The Human Scavenger Receptor CD36

Human CD36 is a class B scavenger receptor expressed in a variety of cell types such as macrophage and adipocytes. This plasma membrane glycoprotein has a wide range of ligands including oxidized low density lipoprotein and long chain fatty acids which involves the receptor in diseases such as atherosclerosis and insulin resistance. CD36 is heavily modified post-translationally by N-linked glycosylation, and 10 putative glycosylation sites situated in the large extracellular loop of the protein have been identified; however, their utilization and role in the folding and function of the protein have not been characterized. Using mass spectrometry on purified and peptide N-glycosidase F-deglycosylated CD36 and also by comparing the electrophoretic mobility of different glycosylation site mutants, we have determined that 9 of the 10 sites can be modified by glycosylation. Flow cytometric analysis of the different glycosylation mutants expressed in mammalian cells established that glycosylation is necessary for trafficking to the plasma membrane. Minimally glycosylated mutants that supported trafficking were identified and indicated the importance of carboxyl-terminal sites Asn-247, Asn-321, and Asn-417. However, unlike SRBI, no individual site was found to be essential for proper trafficking of CD36. Surprisingly, these minimally glycosylated mutants appear to be predominantly core-glycosylated, indicating that mature glycosylation is not necessary for surface expression in mammalian cells. The data also show that neither the nature nor the pattern of glycosylation is relevant to binding of modified low density lipoprotein. Human CD36 is a class B scavenger receptor expressed in a variety of cell types such as macrophage and adipocytes. This plasma membrane glycoprotein has a wide range of ligands including oxidized low density lipoprotein and long chain fatty acids which involves the receptor in diseases such as atherosclerosis and insulin resistance. CD36 is heavily modified post-translationally by N-linked glycosylation, and 10 putative glycosylation sites situated in the large extracellular loop of the protein have been identified; however, their utilization and role in the folding and function of the protein have not been characterized. Using mass spectrometry on purified and peptide N-glycosidase F-deglycosylated CD36 and also by comparing the electrophoretic mobility of different glycosylation site mutants, we have determined that 9 of the 10 sites can be modified by glycosylation. Flow cytometric analysis of the different glycosylation mutants expressed in mammalian cells established that glycosylation is necessary for trafficking to the plasma membrane. Minimally glycosylated mutants that supported trafficking were identified and indicated the importance of carboxyl-terminal sites Asn-247, Asn-321, and Asn-417. However, unlike SRBI, no individual site was found to be essential for proper trafficking of CD36. Surprisingly, these minimally glycosylated mutants appear to be predominantly core-glycosylated, indicating that mature glycosylation is not necessary for surface expression in mammalian cells. The data also show that neither the nature nor the pattern of glycosylation is relevant to binding of modified low density lipoprotein. Human CD36, originally identified in platelets as glycoprotein IV (1Tandon N.N. Kralisz U. Jamieson G.A. J. Biol. Chem. 1989; 264: 7576-7583Abstract Full Text PDF PubMed Google Scholar), is a class B scavenger receptor localized to the plasma membrane. It is not expressed ubiquitously but is present in a variety of different cells and tissue types including epithelial cells (2Greenwalt D.E. Watt K.W. So O.Y. Jiwani N. Biochemistry. 