lysis mediated by TLF1. We also show that a mammalian scavenger receptor facilitates lipid uptake from TLF1 in a manner similar to the trypanosome scavenger receptor. Based on these results we propose that HDL, LDL, and TLF1 are all bound and taken up by a lipoprotein scavenger receptor, which may constitute the parasite's major pathway mediating the uptake of essential lipids. low density lipoprotein apolipoprotein A-I haptoglobin-related protein haptoglobin type 1-1 trypanosome lytic factor high density lipoprotein scavenger receptor class B type I murine SR-BI ldlA (clone 7) LDL receptor-negative CHO cell mutant clone Chinese hamster ovary concanavalin A cholesterol/cholesterol ester phosphatidylethanolamine phosphatidylcholine Dulbecco's modified medium bovine serum albumin fluorescein isothiocyanate 4′,6-diamidino-2-phenylindole Exogenous lipids play indispensable roles in trypanosome cell structure and metabolism. African bloodstream-form trypanosomes are single-celled parasites that appear not to synthesize fatty acidsde novo (1Dixon H. Ginger C.D. Williamson J. Comp. Biochem. Physiol. 1971; 39B: 247-266Google Scholar, 2Dixon H. Ginger C.D. Williamson J. Comp. Biochem. Physiol. 1972; 41: 1-18Crossref Scopus (20) Google Scholar, 3Mellors A. Samad A. Parasitol. Today. 1989; 5: 239-244Abstract Full Text PDF PubMed Scopus (48) Google Scholar), with the exception of myristate ([14C]fatty acid). Trypanosomes have an atypical type II fatty acid synthase that utilizes exogenously supplied butyrate to generate myristate, which is used exclusively for glycosylphosphatidylinositol anchor biosynthesis (4Morita Y.S. Paul K.S. Englund P.T. Science. 2000; 288: 140-142Crossref PubMed Scopus (108) Google Scholar, 5Paul K. Jiang D. Morita Y. Englund P. Trends Parasitol. 2001; 17: 381-387Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Despite having a variety of enzymes that catalyze metabolic lipid-modifying pathways (6Doering T.L. Pessin M.S. Hoff E.F. Hart G.W. Rabens D.M. Englund P.T. J. Biol. Chem. 1993; 268: 9215-9222Abstract Full Text PDF PubMed Google Scholar, 7Low P. Dallner G. Mayor S. Cohen S. Chait B.T. Menon A.K. J. Biol. Chem. 1991; 266: 19250-19257Abstract Full Text PDF PubMed Google Scholar, 8Yokoyama K. Lin Y. Stuart K.D. Gelb M.H. Mol. Biochem. Parasitol. 1997; 87: 61-69Crossref PubMed Scopus (34) Google Scholar, 9Kubata B.K. Duzenko M. Kabututu Z. Rawer M. Szallies A. Fujimori K. Inui T. Nozaki T. Yamashita K. Horii T. Urade Y. Hayaishi O. J. Exp. Med. 2000; 192: 1327-1338Crossref PubMed Scopus (93) Google Scholar), trypanosomes are lipid auxotrophs. They require lipoproteins in addition to other serum components in order to multiply under axenic culture conditions (10Coppens I. Baudhuin P. Opperdoes F.R. Courtoy P.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6753-6757Crossref PubMed Scopus (144) Google Scholar, 11Black S. Vandeweerd V. Mol. Biochem. Parasitol. 1989; 37: 65-72Crossref PubMed Scopus (60) Google Scholar). Delipidation of the lipoproteins abrogates their capacity to support trypanosome growth. Both major classes of serum lipoproteins, LDL1 and HDL, are primary sources of lipids, delivering cholesterol esters, cholesterol and phospholipids to trypanosomes (12Coppens I. Opperdoes F.R. Courtoy P.J. Baudhuin P. J. Protozool. 1987; 34: 465-473Crossref PubMed Scopus (179) Google Scholar, 13Gillett M.P.T. Owen J.S. Biochim. Biophys. Acta. 1992; 1123: 239-248Crossref PubMed Scopus (41) Google Scholar). Trypanosomes endocytose HDL and LDL through their flagellar pocket (10Coppens I. Baudhuin P. Opperdoes F.R. Courtoy P.