1990; 29: 7054-7059Crossref PubMed Scopus (80) Google Scholar), macrophages (3Endemann G. Stanton L.W. Madden K.S. Bryant C.M. White R.T. Protter A.A. J. Biol. Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar), endothelial cells of the microvasculature (4Greenwalt D.E. Lipsky R.H. Ockenhouse C.F. Ikeda H. Tandon N.N. Jamieson G.A. Blood. 1992; 80: 1105-1115Crossref PubMed Google Scholar), and smooth muscle (5Harmon C.M. Abumrad N.A. J. Membr. Biol. 1993; 133: 43-49Crossref PubMed Scopus (164) Google Scholar). Its function is complex, and its involvement in different disease scenarios, such as cancer (6Rutella S. Rumi C. Di Mario A. Leone G. Eur. J. Histochem. 1997; 41: 53-54PubMed Google Scholar), atherosclerosis (3Endemann G. Stanton L.W. Madden K.S. Bryant C.M. White R.T. Protter A.A. J. Biol. Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar, 7Ma X. Bacci S. Mlynarski W. Gottardo L. Soccio T. Menzaghi C. Iori E. Lager R.A. Shroff A.R. Gervino E.V. Nesto R.W. Johnstone M.T. Abumrad N.A. Avogaro A. Trischitta V. Doria A. Hum. Mol. Genet. 2004; 13: 2197-2205Crossref PubMed Scopus (159) Google Scholar, 8Febbraio M. Podrez E.A. Smith J.D. Hajjar D.P. Hazen S.L. Hoff H.F. Sharma K. Silverstein R.L. J. Clin. Invest. 2000; 105: 1049-1056Crossref PubMed Scopus (823) Google Scholar), malaria (9Serghides L. Smith T.G. Patel S.N. Kain K.C. Trends Parasitol. 2003; 19: 461-469Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and insulin resistance (10Aitman T.J. Glazier A.M. Wallace C.A. Cooper L.D. Norsworthy P.J. Wahid F.N. Al-Majali K.M. Trembling P.M. Mann C.J. Shoulders C.C. Graf D. St Lezin E. Kurtz T.W. Kren V. Pravenec M. Ibrahimi A. Abumrad N.A. Stanton L.W. Scott J. Nat. Genet. 1999; 21: 76-83Crossref PubMed Scopus (641) Google Scholar), most likely reflects the interaction of the receptor with a particular ligand in a specific cell type. For example, CD36 expressed in monocytic macrophages functions as a scavenger receptor for the uptake of oxidized LDL 2The abbreviations used are: LDLlow density lipoproteinSRBIscavenger receptor class B, type IOGn-octyl-β-d-glucopyranosidePNGase Fpeptide N-glycosidase FEndo Hendoglycosidase HSf21Spodoptera frugiperda 21non-gnon-glycosylatedNi-NTAnickel-nitrilotriacetic acidBSAbovine serum albuminFT-ICRFourier transform ion cyclotron resonanceMSmass spectroscopyFACSfluorescence-activated cell sorterPBSphosphate-buffered salinemAbmonoclonal antibodyQ-Tofquadrupole-time of flight.2The abbreviations used are: LDLlow density lipoproteinSRBIscavenger receptor class B, type IOGn-octyl-β-d-glucopyranosidePNGase Fpeptide N-glycosidase FEndo Hendoglycosidase HSf21Spodoptera frugiperda 21non-gnon-glycosylatedNi-NTAnickel-nitrilotriacetic acidBSAbovine serum albuminFT-ICRFourier transform ion cyclotron resonanceMSmass spectroscopyFACSfluorescence-activated cell sorterPBSphosphate-buffered salinemAbmonoclonal antibodyQ-Tofquadrupole-time of flight. (3Endemann G. Stanton L.W. Madden K.S. Bryant C.M. White R.T. Protter A.A. J. Biol. Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar, 11Love-Gregory L. Sherva R. Sun L. Wasson J. Schappe T. Doria A. Rao D.C. Hunt S.C. Klein S. Neuman R.J. Permutt M.A. Abumrad N.A. Hum. Mol. Genet. 2008; 17: 1695-1704Crossref PubMed Scopus (150) Google Scholar). Under certain physiological conditions, this results in the lipid loading of macrophages at the site of tissue damage in the arterial wall, leading to foam cell formation and plaque development, a key early stage in the pathogenesis of atherosclerosis (8Febbraio M. Podrez E.A. Smith J.D. Hajjar D.P. Hazen S.L. Hoff H.F. Sharma K. Silverstein R.L. J. Clin. Invest. 2000; 105: 1049-1056Crossref PubMed Scopus (823) Google Scholar, 12Yamashita S. Hirano K. Kuwasako T. Janabi M. Toyama Y. Ishigami M. Sakai N. Mol. Cell Biochem. 2007; 299: 19-22Crossref PubMed Scopus (108) Google Scholar). In fat and muscle cells, CD36 plays an essential role in lipid homeostasis by uptake of long chain fatty acids (13Abumrad N.A. el-Maghrabi M.R. Amri E.Z. Lopez E. Grimaldi P.A. J. Biol. Chem. 1993; 268: 17665-17668Abstract Full Text PDF PubMed Google Scholar). In this case CD36 deficiency has been linked to disorders in lipid metabolism, giving rise to increased incidences of insulin resistance and cardiomyopathies (11Love-Gregory L. Sherva R. Sun L. Wasson J. Schappe T. Doria A. Rao D.C. Hunt S.C. Klein S. Neuman R.J. Permutt M.A. Abumrad N.A. Hum. Mol. Genet. 2008; 17: 1695-1704Crossref PubMed Scopus (150) Google Scholar, 14Koonen D.P. Febbraio M. Bonnet S. Nagendran J. Young M.E. Michelakis E.D. Dyck J.R. Circulation. 2007; 116: 2139-2147Crossref PubMed Scopus (91) Google Scholar, 15Hwang E.H. Taki J. Yasue S. Fujimoto M. Taniguchi M. Matsunari I. Nakajima K. Shiobara S. Ikeda T. Tonami N. J. Nucl. Med. 1998; 39: 1681-1684PubMed Google Scholar). low density lipoprotein scavenger receptor class B, type I n-octyl-β-d-glucopyranoside peptide N-glycosidase F endoglycosidase H Spodoptera frugiperda 21 non-glycosylated nickel-nitrilotriacetic acid bovine serum albumin Fourier transform ion cyclotron resonance mass spectroscopy fluorescence-activated cell sorter phosphate-buffered saline monoclonal antibody quadrupole-time of flight. low density lipoprotein scavenger receptor class B, type I n-octyl-β-d-glucopyranoside peptide N-glycosidase F endoglycosidase H Spodoptera frugiperda 21 non-glycosylated nickel-nitrilotriacetic acid bovine serum albumin Fourier transform ion cyclotron resonance mass spectroscopy fluorescence-activated cell sorter phosphate-buffered saline monoclonal antibody quadrupole-time of flight. Although much is known about the function of CD36, less is known about its structure. CD36 has no bacterial homologues but is a member of a protein family that also includes the mammalian proteins LIMPII (16Vega M.A. Seguí-Real B. García J.A. Calés C. Rodríguez F. Vanderkerckhove J. Sandoval I.V. J. Biol. Chem. 1991; 266: 16818-16824Abstract Full Text PDF PubMed Google Scholar), CLA-1 (17Calvo D. Vega M.A. J. Biol. Chem. 1993; 268: 18929-18935Abstract Full Text PDF PubMed Google Scholar), SRBI (18Acton S.L. Scherer P.E. Lodish H.F. Krieger M. J. Biol. Chem. 1994; 269: 21003-21009Abstract Full Text PDF PubMed Google Scholar), and the Drosophila proteins Croquemort (19Franc N.C. Dimarcq J.L. Lagueux M. Hoffmann J. Ezekowitz R.A. Immunity. 1996; 4: 431-443Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar) and emp (20Hart K. Wilcox M. J. Mol. Biol. 1993; 234: 249-253Crossref PubMed Scopus (55) Google Scholar). The sequence of 471 amino acids has two short hydrophobic regions at the carboxyl and amino termini separated by a large hydrophilic region (21Oquendo P. Hundt E. Lawler J. Seed B. Cell. 1989; 58: 95-101Abstract Full Text PDF PubMed Scopus (400) Google Scholar); however, the topology of the protein is unclear with both ditopic (22Gruarin P. Thorne R.F. Dorahy D.J. Burns G.F. Sitia R. Alessio M. Biochem. Biophys. Res. Commun. 2000; 275: 446-454Crossref PubMed Scopus (37) Google Scholar) and type I (23Pearce S.F. Wu J. Silverstein R.L. Blood. 1994; 84: 384-389Crossref PubMed Google Scholar) topological models proposed. Both are consistent in predicting that the large hydrophilic region is extracellular, which is clearly supported by epitope mapping studies (24Asch A.S. Liu I. Briccetti F.M. Barnwell J.W. Kwakye-Berko F. Dokun A. Goldberger J. Pernambuco M. Science. 