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6753-6757Crossref PubMed Scopus (144) Google Scholar,13Gillett M.P.T. Owen J.S. Biochim. Biophys. Acta. 1992; 1123: 239-248Crossref PubMed Scopus (41) Google Scholar). All endocytosis and exocytosis in trypanosomes occurs at this site. It is a specialized invagination in the cell membrane, which is not lined with the microtubule network that encases the rest of the cell that precludes any vesicular fusion or fission. At physiological concentrations (∼1 mg/ml), specific binding and uptake of the protein component of both LDL and HDL has been demonstrated (12Coppens I. Opperdoes F.R. Courtoy P.J. Baudhuin P. J. Protozool. 1987; 34: 465-473Crossref PubMed Scopus (179) Google Scholar, 13Gillett M.P.T. Owen J.S. Biochim. Biophys. Acta. 1992; 1123: 239-248Crossref PubMed Scopus (41) Google Scholar, 14Lorenz P. Owen J.S. Hassall D.G. Mol. Biochem. Parasitol. 1995; 74: 113-118Crossref PubMed Scopus (11) Google Scholar). In contrast, at subphysiological concentrations (1–50 μg/ml) there was no detectable uptake of the apolipoproteins themselves, whereas the lipid components of HDL and LDL were taken up at rates that exceeded fluid phase endocytosis by 1000-fold, suggesting that specific binding sites were probably involved (15Vandeweerd V. Black S.J. Mol. Biochem. Parasitol. 1989; 37: 201-211Crossref PubMed Scopus (17) Google Scholar). A putative LDL receptor protein has been purified but not yet cloned (16Coppens I. Bastin P. Courtoy P.J. Baudhuin P. Opperdoes F.R. Biochem. Biophys. Res. Commun. 1991; 178: 185-191Crossref PubMed Scopus (36) Google Scholar, 17Bastin P. Stephan A. Raper J. Saint-Remy J.-M. Opperdoes F.R. Courtoy P.J. Mol. Biochem. Parasitol. 1996; 76: 43-56Crossref PubMed Scopus (32) Google Scholar), while there has been no molecular identification of an HDL receptor in bloodstream-form trypanosomes. Trypanosome lytic factors are HDL-related particles found in human plasma. TLF1 contains lipid, apolipoprotein A-I (apoA-I), paraoxonase, and haptoglobin-related protein (Hpr) (18Smith A.B. Esko J.D. Hajduk S.L. Science. 1995; 268: 284-286Crossref PubMed Scopus (145) Google Scholar), while TLF2 is a lipid-poor molecule that contains apoA-I, Hpr, and IgM (19Raper J. Fung R. Ghiso J. Nussenzweig V. Tomlinson S. Inf. Immun. 1999; 67: 1910-1916Crossref PubMed Google Scholar). Both high and low affinity binding sites for TLF1 on trypanosomes have been reported in experiments using purified preparations of TLF1 (20Hager K.M. Pierce M.A. Moore D.R. Tytler E.M. Esko J.D. Hajduk S.L. J. Cell Biol. 1994; 126: 155-167Crossref PubMed Scopus (111) Google Scholar). The low affinity binding site can be competed by HDL whereas the high affinity binding site is partially competed by reconstituted nonlytic HDL containing Hpr (21Drain J. Bishop J.R. Hajduk S.L. J. Biol. Chem. 2001; 276: 30254-30260Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), which led to the proposal that Hpr can mediate TLF1 binding to trypanosomes through a haptoglobin-like receptor. Many lipoprotein receptors have been characterized in eukaryotes, to date only cubilin (22Hammad S.M. Barth J.L. Knaak C. Argraves W.S. J. Biol. Chem. 2000; 275: 12003-12008Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar) and members of the CD36 superfamily of scavenger receptors (23Calvo D. Gomez-Coronado D. Lasuncion M.A. Vega M.A. Arterioscler. Thromb. Vasc. Biol. 1997; 17: 2341-2349Crossref PubMed Scopus (213) Google Scholar, 24Calvo D. Gomez-Coronado D. Suarez Y. Lasuncion M.A. Vega M.A. J. Lipid Res. 1998; 39: 777-788Abstract Full Text Full Text PDF PubMed Google Scholar, 25Acton S. Rigotti A. Landschulz K.T., Xu, S. Hobbs H.H. Krieger M. Science. 1996; 271: 518-520Crossref PubMed Scopus (2001) Google Scholar) bind native HDL (without requiring ApoE as a component). The CD36 superfamily of scavenger receptors bind and take up both native HDL and LDL as well as other polyanionic ligands, including oxidized and acetylated LDL (26Krieger M. J. Clin. Invest. 