1993; 262: 1436-1440Crossref PubMed Scopus (185) Google Scholar). The protein is heavily modified post-translationally. The six extracellular cysteines, which are highly conserved within the orthologous CD36 subfamily, have been shown to be linked by disulfide bonds in bovine Cd36 (25Rasmussen J.T. Berglund L. Rasmussen M.S. Petersen T.E. Eur. J. Biochem. 1998; 257: 488-494Crossref PubMed Scopus (62) Google Scholar), and the remaining four cysteines, two at each terminus, are palmitoylated (26Tao N. Wagner S.J. Lublin D.M. J. Biol. Chem. 1996; 271: 22315-22320Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), lending credence to the ditopic topological model. CD36 is also modified by N-linked glycosylation, which accounts for the observation that the protein migrates with an apparent molecular mass of 78–94 kDa on SDS-PAGE (4Greenwalt D.E. Lipsky R.H. Ockenhouse C.F. Ikeda H. Tandon N.N. Jamieson G.A. Blood. 1992; 80: 1105-1115Crossref PubMed Google Scholar, 27Alessio M. Ghigo D. Garbarino G. Geuna M. Malavasi F. Cell. Immunol. 1991; 137: 487-500Crossref PubMed Scopus (20) Google Scholar) despite a theoretical mass for the polypeptide of 53 kDa. N-Linked glycosylation is a common modification of extracellular and secreted proteins, and defects in the glycosylation pathways lead to a wide range of serious diseases known collectively as congenital disorders of glycosylation (28Freeze H.H. Nat. Rev. Genet. 2006; 7: 537-551Crossref PubMed Scopus (387) Google Scholar). Glycosylation can be important for correct folding of proteins (29O'Connor S.E. Imperiali B. Chem. Biol. 1996; 3: 803-812Abstract Full Text PDF PubMed Scopus (160) Google Scholar, 30Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (560) Google Scholar) either by directly inducing and/or stabilizing the tertiary fold of the polypeptide (31Mitra N. Sinha S. Ramya T.N. Surolia A. Trends Biochem. Sci. 2006; 31: 156-163Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) or via an affinity for lectin chaperones such as calnexin or calreticulin (32Trombetta E.S. Helenius A. Curr. Opin. Struct. Biol. 1998; 8: 587-592Crossref PubMed Scopus (211) Google Scholar). Glycosylation has also been shown to be important for the trafficking of certain glycoproteins through affinity for lectin transport machinery (33Nufer O. Guldbrandsen S. Degen M. Kappeler F. Paccaud J.P. Tani K. Hauri H.P. J. Cell Sci. 2002; 115: 619-628Crossref PubMed Google Scholar). The glycosylation status of bovine Cd36 has already been determined with all eight putative sites shown to be glycosylated (34Berglund L. Petersen T.E. Rasmussen J.T. Biochim. Biophys. Acta. 1996; 1309: 63-68Crossref PubMed Scopus (26) Google Scholar). Human and bovine CD36 are 83% identical (93% when similar residues are included) and share 7 glycosylation sites (human has 10 putative glycosylation sites). In the related mouse SRBI, which is 33% identical (54% similar) to human CD36, there are 11 putative N-linked glycosylation sites, only 3 of which are shared with the human protein. Site-directed mutagenesis of each of the 11 sites independently in SRBI in an otherwise wild type protein indicates that all are glycosylated, with two (Asn-108 and Asn-173) important for either trafficking or folding. Mutagenesis of either of these two residues resulted in very little cell surface expression of the protein (35Viñals M. Xu S. Vasile E. Krieger M. J. Biol. Chem. 2003; 278: 5325-5332Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar); however, neither site is conserved in human CD36. To gain further understanding of the role of glycosylation of CD36, we used mutagenesis and biophysical analysis (mass spectrometry and gel electrophoresis) to identify unequivocally which glycosylation sites are occupied in human CD36. Antibody and ligand binding studies with these mutant proteins also provided insights into the role of glycosylation and site occupancy in the trafficking and function of the protein. The detergent n-octyl-β-d-glucopyranoside (OG) was purchased from Merck. The nickel-NTA-agarose was purchased from Qiagen Ltd, UK, and BODIPY Acetylated LDL FL® (BODIPY Ac-LDL) was from Invitrogen. All the protease inhibitors, fatty acid-free BSA, and tunicamycin were purchased from Sigma-Aldrich. Amicon Ultra 15 (molecular weight cutoff 50) centrifugal devices were from Millipore, and peptide N-glycosidase F (PNGase F) and endoglycosidase H (Endo H) were from New England Biolabs. The primary antibodies, mouse mAb1258 (Chemicon International), and rat mAb1955 (R and D Systems), both recognize folded CD36 for use in flow cytometry, but only mAb1955 recognizes the denatured product after SDS-PAGE and Western analysis. NcoI and BstEII restriction sites were introduced into the 5′ and 3′ ends, respectively, of the CD36 cDNA (ATCC clone MGC-14530) by mutagenic polymerase chain reaction using oligonucleotides 5′-TTG GTA CAT ACG GTG ACC TTT TAT TGT TTC G-3′ (NcoI) and 5′-CCT GAA CAA GAA CCA TGG GCT GTG ACC-3′ (BstEII). These two restriction enzyme sites were used to subclone CD36 into the baculoviral transfer vector BlueBac4.5 (Invitrogen) containing a 12-histidine tag (36Martin C.A. Longman E. Wooding C. Hoosdally S.J. Ali S. Aitman T.J. Gutmann D.A. Freemont P.S. Byrne B. Linton K.J. Protein Sci. 2007; 16: 2531-2541Crossref PubMed Scopus (24) Google Scholar) to generate BlueBac-CD36–12His. The transfer vector was used to engineer a recombinant baculovirus using the Bac-N-Blue system (Invitrogen) as directed. For expression in mammalian cells, the modified CD36 cDNA was subcloned, replacing the rodent sequence in pCI-Cd36–12His (36Martin C.A. Longman E. Wooding C. Hoosdally S.J. Ali S. Aitman T.J. Gutmann D.A. Freemont P.S. Byrne B. Linton K.J. Protein Sci. 2007; 16: 2531-2541Crossref PubMed Scopus (24) Google Scholar) using the restriction enzymes NcoI and BstEII to generate pCI-CD36–12His. Site-directed mutagenesis was performed on pCI-CD36–12His using the QuikChange multisite mutagenesis kit (Stratagene) according to the manufacturer's instructions. Primers were designed to replace the asparagine codon in each putative glycosylation site with a codon for glutamine. The entire coding sequence of each mutant was confirmed by DNA sequencing. The sequences of mutagenic primers for the asparagine to glutamine mutants were: N79Q, 5′-CAC AGG AAG TGA TGA TGC AGA GCT CCA ACA TTC AAG TTA AGC-3′; N102Q, 5′-TCG TTT TCT AGC CAA AGA ACA GGT AAC CCA GGA CGC TG-3′; N134Q, 5′-TGG AAC AGA GGC TGA TCA GTT CAC AGT TCT CAA TC-3′; N163Q, 5′-GTT CAA ATG ATC CTC AAT TCA TTA ATT CAG AAG TCA AAA TCT TCT ATG TTC C-3′; N205Q, 5′-GGT CTG TTT TAT CCT TAC CAG AAT ACG GCA GAT GGA GTT TAT AAA G-3′; N220Q, 5′-GTT TTC AAT GGA AAA GAT CAG ATC TCT AAA GTT GCC ATA ATC G-3′; N235Q, 5′-CAT ATA AAG GTA AAA GGC AGC TGT CCT ATT GGG AAA G-3′; N247Q, 5′-CAC TGC GAC ATG ATT CAG GGT ACA GAT GCA GCC-3′; N321Q, 5′-GAA AAA ATT ATC TCA AAG CAA TGT ACA TCA TAT GGT GTG CTA G-3′; N417Q, 5′-TGT GCC TAT TCT TTG GCT CCA GGA GAC TGG GAC CAT TGG-3′. HEK293T cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Invitrogen) at 37 °C in 5% CO2. The cells were transfected transiently with wild type and mutant pCI-CD36–12His or pCI-ABCB1–12His, as described previously (37Dixon P.H. Weerasekera N. Linton K.J. Donaldson O. Chambers J. Egginton E. Weaver J. Nelson-Piercy C. de Swiet M. Warnes G. Elias E. Higgins C.F. Johnston D.G. McCarthy M.I. Williamson C. Hum. Mol. Genet. 