2001; 108: 793-797Crossref PubMed Scopus (333) Google Scholar). Some of these scavenger receptors mediate bi-directional lipid flux and exhibit a process called selective lipid uptake. In polarized cells selective lipid uptake is characterized by receptor-mediated uptake of the lipoprotein, distribution of the lipid within the cell, and recycling of the apolipoprotein to the cell surface (27Silver D.L. Tall A.R. Curr. Opin. Lipidol. 2001; 12: 497-504Crossref PubMed Scopus (75) Google Scholar). In non-polarized cells there does not appear to be any uptake of the holoparticle, rather binding to the surface of the cell via lipoprotein scavenger receptors facilitates the transfer of lipid from the lipoprotein into cell membranes and intracellular vesicles (28Thuahnai 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). After lipid transfer, the lipid-depleted particle is released intact from the cell. One of the members of this family, SR-BI (scavenger receptor class BI), mediates the highest level of selective lipid uptake analyzed to date (29Connelly M.A. Klein S.M. Azhar S. Abumrad N.A. Williams D.L. J. Biol. Chem. 1998; 274: 41-47Abstract Full Text Full Text PDF Scopus (192) Google Scholar, 30Gu X. Trigatti B., Xu, S. Acton S. Babitt J. Krieger M. J. Biol. Chem. 1998; 273: 26338-26348Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). While studying trypanosome lytic factors, which are by definition lipoproteins, we decided to revisit lipoprotein receptors. We found evidence that Trypanosoma brucei brucei has a lipoprotein scavenger receptor that mediates the selective uptake of lipid over the protein component of both HDL and LDL. The same receptor can also mediate the uptake of oxidized lipoproteins. TLF1 is also bound and endocytosed by this lipoprotein scavenger receptor. We show that HDL and LDL compete for the binding and uptake of TLF1 and therefore attenuate the trypanosome lysis mediated by TLF1. The fluorescent probes: Alexa Fluor-488, 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), NBD-PtdCho, NBD-PtdEth, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine percholrate (DiI), and Calcein-AM were purchased from Molecular Probes (Eugene, OR). Tissue culture reagents: Ham's F12, DMEM, cell stripper solution, and G418 were obtained from Cellgro. Rhodamine-concanavalin A and Vectashield mounting medium with DAPI were obtained from Vector Labs. Protease inhibitors were purchased from Roche Molecular Biochemicals. Polyclonal rabbit anti-mouse SR-BI was obtained from Novus Biologicals. [125I]Iodine was purchased from Amersham Biosciences. Sephadex G-25 columns were bought from Isolab Inc. Mouse monoclonal anti-human haptoglobin, anti-rabbit IgG-FITC (F-0511), and all other chemicals and reagents were purchased from Sigma Chemical Co. T. b. brucei Etat 1.9s were kindly provided by Dr. Miki Rifkin. CHO ldlA clone 7 and ldlA[mSR-BI] cells were generously provided by Dr. Monty Krieger. Normal human serum, plasma or bovine serum was adjusted to a density of 1.25 g/ml with KBr and ultracentrifuged in a near vertical NVTi 65 rotor (Beckman) for 16 h at 49,000, 10 °C (31Poumay Y. Ronveaux-Dupal M.-F. J. Lipid Res. 1985; 26: 1476-1480Abstract Full Text PDF PubMed Google Scholar). The top 2 ml (ρ = 1.0–1.26 g/ml) were collected and size-fractionated on a Superose 6 16/50 column equilibrated with TBS (50 mm Tris-HCl, pH 7.5, 150 mm NaCl). Proteins from fractions were separated on a 4–15% Tris-HCl gel and stained with Coomassie Blue. Fractions containing either ApoA-I (HDL) or Apo-B (LDL) were separately pooled and concentrated. Isolation of human HDL and purification of TLF1 from normal human serum (Hp 1-1) was performed as described by Raper et al. (19Raper J. Fung R. Ghiso J. Nussenzweig V. Tomlinson S. Inf. Immun. 