2000; 9: 1209-1217Crossref PubMed Scopus (266) Google Scholar). 24 h after transfection the cells were treated with 33 μm butyric acid (Sigma) and cultured for a further 24 h before harvesting using trypLETM Express (Invitrogen) for use in flow cytometry and immunoblotting. Tunicamycin (500 ng/ml) was added 5 h post-transfection where indicated. Suspension cultures of Spodoptera frugiperda 21 (Sf21) cells were grown in SF900II serum-free medium (Invitrogen) at 27 °C with shaking at 100 rpm. Cells at a density of 2 × 106 cells/ml were infected with recombinant baculovirus encoding wild type or non-glycosylated CD36 using a multiplicity of infection of at least 3 viruses per cell. After several hours the culture was diluted to a density of 1 × 106 cells/ml with fresh SF900II media. At 72 h post-infection, the insect cells were harvested by centrifugation at 1000 × g, 4 °C for 10 min and washed in ice-cold buffer 1 (10 mm Tris-HCl, pH 7.5, 250 mm sucrose, 0.2 mm CaCl2, 2 mm benzamidine, 40 μm leupeptin, and 1 μm pepstatin A). The cells were resuspended in 10 ml of buffer 1 and frozen at −20 °C. Once thawed, the cells were homogenized at 4 °C by 5 × 30-s bursts at 24,000 rpm (DI 25 homogenizer; Yellow Line). The sample was centrifuged at 500 × g for 10 min at 4 °C to pellet the large organelles and unbroken cells. The supernatant was recovered and centrifuged at 100,000 × g in a TLA100.3 rotor (Beckman Coulter) for 50 min at 4 °C to obtain pelleted membranes. The crude membrane fraction was resuspended in buffer 2 (buffer 1, minus CaCl2) supplemented with 10% (v/v) glycerol and stored at −80 °C. Total protein concentrations of the membrane fractions were determined by DC Protein Assay (Bio-Rad). Membrane fractions were pelleted by centrifugation at 100,000 × g for 30 min in a TLA100.3 rotor at 4 °C. The pellets of wild type CD36 were resuspended in solubilization buffer (20 mm Tris-HCl, pH 6.8, 2% (w/v) OG, 150 mm NaCl, 1.5 mm MgCl2, 5% (v/v) glycerol, 2 mm benzamidine, 40 μm leupeptin, and 1 μm pepstatin A) at 5 mg protein/ml, homogenized by extrusion in a 21-gauge needle, and constantly mixed for 90 min at 4 °C. The insoluble fraction was pelleted by ultracentrifugation at 100,000 × g for 30 min in a TLA100.3 rotor at 4 °C. Ni-NTA resin was pre-equilibrated in equilibration buffer (solubilization buffer where 2% OG was replaced with 1% OG in the presence of 20 mm imidazole). Imidazole (20 mm) was added to the solubilized fraction of wild type CD36 membranes and incubated with the Ni-NTA resin using a protein:resin ratio of 8:1 with continuous mixing for 1 h at 4 °C. The resin was washed 4 times with 20 bed volumes and a stepwise gradient of imidazole (60–120 mm) in wash buffer (20 mm Tris-HCl pH 8.0, 150 mm NaCl, 1.5 mm MgCl2, 5% (w/v) glycerol, 1% (w/v) OG, 2 mm benzamidine, 40 μm leupeptin, and 1 μm pepstatin A) to eliminate proteins bound non-specifically to the resin. Wild type CD36 was eluted using equilibration buffer plus 250 mm imidazole. The purification efficiency was visualized by SDS-PAGE stained with colloidal blue. An identical procedure was used for purification of non-glycosylated CD36 (CD36non-g) except 0.6% SDS substituted 2% OG in the solubilization buffer and 0.3% SDS replaced 1% OG in the equilibration and wash solutions. The eluted protein was concentrated using centrifugal devices with a 50-kDa cut off as directed (Amicon Ultra 15, Millipore). For use in mass spectrometry, ∼10 pmol of wild type CD36 was denatured at 100 °C for 10 min and deglycosylated using PNGase F for 1 h at 37 °C as directed (New England Biolabs). Approximately 10 pmol of purified wild type CD36 (pre and post-deglycosylation) were separated by SDS-PAGE and stained with colloidal blue. The protein bands were excised and digested with trypsin using MassPREP Station (Waters) for the liquid chromatography/tandem mass spectroscopy (LC/MS/MS) or BioRobot 3000 (Qiagen) for the Fourier transform ion cyclotron resonance (FT-ICR MS). Peptides were extracted using 0.1% formic acid, and the tryptic peptide mixture was analyzed by automated LC/MS/MS (CapLC, LC Packings, Q-ToF II, Waters) as described (38Gavin A.C. Bösche M. Krause R. Grandi P. Marzioch M. Bauer A. Schultz J. Rick J.M. Michon A.M. Cruciat C.M. Remor M. Höfert C. Schelder M. Brajenovic M. Ruffner H. Merino A. Klein K. Hudak M. Dickson D. Rudi T. Gnau V. Bauch A. Bastuck S. Huhse B. Leutwein C. Heurtier M.A. Copley R.R. Edelmann A. Querfurth E. Rybin V. Drewes G. Raida M. Bouwmeester T. Bork P. Seraphin B. Kuster B. Neubauer G. Superti-Furga G. Nature. 2002; 415: 141-147Crossref PubMed Scopus (3993) Google Scholar) or Fourier transform mass spectrometry (LTQ-FT hybrid linear trap/7-T FT-ICR mass spectrometer (Thermo Electron, Bremen, Germany)) as described (39Peterman S.M. Dufresne C.P. Horning S. J. Biomol. Tech. 2005; 16: 112-124PubMed Google Scholar). Data from LC/MS/MS and FT-ICR MS were analyzed in conjunction with the MSDB data base using the software tool Mascot (Matrix Services) and Sequest data base using Bioworks software (Thermo Scientific), respectively. After harvest, transiently transfected HEK293T cells were washed in FACS buffer (PBS and 1% fatty acid-free BSA) and resuspended at 1 × 107 cells per ml. A saturating concentration (2 μg) of mouse anti-CD36 mAb1258 (or mAb1955) was added to 50 μl of cells and incubated for 30 min at 4 °C (0.5 μg of monoclonal antibody 4E3 (DAKO) was substituted when staining for P-glycoprotein). The cells were centrifuged at 400 × g for 1 min at 4 °C and resuspended in 1 ml of FACS buffer. The wash step was repeated twice more, and the cells were resuspended in 50 μl of FACS buffer. A saturating concentration (4 μg) of goat, anti-mouse IgG secondary antibody conjugated to R-phycoerythrin (DAKO) was added to the cells and incubated in the dark for 30 min at 4 °C. The cells were recovered by centrifugation and washed as before and then resuspended in 400 μl of FACS buffer. During flow cytometry, 10,000 cells of normal size and granularity were analyzed for CD36–12His surface expression measuring R-phycoerythrin fluorescence (Ex 565 nm and Em 578 nm). The cell surface expression of CD36–12His was analyzed by FlowJo (Treestar). The cells typically exhibited a biphasic staining pattern, likely dependent on whether the individual cell was just about to, or had recently divided, before transfection. The heights of these peaks (reflecting cell number) relative to each other sometimes varied, but the fluorescence intensity of each peak (reflecting CD36 density) remained consistent relative to the positive and negative controls (wild type CD36 and CD36non-g, respectively). The maximal expression level of the receptor (the peak with the higher fluorescence intensity) was, therefore, gated, and the median was calculated and compared in all experiments. The expression level of mutant CD36 proteins was always compared with the expression levels of wild type and non-g proteins performed contemporaneously to control for minor variability in transfection efficiency on different days. -Fold reduction in expression was calculated by dividing the percentage surface expression of the wild type protein by the percent surface expression of the mutant. The synergy factor was calculated by dividing the -fold reduction of the multiple mutant by the product of the -fold reduction of the individual mutants, as described (40Klinkenberg L.G. Webb T. Zitomer