1999; 67: 1910-1916Crossref PubMed Google Scholar). Purified lipoproteins were incubated with Alexa Fluor-488 protein labeling kit (Ex 494, Em 519) according to manufacturers instructions. In order to label lipoprotein lipids, 20 ml of normal human serum were incubated with 50 μl of 1 mg/ml NBD-cholesterol (Abs 469, Em 537) in DMF, NBDC12 PtdCho (Abs 465, Em 534) in Me2SO, or NBD PtdEth (Abs 463, Em 536) in MeOH for 16 h at 37 °C. Lipoproteins were purified according to the above protocol. The specific activity (absorbance/mg of protein) of each fluorophore incorporated into HDL was determined in a 96-well fluorometer (Labsystems Fluoroskan II). Labeling of human HDL with DiI was carried out according to Calvoet al. (24Calvo D. Gomez-Coronado D. Suarez Y. Lasuncion M.A. Vega M.A. J. Lipid Res. 1998; 39: 777-788Abstract Full Text Full Text PDF PubMed Google Scholar). Briefly, lipoproteins were incubated with the DiI probe in lipoprotein-deficient serum for 12 h at 37 °C, using the following relative amounts: 300 μg DiI, 3 mg of lipoprotein, and 2 ml of lipoprotein-deficient serum. The labeled lipoproteins were subsequently re-isolated by ultracentrifugation at 100,000 × gfor 2.3 h at 10 °C in a Beckman table top ultracentrifuge (TLA 1.3 rotor). DiI-labeled HDL was then sized on a Superdex HR 200 column (Amersham Biosciences). DiI-labeled TLF1 was obtained by affinity purification of DiI-labeled HDL using a mouse anti-human haptoglobin monoclonal (H-6395,Sigma) coupled to a HiTrap column (Amersham Biosciences). The fractions containing Hpr were pooled and concentrated. Purified TLF1 was radiolabeled by the [125I]ICl technique with 125I to a specific activity of 300–600 dpm/ng. Bound 125I was separated from unbound by gel filtration on a PD10 column. Trichloroacetic acid-precipitable counts were >95%. The radiolabeled proteins were used on the day they were labeled. Swiss Webster mice were inoculated intraperitoneally with serum-sensitive T. b. brucei ETat 1.9s, and the trypanosomes were harvested 2 days later from infected mouse blood as described previously (32Lanham S.M. Godfrey D.G. Exp. Parasitol. 1970; 28: 521-534Crossref PubMed Scopus (1122) Google Scholar). Parasites were resuspended at 2 × 107/ml in Dulbecco's modified medium (DMEM) supplemented with 0.2–2% BSA. Increasing concentrations of labeled lipoprotein were incubated at 37 °C for various times with 2 × 107 trypanosomes in DMEM containing 0.2–2% BSA supplemented with the following protease inhibitors: 0.3 mg/ml antipain-HCl, 0.05 mg/ml bestatin, 0.1 mg/ml chymostatin, 0.3 mg/ml E-64, 0.05 mg/ml leupeptin, 0.05 mg/ml pepstatin, 0.3 mg/ml phosphoramidon, 2.0 mg/ml Pefabloc SC, 1.0 mg/ml EDTA, and 0.05 mg/ml aprotinin (miniTab, Roche Molecular Biochemicals). Cells were washed twice with bicine-buffered saline with glucose (BBSG), transferred to a black 96-well plate, and lysed in 0.5% SDS. Lysates were read in a Fluoroskan II at the wavelengths for the specific fluorophore. The ligand under study was mixed with competitors at 30–100-fold excess in DMEM/0.2–2% BSA. Then prewarmedT. b. brucei (2 × 107) were added and incubated at 37 °C for 30 min. For radioactive ligands, cells were washed three times with DMEM/BSA and transferred to a clean tube prior to quantitating radioactivity in a gamma counter. Fluorescent-labeled cells were washed twice with BBSG, transferred to black 96-well plates, and lysed and read as above. Live trypanosomes were incubated in DMEM/0.2% BSA with either 300 μg/ml of NBD cholesterol/cholesterol ester-labeled plasma HDL or Alexa-labeled plasma HDL, 150 μg/ml of rhodamine-concanavalin A, or 300 μg/ml Alexa-HDL combined with 150 μg/ml rhodamine-concanavalin A at 37 °C for 30 min. Cells were washed three times in PBSG and resuspended in 3% paraformaldehyde/PBSG. Cells were washed with PBSG and dried onto 12-well slides (Erie Scientific Co.). DAPI-containing mounting medium was used to adhere coverslips to slides. Slides were viewed on a Nokia fluorescent microscope. To measure lytic activity, 2 × 106 trypanosomes were incubated in the presence of TLF1 (1–1.5 LU (5–40 μg/ml), or TLF1 combined with human LDL (0.75–1 mg/ml) or bovine HDL (1–1.6 mg/ml). LU is the lytic unit wherein 50% of 2 × 106 trypanosomes are lysed in 150 min at 37 0C in a final volume of 200 μl. Following incubation for 150 min at 37 °C, parasite lysis was determined using a previously described calcein fluorescence-based assay (33Tomlinson S. Jansen A.M. Koudinov A. Ghiso J.A. Choi-Miura N.H. Rifkin M.R. Ohtaki S. Nussenzweig V. Mol. Biochem. Parasitol. 