R.S. Eukaryot. Cell. 2006; 5: 1007-1017Crossref PubMed Scopus (16) Google Scholar). For purified protein, 1 μg of wild type CD36–12His was added to Ni-NTA-coated plates (Qiagen) in 100 μl of protein binding buffer (20 mm Tris-HCl, pH 6.8, 150 mm NaCl, 1.5 mm MgCl2, 5% (v/v) glycerol, 0.5% (w/v) OG, 2 mm benzamidine, 40 μm leupeptin, and 2 μm pepstatin A) and bound at 4 °C overnight with gentle rocking. Unbound protein was removed, and each well was washed with 2 × 150 μl of ligand binding buffer 1 (LBB1; PBS, 1 mm MgCl2, 1 mm CaCl2, 0.5% (w/v) OG, 0.2% fatty acid-free BSA). Increasing concentrations of BODIPY Ac-LDL were added in LBB1 to a total volume of 100 μl and incubated at room temperature in the dark with gentle rocking for 2 h. Unbound ligand was removed, and the wells were washed with 3 × 200 μl of ice-cold wash buffer 1 (PBS, 1 mm MgCl2, 1 mm CaCl2, 0.5% fatty acid-free BSA). 100 μl of PBS was added per well before determining the bound fluorescence using a fluorescent plate reader (SpectraMax, Gemini EM, Molecular Devices, Ex 485 nm, Em 530 nm). Specific binding of Ac-LDL was calculated after subtraction of the level of BODIPY Ac-LDL bound non-specifically to empty wells. For the whole cell assay, 1 × 105 cells were seeded per well of polylysine-coated flat-bottomed 96-well plates and transfected with plasmid DNA, as described above. 48 h after transfection, the cells were washed with 3 × 200 μl wash buffer 2 (WB2; PBS, 1 mm MgCl2, 1 mm CaCl2), then 150 μl of block buffer (WB2 with 1% fatty acid-free BSA) was added to each well and incubated at room temperature for 45 min with gentle rocking. Increasing concentrations of BODIPY Ac-LDL were added in WB2 plus 0.2% fatty acid-free BSA to a total volume of 100 μl and incubated at 4 °C in the dark with gentle rocking for 2 h. Unbound ligand was removed, and the wells were washed with 3 × 200 μl of ice-cold WB2. 100 μl of PBS was added per well before determining the bound fluorescence as described above and calculation of the specific binding by subtraction of the level of BODIPY Ac-LDL bound non-specifically to mock-transfected cells. Data were analyzed using Graphpad Prism software Version 4.0, and saturation binding data were best fitted by Langmuir adsorption isotherm (Equation 1), which describes binding of ligand to a single class of binding site asB=Bmax×[L]Kd+[L](Eq. 1) where B is bound ligand (relative fluorescent units), [L] is concentration of ligand (μg/ml), and Kd is the concentration of ligand giving half-maximal binding and a measure of the affinity of ligand-receptor interaction. HEK293T cell lysates (50 μg) (untreated or treated with 1 unit of Endo H or 1 unit of PNGase F as directed (New England Biolabs)) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore). Western blots were probed with rat anti-CD36 primary mAb1955 (or mouse anti-P-glycoprotein primary C219) and rabbit anti-rat (or goat anti-mouse as appropriate) secondary antibody-conjugated to horseradish peroxidase (DAKO) before visualization by ECL chemiluminescent detection system (Amersham Biosciences) as directed. Using the consensus sequence N-X-Ser/Thr (where X is any amino acid except proline), asparagines 79, 102, 134, 163, 205, 220, 235, 247, 321 and 417 (henceforth designated N1—N10 for convenience) were identified as possible sites of modification. Mass spectrometric analysis of purified protein can be used to determine the occupancy of these sites; therefore, recombinant wild type CD36 was engineered with a 12 histidine, carboxyl-terminal tag and expressed in Sf21 insect cells using a baculoviral system. Proteins were solubilized in OG from insect cell membrane fractions, and the recombinant CD36 was purified