1995; 70: 131-138Crossref PubMed Scopus (44) Google Scholar). LdlA[mSR-BI] and ldlA7 cells were grown in Ham's F-12 medium supplemented with 5% (v/v) fetal calf serum, 2 mm glutamine with or without 500 μg/ml G-418, respectively. All cells were maintained in a 37 °C humidified 95% air, 5% CO2 incubator. LdlA[mSR-BI] and parental ldlA7 cells were plated overnight in 6- or 12-well plates washed and incubated for 2–4 h in Ham's medium supplemented with 0.2% BSA. The cells were then incubated at 37 °C for 2 h with 2.5 μg/ml DiI-HDL or DiI-TLF1 in Ham's F12 supplemented with 0.2% BSA. Cells were washed twice with PBS, detached from the plate with cell stripper solution and resuspended in PBS, then fixed with an equal volume of 4% paraformaldeyde. For immunofluorescence, LdlA[mSR-BI] and ldlA7 cells were resuspended in 100 μl of Ham's F12, 5% FCS to 106/ml. Anti-SR-BI antibody (100 μl) to a final dilution 1:1000 was added and incubated for 30 min on ice. Cells were then washed three times, resuspended to 100 μl in PBS, 0.1% BSA, and 100 μl of anti-rabbit IgG-FITC-labeled antibody was added to a final dilution of 1:50. After a 30-min incubation on ice, the cells were washed three times with PBS, then resuspended to 100 μl in PBS and fixed with 100 μl of 4% paraformaldehyde. Samples were subjected to flow cytometric analysis using a Becton Dickinson FACScan flow cytometer. The following excitation/emission were used: 488/525 nm for FITC-conjugated antibody and 488/575 nm for DiI-labeled HDL and TLF1. Mean relative fluorescence of cells was recorded and results are expressed as a percentage of control. We labeled HDL and LDL with Alexa, a fluorophore that conjugates to the free amino groups in the protein components of these lipoproteins. We found that trypanosomes accumulated HDL protein (2.25 pmol, calculated based on a molecular mass of 350,000 Da by size exclusion chromatography, 50% of which is protein) and LDL protein (2 pmol, calculated based on the molecular mass of apoB, 550,000 Da) (Fig. 1 A) with similar kinetics, approximating steady state within 30 min. Trypanosomes also accumulated NBD-cholesterol/cholesterol ester (NBD-C/CE) from HDL (lipid uptake equivalent to 350 pmol of protein) and LDL (lipid uptake equivalent to 425 pmol of protein; Fig. 1 B), approximating steady state within 30 min. TLC analysis indicated that the NBD cholesterol labeled both free cholesterol and cholesterol esters in the lipoproteins (data not shown). Therefore, results shown in Fig. 1 B represent total cholesterol uptake. As HDL and LDL have similar kinetics of protein and lipid uptake (Fig. 1, A and B), they were used in competition assays. The competition was assessed at 30 min, such that the uptake was close to steady state (Fig. 1,A and B). Both unlabeled HDL and LDL were effective competitors of protein (Fig. 1 D) and lipid (Fig. 1 C) uptake from labeled HDL. Fig. 1, panel Dshows that 4 times more HDL than LDL was needed to give a 50% reduction in the uptake of HDL protein. This suggests that the putative lipoprotein receptor has higher affinity for LDL than HDL. Although Fig. 1, panel C displays a similar trend in LDLversus HDL competition, we found that some of the NBD-cholesterol was transferred to the non-labeled lipoprotein, which may contribute to the enhanced competition of lipoprotein lipid uptake. This desorption diffusion process may also occur to some extent when NBD-cholesterol HDL is incubated with trypanosomes. To date the only characterized eukaryotic lipoprotein receptors that bind both native HDL and LDL have the unique characteristic of selective lipid uptake over protein. In light of the above findings we examined if there was a differential uptake of other lipid components over the protein components of HDL. We extended this study to include HDL labeled with fluorescent phospholipids NBD-phosphatidylcholine (NBD-PtdCho), NBD-phosphatidylethanolamine (NBD-PtdEth) as well as NBD-cholesterol/cholesterol ester (NBD-C/CE). The specific activity (fluorescence/μg of protein) of each fluorescent-labeled HDL was taken into consideration, such that the uptake of each fluorescent lipid was calculated from a standard curve of μg of proteinversus fluorescent units. The results are expressed as microgram of protein equivalents taken up rather than fluorescent units. We found that all classes of lipid molecules in HDL were taken up 50–100-fold more than the protein component (Fig. 2). It is apparent that the uptake of free cholesterol and cholesterol ester exceeds that of phospholipids. In addition NBD-PtdEth was taken up almost 2-fold more than NBD-PtdCho. The uptake of individual lipid species does not correlate with their concentrations found in a typical human HDL, which are 55, 7, and 27 weight% for phosphatidylcholine, cholesterol, and cholesterol esters respectively (34Shen B.W. Scanu A.M. Kezdy F.J. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 837-841Crossref PubMed Scopus (290) Google Scholar). Since all classes of lipid molecules were taken up more avidly than protein, we examined the distribution of the fluorescent lipid and protein molecules by fluorescent microscopy. NBD-cholesterol and cholesterol esters (NBD-C/CE) were rapidly distributed throughout the parasite, and all of the parasites were labeled (Fig. 3 A). Confocal analysis revealed diffuse staining throughout the cytosol of the parasites (data not shown). The labeled phospholipids, NBD-PtdCho and NBD-PtdEth also demonstrated a staining pattern similar to that of NBD-C/CE (data not shown). In contrast, the apolipoprotein (Alexa-HDL) uptake by trypanosomes was visualized in the flagellar pocket and intracellular vesicles (Fig. 3 B) with a distribution similar to endocytosed concanavalin A (ConA) (Fig. 3 C). All of the parasites accumulated detectable Alexa-HDL. Concanavalin A has been shown to distribute within endocytic vesicles of trypanosomes when endocytosed by live trypanosomes (35Morgan G.W. Allen C.L. Jeffries T.R. Hollinshead M. Field M.C. J. Cell Sci. 2001; 114: 2605-2615Crossref PubMed Google Scholar, 36Jeffries T.R. Morgan G.W. Field M.C. J. Cell Sci. 2001; 114: 2617-2626Crossref PubMed Google Scholar); in contrast ConA labels the VSG coat when used on fixed trypanosomes presumably due to the exposure of carbohydrate epitopes upon fixation. Coincubation with rhodamine-ConA and Alexa-HDL revealed colocalization in some endocytic vesicles (yellow) near the flagellar pocket but not all endocytic vesicles (red) (Fig. 3 D). All of the data thus far point to the presence of a lipoprotein receptor in trypanosomes that can facilitate the uptake of both native HDL and LDL. TLF1 is a subclass of HDL and is composed of lipids, apolipoprotein A-I, paraoxanase, and haptoglobin-related protein. Lipids and apolipoprotein A-I are the common components of all HDLs, whereas Hpr is unique to TLF particles. There are studies documenting the specific and saturable binding of TLF1 and HDL to the flagellar pocket of African trypanosomes (14Lorenz P. Owen J.S. Hassall D.G. Mol. Biochem. Parasitol. 1995; 74: 113-118Crossref PubMed Scopus (11) Google Scholar, 20Hager K.M. Pierce M.A. Moore D.R. Tytler E.M. Esko J.D. Hajduk S.L. J. Cell Biol. 1994; 126: 155-167Crossref PubMed Scopus (111) Google Scholar). We found that TLF1 (20 μg/ml (60 pmol, calculated based on a molecular mass of 550,000 Da by size exclusion chromatography, 60% of which is protein)) and HDL (200 μg/ml (1440 pmol) could compete for binding of 125I-TLF1 to T. b. brucei (Fig. 4). We did not investigate the effect of LDL on the binding of TLF1 to T. b. brucei, because LDL takes 6 h to reach equilibrium binding to trypanosomes whereas HDL and TLF take 30 min. Therefore we could not have a fair competition for binding and evaluated uptake only. Given that we observed competition for binding of TLF1 to trypanosomes by HDL, we evaluated the effect of non-lytic bovine HDL on TLF1-mediated trypanolysis. We observed that non-lytic bovine HDL was able to attenuate trypanosome lysis by purified TLF1 (Fig. 5). Non-lytic human LDL was also effective in attenuating trypanolysis by TLF1. Our results support the presence of a lipoprotein scavenger receptor in trypanosomes that can bind multiple lipoprotein ligands such as TLF1, HDL, LDL, and oxidized LDL (not shown) and exhibit a process called selective lipid uptake similar to eukaryotic lipoprotein scavenger receptors. To directly address whether TLF1 could bind to a eukaryotic lipoprotein scavenger receptor with the same ligand binding characteristics as the trypanosome lipoprotein receptor, we examined the binding of TLF1 to a CHO cell line that overexpresses mouse Scavenger Receptor-Class BI (mSR-BI). The parental CHO ldlA[clone 7] cells do not express the LDL receptor and were stably transfected with a vector expressing mouse scavenger receptor-class BI to create ldlA[mSR-BI] cells (37Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar). These cell lines were first validated with antibodies raised against mSR-BI; the parental ldlA cells showed little staining, while the transfected ldlA[mSR-BI] cells stained readily with anti-mSR-BI (Fig. 6, inset). HDL labeled with the fluorescent lipid DiI exhibited lipid uptake into cells expressing mSR-BI that was 30-fold greater than the uptake by the parental ldlA cells (Fig. 6). TLF1 labeled in the lipid component with DiI to the same specific activity as DiI-HDL, was taken up to a 5-fold greater extent by cells expressing mSR-BI than that shown by the parental ldlA cells (Fig. 6). Lipoprotein receptors that can bind more than one ligand are known as lipoprotein scavenger receptors. Our results suggest that bloodstream-form trypanosomes have a single lipoprotein scavenger receptor that can facilitate the uptake of all major lipoprotein classes including HDL, LDL, oxidized LDL (results not shown), and TLF1. Moreover, like eukaryotic scavenger receptors, the trypanosome receptor shows selective uptake of lipid over protein from the lipoprotein particle. Trypanosomes are lipid auxotrophs, and host lipoproteins are required for their survival. This putative lipoprotein scavenger receptor may well constitute the primary pathway by which the parasite acquires essential host lipids, and would therefore represent an important therapeutic target. Very few bloodstream-form trypanosome receptors have been previously characterized biochemically. These include an LDL receptor (10Coppens I. Baudhuin P. Opperdoes F.R. Courtoy P.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6753-6757Crossref PubMed Scopus (144) Google Scholar) and a HDL receptor (13Gillett M.P.T. Owen J.S. Biochim. Biophys. Acta. 1992; 1123: 239-248Crossref PubMed Scopus (41) Google Scholar) both of which may be identical to the scavenger receptor described here (see below), a haptoglobin-like receptor, which may also be a TLF receptor (21Drain J. Bishop J.R. Hajduk S.L. J. Biol. Chem. 2001; 276: 30254-30260Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), and a receptor for transfe" @default.
• W2029360079 created "2016-06-24" @default.
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